107

CHAPTER 3

SYNTHESIS OF 3-ADAMANTYL-5-BENZOYL-

TETRAHYDROPYRIMIDINE HYBRIDS*

Introduction

The chemistry of heterocyclic compounds has been an interesting field of study for long

time. In recent scenario heterocycles play a significant role in drug synthesis as well as in

drug discovery. The synthesis of novel tetrahydropyrimidine derivatives and investigation

of their chemical and biological behavior has gained more importance in recent decades for

biological and pharmaceutical reasons. From the literature survey report of Chapter 1, it

has been found that in modern years 1,2,3,4-tetrahydropyrimidines have engrossed

significant awareness because of their therapeutic and pharmacological properties more

than a few of them found to demonstrate a wide spectrum of biological properties

including antimicrobial, antitumor, antiviral, antihypertensive, alpha-1a adrenergic

antagonist, neuropeptide antagonist.

*Novel tetrahydropyrimidine-adamantane hybrids as anti-inflammatory agents: synthesis, structure and

biological evaluation, Utpalparna Kalita, Shunan Kaping, Revinus Nongkynrih, Laishram Indira Singha, Jai

Narain Vishwakarma, Med Chem Res, 2015, doi 10.1007/s00044-015-1332-x.

108

3.1 Synthesis of 1,2,3,4-Tetrahydropyrimidines

As part of our interest and keeping in view the immense biological properties of 1,2,3,4-

tetrahydropyrimidines, our research group have been involved in the development of newer

synthetic methodologies and evaluation of biological activities of this class of compounds.

3.1.1 A few years back researchers from our group in connection with the application of 3-

(aryl/benzyl)amino-3-methylthio-1-arylprop-2-en-1-one (1) and also for the construction of

tetrahydropyrimidines ring using formaldehyde and primary amines reported [48] the

synthesis of 1-aralkyl/aryl-3-alkyl/aralkyl/ary/-5-aroyl-6-methylthio-1,2,3,4-

tetrahydropyrimidines (Scheme 1).

3.1.2 Further, envisaging that the absence of thiomethyl group in position 6 of the

pyrimidine ring could alter the biological activities of the molecules, our group has also

reported [49] the synthesis of 1-(aralkyl/aryl)-3-(alkyl/aralkyl)-5-aroyl-1,2,3,4-

tetrahydropyrimidines (4) by dethiomethylation of 5-aroyl-6-methylthio-1,2,3,4-

tetrahydropyrimidines (3) and also to achieve the tetrahydropyrimidine (4) in single step,

an alternative synthetic strategy using enaminones of type 5, primary amine and

formaldehyde has been developed (Scheme 2).

109

3.1.3 Prompted by the absence of literature reports on bis-1,2,3,4-tetrahydropyrimidines

and their biological properties, a facile one-pot synthesis of novel bis-

tetrahydropyrimidines, bis-pyrazolotetrahydropyrimidines and bis-

pyrazolotetrahydrotriazines were reported [50,51] envisaging the fact that molecules with

two tetrahydropyrimidine rings linked through flexible aliphatic chains or through rigid

aromatic chains could have enhanced biological activities (Scheme 3 and Scheme 4).

110

3.2 Synthesis and biological importance of Adamantane derivatives

Further literature survey on bioactive heterocyclic systems, reveals that adamantane

derivatives have received considerable attention because of their diverse biological

activities. Synthesis and characterization of several heterocyclic systems containing

adamantane derivatives continue to be an interesting area of exploration because of their

exceptional qualities. The symmetrical fusion of the cyclohexyl unit in adamantane not

only results in virtually strain-free molecule [174], but this configuration also affords

uniquely beautiful and conceptually pleasing structural motif giving a rigid cage structure

which becomes an interesting building block in many fields [175] such as catalysis, supra-

molecular chemistry, material science and medicinal chemistry. Adamantane derivatives

have attracted a great deal of interest among researchers due to their wide applications in

the field of pharmaceuticals.

111

Adamantane 13 is the trivial name of the hydrocarbon tricyclo[3.3.1.1]decane.

Adamantane nucleus was first built up by Prelog and Seiwerth in 1941 via aluminum

chloride-catalyzed isomerization of tetrahydrodicyclopentadiene [176], this chemical

synthesis was then improved via catalytic hydrogenation of dicyclopentadiene in the

presence of aluminum chloride [177]. Adamantane is a highly lipophilic compound; it is

readily soluble in organic solvents and due to the high lipophilicity of adamantane, the

incorporation of the adamantyl moiety into several molecules results in compounds with

relatively high lipophilicity, which in turn can modify the biological availability of these

molecules [63]. After the discovery of amantadine in 1960 as antiviral and

antiparkinsonian drug, adamantine derivatives attracted the attention of several scientists as

potential chemotherapeutic agents. As a result of this intensive search, thousands of

adamantane derivatives were synthesized and tested for several biological activities. This

resulted in the discovery of several drugs which are now available in market. Adamantane

derivatives have long been known to possess enormous biological properties like anti-

inflammatory [178], analgesic [179], CNS activity [63], hypoglycemic [63], antagonists of

the N-methyl D-aspartate (NMDA) type of glutamate receptors which play an important

role in CNS function [180], trypanocidal activity [181], antibacterial property [182] and as

11β HSD1 inhibitors [183]. 2-(1-Adamantyl)-4H-3, 1-benzoxazin-4-one and 3,4-

dihydroquinazolin-4-one analogues were reported to have marked antitumour activity

[184,185]. N-Adamantylphthalimide showed a potent tumour necrosis factor (TNF-α)

production enhancing activity in a human leukemia HL-60 cell line [186]. An adamantane

derivative, 2,2-bis (4-(4-amino-3-hydroxyphenoxy) phenyl)adamantane (DPA), was found

to inhibit the growth of several cancer cell lines and the combination of DPA with a

chemotherapeutic agent, CPT-11 has significant synergistic effect against human colon

cancer [187]. In a study on a novel immune modulator, 2-(1-adamantylamino)-6-

methylpyridine (AdAMP) on normal and neoplastic human cells it was shown that in a

panel of several human ovarian cancer cell lines, almost half of them spontaneously

112

secreted significant amounts of TNF, a stimulator of cytokine secretion, when incubated

with AdAMP. The results suggest that AdAMP, as a stimulator of cytokine secretion, may

have potential application in tumor therapy [188]. Also, derivatives of adamantane have

long been known for their antiviral activity against the Influenza A [189,190] and HIV

virus [63,69,191]. Several adamantane derivatives were also associated with antimicrobial

properties [192,73-75].

The synthesis of a few such heterocycles is discussed in the following sections.

3.2.1 Z. Kazimierczuk and coworkers reported [193] the synthesis of a series of (1-

adamantyl) aminopyrimidine and -pyridine derivatives and the adamantylated compounds,

particularly 2-(1-adamantyl) amino-6-methylpyridine, were found to be potent TNF-α

inducers in murine melanoma cells transduced with gene for human TNF-α. The synthetic

route to (1-adamantyl)aminopyrimidines and -pyridines is based upon the reaction of

adamantyl cation formed from 1-adamantanol in refluxing trifluoroacetic acid.

Aminopyrimidines and -pyridines are usually protonated at their ring heteroatoms,

therefore, the acidic medium does not hinder the trapping of the adamantyl cation by the

exocyclic amino group (Scheme 5).

3.2.2 The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising

new therapy in the treatment of hypertension and inflammation. The problems of limited

water solubility and high melting points commonly displayed by the active 1,3-

disubstituted ureas prevent the further development of potent urea-based sEH inhibitors.

Therefore, a new class of potent inhibitors of sEH were designed and synthesized by the

113

introduction of a polar constrained piperazino group in the right side of adamantyl urea to

increase the water solubility. H. Y. Li et al. reported [194] a facile and general method to

prepare a series of 1-adamantan-1-yl-3-(2-piperazin-2-yl-ethyl)-ureas (23a-d) with various

5-substitutions on the 2-piperazino ring, which has advanced the SAR study by the

efficient making of structurally diverse analogs (Scheme 6). The effect of the 5-

substitution on the activity and the water solubility was examined. The best potency was

exhibited by the 5-benzyl-substituted-piperazine-containing urea (23a) with an IC50 value

of 1.37 µM against human sEH and good water solubility (S = 7.46 mg/mL) and low

melting point, in which the 5-substituted piperazine serves as a favorable secondary

pharmacophore and a water-solubility enhancing group.

3.2.3 G. Zoidis et al. synthesized [195] 1,2-annulated adamantine analogues such as

adamantanopyrrolidines 30, 31 and 32 (Scheme 7), adamantanopyrrolidines 33 and 34,

adamantanoxazolone 35, adamantanopyrazolone 36, adamantanopyrazolothione 37 and

adamantanocyclopentanamine 38 and tested for anti-influenza A virus and trypanocidal

activity.

114

The stereoelectronic requirements for optimal antiviral and trypanocidal potency were

investigated. Pyrrolidine 33 proved to be the most active of the compounds tested against

influenza A virus, being 4-fold more active than amantadine, equipotent to rimantadine and

19-fold more potent than ribavirin. Oxazolone 35 showed significant trypanocidal activity

against bloodstream forms of the African trypanosome, Trypanosoma brucei, being

approximately 3 times more potent than rimantadine and almost 50-fold more active than

amantadine.

115

3.2.4 A. Nefzi and co workers presented [196] parallel synthesis of chiral pentaamines and

pyrrolidine containing bis-heterocyclic libraries which were multiple scaffolds with

multiple building blocks and identified as new anti-tubercular compounds.

Chiral pentaamines and bis-heterocyclic compounds with 90–100% inhibition against

Mycobacterium tuberculosis strain H37Rv were identified. Some of the identified

compounds were more active than the existing drug ethanbutol. The four scaffolds (39-42)

bearing the same hydrophobic R groups showed good activities which strengthen the

suggestion that the observed activities were mainly due to the hydrophobicity of the

substituents. It is worth noting that the adamantly group is present in all templates, this

group exists in SQ109 [N-geranyl-N’-(2-adamantyl)ethane-1,2-diamine], a novel 1,2-

diamine-based EMB analog, which is in advanced clinical trials for the development of

new drug for the treatment of pulmonary tuberculosis (TB).

116

3.2.5 Yu et al. have reported [197] that the cellular activity of N370S and G202R GCase in

fibroblasts is increased by 2.5- and 7.2-fold with D-IFG-based pharmacological chaperones

N-adamantanyl-4-((3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)butanemi-

de (43) and N-adamantanyl-4-((3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-

yl)pentanamide (44) respectively, the best enhancements observed to date.

3.2.6 A series of 3-(1-adamantyl)-4-substituted-5-mercapto-1,2,4-triazoles of the general

structure 45, 46 and 47 were prepared [198] by A. A. El-Emam and T. M. Ibrahim and

tested for anti-inflammatory and analgesic activities. The derivatives 45 were found to be

the most potent among these derivatives. The activity of the derivatives 45 with a methyl,

ethyl or benzyl substituents was found comparable to the activity of Indomethacin. The

analgesic activity of these compounds correlated to their anti-inflammatory activity.

117

3.2.7 L. V. Anikina and coworkers reported [199] the synthesis of a series of 3-

spiro[adamantane-2,3′-isoquinolines] and evaluated their neurotropic activity and

cytotoxicity.

3.2.8 Shortly after the discovery of the selective 11β-HSD1 inhibitory activity of

adamantane derivatives, several structural modifications were carried out to improve the

selectivity, potency, and pharmacodynamic profiles. The most interesting of these

modifications is the introduction of an amide function into the adamantane structure

reported [78] by J. R. Patel.

118

Thus, the adamantane amide ether 51 was found to possess potent and selective inhibitory

activity against human and mouse 11β-HSD1. The highly active compound 51 was

modified [79] by V. S. C. Yeh and coworkers via replacement of the amide function with

carboxy alkyl group 52 or a heterocyclic nucleus 53. These structural modifications

resulted in marked increase in both the potency and 11β-HSD1 inhibitory selectivity.

Recently, novel related series of adamantane ethers of the structures 54, 55, 56 and 57 were

prepared [200] by J. R. Patel and coworkers. Although these compounds have lower

119

metabolic stability, their potency and selectivity to human and mouse 11 β -HSD1 is more

superior. Then, the highly potent, 11β-HSD1-selective inhibitor N-(2-adamantyl)acetamide

derivative 58, and its (±)-methyl derivative 59 were discovered by J. J. Rohde et al. [80].

The methyl analogue 59 has excellent potency, 11β-HSD1 selectivity and improved

microsomal stability in both in vitro studies on human and mouse 11 β -HSD1 and in vivo

studies on mouse and rats.

3.2.9 Antitumour activity was also reported [179] for some adamantane derivatives, of

these, the (S)-1-(3- and 4-pyridyl)ethyl adamantane-1-carboxylate 60 and 61, which were

characterized by potent inhibitory activity towards 17α-hydroxylase and C17,20-lyase

activities of human testicular cytochrome P45017α. In addition, these derivatives were

found to be resistant to degradation by esterases. The 2-(1-adamantyl)-4H-3,1-benzoxazin-

4-one 62, and the 3,4-dihydroquinazolin-4-one analogues 63, were found to display

marked antitumour and anti-HIV-1 activities [185].

3.3 Molecular hybrids of 1,2,3,4-Tetrahydropyrimidine

As shown in the introductory part, the adamantane nucleus was found to be a very

important pharmacophore in many therapeutic agents. The incorporation of an adamantyl

moiety into a pharmacologically-active molecule resulted in many cases in improving the

therapeutic profile of the parent drug. Since the discovery of amantadine in 1966 as the

first antiviral therapy for systemic use, several hundreds or even thousands of adamantyl

derivatives were synthesized and proved to be effective against several pathogenic

microorganisms and beneficial in improving various physiological disorders.

120

One of the most important concepts of drug design is the covalent conjugation of

biologically active moieties, acting by different mechanisms that would lead, in a favorable

case, to synergism that leads to compounds with an improved activity and reduced toxicity.

In the design of new drug prototypes, the concept of molecular hybridization is a useful

tool and is based on the combination of pharmacophoric moieties of different bioactive

substances to produce a new hybrid compound with improved affinity and efficacy. This

strategy has resulted in compounds with modified selectivity profile, different and/or dual

mode of action and reduced undesired side effects. Hybrid molecules that combine two

heterocyclic structural units of different nature generally lend themselves well to rational

drug design and often possess improved biological activities [201,202]. A number of

biologically potent hybrids have been described in the literature such as steroid antibiotics

[203], steroid nucleosides [204], triterpenoid peptides [205] and DNA cleaving-agent

amino acids [206]. However, to the best of our knowledge, 1,2,3,4-tetrahydropyrimidine

hybrids are unknown in the literature except for a few reports, which are described herein

and hence their synthetic potential and biological properties remain unexplored.

3.3.1 T. N. Akhaja and J. P. Raval designed [60] the synthesis and in vitro evaluation of

tetrahydropyrimidine–isatin hybrids as potential antitubercular and antimalarial agents. A

series of 5-substituted-3-[{5-(6-methyl-2-oxo/thioxo-4-phenyl-1,2,3,4-

tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-yl}-imino]-1,3-dihydro-2H-indol-2-one (67),

the hybrids of tetrahydropyrimidine–isatin were synthesized, characterized and screened

for their anti-tubercular and antimalarial activity (Scheme 8). 1,3-Dihydro-2H-indol-2-one

nucleus is used as a versatile lead molecule for designing potential antitubercular, antiviral,

anticonvulsant and anti-tumor agents. Similarly, imine bases of 1,3-dihydro-2H-indol-2-

ones were reported for various biological activities. It was found that the introduction of

electron-withdrawing groups at positions 5, 6, and 7 greatly increased activity from that of

1,3-dihydro-2H-indol-2-one, with substitution at the 5th

position being most favorable and

C-5 substitution has been associated with increased biological activity and, the presence of

substituted aromatic/heteoaromatic ring at 3rd

position has been reported to be associated

with antimicrobial properties.

121

Also 3-substituted indolin-2-ones have been identified as a versatile scaffold for the

development of protein kinase inhibitors which exhibit selectivity towards different

receptor tyrosine kinases (RTKs) by altering the substituents. It was reported that several

3-substituted indolin-2-one derivatives containing bulky groups in the phenyl ring at the C-

3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs. The

various substituents at 3rd

position of the indolin-2-ones were various substituted phenyl

ring, heterocyclic ring and aliphatic system. Some of the pyrimidinone-

oxadiazolylindolinones as promising antimicrobials together with antioxidant activities

were also reported. This observations led them to synthesis 5-substituted-3-[{5-(6-methyl-

2-oxo/thioxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-yl}imino]-1,3-

dihydro-2H-indol-2-ones, using 5-(5-amino-1,3,4-oxadiazol-2-yl)-6-methyl-4-phenyl-3,4-

dihydropyrimidin-2(H)-one/thiones and different 5-substituted indoline-2,3-dione. All the

newly synthesized compounds were evaluated in vitro to study its activity against

Mycobacterium tuberculosis H37 Rv and in vitro for antimalarial assay against

Plasmodium falciparum 3D7 chloroquine-sensitive strain. All the compounds displayed

moderate to good activity and the presence of nitro and presence of chloro displayed

excellent antimalarial potency. Overall, among the various substitution, the order of

highest antimalarial potency is NO2 ≥ F > Br ≥ H. It was well known from the literature

that the presence of these groups imparts a variety of properties including steric, electronic

properties, enhanced binding interactions, metabolic stability, changes in physical

122

properties and selective reactivities. In this study it was explained that this promising

antitubercular and antimalarial activity may be due to sufficient hydrogen bonding capacity

with desired lipophilicity or with favorable steric hindrance.

3.3.2 K. V. Sashidhara et al. reported [62] the discovery of coumarin-monastrol hybrid as

potential anti-breast tumor-specific agent. The application of potential coumarin

derivatives in the pharmacotherapy of breast cancer using the molecular hybridization

concept was reported in the literature. Also , it has been reported that the isolation of

neotanshinlactone, coumarin containing compound, showing significant inhibition activity

against two ER+ human breast cancer cell lines in comparison to TAM (i.e 10-fold more

potent and 20-fold more selective than TAM). On the other hand monastrol, a structurally

simple dihydropyrimidinone (DHPM), has been identified as a novel cell permeable

molecule that caused mitotic arrest by blocking bipolar mitotic spindle in mammalian cells.

It was the first mitotic kinesin Eg5 (also called kinesin-5 or kinesin spindle protein [KSP])

inhibitor (IC50=14μM) causing a specific and reversible cell cycle block. Moreover, it was

considered as a new lead in anticancer drug development as it specifically inhibits mitotic

kinesin Eg5 motor protein. Although, the antimitotic activity of monastrol was not very

high, the development of more potent, specific and cell permeable monastrol analogues

with enhanced kinesin inhibitory and anti-proliferative properties has been carried out with

encouraging results. The concept of molecular hybridization led them to discover a novel

class of coumarin-monastrol hybrid, as a novel breast cancer agent which selectively

induces apoptosis in both primary and metastatic breast cancer cell lines. The target

compounds were synthesized via the Biginelli reaction involving 3-aryl coumarin

aldehydes, ethyl acetoacetate and thiourea/urea in ethanol in the presence of catalytic

amount of pTSA to afford coumarin-monastrol hybrid compounds (Scheme 9).

123

Our literature survey shows that there are only two reports as mentioned in section 3.3.1

and 3.3.2 on the synthesis of molecular hybrids of 1,2,3,4-tetrahydropyrimidine.

124

3.4 Synthesis and biological properties of 1,2,3,4-tetrahydropyrimidine-adamantane

hybrids

Literature survey at this stage revealed that 1,2,3,4-tetrahydropyrimidine-adamantane

hybrids are unknown in the literature and hence their biological activities remain

unexplored. Prompted by the absence of literature reports on 1,2,3,4-tetrahydropyrimidine-

adamantane hybrids and taking into consideration the pharmacological activities shown by

various adamantane derivatives, we devised a facile one-pot synthesis of hitherto

unreported (3-((3s,5s,7s)-adamantan-1-yl)-1-alkyl/aralkyl/aryl-1,2,3,4-tetrahydropyrimid-

in-5-yl)(aryl)methanone and to evaluate their anti-inflammatory activity (Scheme 10).

Compd Ar R

73a C6H5 4-CH3C6H4

73b C6H5 C6H4

73c C6H5 4-CH3OC6H4

73d C6H5 4-BrC6H4

73e C6H5 CH2C6H5

73f C6H5 CH3

73g 4-ClC6H4 CH3

73h 4-ClC6H4 4-CH3C6H4

73i 4-CH3C6H4 CH3

73j 4-CH3C6H4 CH2C6H5

Scheme 10

125

3.5 Results and Discussions

Chemistry

In order to synthesize the desired tetrahydropyrimidine-adamantane hybrids, enaminones

of type 72 derived from acetophenone were required. Their synthesis is described in

chapter II (Scheme 51). Synthesis of the desired tetrahydropyrimidine-adamantane hybrids

was subsequently undertaken. Thus, when a mixture of enaminone 72a, adamantanamine

and formaldehyde (1:1:2) in methanol (4ml) was heated at reflux for 4 hours, work up of

the reaction mixture gave 73a in 96% yields, the structure of which was proposed to be (3-

((3s,5s,7s)-adamantan-1-yl)-1-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)(phenyl)methanone

on the basis of spectral and analytical data. The reaction conditions could easily be

extrapolated for the synthesis of 73b-j in 85-96 % overall yields in 2.5-6 hours (Table I).

Table I. Synthesis of (3-((3s,5s,7s)-adamantan-1-yl)-1-alkyl/aralkyl/aryl-1,2,3,4-

tetrahydropyrimidin-5-yl)(aryl)methanones 73 (a-j)

Compd Ar R Reaction

Time (hr)

Yield (%) m.p. (oC)

73a C6H5 4-CH3C6H4 4 96 143-144

73b C6H5 C6H5 2.5 88 132-133

73c C6H5 4-CH3OC6H4 4 85 105-106

73d C6H5 4-BrC6H4 5 86 163-165

73e C6H5 CH2C6H5 5 90 153

73f C6H5 CH3 5 92 125-126

73g 4-ClC6H4 CH3 3 94 163

73h 4-ClC6H4 4-CH3C6H4 6 90 155-156

73i 4-CH3C6H4 CH3 5 96 115

73j 4-CH3C6H4 CH2C6H5 6 91 131-132

The structures of the products were explicitly established with the help of spectral and

analytical data. The infrared spectra of 73a-j showed strong peaks in the region of 1500 to

1630 cm-1

due to extensively delocalised double bonds and carbonyl groups. The C-H

126

stretching due to adamantane gave characteristic bands close to 2900 and 2840 cm-1

. In the

1H NMR spectra of 73a-j, the signals due to aromatic protons appear between 6.80 and

7.56 ppm. The C6-H proton of the tetrahydropyrimidine ring resonated as singlet close to

6.99 ppm in 73b, 73e, 73f, 73g and 73i whereas it remains obscured by the aromatic

protons in 73a, 73c, 73d, 73h and 73j. The signals due to benzylic CH2 protons in 73e and

73j appeared as singlet at 4.30 and 4.28 ppm respectively. The CH2 protons at C-2 of the

tetrahydropyrimidine ring appeared between 3.94 and 4.65 ppm while those at C-4 gave

their signals in the range of 3.73-3.93 ppm. The three sets of protons of adamantyl group

resonated as three distinct multiplets in ranges 1.64-1.73, 1.84-1.92 and 2.14-2.18 ppm. In

the 13

C NMR spectra of the tetrahydropyrimidines, the most striking signal was due to

carbonyl carbon (close to 190 ppm) and those due to adamantyl group carbon atoms

appearing in the ranges of 29.4 -29.6, 36.0-36.1, 43.1-43.4 and 53.2-53.8 ppm. The

proposed cyclic structures for the tetrahydropyrimidines are further supported by the

absence of NH (~12.00 ppm) and vinylic C-H (~ 6.00 ppm) proton signals of the starting

enaminones in the spectra of 73a-j. The mass spectra of the products were in also

conformity with the proposed structures. The spectra of two candidates (73a and 73g) of

this series are presented in the following sections.

A plausible mechanism for the formation of these products has been rationalized herein.

127

X-ray crystallography

The proposed structure of (3-((3s,5s,7s)-adamantan-1-yl)-1-methyl-1,2,3,4-

tetrahydropyrimidin-5-yl)(4-chlorophenyl) methanone (73g) was unambiguously

confirmed by single crystal X-ray crystallography (Figure 1) with CCDC no. 976466. The

crystals of 73g suitable for X-ray analysis were obtained by crystallization from

ethylacetate. The crystal belongs to monoclinic, space group P2(1)/c with a = 14.1482 (4)

Å, b = 15.0308 (4) Å, c = 9.1382 (2) Å, β = 92.645 (1)°, V = 1941.25 (9) Å3 and Z = 4.

The molecular graphic was performed using ORTEP-3 and displacement ellipsoids are

drawn at 30% probability level.

Figure 1. Single Crystal X-ray Structure of 73g

128

IR spectrum of compound 73a

4000 3500 3000 2500 2000 1500 1000 500

20

40

60

80

100

T

cm-1

MASS spectrum of compound 73a

129

1H NMR spectrum of compound 73a

130

13C NMR spectrum of compound 73a

131

IR spectrum of compound 73g

MASS spectrum of compound 73g

132

1H NMR spectrum of 73g

133

13C NMR spectrum of 73g

134

Biology

Anti-inflammatory activity

1. Inhibition of FCA-induced paw edema

Compounds 73a-j were examined for their ability to reduce FCA-induced paw edema as

given in Table II. Except 73a, 73b and 73c, all the other compounds exhibited ability to

reduce paw thickness. Compounds 73d and 73g showed the highest reduction after 24

hours and are followed by 73i, 73e, 73h and 73j in decreasing order.

2. Nitric oxide level in the paw exudates

Treatment with all the compounds resulted in reduction of nitric oxide levels in paw

exudates of mice bearing paw edema. Measurements of NO in paw exudates of mice

showed the highest reduction in mice treated with 73b which is followed by 73a, 73g and

73i (Figure 2).

3. Level of Nitric oxide concentration in whole blood

Nitric oxide concentration in blood in response to the test compounds is given in Figure 3.

Except for 73b, 73d and 73g, all the other compounds showed reduction in NO levels.

Compounds 73h and 73j showed the highest reduction, followed by 73e.

4. Differential leukocyte count in blood

Anti-inflammatory activity of the ten compounds was also analyzed by performing a

differential leukocyte count in the blood smear as given in Figure 4. Most of the

compounds resulted in lower counts of both basophils and eosinophils or either basophils

or eosinophils as compared to control mice. Blood from mice treated with compounds 73e

and 73j resulted in the lowest levels of these cells.

135

Table II: Paw thickness of mice bearing FCA-induced paw edema followed by intraperitoneal

administration of 50mg/kg bw of test compounds 73a-j at different time intervals in comparison to

untreated control.

Treatment

group

Time Paw edema (mm) Increase in paw

thickness from 0hr

Percentage

Increase/decrease of

paw thickness

CONTROL 0hr 3.08 ±0.07 0 0.00

1hr 3.27 ±0.28 0.19 6.17

3hr 3.42 ±0.29 0.35 11.24

24hr 3.69 ±0.27 0.62 20.02

73a 0hr 3.83 ±0.29 0 0.00

1hr 3.83 ±0.29 0 0.00

3hr 3.07 ±0.12 -0.77 -19.98

24hr 3.83 ±0.29 0.00 0.00

73b 0hr 3.67 ±0.29 0 0.00

1hr 3.17 ±0.29 -0.5 -13.64

3hr 3.53 ±0.46 -0.13 -3.65

24hr 4.00 ±0.00 0.33 9.08

73c 0hr 3.87 ±0.06 0 0.00

1hr 3.77 ±0.25 -0.10 -2.59

3hr 3.57 ±0.12 -0.3 -7.76

24hr 3.87 ±0.12 0.00 0.00

73d 0hr 3.87 ±0.12 0 0.00

1hr 3.43 ±0.12 -0.43 -11.22

3hr 3.23 ±0.25 -0.63 -16.40

24hr 3.47 ±0.29 -0.40 -10.27

73e 0hr 3.77 ±0.25 0 0.00

1hr 3.47 ±0.29 -0.3 -7.96

3hr 3.13 ±0.12 -0.64 -16.98

24hr 3.53 ±0.25 -0.24 -6.37

73f 0hr 3.87 ±0.06 0 0.00

1hr 3.87 ±0.06 0 0.00

3hr 3.13 ±0.12 -0.73 -18.98

24hr 3.93 ±0.06 0.07 1.71

73g 0hr 3.90 ±0.10 0 0.00

1hr 3.50 ±0.00 -0.4 -10.26

3hr 3.13 ±0.12 -0.77 -19.67

24hr 3.33 ±0.29 -0.57 -14.54

73h 0hr 3.97 ±0.06 0 0.00

1hr 3.77 ±0.23 -0.20 -5.04

3hr 3.00 ±0.00 -0.97 -24.43

24hr 3.87 ±0.12 -0.10 -2.52

73i 0hr 3.70 ±0.17 0 0.00

1hr 3.77 ±0.25 0.07 1.81

3hr 3.17 ±0.29 -3.53 -14.41

24hr 3.43 ±0.12 -0.27 -7.22

73j 0hr 4.00 ±0.00 0 0.00

1hr 3.77 ±0.23 -0.23 -5.75

3hr 3.27 ±0.06 -0.73 -18.25

24hr 3.93 ±0.06 -0.07 -1.75

136

Figure 2: Concentration of the NO (in µM) in the paw exudates of different treatment groups.

Mice were injected with 50 µl of FCA in plantar side of hind paw followed by intraperitoneal

administration of test compounds 73a-j (50mg/kg bw) one hour later. Mice were sacrificed after

24hr following FCA injection, and the hind paw was excised and homogenized in 1ml normal

saline. NO level was measured by using the Griess reaction with standard nitrite reference curve.

Each group represents the mean ± S.E.M (n=3).

*P < 0.05 statistical significance compared to control (unpaired Student’s t-test).

Figure 3: Concentration of the NO in whole blood of the different treatment groups. Paw edema

was induced by injecting FCA in all groups of mice. Mice were then given intraperitoneal

injections of test compounds 73a-j (50mg/kg bw). Blood was collected by retro-orbital bleeding

and the whole blood was used to measure the level of NO by using the Griess reaction with

standard nitrite reference curve. Each group represents the mean ± S.E.M (n=3).

*P < 0.05 statistical significance compared to control (unpaired Student’s t-test).

±0.038

±0.007 ±0.028

±0.012 ±0.011

±0.193

±0.006 ±0.009 ±0.062

±0.082

±0.007

±0.092 ±0.029

±0.057 ±0.014

±0.234

±0.068

±0.036

±0.017

±0.041*

±0.034

±0.141*

137

Figure 4: Percentage counts of different types of leukocytes in mice treated with test

compounds in comparison to untreated control. Mice were sacrificed 24hr after FCA

injection. Blood smear was prepared and the slides were stained with Wright’s stain and

cells were counted under a microscope.

Paw thickness was taken as a physical parameter for this investigation. From the reduction

in paw thickness it is clear that 73d, 73g, 73i and 73e showed the highest ability to reduce

paw edema (Table 1). While compounds 73h, 73a, 73c, 73f and 73b showed lower

reduction in paw edema. The other inflammatory parameters used for the investigation

were NO level in paw exudates and whole blood. NO is an effective mediator of

inflammation in many tissues of mammals. High levels of NO indicate inflammatory

reaction and hence the test compounds with ability to bring down NO levels can be

considered as a potential anti-inflammatory agent. Measurements of NO in paw exudates

of mice showed the highest reduction in mice treated with 73b which is followed by 73a,

138

73g and 73i (Figure 2). Compounds 73c, 73d, 73e and 73f showed no reduction of NO

levels. Levels of NO in blood showed the highest reduction in mice treated with 73h, 73j

and 73e (Figure 3). Compounds 73b, 73d and 73g showed no reduction in NO levels.

Leukocytes like basophils and eosinophils indicate inflammation. Hence a reduction in

their counts in blood suggests a reduction in inflammation. Basophil and eosinophil counts

were lower in mice treated with 73e and 73j (Figure 4).

3.6 Conclusion

We have developed a facile one pot synthetic strategy for the synthesis of novel 1,2,3,4-

tetrahydropyrimidine-adamatane hybrids. This study will lead to the development of

simpler, newer environment friendly and versatile synthetic methodologies for novel

tetrahydropyrimidine-adamantane hybrids possessing anti-inflammatory properties and

find use of their application in pharmaceutical industries and will be of synthetic value in

the preparation of various drug and bio-active molecules.

3.7 Experimental

Melting points were recorded by open capillary method and are uncorrected. The IR

spectra were recorded on a Perkin-Elmer 983 spectrometer (Perkin-Elmer). 1H and

13C

NMR spectra were recorded on a JEOL JNM-ECS 400 taking Me4Si as the internal

standard in CDCl3. The X-ray diffraction data were collected at 296 K with Mo Kα

radiation (λ = 0.71073 Å) using a Bruker Nonius SMART APEX II CCD diffractometer

equipped with a graphite monochromator. The structures were solved by direct methods

(SHELXS97) and refined by full-matrix least-squares based on F square. All calculations

were carried out using WinGX system version 1.80.05. All the non-H atoms were refined

in the anisotropic approximation: H-atoms were located at calculated positions. The

electronspray mass spectra were recorded on a THERMO Finnigan LCQ Advantage max

ion trap mass spectrometer. Elemental analysis was performed on a Vario-EL III

instrument.

139

3.8 General Procedure

Synthesis of (3-((3s,5s,7s)-adamantan-1-yl)-1-alkyl/aralkyl/aryl-1,2,3,4-tetrahydropyr-

imidin-5-yl)(aryl)methanone (73a-j)

A mixture of adamantanamine (1mmol) and formaldehyde (2 mmol) in 1 ml of methanol

was stirred at room temperature for 5-10 minutes. To this was added a solution of the

enaminone (1 mmol) in 4ml of methanol and the resulting solution was refluxed for 2.5-6

hours. On completion of the reaction (monitored by TLC), methanol was distilled off to

give a gum. This gum, on trituration with hexane, gave a solid which was collected by

filtration. The practically pure product thus obtained in 85-96% overall yields were

practically pure and further purification was achieved by column chromatography (silica

gel, 20% EtOAc-Hexane).

Analytical data

(3-((3s,5s,7s)-adamantan-1-yl)-1-(p-tolyl)-1,2,3,4-tetrahydropyrimidin-5-yl)(phenyl)

methanone (73a)

Yellow solid, yield 96%; M.P: 143-144 °C; IR (KBr) ν:

2904, 2853, 1581, 1500, 1244, 1110, 704 cm-1

; 1H NMR

(400 MHz, CDCl3) δ: 1.58-1.67 (m, 6H, Ad), 1.80-1.81 (m,

6H, Ad), 2.09 (s, 3H, Ad), 2.30 (s, 3H, CH3), 3.93 (s, 2H,

CH2), 4.61 (s, 2H, CH2), 6.84 (d, 2H, J = 8.8, ArH), 7.12 (d,

2H, J = 8.8, ArH), 7.36-7.44 (m, 4H, 3H-ArH, 1H-C6H),

7.53-7.55 (m, 2H, ArH); 13

C NMR (400 MHz, CDCl3) δ:

20.7, 29.6, 36.5, 40.0, 41.2, 54.6, 62.2, 113.1, 118.1, 128.0, 128.3, 129.8, 130.1, 133.7,

140.0, 141.5, 146.9, 193.1; Mass [ESI] m/z (%) 413 [MH]+; Anal. Calcd. for C28H32N2O

(412.57): C, 81.51; H, 7.82; N, 6.79. Found: C, 81.55; H, 7.76; N, 6.79%.

140

(3-((3s,5s,7s)-adamantan-1-yl)-1-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)(phenyl)-

methanone (73b)

Yellow solid, yield 88%; M.P: 132-133 °C; IR (KBr) ν:

2900, 2846, 1598, 1482, 1251, 1002, 734 cm-1

; 1H NMR

(400 MHz, CDCl3) δ: 1.58-1.67 (m, 6H, Ad), 1.80-1.81 (m,

6H, Ad), 2.09 (s, 3H, Ad), 3.94 (s, 2H, CH2), 4.65 (s, 2H,

CH2), 6.94 (d, 2H, J = 8.8, ArH), 7.07-7.1 (t, 1H, ArH), 7.34

(d, 2H, J = 8.8, ArH), 7.39-7.43 (m, 3H, ArH), 7.48 (s, 1H,

C6H), 7.54-7.56 (m, 2H, ArH); 13

C NMR (400 MHz, CDCl3) δ: 29.5, 36.4, 40.0, 41.3, 54.7,

62.0, 113.7, 118.1, 123.8, 128.1, 128.3, 129.6, 130.0, 139.9, 143.8, 146.5, 193.5; Mass

[ESI] m/z (%) 399 [MH]+; Anal. Calcd. for C27H30N2O (398.54): C, 81.37; H, 7.59; N,

7.03. Found: C, 81.40; H, 7.53; N, 7.03%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-(4-methoxyphenyl)-1,2,3,4-tetrahydropyrimidin-5-yl)

(phenyl)methanone (73c)

Yellow solid, yield 85%; M.P: 105-106 °C; IR (KBr) ν:

2906, 1627, 1568, 1512, 1259, 1116, 826 cm-1

; 1

H NMR

(400 MHz, CDCl3) δ: 1.58-1.67 (m, 6H, Ad), 1.81-1.82 (m,

6H, Ad), 2.09 (s, 3H, Ad), 3.77 (s, 3H, OCH3), 3.93 (s, 2H,

CH2), 4.58 (s, 2H, CH2), 6.84-6.92 (m, 4H, 3H-ArH, 1H-

C6H), 7.36-7.42 (m, 4H, ArH), 7.52-7.54 (m, 2H, ArH); 13

C

NMR (400 MHz, CDCl3) δ: 29.6, 36.5, 40.0, 41.1, 54.6,

55.5, 62.7, 112.6, 114.7, 120.4, 128.0, 128.2, 129.8, 137.6, 140.1, 147.3, 156.5, 192.9,

Mass [ESI] m/z (%) 429 [MH]+; Anal. Calcd. for C28H32N2O2 (428.57): C, 78.47; H, 7.53;

N, 6.54. Found: C, 78.50; H, 7.47; N, 6.54%.

141

(3-((3s,5s,7s)-adamantan-1-yl)-1-(4-bromophenyl)-1,2,3,4-tetrahydropyrimidin-5 yl)

(phenyl)methanone (73d)

Yellow solid, 86%; M.P: 163-165 °C; IR (KBr) ν: 2908,

2847, 1636, 1493, 1248, 1149, 643 cm-1

; 1H NMR (400

MHz, CDCl3) δ: 1.60-1.64 (m, 6H, Ad), 1.78-1.79 (m, 6H,

Ad), 2.09 (s, 3H, Ad), 3.92 (s, 2H, CH2), 4.61 (s, 2H, CH2),

6.80 (d, 2H, J = 8.8, ArH), 7.41-7.43 (m, 6H, 5H-ArH, 1H-

C6H), 7.54 (d, 2H, J= 8.8, ArH); 13

C NMR (400 MHz,

CDCl3) δ: 29.5, 36.4, 40.0, 41.2, 54.7, 61.9, 114.5, 116.5,

119.4, 128.1, 128.3, 130.2, 132.5, 139.7, 142.8, 145.6, 193.3; Mass [ESI] m/z (%) 477

[MH]+; Anal. Calcd. for C27H29BrN2O (476.44): C, 67.92; H, 6.12; N, 5.87. Found: C,

68.06; H, 6.09; N, 5.88%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-benzyl-1,2,3,4-tetrahydropyrimidin-5-yl)(phenyl)-

methanone (73e)

Yellow solid, yield 90%; M.P: 153 °C; IR (KBr) ν: 2904,

2853, 1562, 1428, 1223, 1100, 926, 711 cm-1

; 1

H NMR (400

MHz, CDCl3) δ: 1.59-1.62 (m, 6H, Ad), 1.66-1.73 (m, 6H,

Ad), 2.06 (s, 3H, Ad), 3.78 (s, 2H, CH2), 3.99 (s, 2H, CH2),

4.30 (s, 2H, CH2), 7.17 (s, 1H, 1H-C6H), 7.22 (d, 2H, J=8.8,

ArH), 7.30-7.40 (m, 6H, ArH), 7.48-7.50 (m, 2H, ArH); 13

C

NMR (400 MHz, CDCl3) δ: 29.5, 36.5, 39.3, 40.7, 54.2, 58.0, 61.0, 109.4, 127.4, 127.9,

128.0, 128.1, 128.9, 129.4, 135.8, 140.1, 151.4, 192.0; Mass [ESI] m/z (%) 413 [MH]+;

Anal. Calcd. for C28H32N2O (412.57): C, 81.51; H, 7.82; N, 6.79. Found: C, 81.55; H,

7.76; N, 6.79%.

142

(3-((3s,5s,7s)-adamantan-1-yl)-1-methyl-1,2,3,4-tetrahydropyrimidin-5-yl)(phenyl)-

methanone (73f)

Yellow solid, yield 92%; M.P: 125-126 °C; IR (KBr) ν:

2899, 2839, 1626, 1557, 1286, 1119, 945, 783 cm-1

; 1H NMR

(400 MHz, CDCl3) δ: 1.62-1.71 (m, 6H, Ad), 1.83-1.84 (m,

6H, Ad), 2.13 (s, 3H, Ad), 2.92 (s, 3H, CH3), 3.76 (s, 2H,

CH2), 4.02 (s, 2H, CH2), 6.96 (s, 1H, 1H-C6H), 7.35-7.40 (m,

3H, ArH), 7.45-7.48 (m, 2H, ArH); 13

C NMR (400 MHz, CDCl3) δ: 29.6, 36.6, 39.4, 40.3,

40.5, 54.3, 62.5, 108.9, 127.9, 128.0, 129.3, 140.6, 151.7, 191.7; Mass [ESI] m/z (%) 337

[MH]+; Anal. Calcd. for C22H28N2O (336.47): C, 78.53; H, 8.39; N, 8.33. Found: C, 78.57;

H, 8.33; N, 8.33%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-methyl-1,2,3,4-tetrahydropyrimidin-5-yl)(4-

chlorophenyl)methanone (73g)

Yellow solid, yield 94%; M.P: 163 °C; IR (KBr) ν:

2914, 2862, 1633, 1541, 1377, 1100, 946, 823 cm-1

; 1H

NMR (400 MHz, CDCl3) δ: 1.61-1.71 (m, 6H, Ad),

1.80-1.83 (m, 6H, Ad), 2.13 (s, 3H, Ad), 2.94 (s, 3H,

CH3), 3.73 (s, 2H, CH2), 4.03 (s, 2H, CH2), 6.93 (s, 1H, 1H-C6H), 7.34 (d, 2H, J= 8.8,

ArH), 7.41 (d, 2H, J= 8.8, ArH); 13

C NMR (400 MHz, CDCl3) δ: 29.5, 36.5, 39.4, 40.2,

40.7, 54.3, 62.5, 108.8, 128.2, 129.5, 135.2, 138.9, 151.6, 190.2; Mass [ESI] m/z (%) 371

[MH]+; Anal. Calcd. for C22H27ClN2O (370.92): C, 71.24; H, 7.34; N, 7.55. Found: C,

71.35; H, 7.29; N, 7.56%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-(p-tolyl)-1,2,3,4-tetrahydropyrimidin-5-yl)(4-chloro-

phenyl)methanone (73h)

Yellow solid, yield 90%; M.P: 155-156 °C; IR (KBr) ν:

2904, 2832, 1572, 1521, 1254, 1110, 814 cm-1

; 1H NMR

(400 MHz, CDCl3) δ: 1.58-1.71 (m, 6H, Ad), 1.79-1.80

(m, 6H, Ad), 2.09 (s, 3H, Ad), 2.31 (s, 3H, CH3), 3.91 (s,

2H, CH2), 4.62 (s, 2H, CH2), 6.84 (d, 2H, J= 8.8, ArH),

143

7.12 (d, 2H, J= 8.8, ArH), 7.35-7.39 (m, 3H, 2H-ArH, 1H-C6-H), 7.48 (d, 2H, J= 8.8,

ArH); 13

C NMR (400 MHz, CDCl3) δ: 20.7, 29.6, 36.5, 40.1, 41.2, 54.7, 62.3, 113.0, 118.5,

128.3, 129.7, 130.2, 134.0, 135.9, 138.4, 141.5, 146.8, 191.5; Mass [ESI] m/z (%) 447

[MH]+; Anal. Calcd. for C28H31ClN2O (446.01): C, 75.23; H, 6.99; N, 6.27. Found: C,

75.33; H, 6.95; N, 6.27%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-methyl-1,2,3,4-tetrahydropyrimidin-5-yl)(p-tolyl)me-

thanone (73i)

Yellow solid, yield 96%; M.P: 115 °C; IR (KBr) ν: 2908,

2847, 1633, 1550, 1387, 1120, 947, 745 cm-1

; 1H NMR

(400 MHz, CDCl3) δ: 1.61-1.70 (m, 6H, Ad), 1.83-1.84

(m, 6H, Ad), 2.12 (s, 3H, Ad), 2.37 (s, 3H, CH3), 2.92 (s,

3H, CH3), 3.75 (s, 2H, CH2), 4.01 (s, 2H, CH2), 6.99 (s,

1H, 1H-C6H), 7.17 (d, 2H, J= 8.8, ArH), 7.38 (d, 2H, J= 8.8, ArH); 13

C NMR (400 MHz,

CDCl3) δ: 21.3, 29.6, 36.6, 39.4, 40.3, 40.5, 54.3, 62.5, 109.0, 128.2, 128.6, 137.8, 139.4,

151.4, 191.7; Mass [ESI] m/z (%) 351 [MH]+; Anal. Calcd. for C23H30N2O (350.50): C,

78.82; H, 8.63; N, 7.99. Found: C, 78.85; H, 8.57; N, 8.00%.

(3-((3s,5s,7s)-adamantan-1-yl)-1-benzyl-1,2,3,4-tetrahydropyrimidin-5-yl)(p-

tolyl)methanone (73j)

White solid, yield 91%; M.P: 131-132 °C; IR (KBr) ν:

2904, 2832, 1561, 1449, 1202, 1110, 926, 731 cm-1

; 1H

NMR (400 MHz, CDCl3) δ: 1.58-1.62 (m, 6H, Ad), 1.65-

1.73 (m, 6H, Ad), 2.05 (s, 3H, Ad), 2.37 (s, 2H, CH3),

3.77 (s, 2H, CH2), 3.94 (s, 2H, CH2), 4. 28 (s, 2H, CH2),

7.18-7.19 (m, 5H, ArH), 7.39-7.41 (m, 5H, 4H-ArH, 1H-

C6H); 13

C NMR (400 MHz, CDCl3) δ: 21.3, 29.6, 36.5, 39.4, 40.8, 54.2, 58.0, 61.0, 109.6,

127.4, 128.6, 128.8, 136.0, 139.5, 138.0, 151.1, 192.0; Mass [ESI] m/z (%) 427 [MH]+;

Anal. Calcd. for C29H34N2O (426.59): C, 81.65; H, 8.03; N, 6.57. Found: C, 81.69; H, 7.98;

N, 6.57%.

144

Biology

Anti-inflammatory activity

Materials

Griess reagent system Promega (USA), Wright stain, anhydrous potassium dihydrogen

phosphate, potassium chloride, disodium hydrogen phosphate, sodium-EDTA and

dimethyl sulfoxide (DMSO) were purchased from Hi-Media. Methanol was purchased

from Merck and Fruend’s complete Adjuvant (FCA) from Genei.

Methodology

Anti-inflammatory activity of the test compounds was analyzed by measuring paw

diameter, NO assay in blood and in paw exudates and by performing a differential WBC

count in mice carrying FCA-induced paw edema and subsequently treated with test

compounds.

1. Induction of paw edema

The anti-inflammatory activity of the test compounds was determined using S.C. Lai et al.

[169] method with little modification. Swiss Albino mice aged between 8-10 weeks of

either sex (3 per group) maintained at controlled temperature with 12 hours light/12 hours

dark conditions, provided with standard mice feed and common tap drinking water were

used in all experiments. About 50µl of the Fruend’s complete adjuvant (FCA) was injected

into the plantar side of left hind paws of the mice [170].

Paw diameter of the FCA induced edema of mice was measured at 0-, 1-, 3-, and 24-hours

after the administration of the FCA by using a caliper. Test compound (dissolved in 10%

DMSO) was administered 1hr after FCA injection. The percentage increase/decrease of the

paw edema is calculated by the formula where ‘a’ is the paw diameter at 0hr and

‘b’ is the paw diameter at different time interval.

After 24hr, blood was collected by retro-orbital bleeding and used for estimating NO and

the mice were sacrificed by cervical dislocation. The left hind paw tissue was rinsed in ice-

cold normal saline, and immediately placed in 1ml of cold normal saline and homogenized.

145

Then the homogenate was centrifuged at 12,000 rpm for 5min. The supernatant was used

for NO assay.

2. NO assay

NO2-

was measured by using the Griess reaction. The assay of NO2-

was performed

according to the manufacturer’s instruction. Three columns in the 96 wells plate were

designated for the nitrite standard reference curve. Six serial twofold dilution of 100 µM

nitrite solution (50µl/well) in triplicate was performed to generate the nitrite standard

reference curve.

50µl of experimental sample was taken in triplicate in test wells. To all wells 50µl of

sulphanilamide solution was added and incubated for 5-10 min at room temperature

protected from light. Thereafter, 50µl of the N-1 napthylethylenediamine dihydrochloride

(NED) solution was dispensed to all wells. The plate was incubated at room temperature

for 5-10min, protected from light. A purple/magenta colour begins to form immediately.

The absorbance was measured within 30min in a plate reader with a filter between 520nm

and 550nm.

The concentration of NO in experimental samples was calculated from the standard curve

obtained from above.

3. Differential WBC count

The differential WBC count was performed according to the method described by B.

Houwen [171]. Blood film was prepared on glass slides by wedge method and air dried.

The blood films were fixed for 30 seconds in absolute methanol. Slides were stained for 2

mins with Wright’s stain and an aliquot of Sorensen’s buffer was added and mixed and

allowed to stand for 3mins. Slides were rinsed with distilled water and air dried. The

prepared slides were observed under the microscope and WBCs were count.