bismillah hirrahman nirraheem in the name of allah, the...
TRANSCRIPT
Bismillah Hirrahman Nirraheem
In the Name of Allah, the Most Gracious, the Most Merciful
All praises for Almighty Allah,
the most kind and cordial. He is ample and the best disposer of all affairs
(for us). I am showing my humble submission to the heart and
soul after The Holy Prophet Hazrat Muhammad
(sallallahualayhewassallum) whose life is an ideal pattern
for all of us .
Phytochemical Studies on the Chemical Constituents of Xanthium
strumarium Linn., Synthesis in addition Bioactivities of
2, 3-Diaminonaphthalenimidazole Derivatives
and Amides of Piperic Acid
Thesis submitted
for
The partial fulfillment of the Degree
of
DOCTOR OF PHILOSOPHY
in
Chemistry
by
AMINA SULTANA
Department of Chemistry
Federal Urdu University of Arts, Science and Technology
Karachi – 75260, Pakistan
2014
CERTIFICATE
Certified that Ms. Amina Sultana has carried out her research work on the topic
entitled Phytochemical Studies on the Chemical Constituents of Xanthium
strumarium Linn., Synthesis in addition Bioactivities of 2, 3-
Diaminonaphtalenimidazole Derivatives and Amides of Piperic Acid at
Department of Chemistry, Federal Urdu University of Arts, Science, and
Technology, Karachi, Pakistan, under the supervision of Dr. Aneela Wahab. Her
research work is original that has not been submitted to any other university.
Some publications have been earned from the course of this research work and the
all are acknowledged.
Dr. Aneela Wahab --------------------------- Assistant Professor
Department of Chemistry
Federal Urdu University, Karachi, Pakistan
Research Supervisor
Dr. Iffat Mahmood --------------------------- Chairperson
Department of Chemistry
Federal Urdu University, Karachi, Pakistan
Dedicated to my loving and caring
Parents whose prayers have
always been a great source of
Strength to me
Contents
Acknowledgement --------------------------------------------------------------------- 1
Summary --------------------------------------------------------------------------------- 3
KHULASA ------------------------------------------------------------------------------- 6
CHAPTER -1
Phytochemical Studies on the Chemical Constituents
of Xanthium strumarium Linn. ----------------------------------------------------- 10
1.0 Introduction -------------------------------------------------------------------------- 11
1.1. General Introduction --------------------------------------------------------------- 12
1.1.1. Classification ------------------------------------------------------------ 17
1.1.2. Introduction About Family Compositeae ---------------------------- 18
1.1.3. Introduction About Genus Xanthium --------------------------------- 20
1.1.4. General Description of Xanthium strumarium Linn. --------------- 21
1.1.4.1. Chemical Constituents of Xanthium strumarium Linn.---------- 21
1.1.4.2. Medicinal Importance of Xanthium strumarium Linn.----------- 22
1.1.4.3. Literature Review ----------------------------------------------------- 24
1.2. Present Work ----------------------------------------------------------------------- 53
1.3. Results and Discussion ------------------------------------------------------------ 55
1.3.1. Lupenyl acetate (1) ----------------------------------------------------- 56
1.3.2. Stigmasterol (2) --------------------------------------------------------- 59
1.3.3. β-Sitosterol (3) ------------------------------------------------------- 60
1.3.4. Palmitic acid (4) ---------------------------------------------------------61
1.3.5. β-Amyrin (5) ------------------------------------------------------------ 62
1.3.6. Oleanolic acid (6) ------------------------------------------------------- 63
1.3.7. β-Sitosterol-3-O-β-D-Glucopyranoside (7) ------------------------- 64
1.3.8. Ferulic acid (8) ---------------------------------------------------------- 65
1.3.9. Biological Activities ---------------------------------------------------- 66
1.3.9.1. In vitro Anti-bacterial Activity ------------------------------------- 66
1.3.9.2. In vitro Anti-fungal Activity ---------------------------------------- 68
1.3.9.3. In vitro Anti-oxidant Activity --------------------------------------- 70
1.4. Experimental ------------------------------------------------------------------------ 71
1.4.1. General Experimental -------------------------------------------------- 72
1.4.2. Collection of Plant Material ------------------------------------------- 72
1.4.3. Extraction and Isolation ------------------------------------------------ 72
1.4.4. Characterization of Compounds -------------------------------------- 79
1.4.4.1. Characterization of Lupenyl acetate (1) -------------------------- 79
1.4.4.2. Characterization of Stigmasterol (2) ------------------------------- 80
1.4.4.3. Characterization of β-Sitosterol (3) -------------------------------- 81
1.4.4.4. Characterization of Palmitic acid (4) ------------------------------ 82
1.4.4.5. Characterization of β-Amyrin (5) ---------------------------------- 83
1.4.4.6. Characterization of Oleanolic acid (6) ---------------------------- 84
1.4.4.7. Characterization of β-Sitosterol-3-O-β-D-
Glucopyranoside (7)-------------------------------------------------- 85
1.4.4.8. Characterization of Ferulic acid (8) ------------------------------- 86
1.5. References ------------------------------------------------------------------------- 87
CHAPTER-2
Synthesis in addition Bioactivities of 2, 3-Diaminonaphthalenimidazole
Derivatives----------------------------------------------------------------------------- 97
2.0. General Introduction For Chapter-2 and 3 ----------------------------------- 99
2.1. Introduction of Benzimidazole -------------------------------------------------- 104
2.1.1 Biological Importance ----------------------------------------------------- 104
2.2. Synthetic Approaches Towards Benzimidazole ------------------------------ 112
2.3. Results and Discussion ----------------------------------------------------------- 123
2.3.1. Chemistry ------------------------------------------------------------------- 124
2.3.2. General Method for the Synthesis of Compounds (65-99) ---------- 124
2.3.3. General Stucture Elucidation --------------------------------------------- 129
2.3.4. Biological Evaluation of Compounds (65-99) ------------------------- 131
2.3.4.1. In Vitro Tyrosinase Inhibitory Activity ----------------------------- 131
2.3.4.2. In Vitro Acetylcholinesterase and Butrylcholinesterase.
Inhibitory Activity ------------------------------------------------------ 133
2.3.4.3. In Vitro Urease Inhibitory Activity -------------------------------- 136
2.3.4.4. In Vitro Anti-bacterial Activity ------------------------------------- 138
2.3.4.5. In Vitro Anti-fungal Activity ---------------------------------------- 146
2.3.4.6. In Vitro Anti-oxidant Activity -------------------------------------- 149
2.3.5. Conclusion ------------------------------------------------------------------- 151
2.4. Experimental ------------------------------------------------------------------------ 152
2.4.1. General Experimental ------------------------------------------------------ 153
2.4.2. General Method for the Synthesis of Compounds (65-99) ----------- 153
2.4.2.1. 2-(1H-indol-3-yl)-1H-naphtho[2,3-d]imidazole (65) --------------- 154
2.4.2.2. 2-(4-ethoxy-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (66)-154
2.4.2.3. 4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (67) -------------------- 154
2.4.2.4. 2-methoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)
phenyl acetate (68) -------------------------------------------------------- 154
2.4.2.5. 2-(5-bromo-2-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (69)- 155
2.4.2.6. 2-(2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (70) ----- 155
2.4.2.7. 4-chloro-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (71) --------- 156
2.4.2.8. 2-(4-trifluoromethylphenyl)-1H-naphtho[2,3-d]imidazole (72) --- 156
2.4.2.9. 2-(4-nitrophenyl)-1H-naphtho[2,3-d]imidazole (73) --------------- 156
2.4.2.10. 2, 6-dimethoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (74) -157
2.4.2.11. 2-(3-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (75) -------- 157
2.4.2.12. 2-(2-fluoro-4-methoxyphenyl)-1H-naphtho[2,3-d]
imidazole (76)------------------------------------------------------------ 157
2.4.2.13. 2-methoxy-5-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (77) ----- 157
2.4.2.14. 2-(4-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (78) ------- 158
2.4.2.15. 2-(3-ethoxy-4-methoxyphenyl)-1H-naphtho[2,3-d]
imidazole (79)------------------------------------------------------------ 158
2.4.2.16. 4-(1H-naphtho[2,3-d]imidazol-2-yl)-3-nitrophenol (80) ---------- 158
2.4.2.17. 2-(thiophen-2-yl)-1H-naphtho[2,3-d]imidazole (81) --------------- 159
2.4.2.18. 2-(3, 4-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (82) ---- 159
2.4.2.19. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1,3-diol (83) ------- 159
2.4.2.20. 2-(2, 3, 4-trimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (84)-- 160
2.4.2.21. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1,2,3-triol (85) ----- 160
2.4.2.22. 2-(4-methylthiophenyl)-1H-naphtho[2,3-d]imidazole (86) ------- 160
2.4.2.23. 2-(2-nitrophenyl)-1H-naphtho[2,3-d]imidazole (87) -------------- 161
2.4.2.24. 2-(naphthalen-2-yl)-1H-naphtho[2,3-d]imidazole (88) ------------ 161
2.4.2.25. N, N-dimethyl-4-(1H-naphtho[2,3-d]imidazol-2-yl)aniline (89)-- 161
2.4.2.26. 3-bromo-6-methoxy-2-(1H-naphtho[2,3-d] imidazol-2-yl)
phenol (90) --------------------------------------------------------------- 162
2.4.2.27. 2-(2-bromo-4, 5-dimethoxyphenyl)-1H-naphtho [2,3-d]
imidazole (91) ----------------------------------------------------------- 162
2.4.2.28. 2-phenyl-1H-naphtho[2,3-d]imidazole (92) ------------------------- 162
2.4.2.29. 2-(2-ethoxyphenyl)-1H-naphtho[2,3-d]imidazole (93) ------------ 163
2.4.2.30. 2-(2, 3-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (94) ---- 163
2.4.2.31. 2-(4-bromo-2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]
imidazole (95) ---------------------------------------------------------- 163
2.4.2.32. 2-(3-bromo-4-methoxyphenyl)-1H-naphtho[2,3-d]
imidazole (96) ---------------------------------------------------------- 164
2.4.2.33. 2-(4-bromo-2-fluorophenyl)-1H-naphtho[2,3-d]
imidazole (97) ---------------------------------------------------------- 164
2.4.2.34. 2-(2-chloro-3-methoxyphenyl)-1H-naphtho[2,3-d]
imidazole (98) ---------------------------------------------------------- 164
2.4.2.35. 2-(3-bromophenyl)-1H-naphtho[2,3-d]imidazole (99) ------------ 165
2.5 References -------------------------------------------------------------------------- 166
CHAPTER-3
Synthesis and Bioactivities of Amides of Piperic Acid------------------------- 173
3.1. Introduction of Amides ----------------------------------------------------------- 175
3.1.1. Importance of Amides ----------------------------------------------------- 177
3.2. Synthetic Approaches Towards Amides of Piperic Acid -------------------- 182
3.3. Results and Discussion ----------------------------------------------------------- 186
3.3.1. Chemistry ---------------------------------------------------------------- 187
3.3.2. General Method for the Synthesis of Compounds (42-56) -------- 187
3.3.3. General Stucture Elucidation ----------------------------------------- 192
3.3.4. Biological Evaluation of Compounds (42-56) ---------------------- 194
3.3.4.1. In Vitro Anti-bacterial Activity ------------------------------------ 194
3.3.4.2. In Vitro Anti-fungal Activity --------------------------------------- 196
3.3.4.3. In Vitro Nematicidal Activity--------------------------------------- 198
3.3.4.4. In Vitro Anti-oxidant Activity-------------------------------------- 200
3.3.5. Conclusion--------------------------------------------------------------- 202
3.4. Experimental------------------------------------------------------------------------ 203
3.4.1. General Experimental-------------------------------------------------- 204
3.4.2. General Method for the Synthesis of Compounds (42-56)-------- 204
3.4.3. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic
acid (Piperic acid, 28) ---------------------------------------------- --- 205
3.4.3.1. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-
yl)penta-2,4-dien-1-one (42)------------------------------------- 206
3.4.3.2. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-morpholinopenta-
2,4-dien-1-one (43)------------------------------------------------- 206
3.4.3.3. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(4-methylpiperazin-
1-yl)penta-2,4-dien-1-one (44)------------------------------------- 206
3.4.3.4. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-
dienamide (45)------------------------------------------------------ 207
3.4.3.5. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenylpenta-
2,4-dienamide (46)-------------------------------------------------- 207
3.4.3.6. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-p-tolylpenta-
2,4-dienamide (47)-------------------------------------------------- 207
3.4.3.7. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-chlorophenyl)
penta-2,4-dienamide (48)------------------------------------------ 208
3.4.3.8. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)
penta-2,4-dienamide (49)------------------------------------------- 208
3.4.3.9. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-propylpenta-
2,4-dienamide (50)-------------------------------------------------- 208
3.4.3.10. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-mesitylpenta-
2,4-dienamide (51)-------------------------------------------------- 209
3.4.3.11. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methylpiperazin-
1-yl)penta-2,4-dienamide (52)------------------------------------ 209
3.4.3.12. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(pyrrolidine-1-yl)
penta-2,4-dien-1-one (53)------------------------------------------ 209
3.4.3.13. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-iodophenyl)
penta-2,4-dienamide (54)------------------------------------------ 210
3.4.3.14. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzylpenta-
2,4-dienamide (55)-------------------------------------------------- 210
3.4.3.15. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-methoxy-4-
methylphenyl)penta-2,4-dienamide (56)------------------------- 210
3.5. References -------------------------------------------------------------------------- 212
CHAPTER–4
Biological Activities Assays---------------------------------------------------------- 216
4.0. Introduction about Biological Activities----------------------------------- 218
4.1. Enzyme Inhibition Activity----------------------------------------- 218
4.2. Anti-microbial Activity---------------------------------------------- 220
4.3. Anti-oxidant Activity------------------------------------------------ 221
4.4. Nematicidal Activity------------------------------------------------- 222
4.5. Protocols--------------------------------------------------------------------------- 223
4.5.1. Enzyme Inhibition Assay---------------------------------------------- 224
4.5.1.1. In Vitro Tyrosinase Inhibitory Assay------------------------------ 224
4.5.1.2. In Vitro Acetylcholinesterase and Butrylcholinesterase
Inhibitory Assay------------------------------------------------------ 224
4.5.1.3. In Vitro Urease Inhibitory Assay------------------------------------ 225
4.5.2. In Vitro Anti-microbial Assay----------------------------------------- 226
4.5.2.1. In Vitro Anti-bacterial Assay---------------------------------------- 226
4.5.2.2. In Vitro Anti-fungal Assay------------------------------------------- 227
4.5.3. In Vitro Anti-oxidant Assay ------------------------------------------- 228
4.5.4. In Vitro Nematicidal Assay-------------------------------------------- 228
4.6. References -------------------------------------------------------------------------- 230
* Research Publications----------------------------------------------------------------- 234
1
Acknowledgement
At very first and foremost, I am much thankful to Almighty Allah, the most
beneficient and merciful who bestow me to accomplish my Ph.D. research work.
Limitless all respect to our beloved last Holy Prophet Hazrat Muhammad
(sallallahoalaihiwasallum) who shower his blessing during the whole research
period in deliberating upon things deeply.
I would like to express my heartfelt and profound acknowledgements to all those
people who helped me out to complete this research work.
I wish to express my high tribute to (Late) Prof.Dr.Salimuzzaman Siddiqui F.R.S.,
H.I. the founding director of H.E.J. Research Institute of Chemistry, for
establishing a renowned institute of chemistry. I am also grateful to Prof.Dr.Atta-
ur-Rahman F.R.S., N.I., S.I., T.I., the chief Patron, International Center for Chemical and
Biological Sciences, University of Karachi for his dedication to the developments
in the field of chemical Sciences. I am thankful to the director of H.E.J Research
Institute, Prof. Dr. M. Iqbal Choudhary S.I., T.I., for providing research facilities in
this institute.
I would like to express my special thanks to my co-supervisor, Prof. Dr. Khalid
Muhammad Khan T.I., H.E.J. Research Institute of Chemistry, University of
Karachi, for his active co-operation, prompt and tactful suggestion and the most
valuable, his ever ready helping attitude that was a motivating factor in finalizing
this study.
I have great pleasure to express my sincere and affectionate gratitude to my
supervisor Dr. AneelaWahab, Assistant Professor, for her skilful guidance, keen
interest and great co-operation at each and every step of this arduous task. Her
encouraging attitude made me self –confident and kept me on the right track.
I am thankful to Dr.Iffat Mahmood (chairperson), Head of the Department of
Chemistry (Federal Urdu University of Arts, Science, and Technology, Karachi)
for providing collaboration with H.E.J. Institute.
I am very grateful to Prof. Dr. Abdul Malik S.I., for his expert guidance to
materialize this work.
2
It is my pleasure to thanks and my sincere appreciation to my colleagues, Dr.
Ghulam Farid, scientific officer, PCSIR Laboratories Complex, Karachi, Mr.
Shafqat Hussain and Dr. Farzana Naz for their valuable, kind and fruitful
suggestions throughout the course of present tenure.
I am pleased to express my sincere regards and appreciation to my dear friend
Miss Shahla Noureen for her bonafide and whole hearted help, prayers, moral
support and nice company.
This dissertation required an extensive bioassay work. I thankfully acknowledge
Dr. Mehrin Lateef, scientific officer, PCSIR Laboratories Complex, Karachi, for
enzyme inhibition screening. Mr. Sikandar Khan Sherwani for nematicidal and
anti-oxidant activities and Dr. Ayesha Irshad, Dr. Saima Faraz and Dr. Zeba
Parveen for the determination of anti- bacterial and anti- fungal activities.
I am also thankful to all technical and non-technical staff members of the institute
for their help and sincere co-operation during this research studies.
My extreme gratitude is also for all my teachers from primary school to university
whom privilege me to submit a Ph.D. dissertation.
Words are restrain to express my immense gratitude to my respected , gracious
parents , my beloved sisters (especially Nasima Nasim for her sincere co-
operation and sacrifice) and brothers for their encouragement , motivation ,
prayers and humble co-operation throughout my this research work.
Amina Sultana
3
SUMMARY
This dissertation has been divided into four chapters. Each chapter has its own
numbering of compounds and references. The general introduction describes the
importance of natural products and the drugs based on them.
The chapter 1 deals with the phytochemical studies on the chemical constituents
of Xanthium strumarium Linn. The introduction provides a review of the earlier
contributions made in the chemistry and pharmacology of the genus Xanthium and
a brief account of the present work.
Studies undertaken on different fractions of methanolic extract (XS-HX, XS-DC,
XS-EA, XS-BU and XS-ME) of the air dried aerial parts of X. Strumarium Linn.
showed weak to moderate antibacterial and weak antioxidant activity except ethyl
acetate fraction (XS-EA) which exhibited moderate to high antibacterial and
antifungal activity (Table-2, 3) while significant antioxidant activity (Table-4)
was observed among all fractions. Studies undertaken on the bioactive
ethylacetate fractions led to the isolation and structure elucidation of eight known
compounds. The known compound (1) is reported for the first time from X.
strumarium Linn. The constituents obtained are listed below.
I. Lupenyl acetate (1)
II. Stigmasterol (2)
III. β-Sitosterol (3)
IV. Palmitic acid (4)
V. β-Amyrin (5)
VI. Oleanolic acid (6)
VII. β-Sitosterol-3-O-β-D- Glucopyranoside (7)
VIII. Ferulic acid (8)
4
The structure of all the isolated compounds have been determined through various
spectroscopic techniques such as, IR, EIMS, HR-EIMS, 1H-NMR,
13C-NMR, 2D-
NMR and also by comparison of their spectral data with those reported in
literature.
Chapter 2 is about characterization and bioassay screening of thirty five (35)
synthesized derivatives of 2, 3-diaminonaphthalenimidazole (65-99). Out of these
thirty five naphthalenimidazoles, twenty six (26) (65, 66, 68, 69, 70, 72, 74, 75,
76, 77, 78, 79, 80, 82, 84, 85, 86, 88, 90, 91, 93, 94, 95, 96, 97, 98) are newly
synthesized compounds. All synthesized derivatives showed interesting in vitro
enzyme inhibitory (urease, tyrosinase, acetylcholinesterase and
butrylcholinesterase inhibitory), antimicrobial and antioxidant activities. Two
compounds 81 and 85 revealed potent in vitro tyrosinase inhibitory activity. On
the other hand compounds 65, 66, 68, 69, 71, 79, 88 and 94 were found
moderately active for this activity. When tested for their in vitro
butrylcholinesterase inhibitory activity, three compounds 65, 66 and 79 exhibited
good activity while compounds 67, 81, 82 and 89 showed moderate
butrylcholinesterase inhibitory activity but all synthesized compounds were found
inactive for acetylcholinesterase inhibitory activity. In urease inhibitory activity
two compounds 71 and 90 revealed good activity while moderate activity was
observed in compounds 65, 66, 68, 81 and 82. All synthesized derivatives when
screened for their anti-microbial activity, only two compounds 90 and 92 were
found exhibiting remarkable activity against bacterial strains B. cereus, B. subtilis,
S. epidermidis, S. paratyphi A, Enterobacter and S.dysenteriae. Significant
activity against Enterobacter and S.dysenteriae was displayed by 99 and moderate
activity was found in compounds 65, 74, 81, 82, 85 and 94 against various tested
bacterial strains. All synthesized compounds showed weak antifungal activity. For
in vitro antioxidant activity, the compounds 65, 68, 77, 90, and 99 revealed
promising whereas compounds 79, 82, 85 and 95 showed good antioxidant
activity.
5
Chapter 3 describes the synthesis, structure elucidation and biological activity of
fifteen (15) amides of piperic acid (42-56). Five compounds (47, 51, 52, 54, 56)
are new amides. All the synthesized derivatives were evaluated for their in vitro
anti-microbial, nematicidal and anti-oxidant activity. In the case of antimicrobial
activity compound 54 was found the most active against all applied bacterial
strains except S. pneumoniae, compound 49 showed excellent activity for P.
vulgaris and 53 was good against P. stutzeri where as compound 44, 46, 47 and
48 showed good activity against P. aeruginosa. It was determined that compound
50 was active against S. aureus, P. stutzeri and P. aeruginosa. Compound 52
showed good activity against P. aeruginosa and E. coli whereas compound 56
exhibited activity against E. coli only. It was observed that among all the
synthesized amides only compound 54 showed antifungal activity against all
applied fungal strains. When screened for nematicidal activity compounds 42, 43,
45, 47, 52 and 56 were found possessing excellent nematicidal activity against
root-knot nematode, Meloidogyne incognita, where as compounds 44, 50 and 54
have significant mortality rate. During antioxidant testing three compounds 44, 49
and 51 showed significant and two compounds 46 and 54 showed moderate DPPH
radical scavenging property.
Chapter 4 deals about introduction of biological activities and all the protocols
used to determine the inhibitory potential of all fractions and synthesized
compounds.
6
Urdu Kholasa
7
8
9
10
CHAPTER-1
Phytochemical Studies on the Chemical
Constituents of Xanthium strumarium
Linn.
11
1.0. Introduction
12
General Introduction
The chemical compounds which derived from living organisms (plants,
animals, insects, and microorganisms) are known as natural products. From the
early history, human beings have been attracted in natural products for their basic
needs (food, shelter, clothing and not least medicines). Plants have formed the
basis of traditional medicine system for thousands of years [1]
Our holy religion ISLAM has superb contribution to the development of
therapy based on the doctrines of AL QURAN and SUNNAH. These two define
the rules for hygiene and sound diet as TIBB AL NABI. The medicinal properties
of date and honey have been described in HOLY QURAN where as our beloved
prophet HAZART MUHAMMAD (sallallahu alaiyhi wassallam) quoted along
with other plants the advantages of nigella sativa and crotalaria juncea [2].
The compounds isolated from plants and even insects had been used in
traditional medicine as hypolipidemic, antiplatelet, antitumor, or immune-
stimulating agents or for the treatment of cardiovascular diseases and cancers, for
thousand of years in China, India, Egypt, and Greece. Many phytochemists have
been formulated healing creams and liniments from plant extracts also. Several
isolated phytochemicals have antimicrobial activity and can also either inhibit or
stimulate the activity of certain enzymes. The volatile essential oils isolated from
herbs and spices suppress cholesterol synthesis and tumor growth [3-14].
This plant-based medication is continuously playing an essential role in
health care. We cannot ignore the importance of plant-derived drugs such as
artemisinin (a sesquiterpene), plant derived alkaloids like morphine, quinine,
digitalis, atropine, reserpine, vincristine, vinblastine, ajmaline and taxol (a
diterpene) for the treatment of malaria, typhoid, hypertension, heart problems, and
certain kinds of cancer [3-14].
As herbal medicine is based on the principle that plants contain
natural substances that can uphold health and lighten illness with no or less side
effects, people are usually interest in the use of herbal remedies over life-
threatening medicines. Due to excess applications, the natural products have
gained major importance in various aspects of human venture especially in health
care [3-14].
13
It has been predicted by the World Health Organization that about 80% of
the world population trust on traditional medicines for their basic health care. It is
apparent in different countries of Asia where phytomedicine is generally under
practice. In short, plants have a significant role in caring human health, improving
their quality of life and also serving humans well with valuable components of
seasonings, beverages, cosmetics and dyes [15].
The physiologically active constituents of medicinal plants have been
studying right through from the development of organic chemistry. It has been
estimated that about 40% of drugs have natural origin. A number of screening
protocols are applied for bioactivity of compounds to led new drugs. Natural
compounds also show an ecological role in maintaining interaction between
plants, microorganisms, insects and animals. They may be defensive, attractants
and pheromones. Chemotaxonomy is another motive for examining the plant
constituents. Phytochemical assessment can reveal natural products as a
“markers” for botanical and evolutionary relationships [16].
Despite the fact that thousands of natural compounds have been isolated,
only less than 100 of them with defined structure are in use all over the world.
Actually the plant material comprises of many chemical components that have
therapeutic effects. These compounds are used for remedy from various ailments.
In view of the above mentioned deliberations, organized efforts have
been done by many researchers in subcontinent Indo-Pakistan to study pharmaco-
chemical properties of physiologically active plant components. Purity of isolated
compounds is highly necessary to investigate their structures, formation, uses and
purpose. Researchers in this respect have been fully facilitated by modern
physical methods of isolation and spectroscopic techniques for structural studies.
Particular concentration is given to determine structure and activity correlation in
physiologically active compounds. These studies have served two purposes, first
to bring new medicines to facilitate theraputic leads and secondly to synthesise
drugs via viable high yield route. Moreover the functional variations in the basic
skeleton of drug have brought to enhance its bioactivity [17].
Taking these facts into account, the work presented in chapter-1 of the present
doctoral dissertation entitled “Phytochemical Studies on the Chemical
14
Constituents of Xanthium strumarium Linn” is related to the brief introduction
about family and genus, review of earlier researchers on the chemical constituents,
their pharmacological significance and uses in indigenous system of medicine.
This is followed by a brief discussion of present work with reference to the
isolation and structure elucidation of isolated 8 known compounds, one of which
was isolated for the first time from this genus. Further the anti-bacterial, anti-
fungal activity along with their MIC values and anti-oxidant activity with EC50 of
different fractions of methanolic extract is also included.
15
References
[1] K. T. Farrell, “Spices, condiments and seasonings”, C. T. Westport, AVI
Publishing Company, p. 17 (1985).
[2] Imam Shamsuddin Muhammad Bin Abi Bkr Ibnulqasim Aljozia, “Tib-e-
Nabwi (S.A.W.W.)” Hakim Aziz ur Rehman Aazami (translator), Muktaba
Muhammadia, Pakistan. www.kitabosunnat.com.
[3] T. Larkin, FDA Consum, 17, 4 (1983).
[4] T. G. Saxe, Am. Fam. Physician, 35, 135 (1987).
[5] I. Nielsen and R. S. Pederson, Lancet, 1, 1305, (1984).
[6] N. Mostefa-Kara, A. Pauwels, E. Pines, M. Biour and V. G. Levy, Lancet,
340, 674 (1992).
[7] T. Y. K.Chan, J.C.N. Chan, B. Tanlinson and J.A. Critchley, Lancet, 342,
1532 (1993).
[8] Encyclopedia Britannica, Halen Hemingway Benton, 9, 1043 (1974) .
[9] J. Bruneton, in “Pharmacognosy, phytochemistry, medicinal plants”.
Hatton, C. K., translator. Paris: Lavoisier Publishers (1995).
[10] G. M.Cragg, S. A. Schepartz, M.Suffness and M. R. Grever, J. Nat. Prod.,
56, 1657 (1993).
[11] W.A. Niering and N. C. Olmstead, in “The Audubon Society field guide to
North American wildflowers, eastern region”. Knopf, A. A., eds., New
York (1979).
[12] J. Mann, “Murder, Magic and Medicine”, Oxford University Press (1994).
[13] K. McNutt, Nutr. Today, 30, 218 (1995).
[14] M.J. Dew, B.K. Evans and J. Rhodes, Br. J. Clin. Pract, 38, 394 (1984).
[15] G. Bodeker and F. Kronenberg, Am J Public Health, 92(10), 1582 (2002)
[16] D.M. Eisenberg, R.C. Kessler, C. Foster, F.E. Norlock, D.R. Calkins and T.
L. Delbanco, N. Engl. J. Med., 328, 246 (1993).
[17] http://www.fas.org/nuke/guide/pakistan/contractor/hejric.htm
16
Xanthium strumarium Linn.
17
1.1.1. Classification*
Kingdom------------------------------------ Plantae (plants)
Subkingdom -------------------------------- Tracheobionta (vascular plants)
Super division ---------------------------- Spermatophyta (seed plants)
Division ------------------------------------ Magnoliophyta (flowering plants)
Class ---------------------------------------- Magnoliopsida (dicotyledons)
Subclass ------------------------------------ Asteridae
Order --------------------------------------- Asterales
Family ------------------------------------- Asteraceae (aster family) or
Compositeae
Genus -------------------------------------- Xanthium L. (cocklebur)
Species ------------------------------------ Xanthium strumarium Linn
Common name -------------------------- Chotagokhru (rough cocklebur)
Trade name ------------------------------ Cocklebur or Burweed
* Natural Resources Conservation Service United State Department of Agriculture
(USDA)
18
1.1.2. Introduction About Family Compositeae
The plant Xanthium strumarium Linn, commonly known as Chotagokhru or
Chotadhatura in Hindi, the trade name is Cocklebur or Bur-weed, belongs to the
sunflower family, commonly known as Compositeae. It is so called due to the
composite flowers on a capitulum, which is their main feature [1]. The
compositeae family members resemble to ‘stars’ in appearance, so these are also
known as Asteraceae, the name comes from Greek term means ‘star’ [2].
The compositeae is the largest family of the angiosperms (flowering plants). It
comprises of about 950 genera and more than 20,000 species. The members of
this family are distributed worldwide in each and every possible habitat. They are
usually found in tropical and in cold arctic alpine area in waste places and along
river banks. They show every form of body known for plants, in Indo-pak the
family is represented by many genera such as chrysanthemum, helianthus,
xanthium, zinnia and others [3].
The plants in compositeae are herbs, shrubs and few of the stature of trees.
Climbers are also reported. Some of the members are xerophytes and hydrophytes.
The root is tapped, branched, sometimes tuberously thickened to store reserved
food (e.g., Helianthus tuberosus). Stem is soft, erect, woody, very rarely climbing.
Most of the plants possess milky or watery, bitter, resinous juice. The leaves are
radical or alternate, simple or compound, of various shapes, hairy, rough surface
exstipulate, some of them contain oil ducts and latex (e.g., sonchus). The
inflorescence racemose comprises of a capitulum with many sessile flowers,
gathered on a common raceptical surrounded by an involucres of bracts. The
florates may be bisexual, unisexual or asexual. The flowers head may be ligulate,
zygomorphic, actinomorphic and epigamous. Disc floret is usually unisexual
while ray floret is either female or sterile. Calyx rudimentary, modified to scaly,
hairy pappus which help in dispersal of fruit. Corolla five, gamopetalous, of
various colours, sometime swollen due to presence of nectar. Androecium
composed of five stamens, epipetalous, free and separate filaments, anthers are
united to a tube (syngenecious), two- celled and superior. Gynoecium two-
carpelled, inferior ovary with one anatropus basal ovule, style simple, stigma two,
19
bifid with many hairs. Fruit is cypsela (single seed fruit). Seed is exalbuminous
with straight embryo [1, 3].
Phytochemical studies of its members have revealed the presence of a number of
class of compounds including terpenoids, flavonoids, alkaloids, steroids,
coumarines, quinones, saponines, glycosides, volatile oils and amino acids [4, 5,
6]. The extracts as well as compounds isolated from the members of compositae
possess pharmacological activities such as anti-HIV, hepatoprotective, cytotoxic
[4], hypoglycemic [5], analgesic, anti-inflammatory [7], anti-bacterial [8], anti-
oxidant [9], anti-cancer, anti-malarial and insecticidal activities [10], cytotoxicity
towards human cancer line cells [11], antifungal activity [12].
1.1.2.1. Economic Value of Family Compositeae
The family Asteraceae has wide spread use in society. Many members of the
family are ornamental plants for their flowers such as chicory, chrysanthemum,
dandelion, daisy, dahlia, marigold, santolina, solidago and zinnia. Some plants are
used as food like artichoke, endive, lettuce, sasifi in addition of safflower,
sunflower and niger seeds which are good sources of oil. Most of the plants of
compositeae are medically important, for example, artemisia (anti-malarial),
calendula (wound healing, anti-spasmodic), echinacea (medicinal tea), chamomile
(herbal tea). The family members of this family have been used for industrial use
like pulicaria and tanacetum species (insecticides), tagetes (orange dye),
parthenium (rubber), bertoni (sweetner), marigold oil (alcoholic beverage
flavoring and cigarette) and wild silver oak and wild camphor ( building material)
[13, 14].
20
1.1.3. Introduction About Genus Xanthium
The genus Xanthium belongs to tribe Ambrosieae of family Compositeae [15].
The members of this genus are annual, tall, branched, coarse and rough herbs.
Their leaves are alternate, rough, palmately dentate. Head monoecious, axillary,
male more apical, sterile, numerous flowers with tubular, 5-toothed corollas,
cylindrical scaly receptical, short involucres, style unarmed and dilated slightly at
the apex, achenes rudimentary; female fetile, apetalous, 2-flowered, involucral
bracts, herbaceous, 2-celled utricle, corolla absent. Achenes enclosed in enlarged
involucres, style branched, pappus absent, anthers and filament free and distinct
[16, 17, 18, 19].
The members of this genus are distributed in nearly all tropical and temperate
regions of the world [20]. The genus Xanthium is represented by 25 speceis, all
are of American origin [21]. Out of these X. macrocarpum, X. strumarium, X.
spinosum, and X. sibiricum are also found in Pakistan and India [16, 17, 22]. The
species of Xanthium have been used as traditional herbal medicine in oriental
countries for the treatment of nasal sinusitis, headache, urticaria, arthritis [23, 24],
fever, scrofula, herpes and cancer [25, 26]. X. spinosum Linn. and X. strumarium
Linn. are of medicinal use in Europe, North America and Brazil, X. canadense
Mill is used in North America and Brazil, X. strumarium is medicinally important
in China, India and Malaysia [27, 28].
21
1.1.4. General Description of Xanthium strumarium Linn.
Xanthium strumarium Linn, a member of Compositeae family, is an annual, erect,
rough, and coarse herb or under shrub, near about 1.5 m tall [29]. Xanthium is the
derivative of Greek word “xanthos” which means yellow and strumarium refers
“cushion like swelling” because the seedpods when ripen turn from green to
yellow. The reason behind its common name chotagokhru is the shape of its fruit
that resemble the cow’s toe (chota- small; go-cow; khuru-toe) [28]. The weed
probably of American origin and found in almost all of the hotter parts of Eurasia
including up to the height of 5000 ft. in the Himalayas region [30]. It is a common
roadside weed found abundantly in plain to 8000 ft. in almost all the four
provinces of Pakistan and in Kashmir [31, 32].
The stem is hispid, slightly branched, erect, stout, rough and hairy. The leaves are
three lobed, toothed, triangular heart shaped, broad, glandular with a long leaf
stalk. Capitulum in terminal and axillary recemes, monoeciou, sessile having
numerous white or green, male and female flowers on separate heads of the same
plant. Sterile (Male) heads uppermost on stem with many flowers which are
globose, hooked bristle, 5-toothed, tubular corolla. Fertile (Female) heads axillary,
2-flowered having no pappus and corolla. Fruits are involucres, ovoid or oblong,
covered with hooked bristles, diverging ending, two strong and hooked beaks,
hard and 2-celled. Achenes ovoid enclosed in a thick, tough and hard bracts
covered with hooked spines [29, 32, 33, 34]. Flowering and fruiting period ranges
from July to October i.e. after rainy season [33, 35]. The weed is propagated by
seed [29, 26, 36].
1.1.4.1. Chemical Constituents of Xanthium strumarium Linn.
The herb analysis reveals beneficial values for crude protein 17.6% ; carbohydrate
31.6% ; cellulose 12.3% ; lignin 12.5% ; nitrogen 2.82% ; phosphorous 1.13% ;
potassium 2.42% ; calcium 3.15%; magnesium 1.40% ; sodium 0.47% and
sulphur 0.61% [37]. The weed is cooked as vegetable in China while in Assam the
floral tops and the leaves just below it are edible after boiling [28, 37, 38]. The
aerial parts contain a mixture of alkaloids xanthinin, xanthatin [36], xanthumin,
22
xanthalol and isoxanthalol [37]. Stem is a source of fuel [37]. Leaves possesses
compounds from class of phytosterols, sesquiterpenes, sesquiterpene lactones like
β-sitosterol, isoxanthanol along with xanthinin and xanthumin, d-limonene, d-
carveol, l-α-ionone, ρ-cymene, β-caryophyllene, isohexacosane, chlorobutanol,
stearyl alcohol, palmitic acid. Leaves are also rich in ascorbic acid and iodine
[39]. Leaves contain a substance due to which they are applied to dye yellow [40].
Fruits have glucose, fructose, sucrose, β-sitosterol, γ-sitosterol, stigmasterol,
strumaroside (glucoside of β-sitosterol), phosphatides, KNO3 and is a good source
of vitamin C [37, 39]. The seeds contain oxalic acid, xanthostrumarine and its
glucoside, iodine [33, 37], toxic agents hydroquinone and choline due to which
they cause poisoning to livestock [37]. The seeds comprises of a semidrying oil of
the same taste like different vegetable oils, it is not only edible but also of
industrial application for soap, paints and as a drying agents [36, 41]. The seed oil
is a combination of saturate fatty acids such as capric, lauric, myristic, palmitic
acids and unsaturated fatty acids oleic, linoleic, stearic acids, behenic [37]. The
cake obtained after extraction of oil is rapidly nitrifying hence may be good
fertilizer [37, 41]. The powdered shell is utilized to make activated carbon [41].
The roots comprises of phytosterols, n-heptacosanol, 3, 4-dihydroxycinnamic acid
[39]. The whole plant can accumulate trace elements (Na, K, Ca, Cl) [42] and
heavy metals (Cd, Cr, Cu, Mn, Ni, Pd) in different concentrations in plant body
parts [43, 44].
1.1.4.2. Medicinal Importance of Xanthium strumarium Linn.
The whole plant is of great importance in medicinal uses, the roots, leaves, fruits
and seed oil all parts are utilized in herbal medications in China, India, Malaysia
[36]. Even though the weed is supposed to be poisonous, the harmful and toxic
substances are washed off by washing and boiling [37]. The herb is astringent,
diaphoretic, diuretic, emollient, sialogogue, sudorific [33]. It is used to treat
mouth ulcer and toothache [32]. The aerial parts composed of substances that
exhibit anti-malarial activity [29], xanthinin is an anti-bacterial agent, xanthumin
is a CNS depressant and also shows anti-bacterial activity [29, 37], xanthatin
isolated from resin is a cytotoxic compound [39] and a hypoglycemic agent,
23
carboxyatractyloside exists in the weed [29]. The leaves exhibit characteristic
properties of astringent, antisyphilitic, diuretic [37]. Their decoction is useful in
scrofula, herpes and venereal sores [39, 41], an extract of leaves with honey is
recommended in cough and fever [29], pounded leaves are applied on ulcer and
for the remedy of snake bite [40, 45], their black pepper and sugar juice is
suggested to cure blood dysentery [45], cocklebur tea has been used to cure fever
[40]. The fruit contains glucoside of β-sitosterol which shows anti-inflammatory
activity. It is given for the relief from kidney pain, scurvy, malaria, small-pox,
recommended for hormonal regulation, for the treatment of urinogenital problems
[41, 45]. The fruit is demulcent and cooling, its ash is applied on sores on lips and
mucous membrane of mouth [33, 37]. The seed composed of anti-inflammatory
agents hence are used to treat inflammatory swelling [37], seed oil is used to treat
bladder infections, herpes, erysipelas, its massage beneficial in rheumatism, seed
paste is useful on wounds [40, 45]. The root gives a bitter tonic which is used
against cancer and scrofula [29]. It possesses anti-tumor and abortifacient
properties [39], their extract is applied for the treatment of ulcer, boils, abscesses,
pulmonary disorders [41], a decoction prepared with sugar is better for diarrhea
[45].
24
1.1.4.3. Literature Review
In the year 1920, B.R. Leland observed the cold-pressed oil do not rancid after 6
months when keep in a cold dark place, he also determined 30.69% of kernel of
burs of X. strumarium Linn [46].
A. Sado in 1937, found the aqueous extract of stem and leaves of X. struarium L.
contained a substance which is weakly toxic to nerves and muscles [47].
I. C. Chopra and co-workers reported the physiologically inactivity of soluble
glucoside of said species in 1946 [48].
Isamu Numato in 1951 obtained 115 – 131 mg % ascorbic acid (vit. C ) from
leaves of X. strumarium L. in addition of other weeds [49].
In the same year, C.V.N. Rao reported pet.ether extract analysis of seeds of X.
strumarium L. composed of oil, 31.5%; crude protein, 29.5%; crude fiber, 10.4%;
carbohydrate, 22.8% and ash, 5.8% [50].
In 1953 R.C. Shrivastava suggested a method to remove hard covering of X.
strumarium L. seed, also calculated its cake value , water, 7.93% ; minerals, 7.06
%; fiber, 2.46 % ; and carbohydrate, 22.11% [51].
In 1954 C.V.N. Rao with his fellow P.D. Kebra analysed seeds composition of the
specie by Mc Cool Pulveriser, found occurance of oleic acid, linoleic acid and
isolinoleic acid [52].
E.F. Leonova and co-workers in 1957 determined the presence of iodine in good
amount in all parts of the said plant [53].
J.R. Plourde and J.A. Mockle in 1960 reported the pet.ether fraction of the fruit
and leaves of Cocklebur composed of viscous oil, lactone and a flavones
derivative. The lactone, C17H22O5, m.p. 121-122º, hydrogenolysed to C15H24O3
and upon deacetylation gives C15H18O3 [54].
D.S. Bhakuni and co-workers in 1961 investigate pet. ether fraction of fruits of
Cocklebur for fattyacids. They found palmitic, stearic, linoleic and oleic acids
whereas β-sitosterol, γ-sitosterol, ϵ-sitosterol and ceryl alcohol from unsaponified
portion. From acetone fraction isolated strumaroside, m.p. 290-292º in addition of
glucose, fructose, amino acid and aqueous extract contained sucrose, aspartic,
fumaric, succinic and tartaric acids [55].
25
Itsuo Nishioka identified stigmasterol by gas chromatography of methanol extract
of fruit of X. strumarium L. in 1965 [56].
In the year 1966, T. Takeo and his group identified a mixture of phytosterols,
stigmasterol, campesterol and β-sitosterol in the methanolic extract of X.
strumarium seeds [57].
In the same year Hitoshi Minato and Isao Horibe found a stereoisomer of
xanthinin, which is a sesquiterpene lactone from the aerial parts of X. strumarium
L. They suggested it xanthumin after elucidating the structure by NMR
spectroscopy and derivatisation to iodoform , m.p. 100- 101º with molecular
formula C17H22O5 [58].
M.M. Pashchenko and G.P. Pivenko, in the same year, isolated xanthatin from
chloroform extract of aerial parts of Burweed, in addition to this, they also
obtained a sesquiterpene lactone, xanthinin, m.p. 110- 120°, turned red in HCl and
yellow in base from Et2O extract of powdered cocklebur [59, 60].
The same author in the year 1967 reported a flavonoid, m.p. 209-211° as 8-(Δ3-
isopentenyl)- 5, 7, 3', 4
'- tetrahydroxy flavone from ethanol extract of Bur weed
[61].
Within the same year, Hitoshi Minato identified, xanthumin from acetone fraction
of aerial parts of cocklebur [62].
S.M. Khafagy and fellows isolated xanthinin, choline and two new lactones,
xanthanol and isoxanthanol from X. strumarium L. in 1974 [63].
In 1975 C. Mc Millan and group determined variant combinations of
sesquiterpene lactones including xanthinin, xanthanol, xanthatin, xanthumin,
xanthumanol, deactoxyxanthumin, xanthinosin and tomentosin in a wild
population of X. strumarium [64].
A.S. Chawla and fellows in 1976 studied pet.ether soluble component of fruit of
bur weed contained n-alkanes, n-alkanols and a mixture of sitosterol, stigmasterol
and campesterol [65].
In the same year, S.M. Khafagy and group studied different fractions of the same
specie they found xanthinin, from non- saponifible portion of petroleum extract a
triterpene alcohol, m. p. 211- 213°, C30H50O2, a crystalline triterpene alcohol,
C30H50O5, m. p. 274- 276° and a phytosterol C28H48O2, m.p. 146-148° [66].
26
Xanthatin
27
28
29
N.P.S. Bisht and R. Singh in 1977 isolated from the leaves of X. strumarium
palmitic acid, β-sitosterol, ϵ- sitosterol, stearyl acohol, isohexacosane in addition
of chlorobutanol which was obtained first time from a natural source [67].
In 1978 same authors found oleic acid and a C-24 epimer of stigmasterol by the
techniques of IR, NMR and Mass spectroscopy as poriferasterol from pet.ether
extract. They also reported 3, 4-dihydroxycinnamic acid and β- sitosterol- D-
glucoside from its leaves extract [68].
In the same year R.G. Patel and V.S. Patel observed the seed oil of the burweed
on heating at 280 for 8 hr under CO2 polymerised and became so viscous that can
be used as drying oil suitable for coating [69].
P. Debetto in 1978 discussed with 12 references the presence of atractyloside in X.
strumarium along with in other plants [70].
N.P.S. Bisht and R. Singh in the same year isolated β-sitosterol, β-sitosterol-D-
glucose, stigmasterol, heptacosanol, 3, 4-dihydroxycinnamic acid, KNO3 and
K2SO4 from the extract of stem of cocklebur [71].
The group of J.R. Cole in 1980 isolated and identified carboxyatractyloside. The
structure of the compound was determined by spectroscopic analysis and a
chemical identity with an authentic sample [72].
In 1982 E. Naidenova and companions isolated xanthatin from aqueous extract of
leaves of X. strumarium in addition of two sesquiterpene lactones [73].
In the same year H.G. Cutler and J.P. Cole identified carboxyatractyloside as a
plant growth inhibitor, isolated from the said specie [74].
In 1985 A. Harada and co-workers isolated xanthatin from burweed and found it
as an anti-attaching repellent against blue mussel [75].
30
31
32
J. Molina-Torres and fellows observed in 1991 the accumulation of α-tocopherol
is associated with the life of leaf while γ-tocopherol present throughout the age of
leaf of X. strumarium Linn [76].
Agata Isao and co-workers in 1993 from fruit of X. strumarium 3,5-di- O-
caffeoylquinic acid accompanied by a new polyphenol, 1,3,5- tri-O-caffeoylquinic
acid [77].
M. S. Malik and fellows in same year reported a new sesquitrpene lactone from
the extract of aerial parts of the same plant as 2-hydroxytomentosin -1β, 5β –
epoxide [78].
In 1994 R. P. Rao analyzed the seed of X. strumarium as a good source of protein
and fat [79].
33
34
V.K. Saxena and fellows in 1994 isolated β–sitosterol, stigmasterol, β-amyrin and
octacosanol and a new xanthanolide, 6β,9β,-dihydroxy-8-epixanthatin from leaves
extract of cocklebur [80].
J.A. Marco and companions reported xanthanol, isoxanthanol and their C-4
epimer from burweed extract within the same year [81].
V.K. Saxena and M. Mishra in 1995 identified a new xanthanolide,
chloroxanthanolide in addition to 8-epi-xanthatin-1β, 5β-epoxide, 2-
hydroxytomentosin and xanthumanol from the extract of same species [82].
P.S Joshi and co-workers in 1997 reported 4 active xanthanolides from acetone
extract of X. strumarium, tomentosin, xanthumin, 8-epi-xanthatin and 8-epi-
xanthatin-1β, 5β-epoxide [83].
A. Ahmed Mahmood et al. in 1999 reported xanthanolid and xanthane type
sesquiterpenoid from cocklebur, identified by high resolution 1D and 2D NMR
and NOE experiments as 11α, 13-dihydroxanthatin, 4β, 5β-epoxy xanthatin-1α,
4α-endoperoxide and 1β, 4β, 4α, 5α-diepoxyxanth-11(13)-en-12-oic acid. Also
identified 11α, 13-dihydroxyxanthatin, a new xanthanolide diol using COSY
NMR analysis along with other techniques [84, 85].
M. Kanauchi et. al in the same year with the help of IR, UV and 1H-NMR
determined the structure of xanthatin, M.W. 246, m.p. 60º. The compound was
acetone soluble and possesses anti-bacterial activity [86].
UI chenko, N.T. in 2000 using first time chromatography and spectral methods,
analysed neutral lipids, normal and hydroxylated fatty acids and lipophilic
components from seeds of X. strumarium linn. [87].
In 2003 Kim Young and co-workers obtained 8-epi-xanthatin and 8-epi-xanthatin
epoxide from the leaves of cocklebur [88].
In the same year S. Shuenn-Jyi and fellows by using HPLC and electrophoretic
methods reported the presence of Potassium 3-O- caffeoylqduinate and 7-
hydoxymethyl-8, 8-dimethyl-4, 8-dihydrobenzo [1, 4] thiazine-3, 5-dione in X.
strumarium linn. [89].
In 2003 B.B.S. Kapoor and companions isolated kaempferol and quercetin as a
major flavonoid from ethanol fraction of extract of leaves and flower of cocklebur
[90].
35
M.S. Kumar and co-workers in 2006 found stigmasterol, they also isolated very
first time stigmasterol-3-O-β-D-glucopyranoside and 2-methylanthraquinone from
methanolic extract of roots of the plant. Their structure was determined by
detailed spectral studies [91].
36
37
38
O
HO
O
O
1,4,4,5-diepoxyxanth-11(13)-en-12oic acid
39
In the same year W. Zwolan and fellows isolated phenolic acids from vegetative
and generative parts of X. strumarium via HPLC technique. They identified them
as caffeic, gallic, protocatechuic, vanillic, syringic and ferulic acid [92].
Han-Ting and co- workers in 2006 obtained xanthiazone, chlorogenic acid, ferulic
acid, fermononetin and ononin from fruit extract of cocklebur along with two new
thiazinediones whose structures were determined to be 7-hydroxymethyl-8,8-
dimethyl-4,8-dihydrobenzol[1,4]thiazine-3,5-dione-11-O-β-D-glucopyranoside
and 2-hydroxy-7-hydroxymethyl-8,8-dimethyl-4,8-dihydrobenzol[1,4]thiazine-
3,5-dione-11-β-D- glucopyranoside [93].
R.N. Yadava and group in same year isolated from leaves of X. strumarium a
novel triterpenoidsaponin, 3-O-[α-L-rhamnopyranosyl-(1-3)-O-β-D-xylopyrano-
syl] manitadiol. The structural determination was done by various spectral
analysis and chemical degradations [94].
In the same year E. Akbar and group analyzed essential oil obtained through
hydrodistillation of X. strumarium stem and leaves via GC and GC/MS contained
bornyl acetate, limonene and β-selinene [95].
R.E. Irving and fellows in 2008 reported first time xanthatin and xanthinosin from
the burs of cocklebur [96].
T. Han and group in same year found caffeoylquinic acid by HPLC method from
n- butanol fraction of the plant [97].
C-L Lin and fellows in 2008 found xanthialdehyde and (-)-xanthienopyrane from
stirr fried seed extract of X. strumarium. The structure was elucidated by
spectroscopic methods [98].
In same year a review study by D. Ying-Hui and others for chemical constituents
found xanthanolide, kaurene glycoside and essential oils are prime components of
xanthium species [99].
V. Kumar and G.S. Rawat in 2008 estimated nitrogen and protein in great amount
in X. strumarium [100].
D.P. Pandey and M.A. Rather in 2012 found in ethyl acetate and methanolic
extracts of air dried, powdered plant over silica gel and sephadex LH20, caffeic
acid, xanthiazone and xanthiazone-(2-O-caffeoyl)-β-D-glucopyranoside. They
40
used 1D and 2D- NMR, Mass, UV and IR spectroscopy and chemical methods for
structural elucidation [22].
Guleryuz and fellows determined heavy metals Cr, Cu, Mn, Ni and Zn
accumulation in different concentrations in X. strumarium Linn. in 2008 and
considered suitable for growing in industrially polluted regions as potential plant
species for cleaning heavy metals from contaminated soil and remediation of
polluted areas [101].
41
42
43
Table – 1: Constituents of Xanthium strumarium Linn.
S.
No Name
M.P
ºC
M.F. Parts References
1 β-amyrin L 80
2 2-methylanthraquinone R 91
3 Ascorbic acid 190 C6H8O6 L 100
4 Aspargine F 55
5 Aspartic acid F 55
6 Bornyl acetate C12H20O2 L 95
7 Borneol L,St 95
8 3,4-dihydroxybenzalde-
hyde
C7H6O3 F 113,114
9 4-oxo-bedfordia acid A.P 84
10 Caffeoylquinic acid B 24
11 Caffeic acid 223 C9H8O4 F 22,24,109
12 Chlorogenic acid F 23,92
13 Pot.-3-O-
Caffeoylquinate
F 24,89
14 1,5-di-O-
Caffeoylquinic acid
F 24
15 1,3,5-tri-O-
Caffeoylquinic acid
C34H30O15 F 24
16 Carboxyatractyloside C31H44O8S2K2 B 70,72,74,103
17 Chalcone derivatives 170 C16H14O4 St 68
18 Campesterol F 57,65
19 Chlorobutanol 96-97 C4H7Cl3O L 67
20 3,4-dihydroxy cinnamic
acid
196-197 C9H9O4 L 68,71,92,109
44
21 Ceryl alcohol F 55
22 Cephalins F 55
23 Fumaric acid F 55
24 Fructose F 55
25 8-(Δ3-isopentenyl) 5,7,
3’, 4’-tetrahydroxy-
flavone
61
26 Formononetin F 23
27 Ferulic acid F 23,92
28 Gallic acid A.P 92
29 Glycine F 55
30 Glucose 148 C6H12O6 F 55
31 Heptacosanol 74 St 71
32 Isohexacosane 60-61 C26H54 L 67
33 4-O-dihydroinusonio-
lide
C15H22O3 A.P 81
34 Kaempferol 90
35 Limonene C10H16 L, St 95
36 Linoleic acid 229(BP) C18H32O2 F,S 52,55
37 Lecithins F 55
38 Leucine F 55
39 Linolenic acid 137(BP) C18H32O2 S 52
40 Malic acid F 55
41 Ononin F 23
42 Octacosanol L 80
43 Oleic acid 11-12 C18H34O2 F 52,55
44 Palmitic acid 61-62 C16H32O2 F,L 55,67
45 Protocatechuic acid 92
46 Phenylalanine F 55
45
47 Quercetin 90
48 3-O[α-L-rhamnopyrano-
syl-O-β-D-
xylopyranosyl]
maniladiol
L 94
49 Є-Sitosterol 144 C29H50O F,L 67
50 β- Sitosterol 137 C29H50O S,F,L 57,65,67,71,80
51 γ- Sitosterol 158 C28H48O F 55,56
52 Stigmasterol 167 C29H48O S,F,L 57,65,71,80,91
53 Strumaroside 290-2 C35H60O5.H2O F 55
54 Sucrose F 55
55 Succinic acid F 55
56 Stearic acid 71-72 C18H36O2 F 55
57 Stearyl alcohol 59-60 C18H38O L 67
58 β- Sitosterol-D-
glucoside
290 C35H66O6 St,L 68,71
59 Stigmasterol-3-O-β-D-
glucopyranoside
R 91
60 β-Selinene L,St 95
61 Syringic acid 92
62 Tomentosin A.P 83
63 Triterpene alcohol 211 C30H50O2 66
64 Triterpene alcohol 274 C30H50O5 66
65 2-hydroxy tomentosin-
1β,5β-epoxide
A.P 78
66 Vanillic acid 92
67 Thiazenedione 186 C26H30O11NS F 93
68 8-epi-tomentosin L 108
69 2-Hydroxytomentosin C15H20O4 L 82
46
70 Xanthialdehyde 156-158 C11H11NO3S S 98
71 (-)-Xanthienopyran 218-220 C17H16O4S S 98
72 Xanthatin 113-115 B,L,F 86,96,108,75,
73,60,59
73 Xanthinosin B 96
74 epi-xanthatin R,L 88
75 8-epi-xanthatin epoxide C15H20O4 L,A.P 82,83,88
76 11α-13-dihydroxy
xanthatin
C15H20O3 A.P 84
77 4β,5β-epoxy-xanthatin-
1α,4α-endoperoxide
C15H18O5 A.P 84
78 1β,4β,4α,5α-diepoxy
xanth-11(13)-en-12-oic
acid
C15H22O4 A.P 84
79 Xanthiazone 159 C11H13O3SN A.P 23,24
80 8-epi-xanthatin 71-72 A.P,L 83,108
81 Xanthumin 100-101 C17H22O5 A.P,L 58,62,83,104
82 Xanthanol L 15,81,85,105
83 Iso-xanthanol A.P,L 81,15
84 6β,9β-dihydroxy-8-epi-
xanthatin
L 80
86 Xanthumanol A.P 82
87 Xanthinin 123 C17H22O5 A.P,L 58,62
88 15-Chloro-2-epi-
xanthanol
C17H23O5Cl A.P 82
89 K2SO4 St 71
90 KCl L 71
91 KNO3 A.P 55,71
92 CaSO4 A.P 55
A.P= Aerial part, B=Burs, F=Fruit, L=Leaves, S=Seed, St=Stem, R=Root, M.P=Melting point
47
1.1.4.3.1 Pharmacological activity
The toxicity of Xanthium strumarium Linn. took attention of many researcher,
hence A. Sado in 1937, analyzed a substance weakly toxic to nerves and muscles,
from the aqueous extract of stem and leaves of X. strumarium Linn [47].
Presence of physiologically inactive soluble glucoside in Xanthium strumarium
Linn was found by I.C. Chopra and co-workers in 1946 [48].
In 1957, E.F. Leonova studied pharmacology on the basis of infusion results of
leaves, stalk and seed tincture injection of X. strumarium, the leaves infusion
increases peristalsis in rabbit intestine, caused cardiac blockade in frog heart,
dilation of blood vessels of rabbit ear, results dilation following constriction of
frog hind leg and a reduction of blood pressure by 20-40 mmHg, depressed
stimulation of spinal cord when intravenously injected to cats whereas respiratory
movements intensified in frog by the tincture of seed [53].
H. Minato in 1967 reported a CNS depressant agent, xanthumin, M.F C17H22O5,
m.p.100.5-101º from acetone extract of aerial parts of cocklebur [62].
In 1980 the group of J.R. Cole identified carboxyatractyloside as a highly toxic
substance, responsible for the poisoning character of cocklebur [72].
In 1982 E. Naidenova with companions communicated pharmacology of
derivatised components. They isolated sesquiterpene lactone after treating
aqueous extract of leaves and root of cocklebur at 80º for 30 min. following
precipitation with lead acetate, chloroform washing, drying organic layer with
sodium sulphate and removal of solvent under vacuum distillation. The residue
was dissolved in aliquot of water. Under 284 nm of spectrophotometer and other
techniques the compound was identified as xanthatin, its derivatives with
hexamethyleneimine and morpholine revealed anti-tumor activity when tested
with leukosis L- R 10 [73].
A. Harada et al. (1985) determined an anti- attaching repellent, xanthatin, from
cocklebur extract, the isolated compound revealed strong repellent property and
weak toxicity against Mytilus edulis (blue mussel) [75].
In 2007 R.N. Yadava and J. Jitendra obtained a novel anti-inflammatory active
triterpene saponin from leaves of X. strumarium. The structure elucidation by
48
different spectral analysis identified it as, 3-O[α-L-rhamnopyranosyl-(1-3)-O-β-D-
xylopyranosyl] manitadiol [94]. T. Han with his group in 2008 studied the polarity
based ethanol extract fractions of X. strumarium amongst them the n-butanol was
the most polar, exhibiting highest anti-inflammatory property in the test of croton
oil induced edema and also possesses strong analgesic effect as it decreases
writhing in mice. They also obtained 10 caffeoylquinic acid and decided them the
cause of polarity of plant extract [21].
C-L Lee and coworkers in 2008 isolated xanthaialdehyde and (-) -
xanthienopyrane from chloroform extract of seed of cocklebur. They found later
one as a superoxide anion generation inhibitor produced by activated neutrophils
showing 1.72µg/ml, IC50 value [98].
Y.H. Dai and group in 2007, research on pharmacological properties of xanthium
species. They analyzed xanthanolide, kaurene glycoside and essential oils as
active principals for antibacterial, antioxidant, anti-inflammatory and antitumor
activities of many species of xanthium [99].
A crystalline hypoglycemic agent was isolated by P.F. Kupiecki and fellows in
1974, from the seeds of cocklebur by boiling mashed seeds in water for 30 min.
following acetone and ethanol fractionation, column chromatography and
crystallization. They analyzed the compound composed of C, H, O and S, it was
active towards rats at a given i.p or s.c [102]. In 1976 J.C. Craige and fellows
identified a glycoside from the burs of cocklebur M.F. C31H48O24S2, m.p. 278-
279o, exhibited hypoglycemic activity when given in 1-5 mg/kg i.v. in lab
animals. The compound was purified by producing its potassium salt,
recrystallisation in water and confirmed by comparing TLC, IR, and 1H-NMR
with a pure and authentic sample as a potassium carboxyatractylate [103].
In the year 2000 M. Kanauchi and fellows isolated antibacterial agent, a white
crystalline compound mp. 60º, MW 246, from leaves extract of X. strumarium.
They determined its structure as xanthatin by IR, UV and 1H-NMR techniques.
The compound exhibited MIC value 25- 100 µg/ml against Candida species,
Pichia species, Sacchromycopsis species and Torulaspora species whereas 12.5-
100 µg/ml MIC against Bacillus species, contaminator of Koji used in
manufacturing of alcoholic beverages. They concluded xanthatin as an anti-
49
bacterial agent that can be used to prevent contamination of Koji while preparing
beverages [86]. Z. Cui et al. (2007) reported antibacterial activity of xanthium
species in their review research about chemical constituents in xanthium species
[99]. C.K. Gupta and D.R. Gupta in 1975 analysed xanthumin as an antibacterial
agent from bioactive guided fraction of pet.ether extract of leaves of Xanthium
strumarium, it was active towards gram positive. The structure was elucidated by
comparison of IR spectrum and Rf values with a reliable sample as C17H22O5, m.p.
100- 101º [104].
In 1988 A.I.M. Jawad determined the anti-microbial activity of MeOH extract of
cocklebur against Proteus vulgaris, Staphylococcus aureus, Basccilus subtilis,
Candila albicans and C. pseudotropicallis. They further reported the property was
due to a sesquiterpene xanthanol isolated from ethyl acetate extract by PTLC
(CHCl3/ EtOAc, 1:1) exhibited similar antimicrobial activity as methanol extract
[105]. V.K. Saxena assessed the medicinally important bactericidal activity of
lipids of cocklebur in the year of 1990 [106]. H.S. Kim and co-workers in the year
1997 isolated compounds A and B from EtOAc extracts of same species. Both
compounds were purified through reverse phase HPLC, stable at 120º but not in
acidic and alkaline medium. When tested with bacteria, yeast and fungi exhibited
growth inhibition against both gram +ve and gram –ve bacteria in agar diffusion
method while in FDA method within esterase compound B inhibited the growth of
bacteria and yeast whereas compound A inhibited the growth of bacteria only
[107].
J-W Ahn and coworkers in 1995 observed cytotoxicity of MeOH extract of leaves
of X. strumarium against human tumor cell line. They realized the property was
due to the presence of α-methylene containing sesquiterpene xanthatin, 8-epi-
xanthatin and 8-epi-tomentosin. They also determined the greater cytotoxicity of
8-epixanthatin in comparison of 8-epi- tomentosin was the result of conjugated
enone moiety in 8-epi-xanthatin [108]. Taking cytotoxicity under consideration
Y.S. Kim and co-workers in 2003 isolated two xanthanolide sesquiterpene
lactones, 8-epi-xanthatin and 8-epi-xanthatinepoxide from leaves extract of
cocklebur and investigated for their cytotoxic effect on A549(non-small lungs),
SK-OV-3(ovary), SK-MEL-2(melanoma), XF498(CNS) and HCT-15(colon).
50
They found the compounds were inhibitors of proliferation of above cultured
human tumor cells and also inhibit fernesylation of human L-amine-B by
fernesyltransferase (FTase) in high dose [88]. Showing keen interest for in vitro
cytotoxicity of medicinally important plants R.E. Irving and group in 2008
recognized two sesquiterpene lactones, xanthatin and xanthinosine from the
chloroform extract of X. strumarium leaves as potent cytotoxic agents having IC50
values 0.1 to 6.2µg/ml for human cancer cell line ATCC (colon), MDA-MB-231
ATCC (breast) and NCI-417 (lungs). These compounds were reported for the first
time in the burs of this specie [96].
In the year 1999 F.L. Hsu and group isolated caffeic acid from fruit extract of
cocklebur. They analyzed the compound as an anti-hyperglycemic after a decrease
in plasma glucose when applying on diabetic rates of streptozotocin induced and
insulin- resistant model through intravenous injection of it. On the other hand the
caffeic acid was inactive on non diabetic, normal specimen [109].
The essential oils of X. strumarium possess strong anthelmintic property. It was
reported by A.K. Gharia and fellows in 2002 when they observed superior
anthelmintic activity of essential oils of said plant against earthworm, tapeworm,
hookworm and nodular worm [110].
Y-H Dai et al. reported in 2007 xanthanolides and kaurene glycosides as prime
substances of X. strumarium that possesses anti- oxidant activity [99]. Next year
(2008) R. Scherer and fellows by applying three concentrations of DPPH
suggested a new antioxidant and index (AAI) for different organic acids, clove
essential oil, eugenol and X. strumarium extract. They observed among them X.
strumarium exhibited strong antioxidant activity with AAI= 1.6 [111].
In 2008 Y. Xu and companions identified X. strumarium Linn as a good source of
preparing environment friendly and green pesticides because the specie contains
such active substances which prevent pests, weed and pathogens growth on plants
[112].
Y.S. Bae along with his group in 2009 recognised 3,4-dihydroxybenzaldehyde as
an active component in X. strumarium extract which induces apoptosis of blood
cancer cells by inhibiting the activity of casein kinase 2 [113].
51
In the same year B.H. Lee with his fellows isolated a CKII inhibitory compound
from X. strumarium fruit. The structure elucidation via different spectroscopy
techniques identified as 3,4-dihydroxybenzaldehyde. It inhibited CKII
phosphotransferase activity of 783µM, IC50. They determined that 50% growth of
human cancer cell U937 by the isolated compound was the result of breaking of
poly (ADP- ribose) polymerase and procarpase-3. The inhibitor also showed
triggered apoptosis by fragmentation of DNA. They used flow cytometry analysis
to confirm all these apoptosis and further concluded that as the CKII takes part in
cell proliferation and oncogenesis, 3, 4 dihydroxybenzaldehyde may inhibit these
diseases by inhibiting the CKII activity [114].
S.P. Joshi and fellows in 1997 isolated four anti-malarial active xanthanolides
from bioactive direct fraction of acetone extract of aerial parts of X. strumarium.
They observed all compounds possesses anti-malarial activity against chloroquine
resistant Plasmodium falciparum strain under IC50 values 7.8, 7.8, 31 and 125
µg/ml for tomentosin, 8-epi-xanthati-1β,5β- epoxide xanthumin and 8-epi-
xanthatin respectively [83]. To evaluate this property, S. Chandel and coworkers
in 2012 determined malarial remedy property of X. strumarium Linn. by testing its
ethanolic extract of leaves for antiplasmodial activity in Plasmodium berghei
infected BALB/c micr. A dose dependent oral administration(500 mg/kg/day)
showed 88.6% chemosuppression during early days of infection that was greater
than that of standard chloroquine drug where as 90.40% chemosuppression was
observed upon taken 350 mg/kg/day concentration during repository infection
comparable to pyrimethamine (92.91% chemosuppression). They found the
survival of mice enhanced from 21 to 26 days when concentration used 250 and
350 mg/kg/day. On the other hand upon 150 mg/kg/day concentration sustain all
mice to 29 days. In the last they concluded that the plant can be used for malarial
remedy [115].
In 2012 F. Khuda and Z. Iqbal with their companions studied solvent fractions of
X. strumarium for bioactivities. They found chloroform extract revealed
insecticidal activity due to which the plant can be a good source of natural
insecticide in comparison of permethrin. They also determined maximum
52
cytotoxicity for brine shrimps with mortality rate 93% at highest dose of n-butanol
fraction hence can apply safely in traditional medicine [116].
H.N. Yoon and coworkers in 2013 determined bioactive components from fruit of
X. strumarium Linn. which are inhibitor of aldose reductase (AR) and galactitol
formation in rat lenses with high glucose level. Methyl -3, 5-di-O-caffeoylquinate
is the most potent inhibitor having IC50 value 0.30 and 0.67 µM for rAR and
recombinant human AR (rh AR) respectively. They further analysed that it is
galactitol formation inhibitor in rat lens and in erythrocytes, as well as the
effectiveness of weed in diabetic applications. They also isolated neochlorogenic
acid methyl ester, 3-hydroxy-1-(4-hydroxy phenyl)-propan-1-one and raffinose
from ethyl acetate fraction by reverse phase C-18 chromatography for the first
time from X. strumarium [117].
53
1.2. Present Work
54
In view of pharmacological characteristics and medicinal uses attributed to
Xanthium strumarium Linn., the present studies were done on the chemical
constituents of air dried aerial parts of this plant. The methanolic extrac (XS-Me)
was subjected to classical methods of separation followed by various
chromatographic techniques such as column chromatography (CC), pencil CC,
preparative thin layer chromatography (TLC) (detail in experimental). The
investigation of this extract led to the isolation of eight known constituents, (1)
lupenyl acetate, (2) stigmasterol, (3) β-sitosterol, (4) palmitic acid, (5) β-amyrin,
(6) oleanolic acid, (7) β-sitosterol-3-O-β-D-glucopyranoside and (8) ferulic acid
among these, compound (1) is reported for the first time from this plant. The
structure of (1) has been determined with detailed spectral studies including IR,
EIMS, HR-EIMS, 1H-NMR,
13C-NMR and 2D-NMR analysis where as other
constituents were recognized by the comparison of their spectral data with those
of literature reported values.
The anti-bacterial, anti-fungal and anti-oxidant activities of crude methanolic
extract (XS-Me) and its fractions (XS-HX, XS-DC, XS-EA and XS-BU) were
analyzed in collaboration with Department of Microbiology, Federal Urdu
University of Arts, Science and Technology, Karachi, Pakistan. All the fractions
possess significant biological activities, however the ethyl acetate fraction (XS-
EA) is the most active fraction as it exhibited moderate to high anti-bacterial and
anti-fungal activity along with this it also showed good anti-oxidant activity.
55
1.3. Results and Discussion
56
1.3.1. Lupenyl acetate (1)
Compound (1) appeared as colourless needles. It showed a molecular ion peak at
m/z 468 [M]+
in EIMS and 468.3970 in HR-EIMS spectrum respectively
corresponding to the molecular formula C32 H52O2. Other peaks in EIMS spectrum
at m/z 453 and 408 were obtained by the loss of CH3 and CH3COOH where as
peaks at m/z 249, 218 along with their counter ions at m/z 393 and 204 revealed
the basic fragmentation pattern of pentacyclic tritrpenes.
In IR spectrum absorption peaks at 1735, 1200 and 1630 cm-1
displayed the
presence of ester and olefinic groups in the molecule.
1H-NMR spectrum of (1) exhibited resonances of seven methyl singlets at δ 1.68,
1.05, 0.98 (Me- 30, 26, 27), 0.85 (9H, Me- 25, 24, 23) and 0.81 (Me- 28). Another
singlet at δ 2.02 identified methyl of ester linkage. However two doublets at δ
4.67 and 4.55 (J = 2.3 Hz) justified two olefinic protons (H-29), double doublets
at δ 4.46 (J = 10.0, 5.7 Hz) attributing a proton with α (alpha) stereo (H- 3α) and
H-19 resonate as multiplet of one proton at δ 2.36. This discussion agreed lup-
20(29)-ene skeleton that was further justified through HMBC (Heteronuclear
Multiple Bond Coherence) assignment of (1) in which the spectrum showed
connectivity of H-19 with C-29 (δ 109.3) and of H-3α with C=O (δ 171.0) of
acetoxy group.
57
The above spectral data was in full agreement of literature values [118]. Hence the
structure of (1) was established as 3β – acetoxylup-20(29) – ene.
58
H3C
Fig. 1: Significant HMBC (H C) interactions of compound (1)
59
1.3.2. Stigmasterol (2)
H
HO
HH
1
67
8
13
14 1516
17
18
2122
23
2425
26
27
29
28
2
34
5
10
1112
20
9
19
The molecular formula C29H48O of (2) was obtained through HR-EIMS (M+
412.3742). The EIMS spectrum of (2) showed peaks at m/z 397 and 394 due to
loss of CH3 and H2O, other remarkable peaks were observed at m/z 379 [C28H43]+,
273 [C20H33]+, 255 [C19H27]
+ and 229 [C18H13]
+ .
The IR spectrum showed the presence of hydroxyl group at 3380 cm-1
and
olefinic group at 1660 cm-1
in the moiety.
The 1H-NMR of compound (2) exhibited resonance as a broad singlet at δ 5.34
(H-6) and a multiplet at δ 3.50 (H- 3α) whereas multiplets at δ 5.17 and 5.03 were
assigned to olefinic H-22 and H-23. It also showed two singlets at δ 0.99 and 0.67
(Me-19, Me-18) two doublets at δ 0.83 (J = 6.7 Hz, Me-27) and δ 0.78 (J = 6.7
Hz, Me-26) and a triplet at δ 0.81 (J = 7.0 Hz, Me-29).
The above spectral data of stigmasterol (2) was in accordance with the reported
values [119, 120]. It could be deduced as stigmasta-5, 22-diene-3β-ol.
60
1.3.3. β-Sitosterol (3)
H
HO
HH
1
67
8
13
14 1516
17
18
2122
23
2425
26
27
29
28
2
34
5
10
1112
19
20
9
The molecular ion [M]+ peak for (3) was observed at m/z 414.3862 in HR-EIMS
spectrum corresponding to the molecular formula C29H50O. In the EIMS spectrum
compound (3) displayed characteristic fragment ions at m/z 396 [M-H2O]+, 381
[M-H2O-CH3]+, 273 [M-side chain]
+ and 255 [M-side chain- H2O]
+.
The IR spectrum showed the hydroxyl band at 3400 cm-1
and band at 1640 cm-1
due to (C=C) double bond.
The 1H-NMR spectrum of (3) was characteristic of a steroidal molecule. It
displayed a signal of broad singlet at δ 5.33 for olefinic proton H-6 and a multiplet
at δ 3.40 for H-3α, two singlets appeared at δ 1.00 and 0.67 were due to the
quaternary Me-18 and Me-19 respectively. 1H-NMR spectrum also displayed
three doublets at δ 0.90 (J = 6.0 Hz), 0.80 (J = 6.5 Hz) and 0.78 (J = 6.5Hz) of
secondary Me-21, Me-26 and Me-27, respectively, whereas a triplet resonating at
δ 0.82 (J = 7.0 Hz) was due to the primary Me-29.
The comparative spectral study with the reported data [119, 121] led to the
assignment of (3) as stigmasta-5-en-3-ol.
61
1.3.4. Palmitic acid (4)
3
4
5
6
7
8
9
10
11
12
13
14
15
161
2HO
O
The molecular formula C16H32O2 of (4) was obtained through HR-EIMS (M+,
256.2389). Its EIMS spectrum showed peak at m/z 45 indicating the presence of
(COOH) group whereas presence of (CH2) groups were exhibited by peaks at m/z
227, 213, and 199.
The IR absorption bands at 3450-2610 cm-1
(COOH) and 1700 cm-1
(C=O) also
confirm the presence of carboxylic group in the molecule.
The 1H-NMR spectrum showed a triplet at δ 0.85 (J = 6.6 Hz) for terminal methyl
(Me- 16) and a triplet of (CH2) located next to COOH at δ 2.25 (J = 7.5 Hz, H-2)
while a broad long singlet at δ 1.26 confirmed the presence of a long chain of
(CH2).
In view of above spectral studies the structure of (2) was assigned as palmitic acid
(Hexadecanoic acid). Its spectral data was in complete agreement with the
reported values [122].
62
1.3.5. β-Amyrin (5)
H
HO
H
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
25
20
21
22
23 24
30
26
19
29
28
27
Compound (5) showed molecular ion peak [M]+ in HR-EIMS at m/z 426.0100
having molecular formula C30H50O. In EIMS spectrum the base peak at m/z 218
and 207 were due to retro Diels Alder fragmentation where as fragments at m/z
411 and 393 were obtained by the loss of CH3 and then H2O from the molecule.
The fragment at m/z 189 indicates that -OH group is present either in ring A or
B. It was placed at C-3 on biogenetic ground. The fragment at m/z 203 located the
CH3 group at C-17. This fragmentation identifies pentacyclic triterpene skeleton
of oleanane and ursane series.
In IR spectrum absorption bands at 3352 and 1648 cm-1
mentioned (OH) and
(C=C) groups.
The 1H-NMR spectrum determined Δ
12 β–amyrin series of the compound as it
displayed signals of eight methyl as singlets in upfield region at δ 1.09, 1.00, 0.99,
0.94, 0.87, 0.86, 0.85 and 0.79 (Me- 27, 26, 23, 25, 29, 30, 28 and 24). Similarly a
signal at δ 3.09 (1H, dd, J = 11.0, 4.0 Hz) was assigned to H-3α and olefinic
proton H-12 exhibited resonance as a triplet at δ 5.15 (J = 4.5 Hz).
All above studies were in full agreement of literature values [123, 124], therefore
structure of (5) was assigned as 3β-hydroxyolean-12-ene.
63
1.3.6. Oleanolic acid (6)
H
HO
COOHH
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
25
20
21
22
23 24
30
26
19
29
28
27
The compound (6) displayed molecular ion peak [M]+
at m/z 456 in EIMS and
456.3410 in HR-EIMS corresponding to the molecular formula C30H48O3. The
retro Diels Alder fragments at m/z 248, 207, 203, 133 and 189 placed the (-
COOH) group at C-17 and –OH group in ring A or B. The later was placed at C-3
on biogenetic ground.
The IR absorption bands at 3410, 2789, 1710 and 1625 cm-1
justified the presence
of hydroxyl, carboxylic and olefinic group in the molecule.
The 1H-NMR spectrum displayed seven tertiary methyl singlets at δ1.20, 1.15,
0.97, 0.94, 0.86, 0.85 and 0.75 (Me-23, 27, 25, 24, 30, 29 and 26), a triplet
resonated at δ 5.28 (J = 3.5 Hz) for olefinic proton (H-12) and δ 2.80 (dd, J= 14.1,
4.6 Hz) for H-18. These data attributed that compound (6 ) belongs to Δ12
β –
amyrin series of pentacyclic triterpenoids and its β-orientation was decided on the
basis of δ value and coupling constant of H-3 (δ 3.40, dd, J = 14.0, 4.0 Hz).
Structure of compound (6) was confirmed by comparing its spectral data with
those reported in literature [125] as oleanolic acid or 3β-hydroxyolean-12-en-28-
oic acid.
64
1.3.7. β-Sitosterol-3-O-β-D-glucopyranoside (7)
The molecular formula C35H60O6 of Compound (7) was assigned by HR-FAB-MS
(+ve) appeared at m/z 577.2877 [M+H]+ whereas in EIMS spectrum the
characteristic peaks appeared at m/z 414 [M-gluc]+ and at m/z 396 [M- gluc-
H2O]+.
IR spectrum of (7) displayed absorption bands for (OH), (C=C), and (C-O) at
3410, 1638 and 1248 cm-1
.
The glycosidic moiety was identified in 1H-NMR by showing a signal of anomeric
proton at δ 4.80 (d, J= 7.5 Hz, H-1') whereas other glucose protons appeared as
multiplet in the range of δ 3.25-4.45. 1H-NMR spectrum showed resonance of
olefinic proton at δ 5.32 (br. s, H-6) and oxymethine proton at δ 3.43 (m, H-3α).
The 1H-NMR spectrum also showed the signals of six methyl groups out of which
two were tertiary (δ 1.08, CH3-19 and δ 0.66, CH3-18), three secondary, CH3-21,
26 and 27 (δ 0.90, δ 0.81 and δ 0.79) and one primary methyl, CH3-29 at δ 0.82.
The above spectral data was in full agreement of reported values [126] to decide
the structure of (7) as β-sitosterol-3-O-β-D-glucopyranoside.
H H
O
H
O
OH
OH
OH
OH
1
2
3
4
56
7
8
9
10
11
12
13
1415
16
17
18
19
20
2122
23
24
25
26
27
29
28
1'2'
3'
4'
5'
6'
65
1.3.8. Ferulic acid (8)
In EIMS spectrum of compound (8) the peak at m/z 194 indicated the molecular
ion [M]+ and 194.0136 in HR-EIMS corresponding to the molecular formula
C10H10O4. The mass spectrum also showed the presence of COOH, OCH3, and
olefinic group in the molecule by showing the peaks at m/z 149, 163 and 123.
The compound displayed 3345-2578cm-1
(OH and COOH), 1675(C=O), 1619
(C=C) and 1439(aromatic C=C) absorption peaks in IR spectrum.
1H-NMR spectrum showed signals of olefinic protons as two doublet at δ 7.48 and
6.01 (J= 14.5 Hz, H-7, H-8) large coupling constant indicated there trans
substitution. Whereas resonance in downfield defined aromatic protons at δ 7.15
(br. s, H-2) and two doublets at δ 7.21 (J =7.8 Hz, H-6) and 6.60 (J = 7.8 Hz, H-
5). It further gave a singlet at δ 3.89 for methoxy (OCH3) moiety attached to
aromatic ring.
On the basis of above spectral studies that coincided with the literature values [23,
127] compound (8) was characterized as ferulic acid.
66
1.3.9. Biological activities
All fractions of methanolic extract of air dried aerial parts of X. strumarium Linn.
(XS- HX, XS-DC, XS-EA, XS-BU and XS-ME) were analysed for their
antimicrobial and anti-oxidant activity by the methods as described in chapter 4.
1.3.9.1. In Vitro Anti-bacterial activity
The anti-bacterial activity of XS-HX, XS-DC, XS-BU and XS-ME fractions was
determined by disc diffusion method whereas agar-well method was applied for
fraction XS-EA.
The anti-bacterial activity of all fractions were determined against Bacillus cereus,
Bacillus subtilis, Bacillus thuringiensis, Corynebacterium xerosis, Mycobacterium
smegmatis, Staphylococcus auereus, Staphylococcus epidermidis, streptococcus
saprophyticus, streptococcus faecalis, streptococcus pyogenes gram positive
bacterial strains and Campylobacter coli, Enterobacter aerugenus, Escherichia
coli, Klebsiella pneumonieae, Proteus mirabilis, Pseudomonas aeruginosa
ATCC 9027, Salmonella typhi , Salmonella typhi A , Salmonella typhi B, Shigella
dysenteriae and Vibrio choleraeae gram negative bacterial strains. The fraction
XS-EA was found to be the most active fraction. It showed remarkable anti-
bacterial activity against Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis
and Klebsiella pneumonieae having zone of inhibition 26, 34, 30 and 22 mm with
MIC = 88, 120, 102 and 56 mg/ml. It further showed good activity against
Campylobacter coli, Enterobacter aerugenus, Salmonella typhi , Salmonella typhi
A , Salmonella typhi B and Vibrio choleraeae creating 19, 17, 18, 19, 20 and 19
mm zone of inhibition respectively (MIC = 200, 200, 89, 90, 52 and 160 mg/ml).
Gentamicin was used as standard. All other fractions exhibited weak to moderate
anti-bacterial activity displaying zone of inhibition in the range of 8-13 mm in
comparison of standard Ampicillin. Results are displayed in Table-2.
67
Table-2: In vitro anti-bacterial activity of different fractions of methanolic extract of X. strumarium Linn
Key: Values are zone of inhibition diameter (mm) and an average of triplicate. (-) indicates inactivity. Fraction : XS= Xanthium strumarium,
HX=n-Hexane, DC= Dichloromethane, BU= n-Butanol, ME= Methanol and EA= Ethyl acetate. MIC = minimum inhibitory concentration
Organisms XS-HX
mm
XS-DC
mm
XS-BU
mm
XS-ME
mm
Ampicillin
mm
XS-EA
mm
Gentamicin
mm
MIC
mg/ml
Gram positive bacteria
Bacillus cereus - 09 - - >25 26 >15 88
Bacillus subtillis - - - 34 >15 120
Bacillus thuringiensis 08 09 - - >45 30 >15 102
Corynebacterium xerosis - - - - - - - -
Mycobacterium smegmatis - - - - - - - -
Staphylococcus aureus 09 - - - >30 - - -
Staphylococcus epidermidis - - - - - - - -
Streptococcus saprophyticus - - 09 - >35 - - -
Streptococcus faecalis - - - 13 >30 - - -
Streptococcus pyogenes - - - - - - - -
Gram negative bacteria
Campylobacter coli - - - - - 19 >15 200
Enterobacter aerogenus - - - - - 17 >15 200
Escherichia coli - - - - - - - -
Klebsiella pneumoniae - - - - - 22 >15 56
Proteus mirabilis - 08 - - >35 - - -
P. aeruginosa ATCC 9027 10 08 - - >30 - - -
Salmonella typhi - - - - >30 18 >15 89
Salmonella para typhi A - - - - - 19 >15 90
Salmonella para typhi B - - - - - 20 >15 52
Shigella dysenteriae - - - - - - - -
Vibrio cholerae - - - - - 19 >15 160
68
1.3.9.2. In Vitro Anti-fungal activity
All the fractions were tested for their anti-fungal activity against Candida albicans,
Candida albicans ATCC (0383), Saccharomyces cerevisiae, Microsporum canis,
Microsporum gypseum, Trichophyton rubrum, Trichophyton mentagrophytes,
Trichophyton tonsurans, Aspergillus flavus, Aspergillus niger, Fusarium species,
Helminthosporium, Penicillium species and Rhizopus species. It was found that XS-
EA was the only fraction that showed excellent anti-fungal activity against
Microsporum canis, Aspergillus flavus, Aspergillus niger, Helminthosporium,
Penicillium species and Rhizopus species showing zone of inhibition 12, 19, 15, 14,
12, and 16 mm (MIC = 312, 320, 100, 212, 149, and 300 mg /ml ) respectively.
Gresiofulvin was used as standard. Results are presented in Table-3.
69
Table-3: In vitro anti-fungal activity of different fractions of methanolic extract of X. strumarium Linn.
Fungi XS-HX
(mm)
XS-DC
(mm)
XS-EA
(mm)
XS-BU
(mm)
XS-ME
(mm) Gresiofulvin
(mm)
MIC
(mg/ml)
Yeast
Candida albicans - - - - - >12
Candida albicans ATCC 0383 - - - - - >12
Saccharomyces cerevisiae - - - - - >12
Dermatophytes
Microsporum canis - - 12 - - >12 312
Microsporum gypseum - - - - - >12
Trichophyton rubrum - - - - - >12
Trichophyton mentagrophytes - - - - - >12
Trichophyton tonsurans - - - - - >12
Saprophytes
Aspergillus flavus - - 19 - - >12 320
Aspergillus niger - - 15 - - >12 100
Fusarium specie - - - - - >12
Helminthosporium - - 14 - - >12 212
Penicillium specie - - 12 - - >12 149
Rhizopus specie - - 16 - - >12 300
Key: Values are inhibition zones (mm) and an average of triplicate. (-) sign indicates no activity. MIC=minimum inhibitory
conc. Fractions: XS=Xanthium strumarium, HX= n-Hexane, DC=Dichloromethane, EA= Ethylacetate, BU= n-Butanol, ME=
Methanol
70
1.3.9.3. In Vitro Anti-oxidant activity
All the methanolic fractions when tested for their anti-oxidant activity in comparison
of ascorbic acid only EtOAc fraction (XS-EA) exhibited significant activity with
70% inhibition (EC50 = 937 µg/ml). The remaining fractions showed less than 50%
inhibition hence were not further studied. Results are displayed in Table-4.
Table-4: In vitro anti-oxidant activity of methanolic fractions of X. strumarium
Fractions % inhibition EC50
(µg/ml)
XS-HX -4.16 -
XS-DC 41.66 -
XS-EA 70 937 ± 0.5
XS-BU 30 -
XS-ME 25 -
Ascorbic acid 80 8.3
Key: Values are inhibition (%) and an average of triplicate.
EC50 = effective concentration to scavenge 50 % of DPPH
Fractions: XS=Xanthium strumarium, HX=n-Hexane,
DC=Dichloromethane, EA= Ethylacetate, BU= n-Butanol,
ME= Methanol.
71
1.4. Experimental
72
1.4.1. General Experimental
Melting points were determined in glass capillary tubes on a Gallenkamp melting
point apparatus and were incorrected. Column chromatography was carried out on
silica gel (70-230 mesh, Merck). TLC was performed on pre-coated silica gel GF-
254.
IR spectra were recorded on a Jasco-302-A spectrophotometer. The mass spectra
were scanned on a Jeol-JMS HX-110 mass spectrometer. The 1H and
13C-NMR
spectra were recorded on a Bruker spectrometer operating at 500, 300 and 75 MHz.
The chemical shift values are reported in δ (ppm) relative to SiMe4 (TMS) as an
internal standard. The coupling constant (J) are given in Hz.
1.4.2. Plant Material
Aerial parts of Xanthium strumarium Linn. was collected by Mohammad Farman Ali
from Lakimarwat (Khyber Pakhtun Khawa) Pakistan and identified by Dr. Sahar. A
voucher specimen (G.H. No.86398, No. 01) has been deposited in the herbarium at
Department of Botany, Faculty of Science, University of Karachi, Karachi, Sind,
Pakistan.
1.4.3. Extraction and Isolation
The air dried aerial parts of Xanthium strumarium Linn (10 kg) were ground to fine
powder and extracted repeatedly with methanol at room temperature. The solvent was
evaporated under vacuum to obtain 250g crude extract (XS-Me). The resultant dark
greenish brown gummy mass was partitioned between EtOAc and H2O (Scheme I).
Aqueous phase was neglected while the EtOAc phase was treated with aq. Na2CO3
solution (4%) to remove acidic portion from neutral fractions. This EtOAc portion
that composed of neutral fractions was again washed with distilled water, dried with
Na2SO4 treated with activated charcoal to remove green colored chlorophyll part and
filtered. The filtrate was concentrated under reduced pressure to obtain 150g neutral
fraction (XS-N). The charcoal bed was eluted with MeOH–C6H6 (1:1) repeatedly.
The total extract was combined and solvent was removed by vaccum distillation.
73
Both fractions were combined after comparison of their TLC on silica gel GF-254
using mobile phase n-Hexane-EtOAc (9.5:0.5) to give total neutral fraction 154g
(150g+4g), (XS-N). The resultant total neutral fraction thus obtained was partitioned
into n-Hexane soluble (XS-HX; 13g) and n-Hexane insoluble portions (Scheme II).
The n-Hexane insoluble fraction was again divided into dichloromethane (DCM)
soluble (XS-DC; 14g) and DCM insoluble portions. The DCM insoluble portions was
again partitioned into EtOAc soluble (XS-EA; 97g) and EtOAc insoluble portion.
The EtOAc insoluble portion was again divided into n-butanol soluble (XS-BU; 21g)
and n-butonal insoluble portion.
All fractions (XS-HX, XS-DC, XS-EA, XS-BU and XS-Me) were analyzed for their
anti-microbial and anti-oxidant activities. Among all these fractions XS-EA was
found to be the most active fraction as it showed high anti-bacterial, moderate anti-
fungal and good anti-oxidant activity whereas all other fractions displayed weak to
moderate anti-bacterial activity (Table-2, 3 and 4).
The EtOAc soluble fraction was subjected to column chromatography (CC) over
silica gel with successive elution with n-hexane and n-hexane- EtOAc in increasing
order of polarity. It ultimately furnished sixteen fractions (FR-1 to FR-16) (Scheme
III). The fraction FR-2 which eluted with n-hexane- EtOAc (9.5 : 0.5) provided white
crystalline solid on slow evaporation which was filtered and recrystallized from same
solvent system to obtain colorless needles of lupenyl acetate (1 ; 40 mg). The mother
liquor (2.0 g) of FR-2 was subjected to pencil CC over silica gel and eluted with n-
hexane, n-hexane –EtOAc in increasing order of polarity to gave 38 fractions (FR-2-1
to FR-2-38). The fraction FR-2-5 obtained with n-hexane–EtOAc (9.5: 0.5) afforded
stigmasterol (2; 20 mg) and FR-2-11 eluted with n-hexane-EtOAc (9:1) gave β-
sitosterol (3; 25 mg). The fraction FR-3 of the main EtOAc fraction which eluted
with n-hexane-EtOAc (9:1) showed two major spots on TLC. Purification through
preparative TLC in the same solvent system provided palmitic acid (4; 22 mg) and β-
amyrin (5; 6.0 mg). The fraction FR-6 was subjected to CC over silica gel (scheme
IV) eluting with n-hexane, n-hexane–EtOAc in increasing order of polarity furnished
10 fractions (FR-6-1 to FR-6-10). The fraction FR-6-3 obtained with n-hexane-
74
EtOAc (8:2) was purified through preparative TLC using mobile phase CHCl3–
MeOH (1:1) to obtain a colorless crystalline compound which was characterized as
oleanolic acid (6; 4.0 mg). On the other hand, FR-6-7 obtained with n-hexane- EtOAc
(1:1) crystallized from CHCl3-MeOH (1:1) gave β-sitosterol-3-O- β–D-
glucopyranoside (7; 5.5 mg). The fraction FR-9 of the main EtOAc fraction was
further subjected to CC over silica gel using CHCl3, CHCl3-MeOH in increasing
order of polarity to obtain 6 fractions (FR-9-1 to FR-9-6). The fraction FR-9-3
obtained with CHCl3-MeOH (9:1) gave colorless solid which was recrystallized from
the same solvent system to afford ferulic acid (8; 3.0 mg).
75
Extraction and Isolation
+ Methanol(5 times repeatedly; R.T.)
Aerial Parts of Xanthium strumarium Linn
Methanolic Extract
Solvent removed under vaccum
Crude Extract (XS- ME)*+EtOAc
+ H2O
EtOAc Phase Aqueous Phase
(Neglected)+Aqueous Na2CO3 (4%)
EtOAc Phase
1) General work up
2) Charcoal
EtOAc eluate Charcoal bed
solvent removed under
reduced pressureMeOH-C6H6 (1:1)
EtOAc residue MeOH-C6H6 eluate
solvent removed under vaccum
MeOH-C6H6 eluate residue
combined
Residue (XS- N)
Aqueous Na2CO3 phase
(Not worked up in the present studies)
Scheme-I
76
DCM
Residue (XS-N)
+ n-Hexane
n-Hexane
solublefraction (XS- HX)*
n-Hexane
insoluble fraction
+ Dichloromethane (DCM)
DCM
soluble fraction (XS- DC)*insoluble fraction
+EtOAc
EtOAc
insoluble fraction EtOAc
soluble fraction+ n- Butanol
n- Butanol
insoluble fraction
(neglected)
n- Butanol
soluble fraction (XS- BU)*
solvent removed under
reduced pressure
Residue
(XS- EA)*
* attributes the fractions exhibiting antimicrobial and antioxidant activities
Scheme-II
77
FR-9 FR-11 FR-12 FR-13 FR-14 FR-15 FR-16FR-8FR-7FR-6FR-5FR-4FR-3FR-2FR-1 FR-10
XS-EA*
CC
(n-Hexane, n-Hexane-EtOAc in
order ofincreasing polarity)
CC
(CHCl3,CHCl3-MeOH;
in order of increasing polarity)
FR-9-1 FR-9-2 FR-9-3 FR-9-4 FR-9-5 FR-9-6
Recrystallization from
CHCl3- MeOH(1:1)
Crystals
Ferulic acid (8)
Preparative TLC
n-Hexane- EtOAc(9:1)
Palmitic acid(4)
amyrin (5)
Kept at room temperature
over night in n-Hexane-EtOAc(9.5:0.5),
filter
Lupenyl acetate (1) Mother Liquor
concentrated in vaccum
CC
(n-Hexane, n-Hexane-EtOAc;
in order of increasing polarity)
FR-2-1 FR-2-2 FR-2-12
-38
FR-2-11FR-2-3 FR-2-6 FR-2-9 FR-2-10FR-2-4 FR-2-5 FR-2-7 FR-2-8
n-Hexane-EtOAc(9.5:0.5)
Stigmasterol (2)
(scheme IV)
-sitosterol (3)
n-Hexane-
EtOAc
(9:1)
CHCl3- MeOH(9:1)
Scheme-III
78
FR-6
CC
(n-Hexane, n-Hexane-EtOAc
in order of increasing polarity)
FR-6-2FR-6-1 FR-6-10FR-6-7FR-6-4FR-6-3 FR-6-8FR-6-6FR-6-5 FR-6-9
Preperative TLC
CHCl3 -MeOH (I:1)
Oleanolic acid (6)
Crystallized from
CHCl3- MeOH (1:1)
sitosterol-3-O-D-glucopyranoside (7)
Scheme-IV
79
1.4.4 Characterization of compounds
1.4.4.1. Characterization of Lupenyl acetate (1)
Colourless needles (40 mg).
M.P: 214-215°C.
IR (KBr) νmaxcm-1
: 1735 (C=O), 1630 (C=C), 1200(C-O).
EIMS: m/z (rel. int., %), 468 (M +
, 51), 453 (17), 408 (13), 249 (18), 218 (91) and
204 (44).
HR-EIMS: m/z 468.3970 (calcd for C32H52O2, 468.3969); 453.3770 [C31H49O2]+,
408.3751 [C30H48]+, 249.1850 [C16H25O2]
+, 218.2030 [C16H26]
+ and
204.1871[C15H24]+.
1H-NMR: (CDCl3, 300 MHz): δ 4.67 and 4.55 (each1H, d, J =2.3 Hz, H-29), 4.46
(1H, dd, J=10.0, 5.7 Hz,H-3α), 2.36 (1H, m, H-19), 2.02 (3H, s, COCH3), 1.68 (3H,
s, H-30), 1.05 (3H, s, H-26), 0.98 (3H, s, H-27), 0.85 (9H, s, H-25, 24, 23), 0.81 (3H,
s, H-28).
13C-NMR: (CDCl3, 75 MHz): δ 38.3 (C-1), 23.8 (C-2), 81.0 (C-3), 37.8 (C-4), 55.3
(C-5), 18.2 (C-6), 34.2 (C-7), 40.8 (C-8), 50.3 (C-9), 37.1 (C-10), 20.9 (C-11), 25.1
(C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.5 (C-16), 43.0 (C-17), 48.0 (C-18),
48.3 (C-19), 150.9 (C-20), 29.8 (C-21), 40.0 (C-22), 27.9 (C-23), 16.5 (C-24), 16.1
(C-25), 15.9 (C-26), 14.5 (C-27), 18.0 (C-28), 109.3 (C-29), 19.2 (C-30), 171.0
(C=O) and 21.3 (COCH3).
80
1.4.4.2. Characterization of Stigmasterol (2)
Colourless needles (20 mg).
M.P: 169-170°C.
IR (KBr) νmax cm -1
: 3380 (OH), 1660 (C=C).
EIMS: m/z (rel. int., %), 412 (M +
, 22), 397 (45), 394 (38), 379 (17), 367 (18), 273
(24), 255 (76), 229 (32), 211 (38), 199 (27), 173 (29), 145 (50) and 119 (40).
HR-EIMS: m/z 412.3742 (calcd for C29H48O, 412.3707); 397.3353 [C28H45O]+,
394.3570 [C29H46]+, 379.3372 [C28H43]
+, 367.3006 [C26H39O]
+, 273.2219 [C20H33]
+,
255.2093 [C19H27]+, 229.1617 [C18H13]
+, 211.1462 [C16H19]
+, 199.1464 [C15H19]
+,
173.1317 [C13H17]+, 145.1019 [C11H13]
+ and 119.0860 [C9H11]
+ .
1H-NMR: (CDCl3, 300 MHz): δ 5.34 (1H, br.s, H-6), 5.17 (1H, m, H-22), 5.03 (1H,
m, H-23), 3.50 (1H, m, H-3), 0.99 (3H, s, Me-19), 0.83 (3H, d, J = 6.7 Hz, Me-27),
0.81 (3H, t, J =7.0 Hz, Me-29), 0.78 (3H, d, J = 6.7 Hz, Me-26) and 0.67 (3H, s, Me-
18).
81
1.4.4.3. Characterization of β-Sitosterol (3)
Colourless solid (25 mg).
M.P: 134-135°C.
IR (KBr) νmax cm-1
: 3400 (OH), 1640 (C=C).
EIMS: m/z (rel. int., %), 414 (M +
, 32), 396 (43), 381 (17), 273 (26), 255 (74), 213
(35), 133 (12) and 55 (36).
HR-EIMS: m/z 414.3862 (calcd. for C29H50O, 414.3864); 396.3773 [C29H48]+,
381.3563 [C28H45]+, 273.2563 [C20H33]
+, 255.2160 [C19H27]
+, 213.1625 [C16H21]
+,
133.1021 [C10H13]+ and 55.6955 [C4H7]
+.
1H-NMR: (CDCl3, 500 MHz): δ 5.33 (1H, br. s, H-6), 3.40 (1H, m, H-3), 1.00 (3H,
s, Me-19), 0.90 (3H, d, J = 6.0 Hz, Me-21), 0.82 (3H, t, J = 7.0 Hz, Me-29), 0.80 (3H,
d, J = 6.5 Hz, Me-26), 0.78 (3H, d, J = 6.5 Hz, Me-27) and 0.67 (3H, s, Me-18).
82
1.4.4.4. Characterization of Palmitic acid (4)
White crystals (22 mg).
M.P: 61-62ºC.
IR (KBr) νmax cm-1
: 3450-2610 (COOH), 2925 and 2850 (CH), 1700 (C=O) and
1120 (C-O).
EIMS: m/z (rel. int., %), 256 (M +
, 12), 227 (17), 213 (34), 199 (16), 171 (27), 129
(61), 73 (90) and 45 (22).
HR-EIMS m/z: 256.2389 (calcd for C16H32O2, 256.2403); 227.2026 [C14H27O2]+,
213.1840 [C13H25O2]+, 199.1705 [C12H23O2]
+, 171.1368 [C10H19O2]
+, 129.0925
[C7H13O2]+, 73.0302 [C3H5O2]
+, 45.0120 [CO2H]
+.
1H-NMR: (MeOD, 300 MHz): δ 2.25 (2H, t, J = 7.5 Hz, H-2), 1.26 (26H, br. s, 13 х
CH2 chain), 0.85 (3H, t, J = 6.6 Hz, Me-16).
83
1.4.4.5. Characterization of β–Amyrin (5)
Colourless solid (6.0 mg).
M.P: 195- 196ºC.
IR (KBr) νmax cm-1
: 3352 (OH), 1648 (C=C), 2720, 2801 (CH).
EIMS: m/z (rel. int., %), 426 (M+, 49), 411 (15), 408 (10), 393 (10), 218 (100), 207
(42), 203 (53), 189 (60), 175 (24), 147 (25), 135 (45), 119 (27), 109 (41) and 69 (55).
HR-EIMS: m/z 426.0100 (calcd. for C30H50O, 426.3862); 411.3412 [C29H47O]+,
408.1502 [C30H48]+, 393.223 [C29H45]
+, 218.0420 [C16H26]
+, 207.3301 [C14H23O]
+,
203.1354 [C15H23]+, 189.3010 [C14H21]
+, 175.0132 [C13H19]
+, 147.1945 [C11H15]
+,
135.1093 [C10H15]+, 119.2203 [C9H11]
+ , 109.0110 [C8H13]
+ and 69.2015 [C5H9]
+ .
1H- NMR: (CDCl3, 500 MHz): δ 5.15 (1H, t, J = 4.5 Hz, H- 12), 3.09 (1H, dd, J =
11.0, 4.0 Hz, H-3α), 1.09, 1.00, 0.99, 0.94, 0.87, 0.86, 0.85 and 0.79 (each 3H, s,
8×Me).
84
1.4.4.6. Characterization of Oleanolic acid (6)
Colourless needles (4.0 mg).
M.P: 195-197C.
IR (CHCl3) νmax cm-1
: 3410-2789 (COOH), 2768 (C-H), 1710 (C=O), 1625 (C=C),
and 1066 (C-O).
EIMS: m/z (rel. int.), 456 (M +
, 4), 248 (100), 207 (23), 203 (31), 189 (10) and 133
(21).
HR-EIMS: m/z 456.3410 (calcd for C30H48O3, 456.3530), 248.1730 [C16H24O2]+ ,
207.1643 [C14H23O]+ , 203.2735 [C15H23]
+ , 189.1549 [C14H21]
+ and 133.1081
[C10H13]+ .
1H-NMR: (CDCl3, 300 MHz): 5.28 (t, J = 3.5 Hz, H-12), 3.40 (1H, dd, J = 14.0,
4.0 Hz, H-3α), 2.80 (1H, dd, J = 14.1, 4.6 Hz, H-18) 1.20, 1.15, 0.97, 0.94, 0.86, 0.85
and 0.75 (3H each, s, 7×Me).
85
1.4.4.7. Characterization of β-Sitosterol-3-O-β-D-glucopyranoside (7)
Colourless crystals (5.5 mg ).
M.P: 284º- 285ºC
IR (KBr) νmax cm-1
: 3410 (OH), 1638 (C=C), 1248 (C-O).
EIMS: m/z (rel. int. %), 414 (19), 396 (100), 255 (36), 135 (20), 120 (15).
HRFAB-MS (+ve): m/z 577.2877 [M + 1]
+ .
1H-NMR: (CDCl3, 500 MHz): δ 5.32 (1H, br. s, H-6), 4.48 (1H, d, J= 7.5 Hz, H-1'),
3.43 (1H, m, H-3), 3.25 – 4.45 (glucose protons) (5H, m, H-2',3',4',5' and 6'), 1.02
(3H, s, Me-19), 0.90 (3H, d, J= 6.3 Hz, Me-21), 0.82 (3H, t, J = 6.9 Hz, Me-29), 0.81
(3H, d, J = 6.4 Hz, Me- 26), 0.79 (3H, d, J = 7.0 Hz, Me-27) and 0.66 (3H, s, Me-18).
86
1.4.4.8. Characterization of Ferulic acid (8)
Colourless solid (3.0 mg ).
M.P: 168-172 ºC.
IR (KBr) νmax cm-1
: 3345 – 2578 (COOH), 1675 (C=O), and 1619 (C=C).
EIMS: m/z (rel. int. %), 194 (M +
, 10), 163 (32), 149 (24), 123 (19) and 92 (5).
HR-EIMS: m/z 194.0136 (calcd. for C10H10O4, 194.2110), 163 [C9H7O3]+, 149
[C9H9O2]+, 123 [C7H7O2]
+ and 92 [C6H4O]
+.
1H-NMR: (CDCl3, 500 MHz): δ 7.48 (1H, d, J = 14.5 Hz, H-7), 7.21 (1H, d, J = 7.8
Hz, H-6), 7.15 (1H, br. s, H-2), 6.60 (1H, d, J = 7.8 Hz, H-5). 6.01 (1H, d, J = 14.5
Hz, H-8) and 3.89 (3H, s, OCH3).
87
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CHAPTER-2
Synthesis in addition Bioactivities of
2,3-Diaminonaphthalenimidazole
Derivatives
98
Introduction
99
2.0. General Introduction For
Chapter 2 and 3
100
General Introduction
Organic chemistry is the study of carbon compounds. The carbon has unique
property of combining with itself and with other atoms (H, O, N, S, P and halogens)
in a number of ways producing acyclic or cyclic compounds. The cyclic organic
compounds are of two types, homocyclic compounds (composed of carbon and
hydrogen only) such as benzene and heterocyclic compounds (containing at least one
atom other than carbon and hydrogen in their cyclic skeleton) like pyridine [1-4].
The heterocyclic compounds are more prominent because of their variable properties.
They play a pivotal role and have great importance in medicinal and pharmaceutical
field. They are abundantly distributed in nature and many of them are essential for
life because these compounds perform significant and vital role in all physiological
functions. For instance, DNA which is fundamental for existence of life, is genetic
code material comprises of heteronuclei i.e. purine and pyrimidine bases. Proteins
that build the external and internal skeleton of a living organism is actually a polymer
of amino acids. Enzymes which are natural biological catalyst assist in many
physiological processes in every second with high level of substrate specificity thus,
maintain, control and speed up body functions. Likewise vitamins such as B12, A, E,
riboflavin, biotin and pyridoxine etc., are essential constituents of daily diet. These
are necessary for better body performance. Hemoglobin transport oxygen to each and
every cell of the body. Similarly, hormones like thyroids balance and regulate body
functions. ATP and ADP (Adenosintri and di phosphate) are energy packets.
Chlorophyll, a green color pigment found in plant is photosynthesizing compound [5-
8].
A vast number of natural and synthetic heterocyclic compounds possesses valuable
pharmacological properties hence, are of clinical uses. For example penicillin is a
potent antibiotic. Chloramphenicol is a protein synthesis inhibitor. Quinine is used to
treat malaria. Caffeine and benzodiazepines are psychopharmacological agents. It is
noticed that nearly all synthetic drugs such as diazepam, barbiturates and
azidothymidine are heterocyclic compounds [9-16].
101
Besides the field of medicine these compounds have found extensive uses in applied
chemistry like dyeing, polymer, rubber, paints, solvents, photographic sensitizers,
valuable synthetics intermediates. In addition, insecticides, pesticides and
rodenticides all are different composition of organic compounds [17-22].
The medicinal chemist takes keen interest in infectious diseases because these
infections are continuously going to rise as the resistance against human pathogens is
increasing. There is a vast range of synthetic organic compounds which possesses
valuable bioactivities due to which they play a crucial role in the development of
medicines. Now there is a list of analgesics, sedatives, anti-aging, anti-inflammatory,
CNS depressants, anti- diabetics etc [23-27].
Organic synthesis has been appeared as an indispensable, influential and valuable
tool for the development of drugs. It is difficult to understand the basics biological
phenomenon without the fundamental knowledge of organic chemistry.
Generally organic synthesis can be divided in to two main groups:
(a) Target oriented synthesis. This is the synthetic route for a natural product, defined
or designed molecule.
(b) Method oriented synthesis. In this group usually an already existing scheme is
used preferably to designed methods in order to get high quality and pure products
[28-30].
The aim of this study was to synthesize different derivatives of 2, 3-
diaminonaphthalenimidaozle and amides of piperic acid then subsequent screening of
their biological activity. The synthetic portion therefore, comprises of synthesis, in
addition of biological screening of derivatives of 2, 3-diaminonaphthalenimidazole in
chapter-2 and of amides of piperic acid dealt in chapter-3 of this dissertation.
102
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[29] L. F. Tietze and T. Eicher, “Reaction and Synthesis in the Organic Chemistry
Laboratory”, D. Ringe (Translator), University Science Books, Mill Valley,
California, p.4 (1998).
[30] W. A. Smit, A. F. Bochkov and R. Caple, “Organic Synthesis, the Science
behind the Art”, The Royal Society of Chemistry, UK, p.232-350 (1998).
104
2.1 Introduction of Benzimidazole
The condensation product of primary amines with an aldehyde or ketone is
known as Schiff base (1). The term is named after Hugo Schiff. The compound
contains a carbon–nitrogen double bond where nitrogen atom connected to an alkyl or
aryl group. The Schiff base which is comprised of a five membered planar aromatic
ring having two nitrogen atoms, one bonded with a hydrogen atom while other shows
pyridine like character is termed as Imidazole (2). When imidazole fused with
benzene ring is called as Benzimidazole (3). It is bicyclic, aromatic heterocycle
compound. The word ‘azole’ represents nitrogen atoms. The protonated nitrogen is
assigned as 1 and tertiary nitrogen as 3.
R1, R2, R3 = alkyl or aryl
Figure 1: Structure of Schiff base (1), Imidazole (2) and Benzimidazole (3)
Imidazoles have much strong chemical stability towards bases, acids and
catalytic hydrogenation. It is observed that benzimidazoles undergo hydrogenation in
the benzene nucleus while imidazole moiety remains unaffected. Benzimidazole
which is an elaborated imidazole exists in natural products possessing biological
importance [1-4].
2.1.1. Biological Importance
Benzimidazole moiety exist as a part of highly significant biomolecule found
in nature such as fifty percent of bases of nucleic acid which are genetic code
105
material, is comprised of benzimidazole moiety i.e adenine (4) and guanine (5) [5].
Vitamin B12, a naturally occurring compound, is essential for blood cells formation
and better function of central nervous system, has cobalt in co-ordination with N-
riboysl-dimethyl benzimidazole [6-9]. Kealiiquinone (6) is a natural benzimidazole
alkaloid which was isolated from Lucetta. It exhibited anti-cancer activity [10].
Figure 2: Structure of Adenine (4), Guanine (5) and Kealiiquinone (6)
The compounds containing benzimidazole moiety attached to a heterocyclic
nucleus revealed a broad spectrum of biological activities. A wide range of synthetic
benzimidazole derivatives have been reported for their physiological and
pharmacological properties hence their scope in remedying several diseases like
epilepsy, influenza, herpes, obesity, infections and depression etc. [11-13].
106
It is also reported by many researchers that the bio-activities of benzimidazole
and its analogues are due to modification in the nucleus by insertion of different
heterocyclic system to exhibit broad spectrum of biological activities [14] such as
anti-bacterial (7) [15-17], anti-fungal (8) [18,19], anti-tubercular (9) [20], anti-
inflammatory (10), analgesic [21], anti-cancer towards human breast (11) and human
colon carcinoma [22], anti-viral (12) [23,24], diuretic, cytotoxic [25], anti-asthmatic,
anti-diabetic [26], anti-oxidant (13) and anthelmintic activities [27].
107
Figure 3: Structures of some biologically active benzimidazoles
108
A wide range of benzimidazoles are in general use like thiabendazole (14)
(anthelmintic), lansoprazole (15) (anti-ulcerative), astemizole (anti-histamine),
albendazole and oxytetracycline (veterinary medicine), as well as (5-substituted
benzimidazole) alkane (16) possesses good anti-leishmanial activity, on the other
hand 2-mercaptobenzimidazole derivatives (17) exhibited analgesic property [28-30].
N
NH
S
N
N
NH
S
H3CO
OCH3
CH3
H3C
N
N NH2
N
N
N
H
S
(14) (15)
(16)
(17)
O
N
N
Figure 4: Structures of generally used benzimidazoles
A series of 2-substituted N-hydroxyacrylamide benzimidazoles were found to
be inhibitor of human histone deacetylase towards A 549, HL 60 and PC 3 cells [31]
where as aminoacridine derived benzimidazoles showed moderate to significant
kinase inhibition against CDK-5, 3 and 1 [32]. Additionally, 1-substituted
109
benzimidazole displayed potential for inhibitory activity in platelet derived growth
factor receptors (PDGFRs) [33].
N-substituted bis-benzimidazole derivatives have been appeared good for
anti-HIV activity as well as cytotoxicity against human erythrocyte kidney cell line
(HET-293T) and human lungs cancer cell line (NCI-H 23) [34]. In addition, a series
of benzylvanilline benzimidazole compound (18) has shown anti-proliferative
property in leukemia cancer cell HL 60 line. [35].
The derivatives of bis-benzimidazole with malonic acid appeared as an anti-
cancer agent because of their DNA topoisomerase I and cytotoxic character against A
431 and MCF 7 cells [36].
It was also observed that 2-amino benzimidazoles which are attached to an
imidazole moiety (19) were active H3-antagonist [37].
N
N
H
O
OCH3
N N
NH
HN
N OCH3H
(18)
(19)
Figure 5: Structures of anti-leukemic (18) and H3-antagonist (19) benzimidazoles
110
Recently many researchers have elucidated naphthoimidazoles for their
biological activities. The derivatives are found as an effective anti-tumor agent-
against human cervical carcinoma (He La), human hepatoma (SMMC -7721) (20)
and some solid tumors [38-40]. Novel naphthoimidazoles (21) have been developed
that reveal considerable potential against Mycobacterium tuberculosis with MIC
value ≤ 0.78 g / ml. The property can be modified against H374 RV (ATCC 27294)
showing MIC values 9.12 and 4.2 M on introducing p.toluyl and indolyl groups of
chalcone [41, 42]. A series of compounds based upon naphthoimidazole derivatives
of natural quinones proved to possess trypanosomal activity against blood stream
form of trypanosoma cruzi, some show EC50 value 15.5 M (22) [43-48].
H3CO
N
NN
O
OHN O
NH
NCF3
(20) (21)
NH
N
O
O
(22)
Figure 6: Structures of biologically active naphthoimidazole containing compounds
Both Benzimidazole and Naphthoimidazoles also have important and
appreciable industrial as well as analytical applications. It is found that aldo-
111
naphthimidazoles can efficiently be used to determine compositional and absolute
configuration in natural polysaccharides through HPLC techniques and by capillary
electrophoresis (CE) methods [49, 50].
Naphthalene based polyimides display high stability and selectivity with
strong electrode surface co-herence due to which they can be used as glucose
immobilization oxidase [51]. These are also analysed to display sensor properties
towards heavy metals captions [52]. A bilayer heterojunction solar cell has been
developed through conjugation of naphthalene-bis-imidazole and zinc phthalocyanin
(23). It serve as an electron acceptor, V = 0.50 V [53]. Some derivatives of
naphthalene benzimidazoles (24) were applied as useful substance in organic dye
sensitized solar cell [54].
N
N
N
N
OO
n
N
N
O
(23)(24)
Figure 7: Structure of naphthoimidazoles used in solar cell
Many compounds with benzimidazole moiety are used in electronics,
photography, as fire retardant and as transition metal corrosion inhibitor [55].
112
2.2 Synthetic Approaches Towards Benzimidazoles
A variety of different methods are reported in literature for the synthesis of
Benzimidazoles and its analogues. Some of them are given below:
2.2.1. Environment friendly synthesis of Benzimidazoles
A library of benzimidazole (26) has been synthesized via copper mediated
intramolecular N-arylation of substituted imides (25). The use of water as a solvent
rendered the method exclusively economical and green (Scheme 1) [56].
N
NHX
R'
H
(25)
CU2O, K2CO3
H2O, 100oC
30hN
HN
R'
(26)
X = Br, I
R' = Me, Ar
Scheme-1
2.2.2. Infra red radiation and clay mediated synthesis of Benzimidazole
A new simple and inexpensive methodology was reported for the synthesis of
benzimidazole (29) by reaction of o-phenylenediamine (27) and carboxylic acid (28)
using clay and infra red radiations in absence of solvent (Scheme-2) [57].
113
NH2
NH2
ClCH2 OH
O
N
N
Cl
H(29)
+
Bentonite
IR, 20 min
(27) (28)
Scheme-2
2.2.3. Synthesis of Benzimidazole via recyclable catalyst
The condensation of o-phenylenediamine (27) and aromatic addehyde (30)
was carried out successfully via oxidation through sulfonic acid functionalized silica
catalyst to afford benzimidazole (31). The recovery and reusing ability of the catalyst
for three reaction cycle with similar reactivity is the advantage of the reaction
(Scheme-3) [58].
NH2
NH2
H Ar
O
N
N
H
Ar
(31)
+
Sulfonic acid
functionalised
silica
CH2Cl2, r. t., 1-2 hr
(27) (30)
Scheme-3
2.2.4 Synthesis of 2-substituted Benzimidazoles
o-phenylenediamine (27) and o-anisaldehyde (32) were allowed to couple
effectively in presence of benzoquinone to afford benzimidazole (33) in appreciable
yield (Scheme-4) [59].
114
NH2
NH2
OCH3
CHO
N
N
H H3CO
(33)(27)
+ Benzoquinone
Ethanol
(32)
Scheme-4
2.2.5 Microwave-assisted synthesis of Benzimidazole
Microwave-assisted condensation of o-phenylenediamine (27) and aldehyde
(30) is a fast and efficient technology to obtain benzimidazole (31) in solvent free
condition (Scheme-5) [60].
NH2
NH2
H Ar
O
N
N
H
Ar
(31)(27)
+
Na2S2O5
microwave
60 sec.
(30)
Scheme-5
2.2.6. Chemoselective synthesis of 2-Arylbenzimidazoles
Ortho substituted aniline (34) and arylaldehyde (30) undergo condensation through
hydrogenperoxide and cericammonium nitrate (CAN) in solvent free condition to
give chemoselective 2-arylbenzimidazole (35) in high yield (Scheme-6) [61].
115
YH
NH2
N
Y
Ar
(35)(34)
+ Ar H
CAN, H2O2
50 o
C, 70 min.
solvent free
(30)
O
Y = S, NH
Scheme-6
2.2.7 Copper catalysed synthesis of Benzimidazoles
One pot condensation of three components 2-halo aniline (36), aryl aldehyde
(30) and sodium azide (37) was efficiently done by using catalytic amount of CuCl in
dimethylsulfoxide (DMSO) to obtain benzimidazole (31) in good yield (Scheme-7)
[62].
X
NH2
N
N
H
Ar
(31)(36)
+ Ar H120
oC, 12 hr.
(30)
O
CUCl, DMSO
X = Br, I
(37)
+ NaN3
Scheme-7
2.2.8 Synthesis of Benzimidazole through functionalized orthoesters
An efficient method for preparation of benzimidazole (40) has been developed
by condensation of ortho-substituted aniline (38) with functionalized orthoester (39)
using BF3.OEt2 at room temperature (Scheme-8) [63].
116
YH
NH2
R'O OR'
OMeR''
BF3. OEt2
N
Y
R"
(40)(38)
+DCM
r.t., 4h
(39)
Y = O, S, NH
R', R'' = alkyl
Scheme-8
2.2.9 Synthesis of Benzimidazole by using lodine as an oxidant
Hypervalent iodine can be used as an oxidant for the condensation of
phenylenediamine (27) and aldehyde (30) in presence of dioxane to obtain
benzimidazole (31) in high yield at ambient temperature in very short time (Scheme-
9) [64].
Scheme-9
2.2.10 Synthesis of Benzimidazole via heterogeneous base catalyst
KF/Al2O3 was found as a favorable solid heterogeneous base catalyst in one-
pot coupling of o-phenylenediamine (27) and carbonyl compound (41) for the
synthesis of benzimidazole (42) at room temperature (Scheme-10) [65].
117
NH2
NH2
NH
N
R
(42)(27)
+CH2Cl2, r.t.
(41)
KF-Al2O3
R X
O
R = Alkyl, Aryl
X = Cl, -OR, -O2CR
Scheme-10
2.2.11 Synthesis of Benzimidazoles from esters
o-phenylenediamine (27) undergo condensation with esters (43) in microwave
(mw) irradiation mediated method to produce benzimidazoles (42) with high yield in
very short time (Scheme-11) [66].
NH2
NH2
R C
O
OEt
(CH2OH)2
NH
N
R
(42)(27)
+mw, 1.5 min.
(43)R = alkyl, aryl
Scheme-11
2.2.12 Phtolysis of protected Benzimidazole
Protected benzimidazole (44) under longer photolysis in presence of dioxane
got deprotected to give benzimidazole (3) (Scheme-12) [67].
118
N
N
NO2
NH
N
(3)(44)
h
Dioxane
Scheme-12
2.2.13 Synthesis of Benzimidazole from a common intermediate
Benzimidazole derivatives (46) have been prepared from cyclization of a
common intermediate i-e arylaminoxime (45) by using triethylamine (TEA) in
dichloromethane. The product obtained in good yield (Scheme-13) [68].
N
N
(46)(45)
23 oC, 6hNH
N
Ar
OHTEA, CH2Cl2
Ar
Scheme-13
2.2.14 Synthesis of 2-substituted Benzimidazole in presence of H2O2
Preparation of 2-substituted benzimidazoles (31) has been carried out in
excellent yield through an efficient one–pot coupling of o-phenylenediamine (27) and
aromatic aldehydes (30) by the use of hydrogen peroxide and HCl in acetonitrile at
room temperature (Scheme-14) [69].
119
NH
N
(31)(27)
NH2
NH2
H Ar
O
+
H2O2 30%
HCl 37%
MeCN, r.t.
30-50 min
Ar
(30)
Scheme-14
2.2.15 Synthesis of Benzimidazoles through reductive cyclisation
The reductive cyclisation of aromatic nitro aniline (47) with formic acid (48)
has been achieved in presence of iron powder and 2-propanol to produce
benzimidazole (49) in appreciable yield. (Scheme-15) [70].
NH
NO2
R
H C
O
OH
N
N
R
(49)(47)
+
(48)
Fe, NH4Cl
2-propanol
80oC, 1-3 hr
R = H, Ph, Et
Scheme-15
2.2.16 Solution-phase synthesis of Benzimidazole
Solution phase synthesis of benzimidazoles (52) was carried out by
condensation of 1,2-phenylenediamine (50) and aldehyde (51) in wet dimethyl
formamide (DMF) and oxone. Products of high purity were obtained by simple
aqueous precipitation (Scheme-16) [71].
120
NH
NH2
R
R'C
O
HN
N
R
(52)(50)
+
(51)
Oxone
DMF/H2O
r.t. 22h
R, R' = alkyl, Aryl
R'
Scheme-16
2.2.17 Synthesis of naphtho [1,2-d] imidazoles
1,2-diaminonaphthalene (53) undergoes condensation with furfural (54) in
presence of organic solvent at room temperature to produce naphtho [1,2-d]
imidazole (55) (Scheme-17) [72].
NH2
NH2
OOHC X
DCM
N
HNO X
(55)(53)
+
(54)
r.t
X = H, Br, NO2
Scheme-17
2.2.18 Synthesis of naphtho [2,3-d] imidazoles
Naphtho [2,3-d] imidazoles (58) was synthesized by simple condensation of
2,3-diamino naphthalene (56) with formazylglyoxylic acid (57) in presence of ethanol
(Scheme-18) [73].
121
EtOH
NH2
NH2
N
N
H
N=NH-Ar
N-NH-Ar
N=NH-Ar
N-NH-ArHOOC
O
(56)
+
(57)
reflux
(58)
Scheme-18
2.2.19 A simple and efficient synthesis of naphtho [2,3-d] imidazoles
2,3-Diaminonaphthalene (56) was effectively condensed with 1-(9-alkyl-9H-
carbazol-3-yl)-4-carboxy-2-pyrrolidinone (59) in DMF at 170-230oC to obtain 2-
substituted naphtho [2,3-d] imidazole (60) in good yield (Scheme-19) [74].
DMF
NH2
NH2
N
N
O
COOH
C2H5
N
N
O
C2H5
N
N
H
(56)
+
(59)
170o - 230
oC
3hr
(60)
Scheme-19
122
2.2.20 Iodine-catalysed synthesis of aldo-naphthimidazole
Aldo-naphthimidazoles (62) were obtained by iodine-catalysed oxidative
condensation of 2,3-diamine naphthalene (56) with different aldoses (61) in presence
of acetic acid at room temperature (Scheme-20) [75].
(56)
+NH2
NH2
N
N
H
OHO
HO
OH
OHOH
I2, CH3COOH
r.t. reflux
(61)OH
OH
HOHO
OH
(62)
Scheme-20
123
2.3. Results and Discussion
124
2.3.1 Chemistry
The above literature study shows that the nitrogen containing heterocyclic
compounds are associated with a number of biological properties, same for
benzimidazole system which is nitrogen containing five–membered heterocycles of
immense biological and clinical uses. Their high therapeutic character switched our
interest towards synthesis of naphthalenimidazole derivatives. With a little difference,
it is almost similar in structure to that of benzimidazole. We assumed it might
possesses biological properties and uses similar to that of benzimidazole moiety.
Very little work is reported on its synthesis. So we aimed to synthesize 2,3-diamino
naphthalenimidazole derivatives first time from conventional method that is generally
used for preparation of imidazoles and to screen them for their biological activities.
2.3.2. General method for the synthesis of compounds (65-99).
2,3-diaminonaphthalenimidazoles (65-99) were synthesized by reacting
commercially available 2,3-diaminonaphthalene with different aromatic aldehydes in
N, N-dimethyl formamide (DMF). The resulted products were obtained in good yield
(Scheme-21).
In a typical reaction, sodium metabisulfite (Na2S2O5) was mixed to stirring
solution of 2,3-diaminonaphthalene (3.12 mmol) and substituted aromatic aldehyde
(3.16 mmol) in DMF (15 ml). The reaction mixture was refluxed at 110oC for 4 hr.
The reaction progress was monitored by TLC. After completion of reaction, the
reaction contents were cooled at room temperature. Then cold distilled water was
added with vigorous shaking till the precipitates of solid residue. It was kept aside on
an ice-bath to settle down the precipitates. The solid was filtered and washed with
distilled water to obtain product in good yield. Recrystallization from ethanol
afforded pure 2,3-diaminonaphthalenimidazole derivatives (65-99).
125
The structures of all synthesized compounds were elucidated with the help of
1H-NMR and EI spectroscopy techniques. All compounds also give satisfactory
elemental (CHN) analysis.
NH2
NH2
N
N
(63)
+
(65 - 99)
H R
O
Na2S2O5
DMF, 110oC
Refluxed
R
H
(64)
1
2
3
6
9
10
11
12
13
Scheme-21
Synthesis of 2,3-diaminonaphthalenimidazole derivatives (65-99)
126
Table-1: Synthesis of 2,3-diaminonaphthalenimidazole derivatives (65-99)
Comp. R Comp. R
65
N
H
1'
2'
5'
6'
7'
8'
72
CF3
2' 3'
6' 5'
66 OCH3
OC2H5
2'
6' 5'
73
NO2
2' 3'
6' 5'
67
OH
3'
6' 5'
2'
74 OCH3
OH
OCH3
2'
6'
68 OCH3
OCOCH3
2'
5'6'
75 OCH2C6H52' 3'
6' 5'
4'
69
Br
H3CO3'
6'
4'
76 F
OCH3
3'
6' 5'
70 H3CO
OCH3
6'
3'
4'
77 OH
OCH3
2'
6' 5'
71
6'
3'
4'
HO
Cl
78
OCH2C6H5
3'
6' 5'
2'
127
79
OCH3
OC2H5
6' 5'
2'
86
SCH3
2' 3'
6' 5'
80
OH
O2N
6' 5'
3'
87 O2N
6' 5'
3'
4'
81
S
4'
5'
3'
88
5'
6'
4'
3'
7'
8'1'
82
OCH3
OCH32'
6' 5'
89
N
CH3
CH35'6'
2' 3'
83 HO
OH
3'
6' 5'
90 HO OCH3
Br
5'
4'
84 H3CO OCH3
OCH3
5'6'
91
OCH3
OCH3
Br
6'
3'
85
OH
6'
HO
5'
OH
92
6' 5'
2' 3'
4'
128
93 C2H5O
6' 5'
3'
4'
94 H3CO OCH3
6' 5'
4'
95 H3CO
OCH3
Br
6'
3'
96
OCH3
Br
5'6'
2'
97
6'
3'
F
Br
5'
98 Cl OCH3
6'
4'
5'
99
6'
4'
5'
Br2'
129
2.3.3. General structure elucidation of compounds (65-99)
N
N
NO2
H
2' 3'
5'6'
12
3
6
913
12
11
10
Figure-8: Structure of compound (73)
The structure of compound (73), taken as a representative example, was
established through spectroscopic techniques. The 1H-NMR was carried out in
DMSO-d6 on 300 MHz. The signal of a singlet at 13.31 confirmed a proton of –NH
and a singlet of two protons, H-6 and 9 was appeared at 8.12. Signals of doublet of
doublet at 8.04 (J = 6.4, 3.2 Hz) and at 7.41 (J = 6.4, 3.2 Hz) identified H-10/13
and H-11/12 respectively of naphthalene moiety where as presence of two doublets
one at 8.44 and other on 8.55 with same coupling constant (J = 8.7 Hz) indicates
two protons H-2/ 6 and next two protons H-3/ 5 of phenyl ring respectively.
The structure of synthetic compound (73) was also determined by
fragmentation patterns, through EI-MS spectra where the molecular ion peak at m/z =
289 leads to molecular formula C17H11N3O2. A peak at m/z = 259 is due to the loss of
NO groups, subsequent loss of CO group from this fragment give a peak at m/z = 231.
Where as the peak appeared due to loss of nitro group (–NO2 ) at m/z = 243. The EI-
MS spectrum also displayed a peak at m/z = 288, obtained by the loss of Hydrogen
from the molecular ion. The loss of nitro phenyl group was determined by the peak at
m/z = 167. The peak at m/z = 141 was obtained by the subsequent loss of nitril group
(CN) from it where as the further loss of hydrogen was indicated by the peak at m/z =
140. A peak at m/z = 122 is due to nitro phenyl ion (Figure-9). From the above
discussion the compound (73) assigned as 2-(4-nitrophenyl)-1H-naphtho[2,3-
d]imidazole. The structures of other synthetic compounds were elucidated in the same
way.
130
N
NH
N
N
NO2
-NO2
-H
N
NH
NO2
N
NH
O
N
NH
NO2
NO2
N
NH
N
NH
-CN
m/z = 243
m/z = 288
+
+
m/z = 289
+
m/z = 259
+
- CO
-
m/z = 167
+
m/z = 122
m/z = 231
N
m/z = 141
+
+
+
-NO
H
Figure-9: Fragmentation patterns of compound (73)
131
2.3.4. Biological Evaluation of Compounds (65-99)
2.3.4.1. In Vitro Tyrosinase Inhibitory Activity
The synthetic compounds (65-99) were screened for their in vitro tyrosinase
inhibitory activity (for method see chapter 4). These compounds showed inhibition in
the range of 52.3-136.2 µM. The compound 81 was found as the most active among
the series exhibiting IC50 = 52.3±0.196 µM. The compound 85 appeared as a second
highest active compound having IC50 = 66.5±0.350 µM. Moreover the compounds
65, 66, 68, 69, 71, 79, 88 and 94 showed moderate tyrosinase inhibitory activity with
IC50 = 106.8 ±0.269, 107.2 ± 0.159, 103.5± 0.210, 102.0± 0.524, 133.6± 0.215,
132.0± 0.125, 136.2± 0.159 and 125.2± 0.212 µM respectively. The activity exhibited
by compounds 81 and 85 might be due to presence of thiophene nucleus and
trihydroxyl groups in phenyl nucleus. The rest of compounds revealed inhibition
under 50%, therefore, were not studied further for IC50. The results are depicted in
Table-2.
132
Table- 2 In vitro tyrosinase inhibitory activity of compounds 65-99
Compounds IC50 ± SEM
(µM)
Compounds IC50± SEM
(µM)
65 106.8±0.269 83 NA
66 107.2±0.159 84 NA
67 NA 85 66.5±0.350
68 103.5±0.210 86 NA
69 102.0±0.524 87 NA
70 NA 88 136.2±0.159
71 133.6±0.215 89 NA
72 NA 90 NA
73 NA 91 NA
74 NA 92 NA
75 NA 93 NA
76 NA 94 125.2±0.212
77 NA 95 NA
78 NA 96 NA
79 132.0±0.125 97 NA
80 NA 98 NA
81 52.3±0.196 99 NA
82 NA Kojic acid 16.67±0.06
Key: SEM = standard error of the mean, IC50 = concentration of compounds that
inhibit 50% enzyme activity, NA= not active
133
2.3.4.2. In Vitro Acetylcholinesterase and Butrylcholinesterase Inhibitory
Activity
All the synthesized compounds (65-99) were screened for their in vitro
acetylcholinesterase and butrylcholinesterase inhibitory activity (for protocol see
chater 4). These compounds were observed possessing IC50 values in the range of
22.7-85.9 µM for butrylcholinesterase inhibitory activity. Among these, compounds
65, 66 and 79 were found to be the most active for butrylcholinesterase inhibitory
activity with IC50= 22.7±0.09, 35.2±0.213 and 29.3±0.121µM respectively. Their
activity may be due to the presence of indole and alkoxy moiety whereas moderate
activity with no structure correlation was observed for compounds 67, 81 82 and 89
(IC50= 85.9 ±0.125, 55.4± 0.121, 58.2 ±0.192 and 52.1± 0.22 µM respectively). The
rest of compounds showed inhibition under 50% therefore, were not further studied.
Unfortunately all compounds were found inactive for acetylcholinesterase activity.
The results are displayed in Table-3 and 4.
134
Table – 3 In vitro butrylcholinesterase inhibitory activity of compounds 65-99
Key: SEM = standard error of the mean, IC50 = concentration of compounds
that inhibit 50% enzyme activity, NA= not active
Compounds IC50± SEM
(µM)
Compounds IC50± SEM
(µM)
65 22.7±0.09 83 NA
66 35.2±0.213 84 NA
67 85.9±0.125 85 NA
68 NA 86 NA
69 NA 87 NA
70 NA 88 NA
71 NA 89 52.1±0.22
72 NA 90 NA
73 NA 91 NA
74 NA 92 NA
75 NA 93 NA
76 NA 94 NA
77 NA 95 NA
78 NA 96 NA
79 29.3±0.121 97 NA
80 NA 98 NA
81 55.4±0.121 99 NA
82 58.2±0.192 Galanthamine 0.5±0.001
135
Table – 4 In vitro acetylcholinesterase inhibitory activity of compounds 65-99
Key: SEM = standard error of the mean, IC50 = concentration of compounds
that inhibit 50% enzyme activity, NA= not active
Compounds IC50± SEM
(µM)
Compounds IC50 ± SEM
(µM)
65 NA 83 NA
66 NA 84 NA
67 NA 85 NA
68 NA 86 NA
69 NA 87 NA
70 NA 88 NA
71 NA 89 NA
72 NA 90 NA
73 NA 91 NA
74 NA 92 NA
75 NA 93 NA
76 NA 94 NA
77 NA 95 NA
78 NA 96 NA
79 NA 97 NA
80 NA 98 NA
81 NA 99 NA
82 NA Galanthamine 0.5±0.001
136
2.3.4.3. In Vitro Urease Inhibitory Activity
The synthesized compounds (65-99) were screened for their urease inhibitory activity
according to the method described in chapter 4. Only seven compounds exhibited
varying degree of urease inhibition. Compound 71 appeared as the most active and
compound 90 as a second most active compound with IC50 values 34.2±0.72 and
42.43±0.65 µM. The highest activity of these compounds is probably due to the
presence of hydroxyl group at position-2 in phenyl nucleus. Other compounds 65, 66,
68, 81 and 82 showed moderate urease inhibitory activity with IC50 = 92.3±0.20,
73.5±0.65, 56.3±0.78, 86.4±0.78 and 67.14±0.67µM respectively. Remaining
compounds showed inhibition less than 50%, so, were not further studied. Results are
displayed in Table -5.
137
Table- 5 In vitro urease inhibitory activity of compounds 65-99
Key: SEM = standard error of the mean, IC50 = concentration of
compounds that inhibit 50% enzyme activity, NA= not active
Compounds IC50± SEM
(µM)
Compounds IC50± SEM
(µM)
65 92.3±0.20 83 NA
66 73.5±0.65 84 NA
67 NA 85 NA
68 56.3±0.78 86 NA
69 NA 87 NA
70 NA 88 NA
71 34.2±0.72 89 NA
72 NA 90 42.43±0.65
73 NA 91 NA
74 NA 92 NA
75 NA 93 NA
76 NA 94 NA
77 NA 95 NA
78 NA 96 NA
79 NA 97 NA
80 NA 98 NA
81 86.4±0.78 99 NA
82 67.14±0.67 Thiourea 21.7±0.12
138
2.3.4.4. In Vitro Anti-bacterial Activity
All synthetic compounds (65-99) were tested for their in vitro anti-bacterial activity
by using disc diffusion method (described in chapter 4). The anti-bacterial activity
was determined in comparison of streptomycine as standard, against twelve gram
positive bacterial strains i.e. Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis,
Corynebacterium diphtheriae, Corynebacterium hoffmanii, Mycobacterium luteus
ATCC 9341, Staphylococcus aureus, Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus faecalis
and Streptococcus pyogenes whereas the nine gram negative bacterial strained used
were Enterobacter, Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B,
Shigella flexeneri and Shigella dysenteriae. The results were reported on the basis of
diameter of zone of inhibition in mm that was appeared around the disc (7mm). All of
the synthesized compounds showed varying degree of anti-bacterial activity. Only
two compounds 90 and 92 showed promising activity against B. cereus creating zone
of inhibition 15mm (MIC= 25 µg/ml) and 11mm zone of inhibition (MIC= 100
µg/ml) was displayed by the compound 81 against the same bacterial strain.
Compounds 82, 90, 92 and 94 were found moderately active against B. subtilis
showing zone of inhibition 11, 13, 13and 11mm respectively (MIC=100, 50, 50 and
100 µg/ml). Only compound 65 showed weak activity (zone of inhibition =10 mm,
MIC= 100 µg/ml) against B. thuringiensis. Compound 74 was found moderately
active for C. diphtheriae creating zone of inhibition=12mm (MIC= 100 µg/ml) and
also exhibited activity with 11mm zone of inhibition against S. aureus. 12 mm zone
of inhibition and MIC= 100 µg/ml was observed by compound 82 against C.
hoffmanii. Furthermore compounds 82, 90 and 92 displayed moderate activity against
S. epidermidis creating 12, 12 and 11mm zone of inhibition respectively ( MIC= 100
µg/ml each). Only compound 65 showed moderate activity for S. saprophyticus with
zone of inhibition=11mm and MIC= 100 µg/ml. Two compounds 72 and 82 showed
moderate activity by creating zone of inhibition 11 and 13mm against S. feacalis with
139
MIC values 100 and 50 µg/ml respectively. The rest of compounds were found
weakly active against tested gram positive bacterial strains.
For the activity against gram negative bacteria compounds 90, 92 and 99 showed
significant activity against Enterobacter , exhibiting zone of inhibition 16, 14 and 15
mm with MIC= 25, 50 25µg/ml respectively. Compound 82 and 85 were moderately
active against E. coli showing zone of inhibition 12 and 10 mm (MIC= 35 and
25µg/ml respectively). Only compound 65 was active against K. pneumoniae
showing zone of inhibition 12mm and MIC=100 µg/ml. Compound 90 was
moderately active against S. paratyphi A with 13 mm zone of inhibition (MIC= 100
µg/ml). Compounds 90, 92 and 99 exhibited good activity with 12, 15 and 13 mm
zone of inhibition against S. dysenteriae (MIC= 100, 25 50 µg/ml). Compound 77
was the only one that was found inactive in this anti-bacterial assay. The rest of
compounds were weakly active, they showed less than 50% anti-bacterial activity,
hence, were not further studied. The results of anti-bacterial assay are displayed in
Table- 6 and 7.
140
Table – 6 In vitro anti-bacterial activity of compounds 65- 99
Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
Gram positive bacteria
Bacillus cereus 9 - 8 - - - 7 9 - 9 7 7 - - 9 - 11 7 -
Bacillus subtilis 8 - 7 9 - 7 9 9 - 9 9 8 - - 10 - 7 11 7
Bacillus thuringiensis 10 7 7 7 7 - 8 9 - 8 - - - - - - - 8 -
Corynebacterium diphtheriae 8 7 8 7 7 9 7 9 7 12 7 8 - - - - - 9 9
Corynebacterium hoffmanii 10 - - - 9 7 7 8 - 10 7 8 - 7 7 8 8 12 -
Mycobacterium luteus ATCC 9341 8 - 8 8 - - 7 8 - 9 - - - 7 7 8 - 7 9 Staphylococcus aureus 7 7 7 8 7 - 7 9 7 11 7 8 - - - - - - -
Staphylococcus aureus (MRSA) 8 8 9 8 7 7 7 7 - 9 8 - - - - - 8 7 -
Staphylococcus epidermidis 8 - 8 - - - 7 - - 8 7 7 - - 8 7 8 12 -
Staphylococcus saprophyticus 11 7 8 - - 7 8 9 7 9 7 8 - 7 7 8 8 7 9
Streptococcus feacalis 9 7 10 7 8 - 7 11 - 10 9 8 - 9 7 8 - 13 -
Streptococcus pyogenes - - - - - - - - - - - - - - 8 - 10 10 10
141
Continue Table-6
Microorganisms 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Streptomycin
Bacillus cereus 7 - - - - - 15 8 15 - - 8 - - - 9 18
Bacillus subtilis - - - - - 9 13 9 13 10 11 9 7 7 9 10 18
Bacillus thuringiensis - 7 - 7 7 7 8 7 9 8 8 7 - - - 9 18
Corynebacterium diphtheriae - - - - 10 7 7 7 10 - - - - - - - 22
Corynebacterium hoffmanii 8 - - - - 7 10 7 9 7 7 7 7 7 7 7 22
Mycobacterium luteus ATCC 9341 7 - 7 10 - - 10 - 10 - - - - - - 10 22
Staphylococcus aureus - - 7 8 9 - 10 10 - - 7 - - - - - 25
Staphylococcus aureus (MRSA) - - - 7 7 9 10 8 10 - 8 7 7 7 8 9 25
Staphylococcus epidermidis - 8 - - - 8 12 7 11 8 10 9 9 7 8 10 18
Staphylococcus saprophyticus 9 - 7 7 7 - - - - - 10 - - - - - 25
Streptococcus feacalis 7 - - - - 8 7 7 8 8 10 8 7 7 7 7 *
Streptococcus pyogenes 8 9 8 7 7 - - - - 8 9 8 7 8 7 7 18
142
Continue Table-6
Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
Gram negative bacteria
Enterobacter 9 - 10 7 - - 7 9 7 9 9 7 - - - 8 8 10 -
Escherichia coli 7 7 8 - - - 7 - - - - - - - - 8 - 12 -
Klebsiella pneumoniae 12 7 11 8 7 8 8 8 7 10 8 8 - - 8 - - 9 -
Pseudomonas aeruginosa - - - - - - - 10 - - - - - 8 7 7 7 7 -
Salmonella typhi - 7 - - - - - - - - - - - 7 7 8 - 8 8
Salmonella paratyphi A 7 - 8 - - - - - - - - - - - - - - 7 7
Salmonella paratyphi B - - - - - - - - - - - - - - - - - 8 -
Shigella flexeneri 7 - - - - - - - - - - - - - - - - - -
Shigella dysenteriae 7 7 8 8 7 7 7 7 7 7 8 8 - - - 8 10 10 9
143
Continue Table-6
Gram negative bacteria 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Streptomycin
Enterobacter - - - 7 - - 16 - 14 - - - - - - 15 22
Escherichia coli 7 10 - 9 8 8 - - - - - - - - - - 10
Klebsiella pneumoniae - - - 7 - - - - - - - - - - - - 18
Pseudomonas aeruginosa - - - - - - 8 7 - - 7 7 - 7 - - 16
Salmonella typhi - 9 7 7 7 - 8 7 - - 7 7 7 - 7 - 15
Salmonella paratyphi A - - - - - - 13 7 9 - 7 - - - - - *
Salmonella paratyphi B - - - - - - - - - - - - - - - - 17
Shigella flexeneri - - - - - - 7 8 8 - - - - - - - *
Shigella dysenteriae - - - 8 - 7 12 7 15 8 10 8 10 10 9 13 10
key: values are zone of inhibition (mm) and an average of triplicate, (-) indicates resistance. * Means not applied.
144
Table-7 MIC (minimum inhibitory concentration) values of compounds
Microorganism MIC values of compounds (µg/ml)
65 67 72 74 79 81 82 83 85 87 88 90 92 93 94 96 97 99
Gram positive
bacteria
Bacillus cereus - - - - - 100 - - - - - 25 25 - - - - - Bacillus subtilis - - - - 100 - 100 - - - - 50 50 100 100 - - - Bacillus
thuringiensis 100 - - - - - - - - - - - - - - - - -
Corynebacterium
diphtheriae - - - 100 - - - - - - 100 - 100 - - - - -
Corynebacterium
hoffmanii 100 - - 100 - - 100 - - - - 100 - - - - - -
Mycobacterium
luteus
ATCC 9341
- - - - - - - - - 100 - 100 100 - - - - -
Staphylococcus
aureus - - - 100 - - - - - - - 100 100 - - - - -
Staphylococcus
epidermidis - - - - - - 100 - - - - 100 100 - 100 - - -
Staphylococcus
saprophyticus 100 - - - - - - - - - - - - - 100 - - -
Streptococcus
feacalis - 100 100 100 - - 50 - - - - - - - 100 - - -
Streptococcus
pyogenes - - - - - - 100 100 - - - - - - - - - -
145
Microorganism MIC values of compounds (µg/ml)
65 67 72 74 79 81 82 83 85 87 88 90 92 93 94 96 97 99
Gram negative
bacteria - - - - - - - - - - - - - - - - - -
Enterobacter - 100 - - - - - - - - - 25 50 - - - - 25 Escherichia coli - - - - - - 35 - 25 - - - - - - - - - Klebsiella pneumoniae 100 100 - 100 - - - - - - - - - - - - - - Pseudomonas
aeruginosa - - 100 - - - - - - - - - - - - - - -
Salmonella paratyphi
A - - - - - - - - - - - 100 - - - - - -
Shigella dysenteriae - - - - - - - - - - 100 25 - 100 100 100 50
146
2.3.4.5. In Vitro Anti-fungal Activity
All the synthetic compounds (65-99) were also tested for their in vitro anti-fungal
activity in comparison of ketoconazole as standard (for methods see chapter 4),
against Aspergillus flavis, Aspergillus nigar, Penicillium species, Rhizopus species,
Candida albicans, Candida albicans ATCC, Sachromyces cerevisiae, Fusarium
species, Helminthosporum, Microsporum canis, Microsporum gypsium,
Trichophyton rubrum, Trichophyton tonsurans and Trichophyton mentagrophytes.
The compounds 65, 66, 67, 68, 74, 80 and 82 were found exhibiting moderate activity
against C. albicans with zone of inhibition in the range of 9-11mm. Compound 74
also showed moderate activity against Fusarium species, M. canis, and T. rubrum
with 9, 9 and 11 mm zone of inhibition. Whereas the compound 92 showed moderate
activity with zone of inhibition 10, 10 and 9 mm against S. cerevisiae, Fusarium
species and T. tonsurans. All the rest of synthesized compounds showed less than
50% zone of inhibition against all the applied fungal strains. Therefore, they were not
further studied. Anti-fungal results are presented in Table-8.
147
Table – 8 In vitro anti-fungal activity of compounds 65- 99
Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
Aspergillus flavis - - - - - - - - - - - - - - - - - - -
Aspergillus nigar - - - - - - - - - - - - - - - - - - -
Penicillium species - - - - - - - - - - - - - - - - - - -
Rhizopus species - - - - - - - - - - - - - - - - - - -
Candida albicans 9 11 10 10 7 8 - 7 - 10 - - - - 7 9 8 10 8
Candida albicans ATCC - - - - - - - - - - - - - - - - - - -
Sachromyces cerevisiae 7 - 7 - - - - - - 7 - - - - - - - - -
Fusarium species 8 7 - - - - - - - 9 - - - - - - - - -
Helminthosporum 7 8 - - - - - - - 8 - - - - - - - - -
Microsporum canis - 7 - - - - - - - 9 - - - - 7 7 - 8 -
Microsporum gypsium 7 - - - - - - - - - - - - - - - - - -
Trichophyton rubrum - 7 7 - - 7 - 8 - 11 - - - 7 - - 7 8 -
Trichophyton tonsurans - - - - - - - - - - - - - - - - - - -
Trichophyton
mentagrophytes - - - - - - - - - - - - - - - - - - -
148
Continue Table-8
Microorganisms 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Ketoconazole
Aspergillus nigar - - - - - - - - - - - - - - - - 24
Penicillium species - - - - - - - - - - - - - - - - 24
Rhizopus species - - - - - - - - - - - - - - - - 22
Candida albicans - 8 - 7 7 8 - - - - - - - - - 7 22
Candida albicans ATCC - - - - - - - - - - - - - - - - 22
Sachromyces cerevisiae - - - - - 7 7 - 10 - 8 - - - - - 24
Fusarium species - - - - - - - - 10 - - - - - - - 22
Helminthosporum - - - - - - - - - - - - - - - - 22
Microsporum canis - - - - - - - - - - - - - - - - 22
Microsporum gypsium - - - - - - - - - - - - - - - - 24
Trichophyton rubrum - - - 8 - - - - - - - - - - - - 24
Trichophyton tonsurans - - - - - - - - 9 - - - - - - - 22
Trichophyton
mentagrophytes - - - - - - - - - - - - - - - - 22
key: values are zone of inhibition (mm) and an average of triplicate, (-) indicates resistance.
149
2.3.4.6. In Vitro Anti-oxidant Activity
All the synthesized compounds (65-99) were screened for their in vitro anti-
oxidant activity against 1, 1-diphenyl-2-picryl hydrazil (DPPH) radical along with
Ascorbic acid as a standard by using protocol described in chapter 4. The
compounds 65, 68, 77, 90 and 99 showed significant anti-oxidant activity having
% inhibition 71.1, 67.4, 71, 71, and 69.3% with EC50 = 37.5, 75, 37.5, 37.5, and
75 µg/ml respectively. Whereas compounds 79, 82, 85 and 95 exhibited moderate
anti-oxidant activity with % inhibition of 57.8, 57.8, 57.9 and 57.7 having EC50 =
100 µg/ml for each compound. The rest of compounds revealed % inhibition less
than 50%, therefore, were not further studied. The results are shown in Table-9.
150
Table–9 In vitro anti-oxidant activity of compounds 65-99
Compds % Inhibition + SD EC50
(µg/ml) Compds % Inhibition + SD EC50
(µg/ml)
65 71.1 + 0.01 37.5 83 43 + 0.01 -
66 43 + 0.01 - 84 13 + 0.01 -
67 27 + 0.01 - 85 57.9 + 0.02 100
68 67.4 + 0.01 75 86 17 + 0.01 -
69 43 + 0.01 - 87 32 + 0.01 -
70 43 + 0.01 88 22 + 0.01 -
71 29 + 0.01 - 89 33 + 0.01 -
72 13 + 0.01 - 90 71.1 + 0.01 37.5
73 47 + 0.01 - 91 45 + 0.01 -
74 12 + 0.01 - 92 43 + 0.01 -
75 19 + 0.01 - 93 29 + 0.01 -
76 29 + 0.01 - 94 43 + 0.01 -
77 71 + 0.01 37.5 95 57.7 + 0.01 100
78 47 + 0.01 - 96 49 + 0.01 -
79 57.8 + 0.01 100 97 23 + 0.01 -
80 47 + 0.01 - 98 27 + 0.01 -
81 32 + 0.01 - 99 69.3 + 0.01 75
82 57.8 + 0.02 100 Ascorbic
acid 80 8.3
Key: SD = standard deviation, EC50 = effective concentration of compounds that
scavange 50% radical, (-) indicates resistance.
151
2.3.5. Conclusion
All the synthesized derivatives of 2, 3-diaminonaphthalenimidazole (65-99) were
screened for their various biological activities. Only ten compounds showed
tyrosinase inhibitory activity in which two compounds 81 and 85 were found most
active and the rest of eight compounds 65, 66, 68, 69, 71, 79, 88 and 94 were
moderately active.
In acetyl and butrylcholinesterase inhibitory assay, total seven compounds
exhibited butrylcholinesterase activity in which three compounds 65, 66 and 79
showed good activity while four compounds 67, 81, 82 and 89 were moderately
active. Unfortunately all candidates were found inactive towards
acetylcholinesterase inhibition activity.
Total seven compounds were found active in urease inhibitory assay. Two
compounds 71 and 90 exhibited good activity and rest of five 60, 65, 66, 81 and
82 were moderately active.
The antimicrobial activity of all the synthesized compounds was also determined.
The compounds 90 and 92 were found significantly active against nearly all
applied bacterial strains, whereas 65, 72, 74, 77, 81, 82, 85, 94 and 99 have
moderate antibacterial activity. It is found that 65, 66, 67, 68, 74, 80, 82 and 92
were the candidates having moderate antifungal activity.
The synthesized compounds were also tested for antioxidant activity. They
displayed varying % inhibition, among them 65, 68, 77, 90 and 99 exhibited
remarkable antioxidant activity.
In view of these biological assays we can conclude that the above cited candidates
may serve as lead compounds for further studies.
152
2.4. Experimental
153
2.4.1 General Experimental
All the reagents and solvents used for synthesis were purchased from E.
Merck, Germany. Melting points were determined in glass capillary using Gallen
Kamp melting point apparatus and are uncorrected. EI-MS spectra were recorded
on Jeol JMS-600H. 1H-NMR spectra were performed on Avance AV-300, 400
and 500 NMR spectrometers operating at 300, 400 and 500 MHz in
Dimethylsulphoxide (DMSO-d6) with trimethylsilane (TMS) as an internal
standard. Thin layer chromatography (TLC) was carried out on precoated silica
gel glass plates (Kieselgel 60, 254, E. Merck, Germany), UV visualized
chromatograms at 254 and 365 nm. The elemental (CHN) analysis was done on a
Carlo Erba Strumentazion-Mod-1106, Italy.
2.4.2 General method for the synthesis of compounds (65-99)
In a typical reaction, sodium metabisulfite (Na2S2O5) was mixed to a
stirring solution of 2,3-diaminonaphthalene (3.12 mmol) and substituted aromatic
aldehyde (3.16 mmol) in DMF (15 ml). The reaction mixture was refluxed at
110oC for 4hr. The reaction progress was monitored by TLC. After completion of
reaction, the reaction contents were cooled at room temperature. Then cold
distilled water was added with vigorous shaking till the precipitated of solid
residue. It was kept aside on an ice-bath to settle down the precipitates. The solid
was filtered and washed with distilled water to obtain product in good yield.
Recrystallisation from ethanol afford pure 2,3-diaminonaphthalenimidizole
derivatives.
All compounds were synthesized applying same methodology. The
structures of all synthesized compounds were determined with the help of 1H-
NMR and EI-MS spectroscopy techniques. All compounds also gave satisfactory
elemental (CHN) analysis.
154
2.4.2.1. 2-(1H-indol-3-yl)-1H-naphtho[2,3-d]imidazole (65)*
Yield: 0.2g (70%); M.P: 289-291ºC; 1H-NMR: (500 MHz, DMSO-d6): 13.71
(1H, bs, -NH), 12.21 (1H, s, H-1′), 8.45 (1H, br.d , J5’,6’ = 6.0 Hz, H-5′), 8.44 (1H,
s, H-2′), 8.13 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12= 6.4, 3.2 Hz, H-10/13), 7.60
(1H, br.d, J8’,7’ = 6.0 Hz, H-8′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.30
(2H, m, H-6′/7′); EI-MS: m/z (rel. abund. %), 283 (M+, 44), 282 (10), 149 (15),
141 (10), 140 (11), 135 (38), 115 (9), 71 (34), 44 (100); Anal. Calcd for C19H13N3
(283. 11): C, 80.54; H, 4.62; N, 14.83; Found: C, 80.52, H, 4.61; N, 14.85
2.4.2.2 2-(4-ethoxy-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (66)*
Yield: 0.18g (58 %); M.P: 265-267ºC; 1H-NMR (400 MHz, DMSO-d6): 13.22
(1H, bs, -NH), 8.16 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
7.87 (2H, dd, J6’/5’,2’ = 8.4, 2.0 Hz, H-2′/6′), 7.43 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-
11/12), 7.23 (1H, d, J5’,6’ = 8.4 Hz, H-5′), 4.14 (2H, q, J = 7.2 H2, - OCH2), 3.92
(3H, s, -OCH3), 1.37 (3H, t, J = 7.2 Hz, - CH3); EI-MS: m/z (rel. abund. %), 318
(M+, 100 %), 303 (6), 289 (57), 288 (15), 275 (13), 273 (7), 261 (31), 260 (13),
168 (5), 141 (7), 140 (18); Anal. Cald for C20H18N2O2 (318.14): C, 75.45; H, 5.70;
N, 8.80; Found: C, 75.43; H, 5.69; N, 8.78.
2.4.2.3 4-(1H-naphtho[2,3-d]imidazole-2-yl)phenol (67)
Yield: 0.15g (47 %): M.P: 268–269ºC; 1H-NMR (400 MHz, DMSO-d6): 10.51
(1H, s, -NH), 8.15 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′) 8.12 (2H, s, H-6/9), 8.07 (2H,
dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.44 (2H, dd, J11/10,13= 6.4, 3.2 Hz, H-11/12),
7.04 (2H, d, J3’,2’ = 8.4 Hz, H-3′/5′); EI-MS: m/z (rel. abund %), 260 (M+, 100),
231 (11), 141 (10), 140 (27), 130 (19), 120 (6); Anal. Calcd for C17H12N2O
(260.09): C, 78.44; H, 4.65; N, 10.76; Found: C, 78.42; H, 4.64; N, 10.78.
2.4.2.4 2-methoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenyl acetate (68)*
Yield: 0.17g (56 %): M.P: 244-245oC;
1H-NMR (400 MHz, DMSO-d6): 10.81
(1H, s, -NH), 8.22 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
155
8.02 (1H, d, J2’/6’ = 2.0 Hz, H-2′), 7.89 (1H, dd, J6’/5’,2’ = 8.4, 2.0 Hz, H-6′), 7.44
(2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.40 (1H, d, J5’/6’ = 8.4 Hz, H-5′), 3.94
(3H, s, OCH3), 2.31 (3H, s, -COCH3); EI-MS: m/z (rel. abund. %), 332 (M+, 87),
298 (6), 291 (56), 290 (100), 289 (54), 275 (23), 192 (5), 141 (8), 140 (17); Anal.
Calcd for C20H16N2O3 (332.12); C, 72.28; H, 4.85; N, 8.43; Found: C, 72.23; H,
4.84; N, 8.44
2.4.2.5 2-(5-bromo-2-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (69)*
Yield: 0.19g (57 %); M.P: 230-232ºC; 1H-NMR (300 MHz, DMSO-d6): 12.27
(1H, s, -NH), 8.49 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 8.19 (1H, s, H-6), 8.05 (1H, s, H-
9), 8.01 (2H, m, H-10/13), 7.96 (1H, dd, J4’/3’,6’ = 8.0, 2.4 Hz, H-4′), 7.35 (2H, m,
H-11/12), 7.28 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 4.05 (3H, s, -OCH3); EI-MS: m/z (rel.
abund. %), 352 (M+, 100), 351 (38), 337 (18), 322 (23), 308 (6), 272 (9), 243 (26),
170 (10), 168 (18), 154 (8), 141 (14), 140 (50), 107 (11), 75 (6); Anal. Calcd for
C18H13BrN2O (352.02); C, 61.21; H, 3.71; N, 7.93; Found: C, 61.23; H, 3.70; N,
7.91
2.4.2.6 2-(2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (70)*
Yield: 0.18g (61%); M.P: 225-226ºC; 1H-NMR (300 MHz, DMSO-d6): 12.22
(1H, s, -NH) 8.18 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
7.92 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 7.42 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12),
7.28 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 7.20 (1H, dd, J4’/3’,6’ = 8.0, 2.4 Hz, H-4′), 4.02
(3H, s, -OCH3), 3.83 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M+, 98),
303 (20), 289 (13), 286 (100), 274 (42), 246 (8), 144 (14), 140 (11), 107 (6), 91
(8); Anal. Calcd for C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C,
74.99; H, 5.30; N, 9.21
156
2.4.2.7 4-Chloro-2-(1H-naphtho[2,3-d]imidazol-2yl)phenol (71)
Yield: 0.19g (67 %); M.P: 289-290ºC; 1H-NMR (400 MHz, DMSO-d6): 13.30
(1H, s, -NH), 8.25 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 8.09 (2H, s, H-6/9), 8.06 (2H, m,
H-10 /13), 7.49 (2H, m, H-11/12), 7.48 (1H, dd, J4’/3’,6’ = 8.8, 2.4 Hz, H-4′), 7.10
(1H, d, J3’,4’ = 8.8 Hz, H-3′); EI-MS: m/z (rel. abund. %), 294 (M+, 100), 258 (10),
140 (26), 111 (8), 76 (9); Anal. Calcd for C17H11ClN2O (294.06); C, 69.28; H,
3.76; N, 9.50; Found: C, 69.26; H, 3.77; N, 9.49
2.4.2.8 2-(4-trifluoromethylphenyl)-1H-naphtho[2,3-d]imidazole (72)*
Yield: 0.17g (60 %): M.P: 255-257ºC; 1H-NMR (300 MHz, DMSO-d6): 13.29
(1H, s, -NH), 8.50 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′), 8.24 (2H, s, H-6/9), 8.10 (2H,
m, H-10/13), 8.06 (2H, d, J3’,2’ = 8.4 Hz, H-3′/ 5′), 7.46 (2H, m, H-11/12); EI-MS:
m/z (rel. abund. %), 312 (M+, 100), 293 (7), 274 (5), 243 (6), 173 (15), 171 (7),
157 (13), 141 (10), 140 (28), 127 (6), 114 (23), 77 (3); Anal. Calcd for
C18H11F3N2 (312.09); C, 69.23; H, 3.55; N, 8.97; Found: C, 69.22; H, 3.53; N,
8.96
2.4.2.9 2-(4-nitrophenyl)-1H-naphtho[2,3-d]imidazole (73)
Yield: 0.19g (67.8%); M.P: 283-285ºC; 1H-NMR (300 MHz, DMSO-d6): 13.31
(1H, s, -NH), 8.55 (2H, d, J3’,2’ = 8.7 Hz, H-3′/5′), 8.44 (2H, d, J2’,3’ = 8.7 Hz, H-
2′/6′), 8.12 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.41 (2H,
dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12); EI-MS: m/z (rel. abund. %), 289 (M+, 100),
288 (5), 259 (36), 243 (73), 231 (11), 167 (9), 141 (5), 140 (15), 122 (9); Anal.
Calcd for C17H11N3O2 (289.09); C, 70.58; H, 3.83; N, 14.53; Found: C, 70.56; H,
3.82; N, 14.54
157
2.4.2.10 2, 6-dimethoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (74)*
Yield: 0.21 g (64 %); M.P: 282-284 ºC; 1H-NMR (400 MHz, DMSO-d6): 10.99
(1H, s, -NH), 8.17 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
7.63 (2H, s, H-2′/6′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 3.92 (6H, s, 2×
–OCH3); EI-MS: m/z (rel. abund. %), 320 (M+, 100), 319 (15), 304 (7), 289 (10),
273 (9), 140 (11), 115 (8); Anal. Calcd for C19H16N2O3 (320.12); C, 71.24; H,
5.03; N, 8.74; Found: 71.22; H, 5.02; N, 8.75
2.4.2.11 2-(3-Benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (75)*
Yield: 0.19g (52 %); M.P: 232 – 234ºC; 1H-NMR (400 MHz, DMSO-d6): 9.96
(1H, s, -NH), 8.17 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
7.96 (1H, br, s, H-2′), 7.87 (1H, d, J6’,5’ = 8.0 Hz, H-6′), 7.56 (1H, m, H-5′), 7.47
(5H, m, Ar-H), 7.41 (2H, m, H-11/12) 7.27 (1H, dd, J4’/5’,6’ = 8.0, 2.0, Hz, H-4′),
5.25 (2H, s, -OCH2); EI-MS: m/z (rel. abund. %), 350 (M+, 60), 349 (6), 259
(10), 231 (12), 140 (11), 92 (10), 91 (100), 65 (8); Anal. Calcd for C24H18N2O
(350.14); C, 82.26; H, 5.18; N, 7.99; Found: C, 82.27; H, 5.19; N, 7.98
2.4.2.12 2-(2-fluoro-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (76)*
Yield: 0.08g (56 %); M.P: 178–179ºC; 1H-NMR (300 MHz, DMSO-d6): 12.46
(1H, s, -NH), 8.24 (1H, d, J6’,5’ =8.2 Hz, H-6′), 8.17 (2H, s, H-6/9), 8.00 (1H, s, H-
3′), 7.98 (2H, m, H-10/13), 7.37 (2H, m, H-11 /12), 7.08 (1H, d, J5’,6’= 8.2 Hz, H-
5′), 3.88 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 292 (M+, 100), 291 (6), 277
(45), 249 (27), 152 (5), 141 (7), 140 (18), 125 (9); Anal. Calcd for C18H13FN2O
(292.10); C, 73.96; H, 4.48; N, 9.58; Found: C, 73.95; H, 4.46; N, 9.57
2.4.2.13 2-methoxy-5-(1H-Naphtho[2,3-d]imidazol-2-yl) phenol (77)*
Yield: 0.2g (85 %); M.P: 287–288ºC; 1H-NMR (400 MHz, DMSO-d6): 10.55
(1H, s, -NH), 8.12 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, Hz, 3.2, H-10/13),
158
7.73 (2H, m, H-2′/6′), 7.43 (2H, dd, J11/10,13 = 6.4, 3.2, Hz, H-11/12), 7.21 (1H, d,
J5’,6’ = 8.4 Hz, H-5′), 3.89 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 290 (M+,
100), 289 (5), 275 (8), 274 (35), 247 (19), 141 (7), 140 (10), 123 (11), 108 (9);
Anal. Calcd. for C18H14N2O2 (290.11); C, 74.47; H, 4.86; N, 9.65; Found: C,
74.45; H, 4.87; N, 9.63
2.4.2.14. 2-(4-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (78)*
Yield: 0.91g (52 %); M.P: 289–290ºC; 1H-NMR (400 MHz, DMSO-d6): 9.85
(1H, s, -NH), 8.23 (2H, d, J2’,3’ = 8.8 Hz, H-2′/6′), 8.14 (2H, s, H-6/9), 8.06 (2H,
dd, J10/11,12 = 6.4, 3.2, Hz, H-10/13), 7.44 (7H, m, Ar-H), 7.35 (2H, d, J3’,2’ = 8.8
Hz, H-3′/ 5′), 5.25 (2H, s, -OCH2); EI-MS: m/z (rel. abund. %), 350 (M+, 48), 270
(6), 259 (41), 231 (28), 141 (7), 140 (26), 92 (15), 91 (100), 65 (32); Anal. Calcd
for Anal. Calcd for C24H18N2O (350.14); C, 82.26; H, 5.18; N, 7.99; Found: C,
82.29; H, 5.20; N, 8.00
2.4.2.15. 2-(3-ethoxy-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (79)*
Yield: 0.09 g (55 %); M.P: 274–275ºC; 1H-NMR (300 MHz, DMSO-d6): 9.99
(1H, s, -NH), 8.16 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2, Hz, H-10/13),
7.86 (2H, br. s, H-2′/6′), 7.45 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.25 (1H,
d, J5’,6’ = 8.8 Hz, H-5′), 4.14 (2H, q, J = 6.9 Hz, -OCH2), 4.19 (3H, s, -OCH3),
1.37 (3H, t, J = 6.9 Hz, -CH3); EI-MS: m/z (rel. abund. %), 318 (M+, 100), 317
(5), 287 (5), 273 (7), 258 (12), 243 (9), 230 (10), 168 (6), 152 (11), 141 (9), 140
(43), 123 (11), 115 (13); Anal. Calcd for C20H18N2O2 (318.14); C, 75.45; H, 5.70;
N, 8.80; Found: C, 75.44; H, 5.71; N, 8.79
2.4.2.16. 4-(1H-naphtho[2,3-d]imidazol-2-yl)-3-nitrophenol (80)*
Yield: 0.09 g (60 %); M.P: 274–276ºC; 1H-NMR (300 MHz, DMSO-d6): 11. 26
(1H, s, -NH), 8.14 (1H, s, H-3′), 8.10 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4,
3.2 Hz, H-10/13), 7.42 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.23 (1H, d, J5’,6’
159
= 8.0, Hz, H-5′), 7.12 (1H, d, J6’,5’ = 8.0, Hz, H-6′); EI-MS: m/z (rel. abund. %),
305 (M+, 55), 304 (52), 288 (19), 259 (8), 257 (7), 141 (13), 140 (100), 138 (5),
120 (9), 114 (24), 107 (10), 92 (12), 74 (20); Anal. Calcd. For C17H11N3O3 (305.
08); C, 66.88; H, 3.63; N, 13.76; Found: C, 66.86; H, 3.65; N, 13.77
2.4.2.17. 2-(thiophen-2-yl)-1H-naphtho[2,3-d]imidazole (81)
Yield: 0.13g (65 %); M.P: 264 – 265ºC; 1H-NMR (300 MHz, DMSO-d6): 11. 06
(1H, s, -NH), 8.04 (2H, s, H-6/9), 8.00 (3H, m, Ar-H) 7.84 (1H, d, J5’,4’= 4.2 Hz,
H-5), 7.38 (2H, dd, J11/10.13 = 6.4, 3.2, Hz, H-11/12), 7.28 (1H, t, J4’,5’ = 4.2 Hz,
H-4); EI-MS: m/z (rel. abund. %), 250 (M+, 100), 249 (5), 141 (18), 140 (22), 114
(43), 109 (15), 97 (13), 83 (14), 64 (57); Anal. Calcd for C15H10N2S (250.06); C,
71.97; H, 4.03; N, 11.19; Found: C, 71.96; H, 4.04; N, 11.20
2.4.2.18. 2-(3, 4-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (82)*
Yield: 0.12g (61 %); M.P: 184 – 185ºC; 1H-NMR (300 MHz, DMSO-d6):
10.61(1H, s, -NH), 8.06 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-
10/13), 7.85 (1H, br. s, H-2), 7.82 (1H, d, J6’,5’ = 8.0 Hz, H-6), 7.38 (2H, dd,
J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.20 (1H, d, J5’,6’ = 8.0 Hz, H-5), 3.90, (3H, s, -
OCH3), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M+, 100), 303
(12), 289 (15), 273 (9), 261 (32), 242 (7), 218 (31), 192 (15), 168 (10), 163 (11),
141 (21) 140 (95), 138 (12), 121 (8), 113 (40), 91 (5), 75 (13); Anal. Calcd for
C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C, 74.96; H, 5.31; N,
9.22
2.4.2.19. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1, 3-diol (83)
Yield: 0.15 g (52 %); M.P: 262 – 264ºC; 1H-NMR (300 MHz, DMSO-d6): 13.10
(1H, br. s, -NH ), 8.06 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10
/13), 7.92 (1H, d, J6,5 = 8.0, H-6), 7.40 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12),
6.50 (1H, dd, J5’/6’,3’ = 8.0, 2.0 Hz, H-5), 6.42 (1H, d, J3’,5’ = 2.0 Hz, H-3); EI-
160
MS: m/z (rel. abund. %), 276 (M+, 100), 275 (6), 219 (61), 168 (10), 141 (12), 140
(27), 115 (25), 109 (16), 79 (22), 75 (8); Anal. Calcd. for C17H12N2O2 (276. 09);
C, 73.90; H, 4.38; N, 10.14; Found: C, 73.91; H, 4.37; N, 10.13.
2.4.2.20. 2-(2, 3, 4-trimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (84)*
Yield: 0.17 (72 %); M.P: 166–167oC;
1HNMR (300 MHz, DMSO-d6):
s, -NH8.20 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.3, 3.1 Hz, H–
10/13), 8.04 (1H, d, J6’,5’ = 8.0 Hz, H– 6), 7.44 (2H, dd, J11/10,13 = 6.3, 3.1 Hz, H–
11/12), 7.12 (1H, d, J5’,6’ = 8.0 Hz, H-5), 4.01 (3H, s, -OCH3), 3.93 (3H, s, -
OCH3), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 334 (M+, 100), 330
(30), 319 (77), 303 (21), 289 (22), 193 (7), 181 (11), 167 (15), 141 (7), 140 (33);
Anal. Calcd for C20H18N2O3 (334.13); C, 71.84; H, 5.43; N, 8.38; Found: C,
71.83; H, 5.40; N, 8.40
2.4.2.21. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1, 2, 3-triol (85)*
Yield: 0.12 g (53 %); M.P: 284–285oC;
1H-NMR (400 MHz, DMSO-d6): 10.82
(1H, s, -OH), 10.65 (1H, s, -OH), 10.27 (1H, s, -OH), 8.70 (2H, s, H-6/9), 8.03
(2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.42 (2H, dd, J11/10,13 =6.4, 3.2 H-11/12),
7.09 (1H, d, J65 = 8.4 Hz, H-6) Hz), 6.51 (1H, d, J5,6 = 8.4 Hz, H-5); EI-MS:
m/z (rel. abund %), 292 (M+, 100) 262 (12), 168 (9), 151 (5), 140 (14), 126 (13),
115 (23), 79 (12); Anal. Calcd for C17H12N2O3 (292.08); C, 69.86; H, 4.14; N,
9.58; Found: C, 69.85; H, 4.14; N, 9.57
2.4.2.22. 2-(4-methylthiophenyl)-1H-naphtho[2,3-d]imidazole (86)*
Yield: 0.16 g (83 %); M.P: 290–291oC;
1H-NMR (400 MHz, DMSO-d6):
br.s.,-NH), 8.20 (2H, d, J2’,3’ = 8.4 Hz, H-2/6), 8.17 (2H, s, H–
6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H–10/13), 7.52 (2H, d, J3,2 = 8.4 Hz, H–
3/5), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H–11/12), 2.58 (3H, s, -CH3); EI-MS:
m/z (rel. abund. %), 290 (M+, 100), 275 (8), 243 (20), 141 (9), 140 (42), 113 (16),
161
75 (5); Anal. Calcd for C18H14N2S (290.09); C, 74.45; H, 4.86; N, 9.65; Found: C,
74.44; H, 4.84; N, 9.66
2.4.2.23. 2-(2-nitrophenyl)-1H-naphtho[2,3-d]imidazole (87)
Yield: 0.12 g (57 %): M.P: 250–252oC;
1H-NMR (400 MHz, DMSO-d6): 13.07
(1H, s, -NH), 8.09 (2H, m, H-3/6), 8.06 (2H, s, H-6/9), 8.01 (2H, m, H-10/13),
7.90 (1H, t, J4/5,6 = 7.6 Hz, H-4), 7.81 (1H, t, J5/4,6 = 7.6 Hz, H-5), 7.40 (2H, m,
H-11/12); EI-MS: m/z (rel. abund. %), 289 (M+, 100), 273 (6), 259 (15), 243 (18),
141 (12), 140 (93), 107 (5), 90 (9), 77 (13), 63 (48); Anal. Calcd for C17H11N3O2
(289.09); C, 70.58; H, 3.83; N, 14.53; Found: C, 70.59; H, 3.82; N, 14.54
2.4.2.24. 2-(naphthalen-2-yl)-1H-naphtho[2,3-d]imidazole (88)*
Yield: 0.15 g (78 %); M.P: 241–243oC;
1H-NMR (400 MHz, DMSO-d6):
br s, -NH8.89 (1H, br. s, H-1), 8.36 (1H, m, H-6), 8.22 (2H, s, H-
6/9), 8.18 (2H, m, H-7/8), 8.09 (2H, m, H-10/13), 7.69 (3H, m, H-3/4/5), 7.55
(2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 294 (M+, 100), 293 (41), 153 (12),
140 (42), 127 (22), 114 (31); Anal. Calcd for C21H14N2 (294.12): C, 85.69; H,
4.79; N, 9.52; Found: C, 85.67; H, 4.80; N, 9.50
2.4.2.25. N, N-dimethyl-4-(1H-naphtho[2,3-d]imidazol-2-yl)aniline (89)
Yield: 0.15g (85 %); M.P: 298–299oC;
1H-NMR (400 MHz, DMSO-d6):
s, -NH 8.12 (2H, d, J2,3 = 8.0 Hz, H-2/6), 8.07 (2H, s, H-6/9),
8.04 (2H, dd, J10/11,12 = 6.5, 3.1 Hz, H-10/13), 7.44 (2H, dd, J11/10,13 = 6.5, 3.1 Hz,
H-11/12), 6.91 (2H, d, J3, 2 = 8.0 Hz, H-3/5), 3.00 (6H, s, -N (CH3)2); EI-MS:
m/z (rel. abund. %), 287 (M+, 100), 286 (20), 272 (15), 271 (21), 244 (13), 146
(12), 141 (8), 140 (31), 121 (10), 113 (23), 105 (8), 90 (6), 76 (7); Anal.Calcd for
C19H17N3 (287.14); C, 79.41; H, 5.96; N, 14.62; Found: C, 80.00; H, 5.90; N,
14.60
162
2.4.2.26. 3-bromo-6-methoxy-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol
(90)*
Yield: 0.17 g (77 %); M.P: 209–210oC;
1H-NMR (400 MHz, DMSO–d6):
s, -NH 8.12 (2H, s, H-6/9), 8.03 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-
10/13), 7.40 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.21 (1H, d, J5’,4’ = 8.0 Hz,
H-5′), 7.09 (1H, d, J4’,5’ = 8.0 Hz, H-4′), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel.
abund. %), 368 (M+, 50), 367 (20), 289 (5), 259 (7), 243 (15), 229 (8), 169 (6),
152 (13), 141 (9), 140 (47), 76 (10); Anal. Calcd. for C18H13Br N2O2 (368.02); C,
58.56; H, 3.55; N, 7.59; Found: C, 57.69 ; H, 3.57; N, 7.58.
2.4.2.27. 2-(2-bromo-4, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole
(91)*
Yield: 0.14 g (58 %): M.P: 241–242oC;
1H-NMR (400 MHz, DMSO–d6):
s, -NH 8.20 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-
10/13), 7.48 (1H, s, H-6′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.39 (1H,
s, H-3′), 3.89 (3H, s, -OCH3), 3.84 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %),
382 (M+, 100), 381 (21), 367 (15), 351 (12), 339 (10), 322 (7), 303 (6), 242 (6),
217 (17), 216 (16), 168 (8), 141 (13), 140 (92), 115 (26), 74 (5); Anal. Calcd for
C19H15Br N2O2 (382.03); C, 59.55; H, 3.95; N, 7.31; Found: C, 59.99; H, 3.97; N,
7.29
2.4.2.28. 2-phenyl-1H-naphtho[2,3-d]imidazole (92)
Yield: 0.14 g (95 %); M.P: 280–281oC;
1H-NMR (400 MHz, DMSO-d6):
s, -NH 8.29 (2H, dd, J2’/3’,4’ = 7.2, 3.6 Hz, H-2′/6′), 8.21 (2H, s, H-
6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.68 (3H, m, H-3′/4′/5′), 7.44
(2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12);EI-MS: m/z (rel. abund. %), 244 (M+,
72), 243 (6), 141 (10), 140 (28), 114 (29), 104 (5), 89 (6), 77 (99); Anal Calcd for
163
C17H12N2 (244.10); C, 83.58; H, 4.95; N, 11.47; Found: C, 83.61; H, 4.91; N,
11.45
2.4.2.29. 2-(2-ethoxyphenyl)-1H-naphtho[2,3-d]imidazole (93)*
Yield: 0.13 g (73 %); M.P: 196–197oC;
1H-NMR (400 MHz, DMSO–d6):
s, -NH8.26 (1H, m, H-6′), 8.12 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12
= 6.3, 3.3 Hz, H-10/13), 7.50 (1H, m, H-5′), 7.38 (2H, dd, J11/10,13 = 6.3, 3.3 Hz,
H-11/12), 7.29 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 7.14 (1H, t, J4’,3’ = 8.0 Hz, H-4′),
4.37 (2H, q, J = 6.8 Hz, -OCH2), 1.47 (3H, t, J = 6.8 Hz,-CH3); EI-MS: m/z (rel.
abund. %), 288 (M+, 69), 287 (14), 273 (83), 259 (4), 244 (100), 243 (7), 231 (17),
168 (7), 141 (17), 140 (82), 107 (5), 91 (9), 76, (6); Anal. Calcd for C19H16N2O
(288.13); C, 79.14; H, 5.59; N, 9.72; Found: C, 79.12; H, 5.63; N, 9.69
2.4.2.30. 2-(2, 3-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (94)*
Yield: 0.18 g (96 %); M.P: 247–248oC;
1H-NMR (400 MHz, DMSO-d6): 12.26
(1H, s, -NH), 8.12 (2H, s, H-6/9), 7.99 (2H, m, H-10/13), 7.90 (1H, m, H-6′), 7.37
(2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.26 (2H, m, H-4′/5′), 3.90 (3H, s, -
OCH3), 3.89 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M+, 100), 303
(16), 289 (25), 274 (6), 273 (19), 258 (10), 243 (11), 169 (8), 140 (9); Anal. Calcd
for C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C, 75.00; H, 5.30; N,
9.19
2.4.2.31. 2-(4-bromo-2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole
(95)*
Yield: 0.18 g (94 %); M.P: 246–248oC;
1H-NMR (400 MHz, DMSO-d6): 12.15
(1H, s, -NH), 8.17 (2H, s, H-6/9), 8.04 (3H, m, H-10/13/6′), 7.58 (1H, s, H-3′),
7.42 (2H, m, H-11/12), 4.05 (3H, s, -OCH3), 3.94 (3H, s, -OCH3); EI-MS: m/z
(rel. abund. %), 384 (M+ 2, 100), 381 (23), 382 (96), 367 (20), 352 (38), 335 (6),
164
242 (9), 217 (31), 168 (5), 141 (6), 140 (26), 74 (7); Anal. Calcd for C19H15 Br
N2O2 (382.03); C, 59.55; H, 3.95; N, 7.31; Found: C, 59.59; H, 3.97; N, 7.35
2.4.2.32. 2-(3-bromo-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (96)*
Yield; 0.20g (95 %); M.P: 218–220oC;
1H-NMR (400 MHz, DMSO-d6): 12.47
(1H, s, -NH), 8.51 (1H, d, J2’,6’ = 2.0 Hz, H-2′), 8.29 (1H, dd, J6’/5’,2’ = 8.4, 2.0 Hz,
H-6′), 8.14 (2H, s, H-6/9), 8.06 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.43 (2H,
m, H-11/12), 7.39 (1H, d, J5’,6’= 8.4 Hz, H-5′), 3.97 (3H, s, -OCH3); EI-MS: m/z
(rel. abund. %), 352 (M+, 100), 351 (5), 337 (12), 309 (6), 273 (4), 258 (13), 154
(6), 141 (7), 140 (21), 74 (3); Anal. Calcd for C18H13BrN2O (352.02); C, 61.21; H,
3.71; N, 7.93; Found: C, 61.18; H, 3.69; N, 7.96
2.4.2.33. 2-(4-bromo-2-fluorophenyl)-1H-naphtho[2,3-d]imidazole (97)*
Yield: 0.15 g (72 %); M.P: 211–213oC;
1H-NMR (300 MHz, DMSO–d6): 10.49
(1H, br s, -NH), 8.28 (1H, d, J6’,5’ = 8.4 Hz, H-6′), 8.22 (2H, s, H-6/9), 8.02 (2H,
m, H-10/13), 7.88 (1H, d, J3’,5’ = 2.1 Hz, H-3′), 7.68 (1H, dd, J5’/6’,3’ = 8.4, 2.1 Hz,
H-5′), 7.39 (2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 340 (M+, 90), 187 (5),
172 (7), 156 (6), 141 (8), 140 (23), 80 (4), 78 (72), 63 (70); Anal. Calcd for
C17H10Br F N2 (340.00); C, 59.85; H, 2.95; N, 8.21; Found: C, 59.83; H, 2.99; N,
8.18
2.4.2.34. 2-(2-Chloro-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (98)*
Yield: 0.15 g (75 %). M.P: 148-149oC;
1H-NMR (400 MHz, DMSO-d6): 10.98
(1H, s, -NH), 8.16 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),
7.52 (2H, m, H-5′/6′), 7.42 (3H, m, H-11/12/4′), 3.95 (3H, s, -OCH3); EI-MS: m/z
(rel. abund. %), 308 (M+, 100), 307 (5), 293 (4), 276 (3), 265 (19), 242 (5), 169
(3), 155 (6), 141 (7), 140 (18), 114 (7), 113 (7), 78 (27); Anal. Calcd for
C18H13ClN2O (308.07); C, 70.02; H, 4.24; N, 9.07; Found: C, 70.00; H, 4.21; N,
9.09
165
2.4.2.35. 2-(3-bromophenyl)-1H-naphtho[2,3-d]imidazole (99)
Yield: 0.16 g (80 %); M.P: 180–181oC;
1H-NMR (300 MHz, DMSO-d6): 11.88
(1H, s, -NH), 8.48 (1H, br. s, H-2′), 8.29 (1H, d, J6’/5’ = 7.8 Hz, H-6′), 8.21 (2H, s,
H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.83 (1H, d, J4’,5’ = 7.8 Hz,
H-4′), 7.62 (1H, t, J5’/4’,6’ = 7.8 Hz, H-5′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-
11/12); EI-MS: m/z (rel. abund. %), 322 (M+, 100), 321 (3), 243 (20), 214 (10);
183 (9), 162 (18), 155 (8), 141 (7), 140 (31), 121 (19), 114 (12), 75 (5); Anal.
Calcd for C17H11BrN2 (322.01); c, 63.18; H, 3.43; N, 8.67; Found: C, 63.20; H,
3.45; N, 8.65
* indicates newly synthesized compounds.
166
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173
CHAPTER-3
Synthesis and Bioactivities of
Amides of Piperic Acid
174
Introduction
175
3.1 INTRODUCTION OF AMIDES
An amide is an organic compound that comprises of a trivalent nitrogen
atom attached to a carbonyl group. The nitrogen may bear substitution [1].
1 2 3
R NH2
O
R NHR'
O
R NR'R''
O
Where: R,R and R′′ = alkyl groups
Figure-1: General structure of amides
An amide is named after the name of its related carboxylic acid by
attaching the word “amide” at the end in place of -oic or -ic acid. Where as the
alkyl substituent attached to nitrogen atom is placed preceded by N- alkyl.
IUPAC:
Trivial:
ethanamide
acetamideN-methyl ethanamide
N-methyl acetamide
4 5
H3C NH2
O
H3C N
O
H
CH3
Figure-2: Naming of amides
Cyclic amide is known as Lactams. The ring size is designated by Greek
letters (3), (4), (5), (6) and (7) [2].
N O
H
6
Figure-3: Structure of Butyrolactam
176
Followings are classes of compounds related to amides.
Class of
Compounds
Basic structure
Urea
Imide
Carbamate
Sulfonamide
N N
O
O
N
O
H
O N
O
O
S N
O
Figure-4: Classes of compounds related to amides
Urea (7) which is excreted by higher animals is the excess nitrogenous
product of metabolism of protein. Lower animals excrete ammonia (8) where as
birds and reptiles excrete guanidine (9). These excreted substances are good and
chief nitrogen fertilizers [1-3].
Figure-5 Structures of Urea, Ammonia and Guanidine
177
3.1.1 Importance of Amides
3.1.1.1 Biological importance of Amides
Amides are very important in biological system. They play a vital role in
protein synthesis which are polyamides and build the body structure as well as
perform various functions in the organism. Glutamine (10) that enhances immune
system is a common amino acid found in proteins. Niacinamide (11) is vitamin
B3, it helps in production of natural emollient to keep skin hydrated [4]. Most of
amides of naturally occurring endogenous fatty acid serve as chemical
messengers. Anandamide (12) is a cannabinoid receptor in mammalian brain and
display analgesic effects [5]. Oleamide (13) that induces physiological sleep is
brain lipid, accumulated in cerebrospinal fluid [6]. Another endogenous fatty acid
amide, erucamide (14) stimulates new blood vessel formation [7].
178
Figure-6: Structures of natural amides
Thyrotropic–hormone releasing factor (TRF) (15) is a tripeptide. It was
isolated from the hypothalamus glands of cattle. It stimulates thyroid hormone
secretion. Another brain peptide is enkephalins (16). It is body natural painkiller,
comprises of at least five amino acid residues [8].
179
HN
N
O
O
N
NH
NH2
O
15
O N
H
Tyr-gly-gly-phe-met
16
Where:
Tyr = tyrosine, gly = glycine, phe = phenylalanine, met = methionine
Figure-7: Structure of TRF and Enkephalins
3.1.1.2 Medicinal importance of Amides
Amide bond and amide derivatives are widely involve in both naturally
occurring and synthetic biologically active compounds. Several researchers
elucidated their biological activities like antimicrobial [9], antitumor [10],
antituberculosis [11], insecticidal [12], anti-inflammatory [13], anticonvulsant
[14] and anti-platelet [15] activities, hence are pharmacologically active in the
treatment of many diseases, for examples, meprobamate (17) is a tranquillizer,
ampicillin (18) has been used as an antibiotics and sulfathiazole (19) is a potent
bacteriostatic sulfadrug [1]. A naturally occurring alkaloid, piperine (20) is the
chief component of Piper nigrum Linn., and is reported to possessed valuable
therapeutic actions like anti-depressent [16], antioxidant [17], anti-inflammatory,
anti-carcinogenic, antimicrobial [18] functions. Some researches also evaluated its
property as bioavailability enhancer for many drugs [19, 20].
180
Figure-8: Structures of biologically active compounds containing amide bond
Piperine (20) also exhibited phytotoxic effect on many vegetables [21].
Not only piperine but also its synthetic analogues have been evaluated for their
biological properties. Some researchers examined isobutylamide containing
analogues exhibited potency towards insecticidal activity [22, 23]. Synthetic
analogue (21) examined for trypanocidal activity against Trypanosoma cruzi
which causes incurable disease in humans ‘chagas’ [24]. A novel synthesized
piperamide (22) is an antioxidant and antidepressant agent [25]. Its derivative
piperlonguminine (23) has shown inhibitory activity against histone deacetylase
and human colon cancerline HCT-116, where as a hydroxamic moiety containing
analogue (24) exhibited in vitro cytotoxicity and HDAC activity [26].
181
Figure-9: Structures of synthetic piperamides
3.1.1.3 Industrial application of Amides
Amides are also utilized in industry for photographic materials, as polymer
stabilizers, pigments, foaming materials, photo developers [27, 28] and for
conformational switching [29].
182
3.2 Synthetic Approaches Towards Amides of Piperic acid
3.2.1 Design and synthesis of bioconjugates of piperic acid–glycine
Bioconjugates of piperoyl glycine were designed and synthesized by
condensation of an activated ester of piperic acid (25) with glycine (26) in
presence of dicyclohexylcarbodiimide (DCC) and dimethylaminopropionitrile
(DMAP). The reaction mixture stirred for 3 hr. at room temp. to get product (27).
All conjugates exhibited good antibacterial and antifungal activity than cefepime
and fluconazole (Scheme-1) [30].
O
O
O
O
25
+ H2NOH
O
DCC/DMAP
r.t., 3hr.
26
O
O
NH
O
OH
O
27
Scheme-1
3.2.2 Synthesis and insecticidal activity of piperamides
Amide derivatives of piperine were synthesized from piperic acid (28)
coupling with amine (29) in presence of oxalyl chloride (COCl)2 in dry
tetrahydrofuran (THF). The reaction mixture was stirred for 6 hr. at room
temperature. The pure product (30) was obtained after purification by flash
column chromatography on silica gel (diethyl ether – Hexane; 1: 2). All the
synthetic amides were tested for their insecticidal property. The results revealed
mortality range from 0 to 97.5% depending on compound and insects (Scheme-2)
[31].
183
O
O
OH
O
28
+ RR'NH
(COCl)2, THF
25 o
C, 6h
29
O
O
N
O
R
R'30
R = H, Et, i-pr
R' = aliphatic or alicyclic
alkyl group
Scheme-2
3.2.3 Synthesis and anti-leishmanial property of bioconjugates of piperoyl-
amino acid
Piperoyl amino acid conjugates were synthesized by reaction of piperic
acid (28) with amino acid methyl ester (31) in dry CH2Cl2 at -15oC with drop wise
addition of triethylamine (TEA) following methanesulfonyl chloride and then
stirred at 0oC for 4 hr. After aqueous work up crude product was subjected to
column chromatography on silica gel (CHCl3 – MeOH with increasing polarity) to
afford pure product as piperoyl–amino acid conjugate (32). Evaluation of
synthetic compounds for their anti-leishmanial activity exhibited better activity
than either amino acid methyl ester and piperine (Scheme-3) [32].
184
O
O
OH
O
H2N R
COOMe
O
O
NH
O
R
COOMe28
+
31
32
R = CH2C6H5, CH(CH3)2
H2C-C6H4-p-OH
CH2Cl2, OoC
CH3SO2Cl, TEA
Scheme-3
3.2.4 Synthesis of piperine analogues and study of their structure activity
relationship
An attempt was made to obtain piperine analogues from piperic acid (28)
and an appropriate amine (33) using SOCl2 as coupling agent in CH2Cl2 (DCM)
with stirring of 1hr. The crude product (34) that was obtained after aqueous
workup was purified by CC over SiO2 with mobile phase pet.ether–EtOAc (4:1).
The synthesized analogues were related with modification in parent molecule for
inhibition of cytochrome P 450 activities. It was found that modification in parent
molecule results lost of inhibitory potential while saturation in side chain
enhanced the property (Scheme-4) [33].
185
O
O
OH
O
28
+ R1R
2NH
33
O
O
NR1R
2
O
34
R1R
2NH = Piperidine,
pyrrolidine,
n-butyl amine
SOCl2, DCM
Refluxed, 1hr.
Scheme-4
3.2.5 Synthesis of piperamides analogues from natural safrole
Piperamide analogues were synthesized from natural safrole (35) by
applying following scheme (Scheme-5) [34].
O
O
O
O
CO2H
O
O
CO2Et
O
O
N
X
O
O
O
COH
O
O
OH
35
1- BF3, THF
R. T., 2Hr.
2- NaOH aq
Reflux 36PCC, DCM
1 hr
37
KH, DME,
triethylphos-
phonoacetate
38
Aq. LiOH, THF
4 hr
39
1- SOCl2, Reflux
1 hr
2- RNH2, DCM
R.T., 30 min
40
X = CH2, O and S
Scheme-5
186
3.3. Results and Discussion
187
3.3.1 Chemistry
The importance and applications of amides of piperic acid have been
revealed from above literature discussion. One of naturally occurring piperidine
amide of piperic acid, commonly known as piperine is well known for various
pharmacological uses. Many researchers have synthesized its analogues and
evaluated them for their application in medicinally and industrially valuable
fields. In view of this literature survey we assumed the possibility of compounds
which are similar in structure to that of piperine and may possesses activities like
it. Here in this research work we aim to synthesise amides of piperic acid by
aliphatic and aromatic amines in order to get compounds which are more potent in
their antimicrobial, antioxidant and nematicidal activities.
3.3.2 General method for the synthesis of compounds (42-56)
The amides (42-56) were synthesized from piperic acid (28) (obtained by
basic hydrolysis of commercially available piperine (20) through the formation of
an active intermediate acid chloride (41) and its subsequent condensation with
appropriate amine. The resulted products were afforded in good yields (Scheme-
6).
Generally the preparation of amides comprises of two steps: (i) conversion
of acid to acid chloride and (ii) condensation of acid chloride with an appropriate
amines.
3.3.2.1 Preparation of piperic acid
Piperine (20) (5g, 0.0175 mol) was refluxed with 300 ml of 20% KOH in
methanol at 150oC for 120 hr. Completion of reaction was monitored on TLC.
After complete hydrolysis excess of methanol was removed under reduced
pressure. The reaction worked up by suspending the residue in hot distilled water
followed by acidification with 2N HCl. The resulted precipitates were filtered,
washed with ice cold water and dried at room temperature. The crude product was
188
purified through recrystallisation from ethanol to afford pale yellow crystals of
pure piperic acid (28), m.p. 215-216 oC, [Lit. 215
oC] yield 94% [32].
3.3.2.2 Conversion of piperic acid to acid chloride
Piperic acid (28), 0.5g (0.00229 mol) was dissolved in dry
dichloromethane (DCM) 15 ml kept over an ice-bath under nitrogen atmosphere,
stirred for 10 min. After that freshly distilled thionyl chloride (SOCl2) 2ml (0.27
mol) was added dropwise and stirred the resultant solution for 2 hr till the reaction
mixture indicated orange-brown colouration. Completion of reaction was
confirmed by TLC. The excess thionyl chloride was then removed under vaccuo
leaving acid chloride (41) as an orange-brown residue.
3.3.2.3 Synthesis of amides of piperic acid by condensation of acid chloride
with an amine
To the resultant solution of crude acid chloride (41) in dry DCM 10 ml,
appropriate amine 0.7 ml (0.585 g, 0.00688 mol) dissolved in 5 ml DCM was
added. The resultant reaction mixture was refluxed at 60oC for 2-2.5 hr.
Completion of reaction was monitored on TLC (n-Hexane-EtOAc, 7:3). The
content was diluted with distilled water. The organic layer was separated out
through separating funnel. Washed with distilled water (2x25 ml), dried with
anhydrous Na2SO4 and concentrated to crude product. The crude product was
subjected to column chromatography (CC) over silica gel using mobile phase n-
Hexane – Ethyl acetate (with increasing order of polarity) to get pure product (42-
56) in good yield. The structures of synthesized compounds (42-56) were
determined by IR, 1H-NMR and EIMS spectroscopy. All compounds give
satisfactory elemental (CHN) analysis.
189
O
O
N
O
20
20 % KOH
150oC, 120 hr
refluxed
O
O
OH
O
28N2 atmosphere,
Dry DCM
SOCl22hr, stirr over ice bath
O
O
Cl
O
Amine
Dry DCM
60o, 2-2.5hr
refluxed
O
O
N
O
R2
R1
12
3
4
5
67
8
910
11
42-5641
Scheme-6
190
Table-1: Synthesis of amides of piperic acid (42-56)
Compd
No.
- NR1R
2 M.P.
oC
M.P. oC [lit]
Yield
%
42 N
2' 3'
4'
5'6'
130-131
132 [35]
74
43 N O
2' 3'
5'6'
159-160
162 [36]
89
44 N N
2' 3'
5'6'
CH3
178-180
185 [36]
61
45
H
N
H
102-105
81
46 N
H 2' 3'
4'
5'6'
173-175
174 [36]
87
47 N
H
CH3
2' 3'
6' 5'
190-191
81
48 N
H
Cl
2' 3'
6' 5'
118-120
80
49 N
H
OCH3
2' 3'
6' 5'
163-165
167 [36]
51
191
50 NH
2'4'
3'
128-129
151 [36]
76
51 NH CH3
H3C
H3C
3'
5'
265-269
92
52 N NNH CH3
2' 3'
6' 5'
70-71
72
53 N
2'
3'
4'
5'
141-143
142 [33]
67
54
115-117
75
55 NH
2' 3'
6' 5'
4'
170-173
177 [36]
36
56 NH CH3
OCH32'
5'6'
201-203
41
192
3.3.3. General Structure Elucidation of Compounds (42-56)
O
O
NH
O
N N CH3
2' 3'
5'6'
2
5
4
3
6
7
8
9
10
11
Figure-10: Structure of compound (52)
The structure of compound (52) (as a representative example) was
established through spectroscopic techniques. The 1H-NMR was carried out in
deutarated methanol on 300 MHz. A singlet at 5.97 indicates two protons of
methylene dioxy group (O-CH2-O). A doublet and a double doublet at 6.10 (J =
15.0 Hz) and 7.38 (J = 15.0, 10.0 Hz) show trans protons H-2 and H-3. Where as
a multiplet in range of 6.79-7.09 indicates olefinic (H-4, H-5) and aromatic
protons (H-7, 10, 11). A singlet at 2.91 indicates three protons of methyl (N-
CH3). It also shows signals at 3.50 and 3.35 as two broad singlets of –N (CH2)2
(H-2′, 6′) and (CH2)2 N (H-3′, 5′) respectively. The synthetic compound (52) was
also confirmed by EIMS showing a molecular ion peak at m/z = 315 which leads
to molecular formula C17H21N3O3. Ion at m/z = 300 appeared due to loss of methyl
group similarly peak at m/z 201 appeared due to loss of amine linkage. It further
gives a peak at m/z 173 by the loss of CO group. In addition a peak at m/z = 114
corresponds to piprazine moiety, subsequent loss of methyl give peak at m/z = 99.
The IR spectrum displayed peaks at 3330 (N-H), 1628-1700 (C=C, C=O) and
1194 (C-O). The structures of other compounds were elucidated in the same way.
193
Figure-11: Fragmentation patterns of compound (52)
194
3.3.4. Biological Evaluation of Compounds (42-56)
3.3.4.1 In Vitro Anti-bacterial Activity
The anti-bacterial activity of all the synthesized compounds 42-56 was
determined by using disc diffusion method (see chapter 4). Susceptibility test in
vitro was performed against bacterial strain i.e. Streptococcus pneumoniae,
Proteus vulgaris, Streptococcus aureus, Pseudomonas stutzeri, Pseudomonas
aeruginosa and Escherichia coli. Streptomycine was taken as standard for
comparison of antibacterial activity under similar conditions. The results of anti-
bacterial activity of synthesized compounds were reported on the basis of
diameter of zone of inhibition in mm that was appeared around the disc. All of the
synthesized compounds showed varying degree of antibacterial activity. None of
the synthesized compounds showed antibacterial activity against S. pneumoniae.
Compound 54 showed significant anti-bacterial activity against all remaining
strains creating highest zone of inhibition 30 mm against S. aureus, second
highest inhibition zone 29 mm against P. stutzeri and 25 mm against P. vulgaris,
20 mm against E. coli and moderate inhibition zone 19 mm against P. aeruginosa.
Compound 49 did not showed anti-bacterial activity against all strains except P.
vulgaris where it produced significant zone of inhibition 30 mm. Compound 50
showed moderate anti-bacterial activity against S. aureus with zone of inhibition
14 mm. It also showed moderate anti-bacterial activity with zone of inhibition 16
mm against P. stutzeri and P. aeruginosa. Compound 53 was found active against
P. stutzeri (zone of inhibition = 16mm). Compound 44 showed anti-bacterial
activity with zone of inhibition 10 mm against P. stutzeri and 14 mm against P.
aeruginosa. Compound 52 showed excellent zone of inhibition against P.
aeruginosa and E. coli (18 and 14 mm). On the other hand compounds 46, 47 and
48 exhibited good anti-bacterial activity against P. aeruginosa with zone of
inhibition in the range of 12-16 mm. Compound 56 showed moderate anti-
bacterial activity with 16 mm zone of inhibition against E. coli. The results of
anti-bacterial activity are given in Table-2.
195
Tabel-2: In vitro anti-bacterial activity of compounds 42-56
Key: Values are diameter of zone of inhibition (mm) and an average of triplicate, (-) indicates resistant.
Compound No.
Microorganisms 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Streptomycin
Streptococcus pneumoniae - - - - - - - - - - - - - - - 35
Proteus vulgaris - - - - - - - 30 - - - - 25 - - 30
Streptococcus aureus - - - - - - - - 14 - - - 30 - - 30
Pseudomonas stutzeri - - 10 - - - - - 16 - - 16 29 - - 15
Pseudomonas aeruginosa - 7 14 - 16 16 12 - 16 - 18 - 19 - - 15
Escherichia coli - - - - - - - - - - 14 - 20 - 16 20
196
3.3.4.2 In Vitro Anti-fungal Activity
All the synthesized compounds (42-56) were evaluated for their anti-
fungal activity against saprophytic fungi (Rhizopus, Aspergillus niger, Aspergillus
flavus) and yeast (Candida albican). The anti-fungal activity was determined by
disc diffusion method as in chapter 4, using ketoconazol as standard. Only
compound 54 showed weak anti-fungal activity while the rest of the compounds
found inactive. So, were not further studied. The results of anti-fungal activity are
displayed in Table-3.
197
Tabel-3: In vitro anti-fungal activity of compounds 42-56
Key: values are diameter of zone of inhibition (mm) and an average of triplicate, (-) indicates resistant.
Compound No.
Microorganisms 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Ketoconazole
Rhizopus - - - - - - - - - - - - 7 - - 22
Aspergillus niger - - - - - - - - - - - - 5 - - 24
Aspergillus flavus - - - - - - - - - - - - 5 - - 24
Candida albican - - - - - - - - - - - - 6 - - 22
198
3.3.4.3 In Vitro Nematicidal Activity
The nematicidal activity of synthesized compounds (42-56) was determined
by applying the method as described in chapter 4. The nematicidal activity was tested
against second stage juveniles of root-knot nematode Meloidogyne incognita while
the toxicity was found by the determination of their lethal concentration (LC) at LC20,
LC50 and LC90 mg/ml (i.e. the lethal concentration at which 20, 50 and 90%
nematodes become immobile). Compound 42 showed highest mortality (98 ± 0.02%)
with good LC50 value 4.2 mg/ml. where as compounds 52 and 56 showed excellent
mortality rate (96 ± 0.03 and 95 ± 0.01 % respectively) having LC50= 3.4 and
3.5mg/ml. The compounds 43, 47 and 45 also exhibited good mortality rate (92 ±
0.03, 92 ± 0.04 and 90 ± 0.02%) with LC50= 5.8, 4.4 and 3.0 respectively. Beside
these the compounds 44, 54 and 50 showed mortality rate 83 ± 0.02, 82 ± 0.02 and
80 ± 0.02 % respectively, with favorable LC50 value 5.8, 4.5 and 3.8 mg/ml
respectively . Results are shown in Table-4.
199
Table-4: In vitro nematicidal activity of compounds 42-56.
Compounds
No.
Mortality
Rate %
LC20
mg/ml
LC50
mg/ml
LC90
mg/ml
42 98 ± 0.02 3.6 4.2 5.2
43 92 ± 0.03 4.1 5.8 6.0
44 83 ± 0.02 4.2 5.8 6.6
45 90 ± 0.02 2.6 3.0 4.4
46 20 ± 0.02 20.2 26.7 31.0
47 92 ± 0.04 3.2 4.4 5.8
48 12 ± 0.02 22.4 34.3 37.6
49 14 ± 0.06 19.3 22.6 34.2
50 80 ± 0.02 3.3 3.8 4.4
51 21 ± 0.04 18.7 22.5 31.8
52 96 ± 0.03 2.5 3.4 4.1
53 13 ± 0.02 28.6 39.0 40.1
54 82 ± 0.02 3.3 4.5 4.9
55 21 ± 0.03 20.1 31.3 39.2
56 95 ± 0.01 2.2 3.5 4.3
Furadan 100
Key: values are mortality rate (%), LC = Lethal concentration (mg/ml)
200
3.3.4.4 In Vitro Anti-oxidant Activity
The compounds (42-56) were screened for their in vitro anti-oxidant activity
by the method used in chapter 4. Free radical solution of DPPH (1,1-diphenyl-2-
picryl hydrazyl) was used to study radical scavenging activity, along with Ascorbic
acid as standard, varying degree of DPPH radical scavenging activity was shown by
these compounds. Compound 49, 51 and 44 exhibited significant % inhibition (80 ±
0.01, 72 ± 0.01 and 70 ± 0.02 respectively). The EC50 value of these compounds is
625, 937.5 and 937 g/ml. Compound 46 and 54 revealed % inhibition (58 ± 0.02 and
57.9 ± 0.01 respectively) showing EC50 value 100 µg/ml hence recognized as
moderate antioxidant. While the rest of compounds revealed inhibition under 50%, so
these compounds were not further studied. The results of anti-oxidant-activity are
given in Table-5.
201
Table-5: In vitro anti-oxidant activity of compounds 42-56.
Compound No. % Inhibition ± SD EC50 g/ml
42 12 ± 0.01 -
43 - -
44 70 ± 0.02 937
45 - -
46 58 ± 0.02 100
47 44 ± 0.02 -
48 - -
49 80 ± 0.01 625
50 15 ± 0.01 -
51 72 ± 0.02 937.5
52 47 ± 0.01 -
53 44 ± 0.01 -
54 57.9 ± 0.01 100
55 - -
56 40 ± 0.02 -
Ascorbic acid 80 8.3
Key: Values are zone of inhibition (%) and an average of triplicate.
EC50 = Effective concentration to scavange 50% of DPPH
(-) indicates inactivity
202
3.3.5. Conclusion
All the synthesized amide derivatives of piperic acid 42-56 were evaluated for
their biological activity. Compounds 44, 46, 47, 48, 49, 50, 52, 53 and 56 exhibited
significant anti-bacterial activity. On the other hand compound 54 showed marvelous
anti-bacterial activity and it was the only compound that showed anti-fungal activity
against all the applied strains. When tested for their nematicidal activity compounds
42, 43, 45, 47, 52 and 56 showed significant mortality rate and LC50 values against
root-knot nematode i.e. Meloidogyne incognita. Hence, these candidates may serve as
a fruitful nematicidal agent. Only three compounds i.e. 44, 49 and 51 showed
excellent and two compounds 46 and 54 showed moderate anti-oxidant activity.
In view of these biological assays we can conclude that the above candidates
may serve as a lead compounds for the further studies.
203
3.4. Experimental
204
3.4.1 General Experimental
All the reagents and solvents used for synthesis were purchased from E.
Merck, Germany. Melting points were determined in glass capillary using Gallen
Kamp melting point apparatus and are uncorrected. EIMS spectra were recorded on
JEOL JMS-600H. 1H-NMR spectra were performed on Avance AV-300, 400, 500
and 600 NMR spectrometers operating at 300, 400, 500 and 600 MHz. in deuterated
methanol (MeOD) with trimethylsilane (TMS) as an internal standard. IR spectra
were done on a JASCO–302-A spectrophotometer. A Carlo Erba Strumentazion-
Mod-1106, Italy was used for elemental (CHN) analysis. TLC (Thin Layer
Chromatography) was carried out on precoated silica gel glass plates (Kieselgel 60,
254, E. Merck. Germany). UV visualized chromatogram at 254 and 365 nm.
3.4.1 General method for the synthesis of compounds (42-56)
The general synthetic procedure of amides of piperic acid comprises of
following steps:
3.4.2.1 Preparation of pipeic acid
Piperine (20) (5g, 0.0175 mol) was refluxed with 300 ml of 20% KOH in
methanol at 150oC for 120 hr. Completion of reaction was monitored on TLC. After
complete hydrolysis excess of methanol was removed under reduced pressure. The
reaction worked up by suspending the residue in hot water followed by acidification
with 2N HCl. The resulted precipitates were filtered, washed with ice-cold water and
dried at room temperature. The crude product was purified through recrystallisation
from ethanol to afford pale yellow crystals of pure piperic acid (28), M. P. 215-216oC
[Lit. 215oC], yield 94% [32].
3.4.2.2 Conversion of piperic acid to acid chloride
Piperic acid (28) (0.5g, 0.00229 mol) was dissolved in dry Dichloromethane
(DCM) 15 ml, kept over an ice-bath under nitrogen atmosphere, stirred for 10 min.
205
After that freshly distilled thionyl chloride (SOCl2) 2ml (0.27 mol) was added drop
wise and stirred the resultant solution for 2 hr. till the reaction mixture indicated
organge-brown colouration. Completion of reaction was confirmed by TLC. The
excess SOCl2 was then removed under vaccuo leaving acid chloride (41) as an
orange-brown residue.
3.4.2.3 Synthesis of amides of piperic acid by condensation of acid chloride with
an amine
To the resultant solution of crude acid chloride (41) in 10 ml dry DCM,
appropriate amine 0.7 ml (0.585 g, 0.00688 mol) dissolved in 5 ml DCM, was added.
The resultant reaction mixture was refluxed at 60oC for 2-2.5 hr. Completion of
reaction was monitored on TLC (n-Hexane-EtOAc, 7:3). The content was diluted
with distilled water. The organic layer was separated out through separating funnel,
washed with distilled water (2x25 ml), dried with anhydrous Na2SO4 and
concentrated to crude product. It was subjected to column chromatography over silica
gel using n-Hexane–EtOAc as mobile phase with increasing order of polarity to get
pure product (42-56) in good yield.
All other compounds were synthesized applying same methodology. The
structures of the synthesized compounds (42-56) were determined by IR, 1H-NMR
and EIMS spectroscopy. All synthesized compounds gave satisfactory elemental
(CHN) analysis.
3.4.3. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid (Piperic acid,
28)
Yield: 94% ; m.p: 215-216ºC; IR (KBr) νmax cm-1
: 2940, 1673, 1597, 1447; 1H-NMR:
(300 MHz, DMSO-d6): δ 12.17 (1H, s, -COOH), 7.01 (1H, dd, J3/2,4 = 15.6, 8.1 Hz,
H-3), 6.89-7.32 (5H, m, olefinic and Ar-H), 6.04 (2H, s, O-CH2-O), 6.01 (1H, d, J2,3
206
= 15.6 Hz, H-2); EI-MS: m/z (rel.abund.%), 218 (M+ , 82), 201 (6), 173 (100), 143
(31), 121(2), 115 (63); Anal. Calcd for C12H10O4 (218.06): C, 66.05; H, 4.62; Found:
C, 66.00; H, 4.63.
3.4.3.1. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)penta-2,4-dien-1-
one (42)
Yield: 74% ; IR (KBr) νmax cm-1
: 1631, 1490, 844; 1H-NMR: (500 MHz, MeOD); δ
7.28 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.94 (2H, m, H-4/5), 6.89 (2H, m, H-
10/11), 6.82 (1H, s, H-7), 6.59 (1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O),
3.60 (4H, m, -N(CH2)2), 1.69 (2H, m, H-4′), 1.59 (4H, bs, H-3′/5′); EI-MS: m/z (rel.
abund. %), 285 (M+, 36), 201 (71), 173 (60), 159 (13), 143 (36), 115 (100), 84 (18);
Anal. Calcd for C17H19NO3 (285.14): C, 71.56; H, 6.71; N, 4.91; Found: C, 17.54;
H, 6.73; N, 4.90.
3.4.3.2. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-morpholinopenta-2,4-dien-1-one
(43)
Yield: 89% ; IR (KBr) νmax cm-1
: 2941, 1633, 1491, 997; 1H-NMR: (500 MHz,
MeOD): δ 7.33 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.95 (2H, m, H-4/5), 6.89 (2H,
m, H-10/11), 6.86 (1H, s, H-7), 6.58 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-
O), 3.70 (4H, m, N(CH2)2), 3.61 (4H, m, O (CH2)2); EI-MS: m/z (rel. abund. %), 287
(M+, 94), 201 (100), 173 (60), 115 (68), 86 (6); Anal. Calcd for C16H17NO4 (287.12):
C, 66.89; H, 5.96; N, 4.88; Found: C, 66.87; H, 5.97; N, 4.86.
3.4.3.3. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(4-methylpiperazin-1-yl)penta-2,4-
dien-1-one (44)
Yield: 61% ; IR (KBr) νmax cm-1
: 2954, 1630, 845; 1H-NMR: (500 MHz, MeOD): δ
7.31 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.78- 7.08 (5H, m, olefinic and Ar-H), 6.59
207
(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 3.29 (4H, m, H-2′/6′), 2.48 (4H,
bs, H-3′/5′), 2.33 (3H, s, N-CH3): EI-MS: m/z (rel. abund. %), 300 (M+, 28), 201 (23),
173 (32), 143 (16), 115 (46), 99 (48), 70 (100); Anal. Calcd for C17H20N2O3 (300.15):
C, 67.98; H, 6.71; N, 9.33; Found: C, 67.97; H; 6.72; N, 9.35.
3.4.3.4. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienamide (45)
Yield: 81% ; IR (KBr) νmax cm-1
: 3021, 1644, 970; 1H-NMR (300 MHz, MeOD): δ
7.24 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.77-7.08 (5H, m, olefinic and Ar-H), 6.12
(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 5.47 (2H, s, -NH2); EI-MS: m/z
(rel. abund. %), 217 (M+, 68), 173 (100), 143 (31), 115 (80), 96 (14), 44 (8); Anal.
Calcd for C12H11NO3 (217.07): C, 66.35; H, 5.10: N, 6.45; Found: C, 66.33; H, 5.12;
N, 6.44.
3.4.3.5. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenylpenta-2,4-dienamide (46)
Yield: 87% ; IR (KBr) νmax cm-1
: 3215, 1639, 890; 1H-NMR: (500 MHz, MeOD): δ
7.61 (2H, d, J2’,3’ = 7.5 Hz, H-2′/6′), 7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 7.29-
7.08 (3H, m, H-3′/4′/5′), 6.79-7.10 (5H, m, olefinic and Ar-H), 6.25 (1H, d, J2,3 = 15.0
Hz, H-2), 5.96 (2H, s, O-CH2O-); EI-MS: m/z (rel. abund, %), 293 (M+, 12) 201 (92),
173 (11), 115 (100), 92 (3), 77 (10); Anal. Calcd for C18H15NO3 (293.11): C, 73.71;
H, 5.15; N, 4.78; Found: C, 73.69; H, 5.13: N, 4.79.
3.4.3.6. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-p-tolylpenta-2,4-dienamide (47)*
Yield: 81% ; IR (KBr) νmax cm-1
: 3198, 1647, 929; 1H-NMR: (500 MHz, MeOD): δ
7.88 (1H, s, -CONH), 7.60 (2H, d, J2’,3’ = 8.5 Hz, H-2′/6′), 7.41 (1H, dd, J3/2,4 = 15.0,
10.0 Hz, H-3), 7.22 (2H, d, J3’/2’ = 8.5 Hz, H-3′/5′), 6.79-7.10 (5H, m, olefinic and
Ar-H), 6.01 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 2.21 (3H, s, Ar-
CH3); EI-MS: m/z (rel. abund, %), 307 (M+, 76), 217 (13), 201 (45), 187 (6), 173
208
(100), 143 (60), 115 (79); Anal. Calcd for C19H17NO3 (307.12): C, 74.25; H, 5.58; N,
4.56; Found: C, 74.24; H, 5.59; N, 4.54.
3.4.3.7. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-chlorophenyl)penta-
2,4-dienamide (48)
Yield: 80% ; IR (KBr) νmax cm-1
: 3291, 1638, 997; 1H-NMR: (300 MHz, MeOD): δ
7.63 (2H, d, J2’,3’ = 8.7 Hz, H-2′/6′), 7.46 (2H, d, J3’,2’ = 8.7 Hz, H-3′/5′), 7.40-6.70
(6H, m, olefinic and Ar-H), 6.23 (1H, d, J2/3 = 15.0 Hz, H-2), 5.97 (2H, s, O-CH2-O);
EI-MS: m/z (rel. abund. %), 327 (M+, 13), 201 (79), 173 (18), 143 (22), 121 (32);
Anal. Calcd for C18H14ClNO3 (327.07): C, 65.96; H, 4.31; N, 4.27; Found: C, 65.93;
H, 4.30; N, 4.25.
3.4.3.8. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)penta-2,4-
dienamide (49)
Yield: 51% ; IR (KBr) νmax cm-1
: 3350, 1649; 1H-NMR: (300 MHz, MeOD): δ 8.17
(1H, s, -CONH-), 6.70-7.47 (10H, m, olefinic and Ar-H), 6.00 (1H, d, J2,3 = 15.0 Hz,
H-2), 5.93 (2H, s, O-CH2-O), 3.76 (3H, s, -OCH3); EI-MS: m/z (rel. abund %), 323
(M+, 64), 307 (8), 293 (6), 201 (14), 173 (6), 143 (10), 121 (13), 115 (17); Anal.
Calcd for C19H17NO4 (323.12): C, 70.58; H, 5.30; N, 4.33; Found: C, 70.57; H, 5.30;
N, 4.34.
3.4.3.9. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-propylpenta-2,4-dienamide (50)
Yield: 76% ; IR (KBr) νmax cm-1
: 3263, 1634, 1539, 787; 1H-NMR: (300 MHz,
MeOD): δ 7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78-7.10 (5H, m, olefinic and
Ar-H), 6.09 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 5.91 (1H, s, -CONH-
), 3.52 (2H, m, H-2′), 1.68 (2H, m, H-3′), 1.00 (3H, t, J4’,3’ = 7.2 Hz, H-4′); EI-MS:
m/z (rel. abund %), 259 (M+, 13), 218 (83), 201 (30), 173 (100), 143 (45), 115 (69);
209
Anal. Calcd for C15H17NO3 (259.12): C, 69.48; H, 6.61; N, 5.40; Found: C, 69.45; H,
6.60; N, 5.41.
3.4.3.10. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-mesitylpenta-2,4-dienamide
(51)*
Yield: 92% ; IR (KBr) νmax cm-1
: 3259, 1633, 845; 1H-NMR: (300 MHz, MeOD): δ
7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.11 (7H, m, olefinic and Ar-H), 6.34
(1H, d, J2,3 = 15.0 Hz, H-2), 5.97 (2H, s, -O-CH2-O) 2.25 (3H, s, p-CH3), 2.16 (6H, s,
2 x o-CH3); EI-MS: m/z (rel. abund %), 335 (M+, 57), 201 (100), 171 (34), 143 (47),
135 (26), 115 (61), 89 (9); Anal. Calcd for C21H21NO3 (335.15): C, 75.20; H, 6.31; N,
4.18; Found: C, 75.19; H, 6.32; N, 4.20.
3.4.3.11. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methylpiperazin-1-yl)penta-
2,4-dienamide (52)*
Yield: 72% ; IR (KBr) νmax cm-1
3330, 1628, 1194; 1H-NMR: (300 MHz, MeOD): δ
7.38 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.09 (5H, m, olefinic and Ar-H), 6.10
(1H, d, J2,3 = 15.0 Hz, H-2), 5.97 (2H, s, O-CH2-O), 3.50 (4H, bs, N-N(CH2)2), 3.35
(4H, bs, (CH2 )2N-), 2.91 (3H, s, N-CH3); EI-MS: m/z (rel. abund %), 315 (M+, 15),
300 (4), 217 (19), 201 (96), 173 (14), 143 (9), 114 (28), 99 (85); Anal. Calcd. for
C17H21N3O3 (315.16): C, 64.74; H, 6.71; N, 13.32; Found: C, 64.72; H, 6.70; N,
13.33.
3.4.3.12. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(pyrrolidine-1-yl)penta-2,4-dien-
1-one (53)
Yield: 67% ; IR (KBr) νmax cm-1
: 1649, 1448, 929; 1H-NMR: (300 MHz, MeOD): δ
7.37 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78-7.09 (5H, m, olefinic and Ar-H), 6.44
(1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 3.59 (4H, m, H-2′/5′), 1.98 (4H,
m, H-3′/4′); EI-MS: m/z (rel. abund %), 271 (M+, 90), 201 (100), 173 (20), 143 (14),
210
115 (53), 70 (6); Anal. Calcd for C16H17NO3 (271.12): C, 70.83; H, 6.32; N, 5.16;
Found: C, 70.81; H, 6.33; N, 5.17.
3.4.3.13. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-iodophenyl)penta-2,4-
dienamide (54)*
Yield: 75% ; IR (KBr) νmax cm-1
: 3321, 1627, 987; 1H-NMR: (600 MHz, MeOD): δ
7.72 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′), 6.90 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.80
(2H, d, J3’,2’ = 8.4 Hz, H-3′/5′), 6.83-7.54 (5H, m, olefinic and Ar-H), 5.96 (2H, s, O-
CH2-O), 5.94 (1H, d, J2,3 = 15.0 Hz, H-2), 3.63 (1H, s, -CONH-); EI-MS: m/z (rel.
abund %), 419 (M+, 8), 418 (9), 218 (41), 173 (47), 143 (23), 115 (62), 43 (100);
Anal. Calcd for C18H14NO3I (419.00): C, 51.57; H, 3.37; N, 3.34; Found: C, 51.55; H,
3.39; N, 3.35.
3.4.3.14. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzylpenta-2,4-dienamide (55)
Yield: 76% ; IR (KBr) νmax cm-1
: 3239, 1623; 1H-NMR: (400 MHz, MeOD): δ 7.29
(1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78- 7.23 (10H, m, olefinic and Ar-H), 6.13
(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 4.45 (2H, s, N-CH2-); EI-MS:
m/z (rel. abund. %), 307(M+, 54), 218 (52), 201 (24), 173 (92), 143 (35), 115 (77),
106 (100), 91 (37), 77 (20), Anal.Calcd for C19H17NO3 (307.12): C, 74.25; H, 5.58;
N, 4.56; Found: C, 74.21; H, 5.59; N, 4.58.
3.4.3.15. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-methoxy-4-methylphenyl)
penta-2,4-dienamide (56)*
Yield: 71% ; IR (KBr) νmax cm-1
: 3257, 1647; 1H-NMR: (300 MHz, MeOD): δ 7.40
(1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.10 (8H, m, olefinic and Ar-H), 6.26 (1H,
d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, -O-CH2-O), 3.82 (3H, s, -OCH3), 2.13 (3H, s, -
CH3); EI-MS: m/z (rel. abund. %), 337 (M+, 35), 215 (25), 201 (100), 143 (34), 115
211
(78); Anal. Calcd for C20H19NO4 (337.13): C, 71.20; H, 5.68; N, 4.15; Found: C,
71.17; H, 5.69; N, 4.17.
* indicates newly synthesized compounds.
212
3.5. References
213
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216
CHAPTER-4
Biological Activities Assays
217
Introduction
218
4.0. Introduction About Biological Activities
4.1. Enzymes Inhibition Activity
The enzymes are biological catalyst. They perform many physiological functions
without being altered in the reactions. They possess high degree of substrate
specificity. They maintain and regulate body functions and also defend against severe
disease [1, 2].
Enzymes inhibition is a prime therapeutic strategy for the treatment of various
infections which are caused by extra ordinary activity of different enzymes via
relevant enzymes inhibitor [1-3].
The enzyme inhibitor is the drug which after binding with the relevant enzyme
inhibits or reduces it’s over activity that is generating ailments. These either kill a
pathogen or regulate the metabolic imbalance. The enzyme inhibitors are valuable
chemotherapeutic agent and are considered as an important tool for the treatment of
diverse state diseases. Chymotrypsin and trypsin (the serine proteases) are digestive
enzymes. They protect cellular protein destruction. Their imbalance may cause
cirrhosis, pancreatic cancer or hepatitis C [1-3].
4.1.1. Urease Inhibition Activity
Urease which is a nickel containing enzyme hydrolyses urea to carbon dioxide and
ammonia. Urea is a basic soil fertilizer, urease help plants, bacteria and algae in
nitrogen assimilation. Ureases are also significant virulence factor implicated in the
pathogenesis of hepatic coma, urinary stones and other infections. Peptic ulcer (the
small erosions in digestive track) is the infection caused by gram-negative bacterium,
Helicobacter pylori (H. pylori). This bacterium can transfer from one to another
person via food and water. Indigestion, abdominal pain, gastric obstruction and ulcer
bleeding are its main symptoms. About 5-10% world population suffer this problem.
This bacterial infection produces enzymes, urease, this increasing concentration
219
causes ulcer. Therefore, the urease inhibitors are of great importance in medicinal
chemistry [4,5].
4.1.2. Tyrosinase Inhibition Activity
Tyrosinase is a copper containing enzyme, widely distributed in every plant and
animal including human. It control and catalyzes the synthesis of melanin from
tyrosine which is a pigment that provides colour to skin, hair and eye. The melanin
protects skin from harmful effects of uv radiations and also plays a vital role in
normal vision. Tyrosinase is further responsible of browning of fruits and vegetables
especially when their tissues are ruptured. Any mutation or uncontrolled activity in
tyrosine gene (TYR gene) may results in impaired production of tyrosinase. It may
lead to over production of melanin (melanogenesis) where it get accumulated
abnormally in different parts of skin to produce pigmented patches on the skin. This
hyperpigmentation of skin is an esthetic problem and the browning of fruit and
vegetables is undesirable as it reduces their commercial values. These problems have
encouraged researchers to develop new effective tyrosinase inhibitors for anti-
browning of fruits and vegetables and to treat hyperpigmention in skin. The
tyrosinase inhibitors prevent over production of tyrosinase, which in turn block
abnormal synthesis of melanin. This blocking of excess formation of melanin is
helpful in controlling of hyperpigmentation and browning action. Now these days’
tyrosinase inhibitors have broad application in cosmetics and food industries as skin
whitening and anti-browning agents [6-9].
4.1.3. Cholinesterase Inhibition Activity
Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) are cholinesterase
enzyme found in mammalian brain.The serum BuChE is produced in liver, lungs,
heart and brain while AChE in brain, muscles and erythrocyte membrane. AChE is
very important part of cholinergic brain synapses. It helps in termination of impulses
through hydrolysis of neurotransmitter acetylcholine. BuChE is abundantly present
in the nervous system and in blood plasma. BuChE (a glycoprotein) is a hydrolase
220
that also catalyse choline hydrolysis. The BuChE participates in nerve conduction and
in CNS function. It also has a role in neurodegenerative disorders. In human brain
BuChE is present in neurons and neuritic plaque. It is found that BuChE takes the
place of AChE after its degradation. BuChE have minor whereas AChE play a major
role in regulating brain acetylcholine. It is found that the Alzheimer’s disease (AD) is
the result of insufficient cholinergic function in the brain. It is observed that BuChE
activity increases in patients of Alzheimer’s disease (AD) whereas AChE activity
decelerated or remains unchanged. Therefore, it is accepted that the inhibition of
brain BuChE may weaken neurodegeneration in AD. So it is suggested that BuChE
inhibitors may be favored for the treatment of AD. Moreover the development of
BuChE inhibitors may lead to an effective treatment and important therapeutic goal in
AD. [10-13]
4.2. Anti-microbial Activity
The anti-microbial resistance is the relative insensitivity of a microorganism
(bacteria, fungi, viruses) to an antimicrobial agent. This resistance is increasing in
various pathogens with the passage of time, allowing them to be able to reduce the
effectiveness of conventional treatment and increases the risk of complications. This
results in the wide spread use of antimicrobial medicines [14, 15].
The Anti-bacterial agents destroy, kill or inhibit the growth of bacteria. In the same
way the drug which selectively removes fungal pathogens with minimal toxicity from
a host is known as antifungal agent. The increased implication of these antimicrobial
agents renders the resistant pathogens to grow and evolve continuously with acquired
resistance such as salmonella species resistant to fluroquinolones, mycobacterium
tuberculosis show resistance to rifampin and staphylococcus aureus to vancomycin.
Moreover, vibrio cholera and shigella species have made difficult to control
infections caused by them. It has been observed that klebsiella speices resistance to
carapenum and enterococcal are resistant towards erythromycin. Furthermore, the
resistant microorganisms diminishes the ability of an antimicrobial medicine to
221
engage with its target like oxyiminocephalalosporin resistant pseudomonas
aeruginosa and aminoglycoside resistant Acinetobacter baumanii, resistance to anti-
malarial drug ( chloroquine) is worldwide problem [16-23].
Similarly some medicines that prepared from aspergillus and penicillum species
possess intestinal ailments. Deep-seated mycosis is due to resistant filamentous fungi
( fusarium species). The plague causing agent yersinia pestis exihibit resistance to
ampicillin, where as candida glabrate shows resistance in both azole and
echinocandin anti-fungal agent. Pseudomonas species have multidrug resistance
while E. coli is third generation (Cephalosporin) resistant. It is also found that the
spoilage and deterioration of food stuff is usually associated with fungi and bacteria.
The emergence of antimicrobial resistance in a variety of pathogens results infectious
diseases as a leading cause of death. So, it is a serious health and economic problem.
It can be controlled by the development and delivery of new antimicrobial agents [16-
23].
4.3. Anti-oxidant Activity
The anti-oxidants are imperative compounds which either inhibit or delay the
oxidation process that takes place in body under the influence of atmospheric oxygen
or by reactive oxygen species. The anti-oxidant species control the generation of free
radical by converting them to stable harmless substance after giving an electron to
them. The free radical formed attack the normal healthy cells and react with lipids,
proteins and other molecules. Hence, lead severe disorders, damages and diseases
associated with them like aging, cancer, heart diseases, CNS disorders, alzheimer,
immune system decline, hypertension, cataract, obesity etc. [24-26].
The antioxidant scavenges and deactivates free radicals to protect body from
oxidative damage and repair damaged tissue and cells. Thus, antioxidants are
essential to health. Moreover they act as an anti- cancer, anti-allergic, vasodilator
agent and prevent body from many ailments. Many free radicals scavenging
222
substances exist within the body like vitamin C and E, β-carotenes, flavonoides,
carotenoids and certain enzymes. Some have dietary sources such as fruits and
vegetables. Gallic acid ester is a synthetic antioxidant. Currently radical scavengers
are used for stabilization of pharmaceuticals, food and polymers [24-26].
4.4. Nematicidal Activity
The nematicide is a substance or chemical pesticide that is used to destroy or kill
plant-parasitic nematodes. The nematodes are simple unsegmented organisms belong
to phylum Nematoda which comrises of nearly 25000 species. They are found in
almost every part of earth. They can adapt all ecosystems and can live in highly
extreme atmosphere. Besides, they have tendency to live on or in a plant and animal
(including humans). In addition, more than half of species are parasitic and are
responsible of many ailments such as stunting shoot of alfa alfa by ditylenchus
depsaci, galling of root by meloidogyne species and ascaris in humans. It has been
found that carbondioxide acts as an attractant for many phyto-parasitic nematodes to
locate their target in complex environment of soil. The phyto-parasitic nematodes
affect worth and mass of crop that results drastic loss of cultivation and so, economy.
These parasitic nematodes must be controlled by means of nematicides, natural bio
control, rotation of plant and soil steaming [27-32].
223
Protocols
224
4.5.0. Biological activities Assays
4.5.1. Enzyme Inhibition Assay
4.5.1.1. In Vitro Tyrosinase Inhibition Assay
Tyrosinase inhibition was determined spectrophotometrically following the method
described in literature [33]. All synthesized compounds were dissolved in DMSO and
screened for o-diphenolase inhibitory activity of tyrosinase. L-Dopa was used as
substrate and kojic acid as a standard inhibitor for tyrosinase. At first 30 units of
mushroom tyrosinase (28 mM) were preincubated with compounds at 25˚ C for 10
min in presence of 50mM sodiumphospahte buffer (pH 6.8). Then L-Dopa (0.5 mM)
was added. The enzyme reaction thus started to produce L-DOPA chrome, was
monitored by measuring absorbance at 475nm (at 37˚C) for 10 min in spectramax
340 microplate reader (Molecular Devices USA). It was compared to the curve of
standard. The percent of inhibition was calculated by following formula:
% Inhibition = [B –S / B] × 100.
Here, B is the absorbance for the blank and S is the absorbance for the samples. The
results were in an average of triplicate and represent means ± SEM (standard error of
the mean).
4.5.1.2. In Vitro Acetylcholinesterase and Butrylcholinesterase Inhibition Assay
Acetylcholinesterase (AChE) and Butrylcholinesterase (BChE) inhibition was
determined spectrophotometrically in a 96 well micro plate on Spectra Max 340
(Molecular device, U.S.A) by slight modification in the method described in literature
[34]. Acetylcholine iodide and butrylcholine chloride were used as substrates for
Acetylcholinesterase and Butrylcholinesterase activity, Electric eel
acetylcholinesterase (EC 3.1.1.7) and horse serum butyrylcholinesterase (EC 3.1.1.8)
were used for enzyme preparations, 5,5′-dithio-bis-nitrobenzoic acid (DTNB) were
225
used to measure cholinesterase activity and galanthamine as standard. 140µL of
sodium phosphate buffer (pH= 8), 20µL of AChE / BChE solutions and 20 µL of test
compounds were mixed then incubated at 25oC for 15 minutes. Subsequent addition
of 10µL of DTNB and 10 µL of Acetylcholine iodide or butrylcholine chloride, in
that order, initiated the reaction. The enzymatic hydrolysis of substrate released
thiocholine which react with DTBN to form yellow 5-thio-2-nitrobenzoate anion. The
hydrolysis was monitored spectrophotometrically at a wavelength of 412 nm. The
results are in average of triplicate. All reagents and chemicals were purchased from
Sigma and of analytical grades.
4.5.1.3. In Vitro Urease Inhibition Assay
Urease activity was determined by using the indophenol method [35]. The results
were obtained after measuring the ammonia produced during the reaction. The
reaction mixtures containing 25 μL of enzyme (Jack bean Urease) solution, 55 μL of
buffers (0.01 M K2HPO4.3H2O, 1 mM EDTA and 0.01 M LiCl2, pH= 8.2) and 100
mM urea were incubated in 96-well plates with 5 μL of test compounds (1 mM) for
15 min at 30oC. Briefly, 45 μL each of phenol reagent (1% w/v phenol and 0.005%
w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1 %
active chloride NaOCl) were added to each well. After 50 minutes the increasing
absorbance was measured at 630 nm by using a microplate reader (Molecular Device,
USA). All reactions were performed in a final volume of 200 μL in triplicate. The
results i.e. change in absorbance per min., were processed on a SoftMax Pro software
(Molecular Device, USA). Thiourea was used as a standard for comparison.
Percentage inhibition was calculated from the formula:
% inhibition = 100 – (ODtestwell / ODcontrol) x 100
226
4.5.2. In Vitro Anti-microbial Assay
Collection of pathogenic organism
All the bacterial and fungal pathogens were obtained from the Department of
Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi,
Pakistan.
Preparation of culture
To culture bacterial strains Muller Hinton agar (Oxoid) and Muller Hinton broth
(Oxoid) were used as a media and Sabourd dextrose agar (SDA) plates for fungal
strains [36].
4.5.2.1. In Vitro Anti-bacterial Assay
The anti-bacterial activity of fractions and all the synthesized compounds was
determined by the disc diffusion method and agar-well method as described in
literaure [37, 38].
Disc Diffusion Method
The suspensions of the applied bacterial strains were prepared in accordance with
0.5McFarland scale and swabbed on to the surface of sterile Mueller Hinton agar
plates. 100 mg/ml of stock solution was prepared by dissolving pure compounds in
DMSO. The sterile filter paper discs (diameter = 7mm) were impregnated with every
tested sample. The discs were allowed to dry at room temperature to evaporate
remaining solvent and placed on the surface of the inoculated plates. The discs
impregnated with DMSO served as the negative control and standards as positive
control. The plates were incubated at 37 °C for 24 hours. The results were recorded in
triplicate on the basis of measurement of the diameter of zone of inhibition appeared
around the disc [37].
227
Agar – well Method
Autoclaved Muller Hinton broth was used to keep bacterial culture in log phase for 2
h with constant agitation then wells were dug onto Muller Hinton Agar. Then 10 µl of
culture were poured into the wells. 10 mg/ml of the test sample was taken for activity
and incubated at 28+ 2°C for 24- 48 hr. After incubation diameter of zone of
inhibition was measured in triplicate to get results [38].
4.5.2.2. In Vitro Anti-fungal Activity Assay
All the fungal strains were checked for purity and maintained on SDA at 4ºC in the
refrigerator until required for use. Antifungal activity of fractions was screened using
agar-well method [39]. Briefly, a little amount of culture was transferred to 2-3 ml
distilled water or normal saline in a screw capped tube with few glass beads of 1 mm
in diameter, vortexes for 5-10 minutes to get a homogeneous suspension of fungal
culture. The fungal spore suspension that was prepared in autoclaved distilled water
transferred aseptically into each SDA plates. Now, the test samples (10 mg/ml) was
taken for activity. All plates were incubated at 28+ 2°C for 24 -48 h. After incubation,
results were recorded in triplicate by measuring diameter of zone of inhibition in mm.
4.5.2.3. Determination of Minimum inhibitory concentration (MIC)
MIC of test samples was determined by Micro broth dilution method using 96-well
microtitre plate. The stock solution of samples (100 mg/ ml) was prepared in distilled
water. Two fold serial dilutions of samples was made in 100 µl broth subsequently 10
µl of 2h refreshed culture matched with 0.5 Mac Farland index was added to each
well. One well served as culture control while other served as antibiotic control.
Microtitre plate was incubated at 37ºC for 24 h. The MIC was found when the well
showing no visible growth. Results were recorded in an average of triplicate [40].
228
4.5.3. In Vitro Antioxidant Assay
Antioxidant activity of the synthesized compounds was determined by using the
procedure described in literature [41]. The stable radical solution of 1, 1-diphenyl-2-
picrylhydrazyl (DPPH) was prepared in ethanol (300 µM). 10 µL of test samples and
90 μL solution of stable radical (DPPH) was added in 96-well microtiter plates and
incubated at 37º C for 30 minutes. Absorbance was measured at 515 nm by means of
a spectrophotometer. Percent inhibition of radicals by treatment of test sample was
found out by comparison with DMSO as negative control. Ascorbic acid was used as
standard control. % inhibition was calculated by following formula:
% Inhibition = (absorbance of the control-absorbance of the test sample) x 100
Absorbance of the control
The EC50 value calculated denotes the concentration (in µg/ml) of sample required to
scavenge 50% of DPPH.
4.5.4. Nematicidal Assay
Harvesting of Meloidogyne incognita
The nematicidal activity of synthesized compounds was determined through the
method reported in literature [42]. The tested nematode species, Meloidogyne
incognita was harvested and the roots of about 3 month old tomato plants, which had
been infected with the nematode, were washed in fresh tap water. After that the roots
were cut into 1-2 cm length and put in a round filter container then gently placed in
the funnel, which had been placed in a mist chamber. Active nematodes passed
through the filter and sank to the bottom of the funnel stem. After 4 days nematodes
could be harvested and used for the experiments.
229
Mortality test and lethal concentration (LC) determination
The test concentrations were prepared by adding 40 µL of synthetic drug stocks to
460 µL of fresh tap water in a 12-well plate. The mixing-plate was gently shaken by
hands around 2 min to allow the drug to mix well. After that, 150 ml of the solution
was transferred into 24 well test plates. Next, 90 µL of the nematode suspension
containing approximately 150 second stage juveniles was added into the wells and
gently mix for another 2 min then kept standing overnight at 24ºC. After 24 h the
dead and alive nematodes were counted under stereoscopic binocular microscope to
evaluate the mortality rate. Nematodes were considered dead when no movement was
observed even after mechanical prodding. The % mortality was calculated from an
average of replicate. The lethal concentration was determined at LC20, LC50 and LC90
mg/ml (the concentration needs to kill 20, 50 and 90% nematodes).
230
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Research Publications
1. Aneela Wahab, Amina Sultana, Khalid M. Khan, Ayesha Irshad, Nida
Ambreen and M. Bilal, Chemical investigation of Xanthium strumarium Linn.
and biological activity of its different fractions, J. Pharm. Res., 5(4), 1984
(2012).
2. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani
and Sandaleen Kanwal, Chemical constituents from the bioactive ethyl acetate
fraction of Xanthium strumarium Linn., Pak. J Pharm. Sci. (Submitted).
3. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani
and Zeba Parveen, Synthesis, Antimicrobial, Antioxidant and Nematicidal
activity of (2E, 4E) 5(benzo[d][1,3]dioxol-5yl)penta-2,4-dienamides, J Saud.
Chem. Soc., (Submitted).
4. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani
and Saima Faraz, Anti-microbial and antioxidant activity of conventionally
synthesized 2, 3-Diaminonaphthalenimidazole Derivatives, (in Process).