chapter iv isolation, characterization and structural...
TRANSCRIPT
Chapter - IVIsolation, characterization and structural modifications of
natural products.
I l l
Chapter - IV
General introduction
Natural products continue to play an important role in the discovery and development
of new pharmaceuticals, as clinically useful drugs, as starting materials to produce
synthetic drugs, or as lead compounds from which a totally synthetic drug is designed.
Discoveiy and development of lead compounds from natural products has
traditionally involved isolation of natural products with biochemical activity of
interest, elucidation of its structure, development of chemical and biosynthetic
methods for producing the compound and related compounds in larger quantities,
structural modifications to produce lead structures and eventually the examination of
structural-activity relationships and pharmacological properties.
Historically, plants were a folkloric source of medicinal agents, and as modem
medicine developed, numerous useful drugs were developed from lead compounds
discovered from medicinal plants. Today, this strategy remains an essential route to
new pharmaceuticals. Since 1961, the various plant-derived compounds that have
been used as anticancer drugs are either the natural products themselves viz
vincristine, vinblastine, taxol etc. or their analogues viz etoposide, teniposide,
topotecan, irinotecan etc.
With this background the research work in this chapter is based on structural
modifications of two well known anticancer natural products, podophyllotoxin and
camptothecin as well as the ̂isolation & characterization of natural products (datura
lactones) from Datura quercifolia.
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Chagter^J^
SECTION A
M odifications of podophyllotoxin as potential anticancer agents.
4.1.0. Introduction:
Podophyllotoxin, a bioactive lignan, was first isolated by Podwyssotzki in 1880 from
the North American plant Podophyllum peltatum Linnaeus (American podophyllum),
commonly known as the ‘American mandrake or May apple.1 Later on, it was isolated
from several other species like P. emodi Wall (Indian Podophyllum, syn. P.
hexandrum Royle) and P. pleianthum (Taiwanese Podophyllum). Other than these, 4-
deoxypodophyllotoxin has also been isolated2 from Anthriscus sylvestris and
Pulsatilla koreana. It is a potent cytotoxic agent. Two of the semi-synthetic
derivatives of PDT, that is, etoposide and teniposide, are currently used in frontline
cancer chemotherapy against various cancers (figure 1).
4.1.0.1. Biological activity:
Podophyllotoxin shows strong cytotoxic activity against various cancer cell lines. It is
effective in the treatment of Wilms tumors, various genital tumors and in non-
Hodgkin’s and other lymphomas and lung cancer.4,5 The attempts to use PDT in the
treatment of human neoplasia were mostly unsuccessful due to complicated side
effects6,7 such as nausea, vomiting, damage of normal tissues, etc. Because of this
reason, PDT as such is not used as a drug. Extensive structure modifications were
performed to obtain more potgnt and less toxic anticancer agents, which resulted in
two semi-synthetic glucosidic cyclic acetals of epipodophyllotoxin, etoposide and
teniposide. These are the most widely used derivatives for the treatment of
lymphomas, acute leukemia, testicular cancer, small cell lung cancer, ovarian,
bladder, brain cancers etc.8
4.1.0.2. Mode of action:
Podophyllotoxin acts as an inhibitor of assembly of microtubules and arrests the cell
cycle in metaphase.9,10 These PDT lignans block the catalytic activity of DNA
topoisomerase II by stabilizing a cleavage enzyme-DNA complex in which the DNA
is cleaved and covalently linked to the enzyme11. It binds at the colchicine site of the
tubulin12'14. From podophyllotoxin to etoposide/teniposide, some chemical
modifications were made that also led to a change in the mechanism of action; from
113
Chapter - IV
the inhibitor of microtubule formation by the parent compound PDT to DNA
topoisomerase II inhibitor by etoposide and congeners.
Figure 1. Structures o f podophyllotoxin (1); etoposide (2); teniposide (3); GL-331; (4) and TOP-53 (5).
4.I.O.3. Structure and activity relationship:
To obtain better therapeutic agents, extensive synthetic efforts have been devoted to
obtain a rationale for the improvement of topoisomerase II inhibition activity of
podophyllotoxin derivatives. Podophyllotoxin contains a five-ring system (i.e., A, B,
C, D and E rings). Molecular area-oriented chemical modification of Podophyllotoxin
has revealed structural features critical for the topoisomerase II inhibition:
• The 4p-configuration is essential with various substitution accommodated at
C-4.
• The free 4'-hydroxy is crucial.
o o
OMe OH
2. R = CH3; Etoposide
OH
4 5
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Chapter - IV
• The trans-lactone D ring with 2a, 3P configuration is very important.
• The dioxolane A ring is optimal.
• The free rotation of ring E is required.15
Such structure-activity relationships (SAR) unambiguously demonstrate that C-4 is
the only molecular area tolerable to significant structural diversification.
Recently two 4p-analogs GL-33116 and TOP-5317 of podophyllotoxin have been
found to have potential anticancer activity. GL-331, a 4p- arylamino derivative, was
more active than 2 both in vitro and in vivo 18 and retained cytotoxicity against
resistant cells. 19 It is currently under phase II clinical evaluation against several forms
of cancer, especially resistant malignancies.20 TOP-53, a 4p-alkylated derivative was
a more potent topoisomerase II inhibitor than 2. It exhibited high activity to nonsmall
cell lung cancer in both tumor cells and animal tumor models 21 and showed nearly
wild-type potency against a mutant yeast type II enzyme highly resistant to 2 .22 TOP-
53 is currently in phase I clinical trials.22 Both GL-331 and TOP-53 showed
topoisomerase II inhibitory activity, antitumor spectra, and drug-resistance profiles
significantly different from those of 2, which suggest an important role of various C-4
substitutions to the activity profiles of 2-related analogs and the feasibility of
optimizing this class of compound through rational C-4 modifications. This
postulation coincides with the composite pharmacophore model proposed by
MacDonald et a l 23 and the comparative molecular field analysis (CoMFA) models
generated by Zhiyan Xiao et a l 24 Both models indicated that the C-4 molecular area
could accommodate considerable structural diversity. The CoMFA model further
demonstrated that bulky substituents at C-4 might be favorable for topoisomerase II
inhibition. Accordingly a lot of derivatization has been done at C-4 position of
epipodophyllotoxin and some of its derivatives were found to be very potent for their25anticancer behavior.
Recently, Yan Guang Wang and co-workers have reported a brief synthesis and
biological evaluation of three compounds of 4p-[(5-substituted)-l,2,3-triazol-l-yl]
podophyllotoxin derivatives as new antitumor compounds.26 later on, the same group
reported the 1,3-Dipolar cycloaddition of 4p-azidopodophyllotoxins with symmetrical
alkynes under solvent-free microwave irradiation to corresponding 4p-l,2,3-triazol-l-
yl podophyllotoxins.27
115
Chanter - IV
4.1.1. Present work:
Keeping in view the excellent biological activity of 4(3-[(5-substituted)-l,2,3-triazol-
1-yl] podophyllotoxins and SAR of podophyllotoxins, a research program has been
initiated directed towards the design and synthesis of lead compounds for potentially
interesting drugs from 4P-azido-podophyllotoxins. Accordingly, a series of novel
epipodophyllotoxin derivatives bearing bulky 4-substituted triazoles at the C-4 side
chain has been synthesized by Cu (I) catalyzed 1,3-dipolar cycloaddition reaction of
4P-azido-4'-(9-demethyl podophyllotoxin and 4p-azido-podophyllotoxin with N-prop-
2-yn-l-ylanilines with the aim to overcome drug resistance and simultaneously
enhance topoisomerase II inhibition. This 1,3-dipolar-cycloaddition reaction of C4(3-
azido-podophyllotoxins with terminal alkynes is considered to be the “cream of the
crop” of click chemistry.
Click chemistry enables a modular approach to generate novel pharmacophores
utilizing a collection of reliable chemical reactions which give products
stereoselectively in high yields, produce inoffensive byproducts, are insensitive to
oxygen and water, utilize readily available starting materials, and have a
thermodynamic driving force of at least 20 kcal m o l1. Two types of click reactions
that have influenced drug discovery are the nucleophilic opening of strained ring
systems and 1,3-dipolar cycloadditions. Of particular interest is the Huisgen [3 + 2]
cycloaddition between a termjnal alkyne and an azide to generate substituted 1,2,3-
triazoles.
All the compounds synthesized by click chemistry protocol were evaluated for their
anticancer activity in vitro against a panel of seven human cancer cell lines. It was
interesting to note that all of them are having promising activity especially against SF-
295 (CNS), HCT-15 (colon) and 502713 (colon) cell lines. Compound lOe was found
to be the most promising in this study.
4.1.1.1. Chemistry:
As illustrated in scheme I, 4p-[(4-substituted)-l,2,3-triazol-l-yl] podophyllotoxin
derivatives were synthesized by the cycloaddition reaction of C4P-azido-4'-<9-
demethyl podophyllotoxin and C4P-azido-podophyllotoxin with ;V-prop-2-yn- 1 -
ylanilines obtained by the reaction of anilines with proporgyl bromide in presence of
K 2C O 3 . 4p-azido-4'-0-demethyl podophyllotoxin and 4P-azido-podophyllotoxin were
116
Chapter - IV
OH
c, b
OH
OH
7b
OH
7a
7a. R., = O C H 3
7b. R, = H
10
Scheme I. a) M eS03H, Nal, DCM; b) H20: acetone, BaC03; c) M eS03H, Nal, MeCN; d) NaN3; CF3COOH; e) CuS04, sodium ascorbate / t-BuOH: H20 (1:2).
117
Chggter^n^
synthesized according to the literature procedure28 in which the podophyllotoxin was
converted to 4'-0-demethyl epipodophyllotoxin by the demethylation of 4 '-OCH3 and
simultaneous inversion of 4-hydroxyl using CH3SO3H and Nal in DCM to
corresponding iododo-derivative followed by weak base hydrolysis (water: acetone,
BaCCb) to C4(3- 4'-0-demethyl podophyllotoxin. It was interesting to note that when
the reaction of podophyllotoxin was carried out in presence of MeCN as solvent, C4p-
epipodophyllotoxin was obtained as a major product. Therefore, by using these two
solvents, the selectivity of 4'-0-demethylation could be manoeuvred by employing
this methodology. The key intermediates 4p-azido-4'-0-demethyl podophyllotoxin
and 4p-azido-podophyllotoxin were obtained in excellent yields by treatment of C4p-
4'-0-demethyl podophyllotoxin and C4P-podophyllotoxin respectively with TFA and
NaN3 in CHCI3. 1,3-dipolar cycloaddition of 4p-azido-4'-0-demethyl
podophyllotoxin and 4p-azido-podophyllotoxin with terminal-alkynes in presence of
CUSO4.5H2O, sodium ascorbate in ^-BuOH and water (1:2) at ambient temperature
resulted in the formation of corresponding triazoles, 4p-[(4-substituted)-l,2,3-triazol-
1-yl] in excellent yield.
The formation of triazoles via the cycloaddition of azide and acetylene was first
reported by Dimroth in the early 1900’s but the generality, scope, and mechanism of
these cycloaddition was not fully realized until the 1960’s.29 The reaction generates a
mixture of 1,4 and 1,5-disubslituted triazoles (scheme II).
Various attempts to control the regioselectivity have been reported without much
success until the discovery of the copper (I)-catalyzed reaction in 2002, which
exclusively yields the 1,4-disubstituted 1,2,3-triazole.30’ 31 The in situ reduction of
copper (II) salts such as CuS04-5H 20 with sodium ascorbate in aqueous alcoholic
solvents allows the formation of 1,4-triazoles at room temperature in high yield.
The copper-catalyzed reaction is thought to proceed in a stepwise manner starting
with the generation of copper (I) acetylide (I). Density functional theory calculations
R1\
N— N = N+
+R2R2 R2
Scheme II. 1,2,3-triazole formation.
118
Chagter^n^
show a preference for the stepwise addition (I - II - III - IV) over the concerted
cycloaddition (I-IV) by approximately 12-15 kcal mol'1, leading to the intriguing six
membered metallocycle III (figure 2).
Comparison of the thermal reaction between benzyl azide and phenyl propargyl ether
with the copper-catalyzed reaction of the same substrates demonstrates the importance
of copper catalysis (scheme III). The thermal reaction leads to the formation of two
disubstituted triazole isomers while the copper (I)-catalyzed reaction selectively
produces the 1,4-isomer in 91% yield.30C u S 0 4
+(H)
Figure 2. Postulated catalytic cycle for azide-alkyne coupling.
-Ph
N = N — Nneat, 92 °C
Ph— O\ —
N N Ph N x N Ph
PhPh"
-Ph
NEEN—N
Ph— O
C uS 0 4.5H20
Sodium ascorbate
H,0 I t-BuOH, 2:1; rt
N x N Ph
VP h '
Scheme III. Thermal and Cu (I) catalyzed 1,3-dipolar cycloaddition.
119
Chapter - IV
OR.
Table 1. Various podophyllotoxin 4(3-[(4-substituted)-l,2,3-triazol-l-yl] derivatives.
a) Yields reported are isolated yields.
120
Chapter - IV
So using the above 1,3-dipolar cycloaddition reaction of 4(3-azido-4'-0-demethyl
podophyllotoxins and 4(3-azido-podophyllotoxins with iV-prop-2-yn-l-ylanilines in
presence of CUSO4.5H2O and sodium ascorbate, a series of 4(3-[(4-substituted)-l,2,3-
triazol-l-yl] derivatives differing in substitution at Rj and R2 has been synthesized in
excellent yield as shown in table 1.
All the products were characterized by 'HNMR, 13C NMR, IR, ESI-.MS and
elemental analysis. In 'HNMR cyclization of azides to triazoles were confirmed by
resonance of H-5 in triazole ring in aromatic region and that of C4a-H around 8 6 .
The structure was further supported by the 13CNMR and DEPT, which showed all the
carbon signals corresponding to triazole derivatives. ESI-MS of all the compounds
showed M+Na adduct ion as the parent molecular ion.
4.1.1.2. Biology:
All the compounds were assayed for in vitro cytotoxicity against a panel of seven
human cancer cell lines including including DU-145, PC-3 (prostrate), SF-295 (CNS),
HCT-15, 502713 (Colon), HEP-2 (liver) and A-549 (lung) using sulforhodamine B.
The cells were allowed to proliferate in presence of test material for 48 hours and the
results are reported in terms of I C 50 values (table II).
Table II. IC50 values (|J.M) o f 4P-[(4-substituted)-l,2,3-triazol-l-yl] podophyllotoxin against a panel of human cancer cell lines
Entry DU-145 PC-3 -* SF-295 HCT-15 502713 HEP-2 A-549
10a 1.06 1.09 1.94 0.36 0.788 9.14 7.88
10b 1.19 1.53 0.962 0.139 0.624 1.5 5.17
10c 0.936 1.18 0.839 0.34 0.714 1.37 4.99
lOd 4.16 1.37 3.11 4.22 0.98 1.22 8.63
lOe 0.651 0.742 0.681 0.0436 0.566 3.65 5.78
10 f 4.37 6.63 4.2 1.55 1.19 4.1 7.43
lOg 1.12 0.878 0.781 0.147 0.607 4.64 6.1
lOh 1.09 1.03 0.704 0.406 0.689 2.96 7.46
lOi 3.57 3.45 1 1.43 1.88 1.26 9.24
10j 2.75 4.49 6.03 1.69 1.34 3.27 7.41
From the I C 50 values, it is clear that all the compounds have significant cytotoxic
activity against prostrate, CNS, colon and liver derived cancerous cell lines. However,
it may be noted that these compounds showed least activity against A-549, a lung
121
Chapter - IV
derived cancerous cell line. Compound lOe showed significant cytotoxicity against
DU-145, PC-3, SF-295, HCT-15 and 502713 cell lines and it was found to be the
most active against HCT-15 cell line with IC50 value 0.0436 (J.M. Compounds such as
10a, 10b, 10c, lOg and lOh were also found to have promising activity against the
above mentioned cell lines. From the IC50 values, it is clear that the compounds with
trimethoxy moiety in ring E are having more cytotoxic activity than those of
dimethoxy moiety compounds.
In conclusion, a series of 4p-[(4-substituted)-l,2,3-triazol-l-yl] podophyllotoxin
derivatives were synthesized and screened for anticancer activity against a panel of
human cancer cell lines. From the data it was found that all the compounds are having
promising anticancer activity, but the compound lOe was found to be the most active
in this study.
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Chapter - IV
4.1.2. Experimental:
4.1.2.1. Synthesis of 4'-0-demethyl-C4P-epipodophyllotoxin (7a).
To a solution of podophyllotoxin (414 mg, 1 mmol) in dry DCM (10 mL), Nal (447
mg, 3 mmol) was added and stirred for 5 min. To this stirred suspension, MeSCbH
(288 mg, 3 mmol) was added dropwise with syringe at 0 °C and the stirring was
continued for 5 h at room temperature. Nitrogen was bubbled through the solution to
drive of the excess hydrogen iodide. This solution was then evaporated in vacuo and
used for the next reaction without further purification. To the above crude product
BaCC>3 (395 mg, 2 mmol), 0.5ml of water was added in 10ml of acetone and stirred
for 8 hours at r.t. The reaction mixture was then filtered, diluted with ethyl acetate and
washed with water, 10% Na2S203 solution dried and purified via column
chromatography on silica gel with ethyl acetate/hexane as eluent. All physico
chemical data was same as reported in literature.32
4.1.2.2. Synthesis of C40- epipodophyllotoxin (7b).
To a solution of podophyllotoxin (414 mg, 1 mmol) in dry MeCN (10 mL), Nal (298
mg, 2 mmol) was added and stirred for 5 min. To this stirred suspension, MeSCbH
(192 mg, 2 mmol) was added dropwise with a syringe at 0 °C and the stirring was
continued for another 15 min at room temperature. Nitrogen was bubbled through the
solution to drive the excess hydrogen iodide. This solution was then evaporated in
vacuo and used for the next reaction without further purification. To the above crude
product BaCC>3 (395 mg, 2 mmol), 0.5ml of water was added in 10ml of acetone and
stirred for 8 hours at r.t. The reaction mixture was then filtered, diluted with ethyl
acetate and washed with water, 10% Na2S2C>3 solution dried and purified via column
chromatography on silica gel with ethyl acetate/hexane as eluent. All physico
chemical data was same as reported in literature.32
4.1.2.3. 4'-0-demethyl- 4/J-azido-4-deoxypodophyllotoxin (8a).
To 1.60g (4 mmol) of 4'-0-demethyl-C4p- epipodophyllotoxin and 1.32 (2 mmol) of
sodium azide in 8mL of C H C I 3 was added TFA (4mL) dropwise. The reaction
mixture was stirred for 15 minutes. Saturated aqueous sodium bicarbonate solution
was added. The organic layer was washed with water and dried over MgSC>4. After
the solvent was removed the crude product was purified by column chromatography
to give 1.5g (94 %) of 4'-0-demethyl-4/?-azido-4-deaoxypodophyllotoxin.
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Chapter - IV
’HNMR (200MHz, CDC13):
IR (KBr):Mass (ESI-MS):4p-Azidopodophyllotoxin (8b).
OH
8 2.95 (m, 1H, H-3), 3.18 ( d d , 1H, J = 13.9 Hz, H-2), 3.79 (s, 6H, 2 x -OCH3), 4.32 ( d , 2H, J =9.2 Hz, 2H-11), 4.64 ( d , 1H, J = 5.2, H-4), 4.78 ( d , 1H, J = 3.7 H-l), 5.43 (s, 1H, OH), 6.02 (s, 1H, -OCH2O-), 6.04 (s, 1H, 0CH20), 6.28 (s, 2H, 2'-H, 6 '-H), 6.62 (s, 1H, H-8), 6.82 (s, 1H, H-5).3400, 2920, 2155, 1720, 1602, 1460 cm’1 448 (M+ Na).
OMe
'HNMR (200MHz, C D C I 3 ) : 5 2.93 (m, 1H, H-3), 3.16 ( d d , 1H, J = 13.9 Hz,H-2), 3.64 (s, 6H, 2 x -OCH3), 3.89 (s, 3H, - OCH3), 4.34 ( d , 2H, J = 9.2 Hz, 2H-11), 4.66 ( d , 1H, J = 5.0 Hz, H-4), 4.78 ( d , 1H, J = 3.7 H-l),6.02 (s, 2H, 0CH20), 6.28 (s, 2H, 2'-H, 6 '-H),6.60 (s, 1H, H-8), 6.82 (s, 1H, H-5).
IR (KBr): 3420, 2910, 2200, 1723, 1611, 1467 cm’1Mass (ESI-MS): 462 (M+ Na).
124
Chapter - IV
4.I.3.4. Preparation of 4(3-[(4-substituted)-l,2,3-triazol-l-yl]podophyllotoxins:
General procedure.
To a solution of 9 (1 mmol) in t-butyl alcohol and water (1:2, 8 mL) was added
CuS04.5H20 (1 mmol), sodium ascorbate (2 mmol) followed by 4P-azido-
podophyllotoxin (44mg, 0.1 mmol). The reaction mixture was stirred at room
temperature for 8 hours. After completetion, the reaction mixture was diluted with 80
mL of water and extracted with ethylacetate (2 x 20 mL). The combined extracts were
washed with brine, dried over Na2SC>4 and evaporated in vaccuo. The crude product
obtained was precipitated in diethyl ether to yield the pure product 10.
9-[4-(o-Tolylamino-methyl)-[l,2,3]triazoI-l-yI]-5-(3,4,5-trimethoxy-phenyI)-
5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one (10a).
OMe
White solid; mp:'HNMR (200MHz, CDC13):
13 C NMR (125 MHz, CDC13):
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C3 2 H 3 2N 4 O7 :
128-130 °C5 2.19 (s, 3H), 3.06 (m, 1H), 3.23 (m, 2H), 3.64 (s, 6H), 3.89 (s, 3H), 4.03 (d, 1H, J = 2.29 Hz),4.40-4.51 (m, 3H), 4.75 (d, 1H, J = 4.76 Hz),6.08 (m, 3H), 6.33 (s, 2H), 6.59-6.76 (m, 4H), 7.13 (m, 3H).5 17.4, 33.7, 37.1, 41.6, 43.5, 56.3, 60.7, 67.4,71.4, 101.9, 108.3, 108.8, 110.5, 118.2, 124.7,127.1, 130.2, 133.3, 134.3, 137.7, 148.09, 149.45, 152.8, 173.1.3425, 1778.6, 1588.5, 1506.3, 1237.8, 1126.4,751.0 cm'1 607 (M+ Na).C, 65.74; H, 5.52; N, 9.58. Found: C, 65.66; H, 5.63; N, 9.88.
125
Chapter - IV
5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-[4-(o-tolylamino-methyl)-[l,2,3]triazol-l-
yl]-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one (10b).
OH
White solid; mp:'HNMR (200MHz, CDC13):
13 C NMR (125 MHz, CDC13):
145-148 UC5 2.16 (s, 3H), 3.02 (m, 1H), 3.18 (m, 2H), 3.79 (s, 6H), 3.99 (d, 1H, J = 2.28 Hz), 4.35 (m, 1H), 4.47(s, 2H), 4.71 (d, 1H, J = 4.82 Hz), 6.02 (m, 3H), 6.32 (s, 2H), 6.59-6.73 (m, 4H), 7.11 (m, 3H).5 18.7, 33.7, 37.6, 40.1, 42.9, 56.99, 60.3, 67.8,103.5, 108.4, 109.5, 112.2, 117.1, 122.1, 127.2,129.1, 130.4, 134.7, 135.09, 144.8, 145.4, 146.8,172.4.3418.1, 1778.2, 1605.1, 1485.07, 1231.9,1115.4, 762.8 cm'1593.1 (M+Na).C, 65.25; H, 5.30; N, 9.82. Found: C, 65.33; H, 5.23; N, 9.89.
9-{4-[(3-Chloro-phenylamino)-methyl]-[l,2,3]triazol-l-yl}-5-(3,4,5-trimethoxy-
IR (KBr):
Mass (ESI-MS): Anal Calcd. for
phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one
(10c).
126
Chanter - IV
13 C NMR (125 MHz, CDC13):
White solid; mp: 136-138 C'HNMR (200MHz, CDC13): 8 3.01 (m, 1H), 3.21 (m, 2H), 3.76 (s, 6H), 3.81
(s, 3H), 4.12-4.21 (m, 1H), 4.40- 4.53 (m, 3H),4.73 (d, 1H, J = 4.84 Hz), 5.99-6.07 (m, 3H), 6.51 (s, 2H), 6.51-6.72 (m, 5H), 7.07 (m, 2H).5 37.1, 39.7, 41.6, 43.6, 56.3, 58.7, 60.7, 67.3,102.0, 108.3, 108.7, 110.5, 111.7, 113.0, 113.2,118.2, 118.5, 124.5, 130.2, 133.2, 134.2, 135.0,137.7, 148.0, 148.5, 149.5, 152.8, 173.1.3389.2, 1778.6, 1598.5, 1505.3, 1485.2, 1238.0,1002.6, 766.7 cm' 1 627 (M+Na).C, 61.54; H, 4.83; N, 9.26. Found: C, 61.46; H, 4.66; N, 9.38.
9-{4-[(3-Chloro-phenylamino)-methyl]-[l,2,3]triazol-l-yl}-5-(4-hydroxy-3,5-
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C31H29CIN4O7:
dimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]-
dioxol-6-one (lOd).
White solid; mp:'HNMR (200MHz, CDCI3):
13 C NMR (125 MHz, CDCI3):
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C30H 27CIN 4O 7:
OH
44-147°C5 3.18 (m, 1H), 3.22 (m, 2H), 3.79 (s, 6H), 3.92 (d, 1H, J = 2.27 Hz), 4.38 (m, 3H), 4.72 (d, 1H, J = 4.66 Hz), 5.99-6.05 (m, 3H), 6.32 (s, 2H), 6.47-6.76 (m, 5H), 7.08 (m, 2H).8 37.1, 39.7, 41.6, 56.3, 58.7, 67.3, 72.8, 102.0,108.3, 108.7, 110.5, 111.7, 113.0, 118.2, 124.5,130.2, 134.2, 135.0, 137.7, 148.0, 149.5, 152.8,173.1.3405.5, 1777.5, 1598.3, 1505.3, 1232.2, 1114.5,1036.9, 765.9 cm' 1 590 (M+Na).C, 60.97; H, 4.60; N, 9.48. Found: C, 61.16; H, 4.66; N, 9.68
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Chapter - IV
9-{4-[(3-Nitro-phenylamino)-methyl]-[l,2,3]triazol-l-yl}-5-(3,4,5-trimethoxy-
phenyl)-5,8,8a,9-tetrahy dro-5aH-furo [3' ,4': 6,7] naphtho [2,3-d] [ 1,3] dioxol-6-one
(lOe).
13 C NMR (125 MHz, CDC13):
White solid; mp: 208-211 C‘HNMR (200MHz, CDC13): 5 3.08 (m, 1H), 3.29 (m, 2H), 3.82 (s, 6H), 3.87
(s, 3H), 4.07-4.21 (m, 1H), 4.40-4.52 (m, 3H), 4.80 (d, 1H, J = 4.82 Hz), 6.10 (m, 3H), 6.37 (s, 2H), 6.61 (s, 1H), 6.70 (s, 1H), 6.98 (d, 1H, J = 8.04 Hz), 7.24-7.45 (m, 3H), 7.62 (d, 1H, J = 9.12 Hz).8 34.9, 37.7, 40.1, 42.7, 56.4, 58.6, 60.0, 70.3,105.5, 107.6, 108.3, 108.6, 110.1, 112.1, 120.3,124.1, 125.2, 130.2, 133.3, 134.1, 137.1, 148.3,149.5, 152.2, 173.2.3389.5, 1778.2, 1588.7, 1530.1, 1348.8, 1237.7,1125.6, 1092.95, 1002.35, 736.4, 675.3 cm"1 638 (M+Na).C, 60.48; H, 4.75; N, 11.38. Found: C, 60.56; H, 4.67; N, 11.77
5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[(3-nitro-phenylamino)-methyl]-
[l,2,3]triazol-l-yl}-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-
d][l,3]dioxol-6-one (lOf).
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C31H29N5O9:
128
Chanter - IV
White solid; mp: 221-223 °C'HNMR (200MHz, CDC13): 5 3.08 (m, 1H), 3.29 (m, 2H), 3.82 (s, 6H), 4.07-
4.23 (m, 1H), 4.40-4.52 (m, 3H), 4.80 (d, 1H, J = 4.82 Hz), 6.10 (m, 3H), 6.37 (s, 2H), 6.59 (s, 1H), 6.68 (s, 1H), 6.98 (d, 1H, J = 7.88 Hz), 7.24-45 (m, 3H), 7.62 (d, 1H, J = 9.12 Hz).
13 C NMR (125 MHz, CDC13): 5 34.9, 37.7, 40.1, 42.7, 56.4, 60.0, 61.7, 70.3,105.5, 107.6, 108.3, 108.6, 110.1, 112.1, 120.3,124.1, 125.2, 130.2, 133.3, 134.1, 137.1, 148.3,149.5, 152.2, 173.2.
IR(KBr): 3389.5,1778.2,1588.7,1530.1,1348.8,1237.7,1125.6, 1092.95, 1002.35, 736.4, 675.3 cm' 1
Mass (ESI-MS): 624 (M+ Na).Anal Calcd. for C30H27N5O9: C, 59.90; H, 4.52; N, 11.64. Found: C, 60.16; H,
4.57; N, 11.57.
9-(4-Phenylaminomethyl-[l,2,3]triazoI-l-yl)-5-(3,4,5-trimethoxy-phenyl)-
5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one (lOg).
White solid; mp:'HNMR (200MHz, CDC13):
13 C NMR (125 MHz, CDC13):
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C31H 30N 4O 7:
OMe
168-171 °C5 2.99 (m, 1H), 3.16 (m, 2H), 3.76 (s, 6H), 3.94 (s, 3H), 3.94 (d, 1H, J = 2.28 Hz), 4.36-4.43 (m, 3H), 4.70 (d, 1H, J = 4.67 Hz), 6.00 (m, 3H),6.31 (s, 2H), 6.58-6.79 (m, 5H), 7.12-7.22 (m, 3H).8 38.1, 41.1, 42.3, 47.1, 57.3, 59.6, 61.7, 103.6,108.9, 110.1, 112.7, 115.8, 119.4, 123.5, 125.4,132.2, 133.7, 134.9, 145.4, 149.5, 150.4, 157.2,173.3.3405.4, 1777.6, 1602.4, 1504.2, 1485.1, 1237.5,1125.7, 752.2 cm' 1 593 (M+ Na).C, 65.25; H, 5.30; N, 9.82. Found: C, 65.36; H, 5.27; N, 10.07.
129
Chanter - IV
5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-(4-phenylaminomethyl-[l,2,3]triazol-l-
yl)-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one (lOh).
OH
White solid; mp:'HNMR (200MHz, CDCI3):
13 C NMR (125 MHz, CDC13):
181-182 UC8 2.98 (m, 1H), 3.19 (m, 2H), 3.80 (s, 6H), 3.95 (d, 1H, J = 2.28 Hz), 4.36-4.44 (m, 3H), 4.72 (d, 1H, J = 4.71 Hz), 5.99-6.06 (m, 3H), 6.32 (s, 2H), 6.59-6.85 (m, 5H), 7.12-7.27 (m, 3H).8 38.1, 41.1, 42.3, 47.1, 57.3, 59.6, 103.6, 108.9,110.1, 112.7, 115.8, 119.4, 123.5, 125.4, 132.2,133.7, 134.9, 145.4, 149.5, 150.4, 157.2, 173.33405.4, 1777.6, 1602.4, 1504.2, 1485.1, 1237.5,1125.7, 752.2 cm' 1 579 (M+ Na).C, 64.74; H, 5.07; N, 10.07. Found: C, 64.86; H, 5.20; N, 10.02.
9-{4-[(2-Chloro-phenylamiiu>)-methyl]-[l,2,3]triazol-l-yl}-5-(3,4,5-trimethoxy-
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C30H28N4O7:
phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxol-6-one
(lOi).
130
Chapter - IV
White solid; mp:'HNMR (200MHz, CDCI3):
13 C NMR (125 MHz, CDCI3):
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C31H29CIN4O7:
167-170°C5 2.96 (m, 1H), 3.20 (m, 2H), 3.77 (s, 6H), 3.82 (s, 3H), 3.93 (d, 1H, J = 2.35 Hz), 4.41 (bs, 3H),4.74 (d, 1H, J = 4.71Hz), 6.01-6.09 (m, 3H),6.32 (s, 2H), 6.48-6.73 (m, 5H), 7.09 (m, 2H).5 36.8, 37.7, 40.4, 42.2, 56.9, 59.3, 61.3, 67.9,102.5, 105.1, 108.8, 110.5, 111.7, 113.0, 113.2,118.2, 118.5, 124.5, 130.2, 133.2, 134.2, 135.0,137.7, 148.0, 148.5, 149.5, 153.8, 173.6.3389.2, 1778.6, 1598.5, 1505.3, 1485.2, 1238.0,1002.6, 766.7 cm' 1627 (M+Na).C, 61.54; H, 4.83; N, 9.26. Found: C, 61.66; H, 4.55; N, 9.33.
9-[4-(p-TolyIamino-methyl)-[l,2,3]triazoI-l-yl]-5-(3,4,5-trimethoxy-phenyl)-
5,8,8a, 9-tetrahydro-5aH-furo[3',4':6,7]naphtho[2,3-d][l,3]dioxoI-6-one (lOj).
White solid; mp:'HNMR (200MHz, CDC13):
13C NMR (125 MHz, CDC13):
IR (KBr):
Mass (ESI-MS):Anal Calcd. for C32H 32N 4O 7:
OMe
146-148°C5 2.19 (s, 3H), 3.06 (m, 1H), 3.23 (m, 2H), 3.64 (s, 6H), 3.89 (s, 3H), 4.03 (d, 1H, J = 2.29 Hz),4.40-4.51 (m, 3H), 4.75 (d, 1H, J = 4.76 Hz),6.08 (m, 3H), 6.32 (s, 2H), 6.63 (s, 1H), 6.66 (s, 1H), 6.79 (m, 4H), 7.17 (m, 1H).5 18.20, 37.78, 42.10, 44.45, 56.96, 59.51, 61.19, 68.01, 71.8, 102.43, 108.81, 109.33, 110.62, 114.65, 125.0, 129.20, 130.81, 132.49, 134.38, 149.31, 151.42, 154.26, 156.04, 173.12. 3437, 2905, 1768, 1505.3, I486,, 1238.0, 1095,1002.6, 766.7 cm' 1 607 (M+ Na).C, 65.74; H, 5.52; N, 9.58. Found: C, 66.01; H, 5.66; N, 9.67.
131
Chapter - IV
SECTION B
Synthesis o f Camptothecin and Podophyllotoxin Conjugate.
4.2.0. Introduction:
Like podophyllotoxin, camptothecin (CPT), first isolated from the Chinese
ornamental tree Camptotheca acuminata 32, 33 is also a very powerful anticancer
compound especially against ovarian cancer. It is a member of the quinolinoalkaloid
group. It consists of a pentacyclic ring structure that includes a pyrrole quinoline
moiety and one asymmetric center within the a-hydroxy lactone ring with 20(S)
configuration.
4.2.0.1. Mode of action:
In the early 1970s, initial studies examining the mechanism of action of CPT
suggested that cytotoxicity might result from its immediate synthesis, which was
found to be reversible following brief exposure to camptothecin, but DNA
topoisomerase I inhibition progressively became irreversible with increasing
concentration and exposure duration. These studies also suggested that camptothecin
is selectively cytotoxic to S-phase cells, arrests cells in the G-2 phase and induces
fragmentation of chromosomal DNA.
CPT was approved by US Food and Drug Administration in the 1970s against colon
carcinoma and thus it was evaluated34,35 as a possible drug in the treatment of human
cancer in phase I and phase II studies. Although camptothecin showed strong
antitumor activity among patients with gastrointestinal cancer, it also caused
unpredictable and severe adverse effects including myelosuppression, vomiting,
diarrhoea, and severe haemorrhagic cystitis. These findings eventually resulted in the
discontinuation of phase II trials in 1972.
4.2.0.2. Synthetic analogues of CPT:
CPT as such could not be used as a drug of choice due to its severe toxicity. Several
groups have tried to synthesize derivatives having lower toxicity. Thus, the
development of these synthetic and semisynthetic strategies have facilitated the study
of the CPT mechanism, as well as the identification of analogues with improved
properties. From its SAR, it has been found that the modifications in rings A and B
are well tolerated and resulted in better activity than CPT in many cases.
132
Chapter - IV
4.2.0.3. Modifications in quinoline A and B rings: The most successful derivatives
of CPT have been obtained due to modifications of rings A and B. Till date, the only
CPT analogues approved for clinical use36,37 are topotecan and irinotecan, which were
obtained by modifications of these rings. Rubitecan, 9-nitro-camptothecin (9-NC)
serves as a metabolic precursor to 9-amino CPT and is currently in phase III clinical
trials for the treatment of pancreatic cancer.
Modifications can involve additions to the quinoline ring or the complete replacement
of the quinoline ring with an alternative ring system. Several other heterocyclic ring
systems have been found to have significant cytotoxicity on replacement of the
quinoline ring38 but the quinoline ring system was found to be the most potent and
hence, most of the modifications were done with retention of the quinoline ring
system.
4.2.0.4. Conjugate analogues:
The use of conjugates has emerged as a frequent strategy in efforts to optimize
therapeutically beneficial properties of CPT, including lactone stability, solubility/
lipophilicity, tumor cell recognition and sequence specificity of DNA damage. Two
predominant methodologies have been utilized for the synthetic preparation of CPT-
conjugates. The first relies on the utilization of the 20(S)-hydroxyl group as the site
for conjugation. The second involves the exploitation of reactive functional groups,
for example, amino, hydroxyl and carboxylic acid groups on modified CPT
analogues. Most of these groups have been part of the quinoline (A/B) ring system.
Perhaps the simplest of these derivatives have been obtained by etherification of the
20(S)-hydroxyl group.
4.2.1. Previous work:
Giovanella et al. reported the synthesis and biological evaluation of a series of alkyl
esters of varying size.39 Specifically, the 20(S)-0-ethyl ester (11) was reported to
possess reduced toxicities while demonstrating enhanced lactone stability and
pronounced in vivo activities against human tumor xenografts in nude mice. Studies
utilizing 20(S)-0-acyl esters resulted in CPT analogues with excellent in vitro
cytotoxicity and antitumor activity equivalent to those of CPT.40 The utilization of
20(S)-0-amino acid analogues as prodrugs has also been studied.41 Interestingly, the
isolation of 20(S)-C)bglucopyranosyl CPT from Mostuea brunonis has been reported,
133
Chagter^U^
suggesting that the 20(S)-hydroxyl group acts as a point of attachment for natural
bioconjugates.42 A biosynthetic precursor of CPT glycosylated at the 20- position has
also been reported.43 These studies have demonstrated the feasibility of using the
20(S)-hydroxyl group to modify CPT. The use of 20(S)-0-esters, amides, carbonates
and carbamates with a variety of linkers have been employed in recent studies in the
preparation of novel CPT conjugates (figure 3).
Figure 3. Various conjugates o f CPT.
The antitumor activity, biodistribution and lactone stabilization have been reported for
a series of polymeric conjugates of CPT, including polyethylene glycol (PEG)
conjugate, which was designed to be CPT delivery vehicle.44 The polymeric PEG-
CPT conjugate (12) was reported to produce improved levels of tumor regression in
HT-29 xenografts and decreased toxicity relative to CPT.
In a recent study, Firestone et al. have reported the synthesis and initial biological
profile of a CPT immunoconjugate designed to target tumor cells specifcally45
Utilizing the tumor-recognizing antibody BR96 attached to CPT through a 20(S)-0-
carbonate linker cleavable by cathespin B, the resulting immunoconjugate (13) was
134
Chapter - IV
intended to eliminate the dose-limiting the parent drug. The in vitro activity of the
BR96-CPT conjugate was demonstrated to be superior to that of CPT alone.
Interestingly, a glutathione-CPT conjugate, 7-(glutathionylmethyl)-10,l 1-
methylenedioxy-CPT (14) showed enhanced stability of the derived ternary
complex.46 Analogue 14 was shown to inhibit topo I and exhibit potent antineoplastic
activity against U937 and P388 leukemia cell lines.
The observation that some CPT resistant cell lines concurrently developed resistance
toward the topoisomerase II inhibitor etoposide, prompted Chang et al. to develop a
CPT conjugate incorporating the 4(3-amino-4-0-demethylepipodophyllotoxin moiety
of etoposide.47 Two bioconjugates were synthesized differing only through the para
(W l) and ortho (W2) phenylenediamine linkers and were shown to exert their
cytotoxic effects primarily through topo I inhibition. Further, Wl (15) was
demonstrated to be a more effective antineoplastic agent against human prostate
cancer cells than either CPT or etoposide (figure 4).
15. L= para linkage (W l)
16. L= ortho linkage (W2)
Figure 4. Podophyllotoxin-CPT conjugate.
Recently, Hironori Ohtsu et. al 48 has synthesized and evaluated five conjugates (17-
21) composed of a paclitaxel and a camptothecin derivative joined by an imine
linkage as cytotoxic agents and as inhibitors of DNA topoisomerase I (figure 5). All
of the conjugates were potent inhibitors of tumor cell replication with improved
activity relative to camptothecin. Significantly, compounds 17-19 were more active
135
Chavter - IV
than paclitaxel and camptothecin against HCT- (colon adenocarcinoma) cell
replication, and the spectrum of activity was different from a simple mixture of
paclitaxel and camptothecin.
ox
P h " '^ N H
Ph'
OH
17. R = (CH^18. R = (CH2)319. R = ( CH2)5
2 0 .R ^ O ^21.R = 0 ^
Figure 5. CPT-Paclitaxol conjugates.
4.2.2.Prsent work:
Keeping in view the magnificent biological relevance of these bioconjugates, mode of
action and SAR of camptothecin and podophyllotoxin, the present work describes the
synthesis of their cross conjugate linked through a succinyl linker. The aim of the
present work was to investigate whether a topoisomerase inhibitor displaying dual
target specificity could be prepared by conjugating derivatives of camptothecin and
podophyllotoxin. For this purpose, a linkage position was chosen based on the known
tolerance of substituents in 4(3-amino-epipodophyllotoxin and the ring A of
camptothecin. The hybrid molecule developed in the present approach overcome the
problem of unstability of the conjugate developed by Chang et al et.al in which 4p-
amino-epipodophyllotoxin and camptothecin are linked by highly unstable Shiffs
base. Besides the linker used in Chang approach is not flexible to allow the
independent action of two molecules at the receptor site.
The present approach describes the synthesis of a bioconjugate of 12-amino-
camptothecin and 4p-amino-epipodophyllotoxin linked by a stable and flexible linker,
136
Chanter - IV
which may allow the independent recognition of the two molecules at the receptor
site. For this purpose, CPT was nitrated by using KNO3 and H2SO4 as a nitrating
mixture to give two regioisomers 9-NCPT (23) and 12- NCPT (24). These compounds
were isolated by column chromatography to give pure 23 (20 %) and 24 (48 %).
Reduction of 23 and 24 was done by anhydrous SnCl2/HCI to give corresponding
amino-CPTs, 25 and 26 as orange yellow solids. Amino CPT (9 or 12) was allowed to
react with succinic anhydride in DCE at reflux temperature to yield their succinyl
derivatives, 27 and 28. Structures of these compounds were confirmed by the
presence of succinyl peaks around 5 2-3 and a carboxylic proton signal around 8
10.25 in their ’HNMR besides a marked chemical shift of the aromatic region. Other
physico-chemical data also supported the structures.
On the other hand C4|3-aminopodophyllotoxin (29) was synthesized from its
corresponding azido derivative by Pd-C catalyzed hydrogenation. This compound is
highly unstable and was subjected to further reaction immediately without any
purification. 29 was subjected to the DIC / HOBT coupling with 12-succinyl-amino-
CPT to yield the product 30, as their conjugate analogue (scheme IV).
Biological evaluation of all the intermediates (23-28) and the final conjugate 30 is in
progress.
137
Chagter^IVm
R1
25. R1 = Nl-̂ ; R2 = H26. R1 = H; R2 = NHj
24. R1 = H; R2 = NO,
27. R1 = NHCOCI-^CHjCOOH; R2 = H28. R1 = H; R2 = NHCOCH,CH2COOH
+ 28
29
Scheme IV. a) K N 03 / H2S 0 4; b) SnCl2 / HC1; c) Succinic anhydride / DCE; d) Pd-C / EtOAc; e) DIC / HOBT / DMF.
138
Chapter - IV
4.2.3. Experimental:4.2.3.1. Synthesis of 9-nitrocamptothecin and 12-nitrocamptothecin: procedure.
CPT (0.50g, 1.4 mmol) and KNO3 (0.50g, 5mmol) were added to concentrated H2SO4
(30 mL) in a lOOmL round-bottom flask equipped with a magnetic stirrer all at once.
The mixture was stirred at r.t for lday and then poured onto ice-water (500mL)
slowly while stirring. The yellow suspension was extracted with DCM (3 x 200 mL).
The combined extracts were dried (anhd. Na2SC>4). The DCM was evaporated by
rotatory evaporator and the residue was refluxed in petroleum ether for 4hrs. After
cooling to r.t, the mixture was filtrated and the yellow powder was subjected to
column chromatography (CHCI3: MeOH; 99: 1 v/v) to give the two regioisomers
isomers; 23 and 24 in 20 % and 48 % respectively in yield.
9-Nitrocamptothecin (23).
'HNMR (200MHz, DMSO-d6):
Mass (ESI-MS):Anal. Calcd. for C20H15N3O6 :
12-Nitrocamptothecin (24).
'HNMR (200MHz, DMSO-d6):
Mass (ESI-MS):
5 0.88 (t, 3H, J = 6.74 Hz), 1.85 (q, 2H, J = 7. 26 Hz), 5.35 (s, 2H), 5.45 (s, 2H), 6.59 (s, 1H, OH), 7.39 (s, 1H), 8.05 (m, 1H,), 8.51 (m, 2H), 9.17 (s, 1H).394 (M+ H).C, 61.07; H, 3.84; N, 10.68. Found: C, 61.36 H, 3.37; N, 10.78.
5 0.87 (t, 3H, J = 7.25Hz), 1.84 (q, 2H, J = 7, 26 Hz), 5.33 (s, 2H), 5.44 (s, 2H), 6.60 (s, 1H, OH), 7.27 (s, 1H), 7.85 (t, 1H, J = 7.82 Hz), 8.41 (m, 2H), 8.89 (s, 1H).394 (M+ H).
139
Chanter - IV
Anal. Calcd. for C20H15N3O6 : C, 61.07; H, 3.84; N, 10.68. Found: C, 61.36 H,3.37; N, 10.78.
4.2.3.2. Reduction of Nitro-CPTs: Synthesis of 9 and 12-aminoCPT.
Nitro analogue 23 or 24 (200mg, 0.509 mmol) was added to a cold (-10 °C) solution
of anhyd. SnCh (350mg, 1.842 mmol) in concd. HC1 (3mL). The mixture was stirred
for 2 hrs. at ambient temperature, and after chilling to -10 °C, the yellow solid was
collected (Buchner) and washed with cold concd. HC1 (lmL). Crude mass was
suspended in H2O and neutralized with solid NaHCCb. The resulting solid was
collected (Buchner) and washed with H2O (2 mL) and then stirred for 1 hr. in
absolute EtOH. (20 mL). The solid was again collected washed with EtOH (3mL) and
Et20 (lOmL) and dried. This martial was extracted with DMF (5x 35 mL) and the
resulting yellow-orange solution was concentrated in vacuo to 1-2 mL volume.
During this time, amino-CPT crystallized as a yellow orange solid in 50 % yield.
9-Aminocamptothecin (25).
'HNMR (200MHz, DMSO-d6): 5 0.89 (t, 3H, 5 = 1. MHz), 1.91 (q, 2H, J = 7.99Hz), 5.33 (s, 2H), 5.47 (s, 2H), 6.16 (bs, OH or NH), 6.86 (d, 1H, J = 7.49 Hz), 7.35 (s, 1H),7.38 (d, 1H, J = 7.49 Hz), 7.56 (m, 1H), 8.89 (s,1H).
Mass (ESI-MS): 386.1 (M+Na).Anal. Calcd. for C2oH17N30 4 : C, 66.11; H, 4.72; N, 11.56. Found: C, 66.36 H,
4.77; N, 11.78.12-Aminocamptothecin (26).
140
Chapter - IV
'HNMR (200MHz, DMSO-d6): 8 0.89 (t, 3H, J = 7.41Hz), 1.85 (q, 2H, J = 8.62Hz), 5.25 (s, 2H), 5.42 (s, 2H), 6.21 & 6.52 (s, - OH & NH), 6.93 (d, 1H, J = 7.45Hz), 7.15 (d, 1H, J = 7.82 Hz), 7.38 (t, 1H, J = 7.82Hz), 7.40 (s, 1H), 8.47 (s, 1H).
Mass (ESI-MS): 386.1 (M+Na).Anal. Calcd. for C2oHi7N304 : C, 66.11; H, 4.72; N, 11.56. Found: C, 66.36 H,
4.77; N, 11.78.4.2.3.3. Reaction of 25 & 26 with Succinic anhydride: Synthesis of 27 & 28.
Amino analogue 25 or 26 (36.3mg, O.lmmol) and succinic anhydride (12mg, 0.12
mmol) was refluxed in dry DCE (small amount of dry DMF to make a clear solution)
for 24 hrs. under nitrogen atmosphere. After completion of reaction (monitored by
TLC), solvent was evaporated and crude mass was subjected to column
chromatography (CHC13: MeOH; 95: 5 v/v) to give the corresponding succinyl
derivative 27 or 28 in 40-44 % yield.
9-Amino (4-oxobutanoic acid) camptothecin (27).
'HNMR (200MHz, DMSO-cfc): 8 0.92 (t, 3H, J = 7.08 Hz), 1.92 (q, 2H, J = 7.4 Hz), 2.66- 3.11 (m, 4H), 5.34 (s, 2H), 5.48 (s, 2H), 7.41 (s, 1H), 7.87 (m, 2H), 8.07 (d, 1H, J = 7.63 Hz), 8.85 (s, 1H), 10.28 (s, 1H).486.2 (M+Na).C, 62.20; H, 4.57; N, 9.07. Found: C, 62.47; H, 4.52; N, 9.18
12-Amino (4-oxobutanoic acid) camptothecin (28).
Mass (ESI-MS):Anal. Calcd. for C24H21N3O7 :
141
Chavter - IV
'HNMR (200MHz, DMSO-d6): 6 0.9 (t, 3H, J = 7.4 Hz), 1.89(q, 2H, J = 7.4 Hz),2.60 (t, 2H, J = 6.5 Hz), 2.93 (t, 2H, J = 6.5 Hz),5.30 (s, 2H), 5.43 (s, 2H), 7.64-7.79 (m, 3H), 8.7 (bs, 2H), 10.25 (s, 1H).486.2 (M+Na).C, 62.20; H, 4.57; N, 9.07. Found: C, 62.47; H, 4.52; N, 7.11
4.2.3.4. Reaction of 12-Amino (4-oxobutanoic acid) camptothecin and C4P-
Mass (ESI-MS):Anal. Calcd. for C24H21N3O7 :
aminopodophyllotoxin: synthesis of cross conjugate, 30.
Crude C4p- aminopodophyllotoxin; obtained by the Pd-C catalyzed hydrogenation of
of C4p- azidopodophyllotoxin (44 mg, 0.1 mmol) in ethylacetate at 1 atmosphere and
12-amino (4-oxobutanoic acid) camptothecin (22.3 mg, 0.05mmol) was dissolved in
dry DMF (5mL). To it was added DIC [2mol eq. with respect to 12-amino (4-
oxobutanoic acid) camptothecin] and catalytic amount of HOBT. The reaction
mixture was stirred for 48 hrs at r.t. The solvent was removed and the crude mixture
was subjected to column chromatography (CHCI3: MeOH) to give 8 mg of 30.
Solid; mp:'HNMR (200MHz, CDC13):
IR (KBr):
M ass (ESI-MS):Anal. Calcd for C4 6 H4 2 N 4 O 1 3 :
> 300 °C5 1.08 (t, 3H, J = 7.08 Hz), 1.88 (q, 2H, J = 7.4 Hz), 2.69-3.14 (m, 9H), 3.78 (s, 6H), 3.84 (s, 3H), 4.18 (m, 1H), 4.38 (m, 1H), 4.64 (m, 1H),5.30 and 5.34 (s x 2, 4H), 5.99 (d, 2H, J = 3.4 Hz), 6.33 (s, 2H), 6.56 (s, 1H), 6.85 (s, 1H), 7.47 (s, 1H), 7.68-7.79 (m, 3H), 8.51 (d, 1H, J = 7.02 Hz).3518.1, 1770.2, 1731.4, 1605.1, 1485.07,1231.9, 1115.4, 762.8 cm' 1 881 (M+Na).C, 64.33; H, 4.93; N, 6.52. Found. C, 64.77; H, 5.02; N, 6.71
142
Chanter - IV
SECTION C
Isolation and Characterization of Datura Lactones from D a tu ra
qu ercifo lia .
4.3.0. Introduction:
Datura lactones are withasteroids obtained from Datura species belonging to the
family of Solanaceae. These withasteroids are a group of naturally occurring C-28
steroidal lactones built on an intact or rearranged ergostane framework. These C-28
steroidal lactones characterized by a nine-carbon chain and a six membered ring
lactone were designated as ‘withanolides’ after the name of well-known Indian
medicinal plant, Withania somnifera 49 from which they were first isolated. The
withanolide skeletons may therefore be defined as 22-hydroxyergostane-26-oic acid
22,26-lactone (Figure 6).
Figure 6. Withanolide skelton
4.3.0.1. Features of withasteroids and their classification:>
Withasteroids are generally poly oxygenated and it is believed that plants elaborating
these compounds possess an enzyme system capable of oxidizing all carbon atoms in
a steroid nucleus. In fact, with the exception of C-8 and C-10, all the carbon atoms
including C-13 (by fission of 13, 14-bond) of the withasteroids have been found to
bear oxygen functions. This wealth of oxygen functions has lead to many
modifications of carbocyclic part as well as of the side chain. A C-9 side chain with a
lactone or lactol ring is the characteristic feature of withasteroids but the lactone ring
may be six -membered or five-membered and may be fused with the carbocyclic part
of the molecule through a carbon-carbon bond or through an oxygen bridge.
Appropriate oxygen substituent may lead to bond scission, formation of new bonds,
aromatization of rings and many other kinds of rearrangements resulting in
compounds with novel features. Thus, in spite of the common framework on all the
27
143
Chanter - IV
withanolides are built, the variety of structures exhibited by these steroidal lactones
necessitates their classification into nine groups. These are:
(I) Withanolides, (II) Withaphysalins, (II) Physalins, (IV) Nicandrenones or ring D
aromatic withanolides (V) Jaborols or ring A aromatic withanolide, (VI) Acnistins,
(VII) Ixocarpalactones, (VIII) Perulactones and (IX) Miscellaneous withsteroids.
VI VII VIII
Figure 7. Carbon-Oxygen framework of withanolides.
4.3.O.2. Biological activity of withanolides:
Steroids are found throughout the evolutionary hierarchy, from microbes to man, and
these are considered to act as architectural components of membrane apart from their
other biological functions. However while the specific role of the complex steroid
molecules with diverse structural features in the plants elaborating these compounds
is often not very clear. Many of these steroids have been proven to be very useful to
man because of their beneficial biological activities which include antistress,
anticonvulsant, CNS-depressant50 antitumor,51 cytotoxic,52 anti-inflammatory,53
144
Chapter - IV
antibacterial,54 hepatoprotective,55 sedative,56 cytostatic57 and immunomodulatory58
properties.
4.3.0.3. Distribution, morphological features and traditional use of Datura
species: Datura is a small genus of about twelve species, distributed throughout the
warm and temperate regions of the world. About ten species of Datura occur in India
and out of them Datura stramonium, D. metel, D. innoxia and D. fastuosa are very
common. The plants are coarse, rank scented, shrub-like herbs or small trees with
entire or coarsely sinuate-dentate leaves. Flower large, white or purple, solitary, calyx
long, tubular, herbaceous, 5-toothed, breaking transversely above the fruit. Corolla
tubular, funnel shaped. Stamens attached near the corolla-base, included. Capsule
ovoid or globose, usually spinous, 4-celled or 4-valved or irregularly breaking up near
the apex. Seeds compressed, rugose or dotted.
Datura has been known for the ages because of its narcotic and hypnotic effect. These
effects have been utilized in religious ceremonies, in oracular divination or in
forecasting of future events.59 In western South America, the herb was used by natives
to induce partial intoxication, to control unruly children and mixed with tobacco given
to women and slaves to deaden their senses before burial alive with their dead
husbands or masters.59 Aztoc Indians used the drug for the treatment of various types
of diseases including paralysis and priests used the plant in the form of drinks to
enable them to communicate with spirits. Some tribes used the plant in ceremonies in
initiating boys into manhood.
4.3.0.4. Chemical Profile of Datura quercifolia:
So far several withasteroidal compounds called datura lactones60 have been isolated
from this plant.
4.3.1. Present work:
Keeping in view the structural features of datura lactones and increase in demand of
withanolides because of their multifaceted pharmacological and medicinal
applications, we re-investigated the chemical constituents of Datura quercifolia plant
and evaluated the compounds for immunomodulatory activity. Immunomodulation
denotes to any change in the immune response and may involve, induction,
expression, amplification or inhibition of any part or phase response. Stimulation of
immune response is required in certain patients, whereas suppression of the immune
,
response is needed in other conditions.61 Novel immunomodulating agents are used
for the treatment of various conditions such as infections, organ transplantation,
cancer, rheumatoid arthritis etc.62'64 Natural products and their derivatives represent a
major breakthrough in these immunological disorders.65
The present work describes the isolation and characterization of a new compound, 31
along with two known compounds, 32 and 33 from Datura quercifolia plant and
determined their influence on various aspects of immune system like antibody
production, T-cell, B-cell activation and cytokine production from spleenocytes. From
our data, it was found that these compounds exerted dose dependent effect on humoral
and cell-mediated immune responses. 31 was found to be an immunosupressor at
lower doses while as 32 and 33 were immunostimulators. Compound 32 was found to
be the most promising immunostimulator in our study.
145
Figure 8i Structure of compound 31, 32 and 33.
4.3.1.1. Chemistry:
The leaves of Datura quercifolia were extracted with benzene and methanol. Column
chromatography of benzene extract yielded 32 and 33 while that of MeOH extract
yielded a mixture of 31 and 33 which was further separated by repeated column
chromatography to give pure 31 and 33. Among them, compound 31 was found to be
a new compound, while as 32 and 33 were reported earlier by our group from the
present institute. Methanol extract of the residue on chromatographic separation in
benzene: ethyl acetate (50: 50, v/v) yielded a colorless silky crystalline solid (31),
m.p 250-252 °C, [a]o +35.4 (CHCI3), formula C28H40O7 according to elemental
analysis and m/z 511.3 (M+Na). This molecular formula indicated nine degrees of
unsaturation; two olefin signals, one carbonyl group and two epoxides accounted for
146
Chapter - IV
four of these degrees and 31 therefore was pentacyclic. The IR spectra exhibited
bands at 1738.1 cm' 1 (six membered lactone ring) and 3508.4 cm' 1 (-OH group/s).
'HNMR (table III) showed characteristic proton signals for five methyl groups of the
withanolide nucleus at 8 1.50 (3H, s, 28- CH3), 1.53 (3H, s, 27-CH3), 1.02 (d, 3H, J =
4.59 Hz, 21- CH3), 0.77 (6H, s, 18 and 19 CH3). Signals at 8 5.74 (dd, 1H, J = 10.13,
2.77 Hz, H-2) and 5.91 (dq, 1H, J = 10.13, 4.23, 2.5 Hz, H-3) was due to olefinic
protons, signals at 8 2.94 (d, 1H, J = 4 Hz, H-6), 3.24 (dd, 1H, J = 4Hz, H-7) was the
protons in epoxy ring while as signals at 8 3.32 (bs, H-l) and 4.03 (bs, 1H, H-12) was
due to the protons attached to the carbon with equatorial and axial -OH groups
respectively. The lone signal at 8 4.58 (m, 1H, H-22) was due the resonance of 8-H of
the lactone ring. It was interesting to note that 19-methyl was located up field at 0.77
which has rarely been observed with other datura lactones or withanolides. This
observation clearly indicated the absence of carbonyl at C-l position. Acetylation
under mild condition (AC2O / pyridine at r.t) yielded a diacetae, 8 2.11 (s, 2 x
OCOCH3) indicated two -OH groups. Upon catalytic hydrogenation the compound
absorbed one mole of hydrogen to give a dihydro derivative, which was identified by
disappearance of two signals in 'HNMR at 8 5.74 and 5.91. ESI of the molecule
showed a peak at m/z at 511.3 (M +Na). Finally the structure and its stereochemistry
were determined by its chemical transformation into known datura lactones. Jone’s
oxidation of 31 at 0 °C yielded 33 while as prolonged oxidation at room temperature
yielded 32. It was interesting to note that 19-CH3 shifted downfield to 8 1.18 and 8
1.26 after oxidation to 32 and 33 respectively, indicating the presence of hydroxyl at
C-l. Since on controlled oxidation 31 gave 33 and 32 of known structures, it was
therefore clear that stereochemistry at C-5 and beyond is same as that in 33. The extra
hydroxyl is located at C-l and in view of chemical shift of C-l, H at 8 3.32 inspite of
being allylic to A2 bond, it was clear that C-l, H is axial (a) rather than equatorial
beyond any shadow of doubt and hence -OH group at C-l is equatorial with P-
configuration. From the above discussion, the structure of 31 was given as shown in
figure 8.
147
Chanter - IV
4.3.1.2. Biological activity:
In order to test the immunomodulatory effect of these compounds, we used many
assays to see the influence on T-cell, B-cell activation with reference to antibody titre,
DTH reaction and T-Cell subtypes, CD4 and CD8. Levamisole and Betamethasone
were used as standards in this study
Table III. ‘H and 13C NMR of 31 in CDC13
Position *H, 6 (J, Hz) I3C, 6
1 3.32 (bs) 70.99
2 5.74 (dd, 1H,J= 10.13, 2.77 Hz) 129.65
3 5.91 (dq, 1H, J = 10.13, 4.23, 2.5 Hz) 124.80
4 2.29 (bs, 2H) 28.24
5 - 72.48
6 2.94 (d, 1H, J = 4 Hz) 57.51
7 3.24 (dd, 1H, J = 4Hz) 57.07
8 1.2-2.33 (m) 35.48
9 do 39.36
10 - 47.34
11 1.2-2.33 (m) 28.48
12 4.05 (br. S, IH) 72.07
13 - 47.34
14 1.2-2.33 (m) 35.71
15 do 22.91
16 do 26.65
17 do 43.99
18 0.77 (3H,s) 12.43
19 0.77 (3H, s) 12.02
20 1.2-2.33 (m) 36.11
21 1.02 (d, 3H, J = 4.59 Hz) 17.94
22 4.58 (m, 1H) 76.69
23 1.2-2.33 (m) 35.48
24 - 62.74
25 - 59.27
26 - 170.15
27 1.50 (s, 3H) 13.66
28 1.53 (s, 3H) 14.66
148
Chapter - IV
4.3.1.3. Effect on antibody titre: The compounds were tested for possible role of B-
cell activation by determining IgM and IgG titre. Results presented in table IV
indicated that all the three compounds showed dose-related increase or decrease of
titre. Levamisole used as a standard drug showed 26.6% and 45.2% increase in
primary and secondary antibody synthesis respectively at a dose of 2.5 mg/kg.
Compound 31 showed maximum suppression at a dose of 0.001 mg/kg. 32 and 33
produced a dose related increase in the primary and secondary antibody synthesis.
Maximum effect of 32 was observed at lmg/kg (93.3 %) and O.Olmg/kg (93.5 %) for
primary and secondary titres while as for 33, the maximum effect was observed at
lOmg/kg, 72.7 % and 86.2 % respectively.
4.3.1.4. Delayed type hypersensitivity (DTH) response: The effect of 31, 32 and 33
on SRBC induced DTH reaction was assessed in mice following various doses. The
results are summarized in table V. The effect was compared to that of an equivalent
dose of betamethasone (BMS) as positive control. Out of the three compounds
evaluated, 32 induced significantly higher DTH response (69.03 %) at a dose of
0.lmg/kg p.o. The values are higher than that observed with BMS.
Since 32 was found to be the most promising stimulator against both humoral and
cell-mediated immune responses, it was further studied for the activation of spleen T-
cell subtypes, CD4 and CD8 using flowcytometer and selective release of cytokines,
IL-2 and TNFa by stimulated mouse spleen cells using ELISA.
4.3.1.5. Effect of 32 on Spleen T-Cell subtyping: Spleen single cell suspension (106
cell/ml) was studied for CD4+/CD8+ T-Cell subtypes by anti-CD4 and CD8
monoclonal antibodies conjugated with Flouresceine-isothio-cyanate (FITC) and
Phycoerthyrin (PE) using flowcytometer. By multiplying differential ratios of each
CD4 and CD8 subtypes to the total spleen cell contents, their total amounts in spleen
were calculated (table VI). Maximum effect of 32 was obtained at 0. lmg/kg p.o dose,
32.2 % CD4+ and 12.6 % CD 8+ T cells. The control values were 20.70 % of CD4+
and 13.3% of CD 8+ T cells. This shows a significant increase in CD4+ T cell count.
Levamisole, a standard T-cell stimulator at 2.5mg/kg oral dose stimulated both CD4+
and CD 8+T cells showed 30.8% of CD4+ and 18.3% of CD 8+T cells.
149
Chapter - IV
Table IV. Effect o f 31, 32 and 33 on SRBC-induced antibody synthesis in mice.
Compound Treatment Primary % Secondary %dose antibody (IgM) Change antibody (IgG) Change(mg/kg p.o) titre titre
Mean + S.E Mean ± S.EControl 6.0 ± 0.20 6.2 ± 0.21bSRBC31 0.001 4.4 ± 0.12a 26 .61 4.2 ± 0.15 32.3 131 0.01 5.0 ± 0.04a 16.6 -!• 5.0 + 0.16 19.3 431 0.1 5.4 ± 0.11 10.0 4 6.6 + 0.12b 06.4 t31 1 6.2 ± 0.24 3.30 t 6.8 ± 0.24 09.7 t32 0.001 9.6 ± 0.25a 60.0 t 10.6 ± 0.33a 70.9 t32 0.01 9.2 ± 0.34 53.3 t 12.0+ 0.31a 93.5 t32 0.1 10.2 ± 0.29 70.0 t 11.2+ 0.42 80.6 t32 1 11.6 ± 0.24b 93.3 t 9.6 + 0.35c 54.8 t32 3 9.5 ± 0.29b 58.3 t 7.0 + 0.25c 12.9 t32 10 5.4 ± 0.18b 10.0 4 6.4 ± 0.25 03.2 t33 0.1 6.5 ± 0.26 8.30 t 7.2 ± 0.26 16.1 t33 1 7.3 ± 0.24c 10.6 t 8.6 ± 0.31a 48.2 t33 3 9.4 ± 0.35 42.4 t 9.2 ± 0.35a 58.6 t33 10 11.4 ± 0.40b 72.7 t 10.8 ± 0.30 86.2 tLevamisole 2.5 7.6 ± 0.19 26.6 t 9.0 ± 0.21b 45.2 t
n = 6 ; Values are m ean ± S.E(a) P < 0.05; (b) P < 0.01; (c) P < 0.001; (4-) reduction; (t) stimulation
Table V. Effect o f 31, 32 and 33 on SRBC-induced delayed type hypersensitivity.
Compound Treatment DTH Response % Change compared todose betamethasone
(mg/kg p.o)Mean ± S.E
Control 1.76 + 0.0631 0.001 1.80 ±0 .04 16.13 t31 0.01 2.01 +0.03 29.67 t31 0.1 2.18 + 0.05a 40.64 t31 1 1.93 ± 0.05a 24.61 t32 0.001 2.04 + 0.09a 31.60 t32 0.01 2.23 + 0.02b 43.87 t32 0.1 2.62 + 0.03b 69.03 t32 1 2.36 + 0.12b 52.25 t32 3 2.08 + 0.10a 34.19 t32 10 1.86 ± 0.05b 20.00 t33 0.01 1.58 + 0.05b 1.93 T33 0.1 1.64 ± 0.10b 5.81 t33 1 1.86 ± 0.10b 20.00 t33 3 2.46 ± 0.14b 58.71 t33 10 2.80 ± 0.06a 80.64 tBetamethasone 0.01 1.55 + 0.04a
n = 6 ; (a) P < 0.05; (b) P < 0.01
150
Chapter - IV
Table VI. Effect o f different doses of 32 on spleen T cell subtypes
Dose of 2 (mg/kg) CD4+ T-Cell (%)
CD8+T-Cell(%)
CD4/CD8Ratio
Spleen CD4+ Content (x 107)
Spleen CD8+Content(xlO7)
Control (Vehicle) 20.7 ± 0 .90 13.3 ±0.34 1.56 ± 0 .09 2.70 ± 0 .12 1.47 ± 0 .04
Levamisole (2.5 mg/kg) 30.8 ± 1.30a 18.3 ± 0.53a 1.68 ± 0.06a 1.48 ± 0.10a 1.38 ± 0.10a
0.1 32.2 ± 0 .59 12.6 ±0.38 2.55 ± 0 .07 1.55 ± 0 .06 0.95 ± 0.03
1 27.2 ± 0 .59 13.6 ±0.38 2.00 ± 0.07 1.31 ± 0 .06 1.02 ±0.03
2.5 20.3 ± 0.63 15.4 ± 0 .69 1.32 ± 0 .07 1.02 ± 0 .07 0.86 ± 0 .04
3 19.7 ±0.63 13.6 ± 0 .69 1.44 ± 0 .06 0.90 ± 0 .02 1.02 ± 0 .07
10 15.1 ± 1.93b 25.8 ± 0.35b 0.58 ± 0 .09 0.72 ± 0.02 1.94 ± 0 .06
n = 6; (a) P < 0. 01; (b) P < 0.05
4.3.1.6. Effect of 32 on IL-2 and TNFa. The effect of 32 was tested on the release of
selected cytokines including IL-2 and TNFa by stimulated mouse spleen cells Results
shown in figure 9 indicated that compound 32 stimulated IL-2 and TNFa release in a
dose related manner. A dose as low as 0.01 mg/kg for TNFa and IL-2 production was
found to be the most effective.
■ I l l ID o s e ( m g / k g )
600
500400
300200
1000 U
J ■ T N F - a l ph a I
OS*'
D o s e ( m g I k g )
Figure 9. Effect o f 32 on IL-2 and TNFa on cytokine production. Each bar represents the mean value o f triplicate readings ± S.E. Mouse Spleen cells 2x l0 6 were stimulated with and without (control) 2|ig/ml o f Con-A in presence of 32 for 48h. Cell,supernatant was collected to see the effect o f 32 on production of IL-2 & TNF-a, measured by commercial kits (quantikine, R&D SYSTEMS.
151
Chanter - IV
The results obtained in the present study showed that 31, 32 and 33 displayed
immunosuppressive or immunopotentiating activity depending on the dosage
selection in relation to antigen. A number of assays were used to investigate the
immunomodulating effects. The effect of the compound 32 and 33 are clearly exerting
immunostimulatory activity on the immune system and could be suitable
immunomodulatory lead compounds for future research. The only difference between
32 and 33 is the dose concentration. 32 at lower doses (lmg/kg) stimulated antibody
titre in comparison to 33, which stimulated at higher doses (lOmg/kg). The possible
structural feature, which influences the activity, is the 12-carbonyl in 32. In contrast
31 has shown immunosuppressive effect on antibody titre. The activity differences
between these three compounds is clearly due to presence or absence of functional
groups ( >C=0 or -OH) at position 1 and 12 which possibly interact with the immune
competent T-helper cells. The results on cell mediated and humoral immune response
suggested that 32 is the most potent compound in comparison to 31 and 33.
152
Chapter - IV
4.3.2. Experimental:
4.3.2.1. Plant material: Leaves of Datura quercifolia were collected from the
experimental fields of RRL, Srinagar- India, in the month of July 2004.
4.3.2.2. Extraction and isolation:
Crushed and air dried leaves of D. quercifolia (3 kg) were extracted with cold
benzene. The extract was concentrated and allowed to stand at 0 °C for 30 hrs; when a
light green crystalline substance was obtained. This was purified by passing it through
a silica gel (60-120 mesh) column. Elution with CHCI3 : EtOAc (3:2, v/v) yielded 32
and 33. Their structures were confirmed by comparing their physicochemical data
with the existing literature. The marc of the crude leaves was then extracted with
MeOH. The extract was concentrated on rotatory evaporator and subjected to column
chromatography on silica gel (60-120 mesh). Elution with CHC13: EtOAc (3:2, v/v)
yielded a mixture of 31 and 33, which was rechromatographed to give pure 31 and 33.
Yield of compounds 31, 32, and 33 were found to be 0.001%, 0.34% and 0. 87 %
respectively.
Ip, 5a, 12a-trihydroxy-6a, 7a, 24a, 25a-diepoxy-20 S, 22 R with-2-enolide (31).
OH ' '0
'H and 13C NMR:Mass (ESI-Ms):Anal. Calcd. For C28H40O7 :
White amorphous solid; m.p: [a]D(CHCl3):IR (KBr):
250-252 °C;+35.4;3508.1, 1738.4, 1685.9, 1391.8, 1304.9, 1143.5, 905.6 cm’1;See table 1.
511.3 (M+Na).C, 68.83; H, 8.24. Found C, 69.08; H, 8.43.
153
Chapter - IV
5a, hydroxy-1,12-dioxo-6a, 7a: 24a, 25a-diepoxy-with-2-enolide (32).
White amorphous solid; m.p: ’HNMR (200 Hz, CDC13):
13,C NMR (50Hz, CDC13):
IR (KBr):Anal. Calcd. for C28H36O7:
303-305 C;8 0.90 (d, 3H, J = 7 Hz, 21- CH3), 1.10 (s, 3H, 18-CH3), 1.26 (s, 3H, 19-CH3), 1.52 and 1.58 (s, 3x2H, 27, 28-CH3), 1.4-2.7(m, CH’s and CH2’s), 3.08 (d, 1H, J = 4 Hz, 6-H), 3.42 (d, 1H, J = 4 Hz, 7-H), 4.55(m, 1H, 22-H), 5.85(d, 1H, J = 10 Hz, 2-H), 6.60 (dq, 1H, J = 10, 4.5, 3 Hz, 3-H).5 11.38, 13.06, 13.64, 14.71 17.94 23.58, 26.97, 28.98, 35.55, 36.64, 37.56, 38.29, 39.47, 42.51,51.44, 52.78, 56.14, 56.86, 57.65, 62.79, 73.18, 76.29, 128.79, 129.91, 169.93,201.36,212.18.3556.6, 1730.04, 1700, 1684.6 cm' 1 C, 69.42; H, 7.44. Found: C, 69.21; H, 7.24.
5a, 12a-dihydroxy-l-oxo-6a, 7a: 24a, 25a-diepoxy-with-2-enoIide (33).
'HNMR (200 Hz, CDC13):
13C NMR (50Hz, CDC13):
8 0.88 (s, 3H, 18-CH3), 1.02 (d, 3H, J = 5 Hz, 21- CH3), 1.18 (s, 3H, 19-CH3), 1.50 and 1.58 (s, 3x2H, 27, 28-CH3), 1.4 - 2.86 (m, CH’s and CH2’s).3.06 (d, 1H, J = 4.1 Hz, 6-H), 3.35 (d,1H, J = 4.1 Hz, 7-H), 4.00 (m, 1H, 12-H), 4.56 (m, 1H, 22-H), 5.81 (dq, 1H, J = 10, 3, 1 Hz, 2- H), 6.60 (dq, 1H, J = 10, 4.5, 3 Hz, 3-H).8 11.50, 12.35, 13.64, 14.70, 17.95, 22.99,26.45, 28.47, 28.64, 29.61, 35.96, 36.72, 38.70,
154
Chapter - IV
42.61, 43.52, 46.98, 50.67, 56.13, 56.85, 59.24, 62.87, 72.35, 73.41, 128.31, 128.76, 140.24, 170.25,203.80.
IR (KBr): 3539.6, 1728.04, 1694.6, c m 1;Anal. Calcd. for CsgFbgOy: C, 69.13; H, 7.82. Found: C, 68.87; H, 7.74.
4.3.2.3. Oxidation of 31 to 32 and 33: To an ice-cold solution of 31 (lOOmg) in dry
acetone (200ml) was added Jone’s reagent dropwise until a brown colour appeared.
The mixture was stirred at 0 °C until the reaction was complete (monitored by TLC).
It was then poured into ice-cold water and extracted with CHCI3. The CHCI3 extract
was washed with 35 % aq. NaHCC>3 and H2O. The extract was dried, evaporated and
crystallized from methanol to give a crystalline solid, which resembled the natural
compound 33 in all physicochemical properties. However when the oxidation was
done for 24 hrs with continuous stirring at r. t, 32 was formed as a major product.
4.3.2.4. Preparation of test material: For in vivo studies in Balb/mice, 31, 32 and 33
were suspended in 1% (w/v) gum acacia while as for in vitro studies a stock solution
of test materials in 10% DMSO was prepared. In the cell culture supernatant (< 0.1%)
did not interfere with the test system.
4.3.2.5. Serum SRBC antibody titre: Mice were immunized by injecting 20|uL of 5
x 109 SRBC/mL i.p on day 0, and the blood samples were collected on day +7 (before
challenge) for primary antibody titre and on day + 14 (7 days after challenge) for
secondary antibody titre. fraemagglutination antibody titres were determined
following the microtitration technique described by Nelson and Mildenhall 66
4.3.2.6. Delayed type hypersensitivity response (DTH): The method of Doherty67
was followed. Test material was administered 2h after SRBC injection and once daily
on consecutive days. Six days later, the thickness of the left hind foot was measured
with a spheromicrometer and was considered as control. The mice were then
challenged by injecting the same amount of SRBC intradermally into the left hind
footpad. The foot thickness was measured again after 24h.
4.3.2.7. Spleen T-Cell subtyping: T-Cell sub-typing was performed as described in
literature 68. Briefly, spleenocyte single cell suspension in RPMI-1640 (106 cell/ml)
was prepared and after counting viable cells by tryphan-blue dye exclusion method,
spleen cellularity was obtained. The CD4+/CD8' and CD4~/CD8+ T-Cell subtypes
^ ^ ^ ^hagter^B^
were measured using flowcytometer and mouse anti-CD4 and CD8 monoclonal
antibody conjugated with Flouresceine-isothiocyanate (FITC) and Phycoerythrin
(PE). By multiplying differential ratios of each CD4 and CD8 subtypes to the total
spleen cell contents, their total amounts in spleen were calculated.
4.3.2.8. Cytokine production from spleenocytes: Cytokines from mouse
spleenocytes were assayed using the cytokine kits (Quantikine R & D SYSTEMS).69
4.3.2.9. Statistical analysis: All the data are presented as mean ± S.E of the mean.
Statistical analysis for all the results was compared using student’s f-test.
155
156
Chanter - IV
4.3.3. References:
1. Podwyssotzki, V. Arch. Exp. Pathol. Pharmakol. 1880,13,29
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PUBLICATIONS
1. Ferrier Rearrangement for the synthesis o f PEG-bound 2,3-unsaturated glycopyranosyl-amino acids by Bilal A. Bhat, Syed Shafi, Basant Pumima, Abid Hussain Banday and H. M. Sampath Kumar. Tetrahedron Lett. 2007, 48, 1041.
2. A novel one-pot rearrangement reaction o f 2,3-epoxydiaryl ketones: Synthesis of (±)5,5-disubstituted-imidazolones and 5,5-disubstituted-hydantoins by Bilal A. Bhat, Kanaya L. Dhar, Satish C. Puri and Michael Spiteller. SynLett. 20 0 6 ,17, 2723.
3. Synthesis and biological evaluation of chalcones and their derived pyrazoles as potential cytotoxic agents by B. A. Bhat, K. L. Dhar, S. C. Puri, A. K. Saxena, M. Shanmugavel and G. N. Qazi. Biorg. Med. Chem. Lett. 20 0 5 ,15, 3177.
4. Isolation, characterization and biological evaluation o f datura lactones as potential immumnomodulators by B. A. Bhat, K. L. Dhar, S. C. Puri, M. A. Qurishi, A Khajuria, Amit Gupta and G. N. Qazi; Biorg. Med. Chem. 2 0 0 5 ,13, 6672.
5. Synthesis o f 3,5-Dipheny-l//-Pyrazoles by B. A. Bhat, S. C. Puri, M. A. Qurishi, K. L. Dhar, and G. N. Qazi; Synthetic Commun. 2005, 35 (8), 1135.
6. Synthesis and biological evaluation of ± 5,5-disubstituted-imidazolones and 5,5- disubstituted-hydantoins as anticancer agents by Bilal A. Bhat, K. L. Dhar, Satyam Agarwal, A. K. Saxena, M. A. Qurishi and G. N. Qazi. (Communicated).
7. Synthesis and biological evaluation of 4(3-[(4-substituted)-l,2,3-triazol-l-yl] podophyllotoxins as potential anticancer agents by Pitta Bhasker Reddy, Satyam Agarwal, A. K. Saxena, Bilal A. Bhat, H. M. Sampath Kumar and G. N. Qazi. (Communicated).
8. Studies on novel 4p-[(4-substituted)-1,2,3-triazol-1 -y 1] podophyllotoxins as potential anticancer agents by Bilal A. Bhat, Pitta Bhasker Reddy, Satyam Agarwal, A. K. Saxena, H. M. Sampath Kumar and G. N. Qazi. (Communicated).
9. Design and synthesis o f membrane permeable signal (MPS) peptide conjugates of anticancer compounds as resistance modifiers by H. M. Sampath Kumar, Bilal A. Bhat and Pitta Bhasker Reddy (Manuscript under preparation).
SYMPOSIA ATTENDED
Participated as a delegate in Indo-US Conference on New Bioactive Molecules
in Pharmaceutical Research-Contribution of Natural Products-2006 at
IICT Hyderabad.
Presented a poster in National Symposium on Magnetic Resonance and its
Applications- 2005 at RRL, Jammu.
Presented a poster in 1st J & K State Science Congress-2005 at University of
Jammu.
Presented a poster in International Conference on Chemistry Biology
Interface: Synergistic New Frontiers-2004 at New Delhi.
Presented a poster in IUPAC International Conference on Biodiversity and
Natural Products: Chemistry and Medical Applications-2004 at New Delhi.
AWARDS
Recipient of RRL best paper award in 2006 to the paper entitled “A novel
one-pot rearrangement reaction of 2,3-epoxydiaryl ketones: Synthesis of (±)
5,5-disubstituted-imidazolones and 5,5-disubstituted-hydantoins” by Bilal A.
Bhat, Kanaya L. Efhar, Satish C. Puri and Michael Spiteller; Syn. Lett. 2006,
77, 2723.
Recipient of RRL best paper award in 2005 to the paper entitled “Synthesis
and biological evaluation of chalcones and their derived pyrazoles as potential
cytotoxic agents” by B. A. Bhat, K. L. Dhar, S. C. Puri, A. K. Saxena, M.
Shanmugavel and G. N. Qazi; Biorg. Med. Chem. Lett. 2005, 15, 3177.