Design, synthesis, physicochemical, and pharmacological evaluation of gallic acid esters as...

ORIGINAL RESEARCH

Design, synthesis, physicochemical, and pharmacologicalevaluation of gallic acid esters as non-ulcerogenicand gastroprotective anti-inflammatory agents

Mamta Sachdeva Dhingra • Sameer Dhingra •

Renu Chadha • Tejvir Singh • Maninder Karan

Received: 22 October 2013 / Accepted: 16 May 2014

� Springer Science+Business Media New York 2014

Abstract In our effort to identify the effective gastric

sparing and protective anti-inflammatory agents, a series of

gallic acid esters were synthesized, characterized, and

studied to assess their physicochemical properties. Subse-

quently, the esters were evaluated for their anti-inflam-

matory activity and effect on gastric mucosa by most active

compounds. All the compounds exhibited promising anti-

inflammatory activity in carrageenan-induced rat paw

edema model. In particular, 7a, 7c, 7f, and 7h emerged as

the most active compounds in the series. The findings of

gastric ulcer and antioxidant studies suggested these com-

pounds as non-ulcerogenic and gastroprotective. Further,

the predicted ADME properties of all the tested compounds

were found to be in the ranges as predicted by QikProp for

95 % of known oral drugs and also satisfy the Lipinski’s

rule of five.

Keywords Gallic acid esters � Physicochemical �Enzymatic hydrolysis on human plasma �ADME profiling � Anti-inflammatory activity �Carrageenan-induced paw edema � Gastroprotective �Ulcerogenic activity

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the

most common therapeutic class of drugs used in medical

practice for the treatment of pain, fever, and inflammation.

However, usefulness of this class of drugs is limited due to

higher incidence of gastrointestinal side effects such as

gastric ulceration, perforation, bleeding, and other associ-

ated complications including cardiovascular, hepatic, and

renal toxicities (Laporte et al., 2004; Wolfe et al., 1999;

Henry and McGettigan, 2003; Boelsterli, 2002; Dogne et al.,

2005). These observations indicated that safety of such

agents is questionable on their long-term use and some of

these agents have already been withdrawn from the market

(Schnitzer, 2001). Thus, the need for the design and devel-

opment of safer anti-inflammatory agents still remains.

During recent years, it has been well known that

excessive free radical generation takes place in inflamma-

tory disorders and these observations indicate that antiox-

idants may be used to prevent free radicals-induced

inflammation (Bandyopadhyay et al., 1999). During the

past few decades, a large number of naturally occurring

phenolic compounds have been identified as antioxidants,

which are viewed as promising therapeutic agents for the

treatment of these free radical-mediated inflammatory

diseases. Several reviews have addressed the anti-inflam-

matory activity of phenols, attributing their property not

only to the antioxidant capacity, but also to inflammatory

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00044-014-1041-x) contains supplementarymaterial, which is available to authorized users.

M. S. Dhingra � R. Chadha � M. Karan (&)

Department of Pharmacognosy and Natural Products, University

Institute of Pharmaceutical Sciences, UGC Center of Advanced

Study (UGC-CAS) in Pharmaceutical Sciences, Panjab

University, Chandigarh 160 014, India

e-mail: [email protected]

S. Dhingra

School of Pharmacy, Faculty of Medical Sciences, The

University of the West Indies, St. Augustine, Eric Williams

Medical Sciences Complex, Uriah Butler Highway, Champ

Fleurs, Trinidad and Tobago

T. Singh

Department of Chemistry, UGC Center of Advanced Study

(UGC-CAS) in Chemistry, Panjab University,

Chandigarh 160 014, India

123

Med Chem Res

DOI 10.1007/s00044-014-1041-x

MEDICINALCHEMISTRYRESEARCH

http://dx.doi.org/10.1007/s00044-014-1041-x

mediators’ modulation, namely cytokines and proinflam-

matory enzymes, such as inducible nitric oxide synthase

and cyclooxygenase (Costa et al., 2012).

Gallic acid (GA, 3,4,5-trihydroxybenzoic acid) and its

derivatives are widely distributed in the plant kingdom and

represent a large family of plant secondary polyphenolic

metabolites. They are present in the form of either meth-

ylated GAs (syringic acid) or galloyl conjugates of catechin

derivatives, i.e., flavan-3-ols, or polygalloyl esters of glucose,

quinic acid, or glycerol (Lu et al., 2006). GA and its deriv-

atives have been widely reported for various biological and

pharmacological activities including anti-inflammatory

activity (Dhingra et al., 2014; Lu et al., 2006; Kroes et al.,

1992). These activities are possibly linked with their antiox-

idant potential due to their ability to prevent damage from

free radicals or to prevent generation of these free radicals

(Perron and Brumaghim, 2009).

Based on these observations and the previous studies

reported from our laboratory (Dhingra et al., 2013; Sawraj

et al., 2012; Madhukar et al., 2010), it is recommended that

there are potential advantages in coadministering these

naturally occurring phenolic/alcoholic compounds together

in the form of mutual prodrugs/codrugs with improved

physicochemical properties such as bioavailability and

release the parent drug at the site of action (Singh and

Sharma, 1994; Bhosle et al., 2006; Leppanen et al., 2002).

Hence, our intention was to synthesize GA esters by con-

jugating GA with naturally occurring phenolic/alcoholic

antioxidant compounds such as eugenol, guaiacol, thymol,

menthol, vanillin, isovanillin, sesamol, and umbelliferone

which can act synergistically with enhanced therapeutic

outcomes as non-ulcerogenic and gastroprotective anti-

inflammatory agents. In silico ADME profiling was carried

out to assess the bioavailability of synthesized compounds.

Results and discussion

Chemistry

As illustrated in Scheme 1, GA (1) was converted to

methyl ester (2), and the phenol groups were protected by

benzylation (3), followed by hydrolysis of the ester group

to afford protected GA (4). This compound (4) was con-

verted into acid chloride (5) in the presence of oxalyl

chloride. The compound (5) was treated with potassium

salts of different naturally occurring antioxidant com-

pounds (1a–1j, Fig. 1) to afford protected GA ester com-

pounds (6a–6j), which were deprotected by debenzylation

using H2/Pd–C to obtain gallates (7a–7j). The structures of

the synthesized compounds were characterized using

spectroscopic techniques such as UV, IR, 1H NMR, 13C

NMR, MS, and elemental analysis. Characteristic spectral

data of synthesized GA esters is shown in Table 1. Com-

pounds 7a, 7b, 7c, 7e, 7f, 7h, and 7j are reported as pat-

ented molecules in the literature (Ihrman and Malec, 1974;

Sakaguchi et al., 1999; Ghisalberti, 2008), but none of

these compounds have been studied before for medicinal

use.

Solubility and partition coefficient

Lipophilicity is an important factor controlling the inter-

action of drugs with biological membranes. It is generally

accepted that good absorption of an orally administered

drug could be obtained when apparent partition coefficient

value (log P) is more than 2 (Yalkowsky and Morozowich,

1980). The evaluation of log P of newly synthesized esters

(7a–7j) was performed by saturation shake flask method

(Hairsine, 1989; Yalkowsky et al., 1992; Streng, 2001).

The standard plots were constructed using UV spectro-

photometer and quantification was carried out accordingly.

To assess the lipophilicity, the log P of GA and its esters

was determined in n-octanol and buffer layers (pH 7.4) and

calculated by correlating the absorbance with the concen-

tration in standard plot. The obtained values of log P and

solubilities at pH 7.4 are shown in Table 2, indicating that

the synthesized derivatives meet the requirement for gas-

trointestinal absorption.

Chemical stability: in aqueous buffer solutions

Prodrugs or ester derivatives should be chemically stable

so that they can be formulated in an appropriate pharma-

ceutical dosage form with optimum half life (Wadhwa and

Sharma, 1995). At the same time, it should be biolabile to

regenerate the parent drug molecule(s) to exhibit thera-

peutic activity. Therefore, the esters (7a–7j) were assayed

in vitro to evaluate their chemical stability. The kinetics of

chemical hydrolysis was studied at 37 �C using buffer

solutions of pH 1.2 and 7.4 by HPLC. The reactivity to

chemical hydrolysis was evaluated by pseudo-first-order

rate constants obtained from slopes of semi-logarithmic

plots of six concentrations against time. The half life (t1/2)

and rate constants (k) are shown in Table 2, indicating the

stability in buffer solutions. The synthesized derivatives

showed considerable chemical stability at pH 1.2 which

implies that these passed unhydrolyzed through the stom-

ach after oral administration. At pH 7.4, the compounds

showed enough stability to be absorbed intact from the

intestine.

Enzymatic hydrolysis (in human plasma)

In 80 % human plasma (human plasma containing 20 %,

0.02 m phosphate buffer, pH 7.4), ester bonds of GA

Med Chem Res

123

derivatives (7a–7j) were found to be cleaved, forming the

parent moieties GA (1) and phytophenols/alcohol (1a–1j).

The degradation process was found to correlate with

pseudo-first-order kinetics for several half lives. The rate of

hydrolysis of ester derivatives (Table 2) indicated that

these compounds are readily hydrolysed in plasma to

OHHO OH

O OH

OHHO OH

O OCH3

OCH2C6H5

C6H5H2CO OCH2C6H5

O OCH3

OCH2C6H5

C6H5H2CO OCH2C6H5

O OH

OCH2C6H5

C6H5H2CO OCH2C6H5

O Cl

OCH2C6H5

C6H5H2CO OCH2C6H5

O OAr/R

OHHO OH

O OAr/R

(i) (ii)

(iii)

(iv)

(v)

(vi)

)3()2(

(4)(5)

(6a-6j) (7a-7j)

(1)

Scheme 1 Steps involved in

the synthesis of gallic acid

esters (7a–7j)

HO OH

HO

HOOCH3

HOOCH3

HO

CHO

OCH3

OH

CHO

OCH3

OH HO

O

O

OHO O

(1b) (1c) (1d)

(1e) (1f) (1g) (1h)

(1i) (1j)

HOOH

(1a)

Fig. 1 Structures of naturally

occurring antioxidants;

2-hydroxyphenol (catechol)

(1a), 3-hydroxyphenol

(resorcinol) (1b),

2-methoxyphenol (guaiacol)

(1c), 4-allyl-2-methoxyphenol

(eugenol) (1d), 5-methyl-2-

isopropylphenol (thymol) (1e),

4-hydroxy-3-

methoxybenzaldehyde (vanillin)

(1f), 3-hydroxy-4-

methoxybenzaldehyde

(isovanillin) (1g),

benzo[d][1,3]dioxol-5-ol

(sesamol) (1h), 7-hydroxy-2H-

chromen-2-one (umbelliferone)

(1i), and (1a,2b,5a)-5-methyl-2-

isopropylcyclohexanol

(menthol) (1j)

Med Chem Res

123

Table 1 Characteristic spectral features of test compounds

Ar =

OHOHHO

Compounds Structure IR (t, cm-1)

(C=O)

13C NMR

(carbonyl carbon,

d, ppm)

Mass ESI-MS (m/z)

7a

OHO Ar

O 1,711 – 261 (M?-1)

7b

O Ar

O

HO

1,715 – 261.1 (M?-1)

7c

OCH3

O Ar

O 1,711 164 277.1 (M??1),

299.1 (M??Na)

7d

O Ar

O

OCH3

1,706 165 341.1 (M??Na)

7e

O Ar

O

1,713 166 325.1 (M??Na),

303.1 (M??1)

7f

O A

O

OCH3

HOH2C 1,717 164 305.1 (M?-1)

Med Chem Res

123

Table 1 continued

Compounds Structure IR (t, cm-1)

(C=O)

13C NMR

(carbonyl carbon,

d, ppm)

Mass ESI-MS (m/z)

7g

O Ar

O

OCH3

CH2OH 1,712 164 329.1 (M??Na,

base peak)

7h O

O O Ar

O1,706 164 289 (M?-1)

7i

OO O Ar

O 1,705 165 337.1 (M??Na)

7j

O Ar

O

1,709 – 331.1 (M??Na)

Table 2 Evaluation of physicochemical properties of test compounds (7a–7j)

Compounds Partition

coefficient

(log P)

Solubility

(lg/mL)

kmax

(nm)

Extinction

coefficient

(E1cm1% )

pH 7.4 pH 1.2 Human plasma (80 %)

k (h-1) t1/2 (h) k (h-1) t1/2 (h) k (h-1) t1/2 (min)

GA (1) 0.53 18,500 258 2,810 – – – – – –

7a 1.98 510 275 260 0.002 347.647 0.041 79.413 0.0037 187.29

7b 2 508 269 210 0.0016 437.144 0.006 125.534 0.0038 182.36

7c 2.14 420 275 290 0.0018 368.01 0.003 262.121 0.0036 192.5

7d 3.21 384 278 300 0.0016 389.172 0.008 98.135 0.0031 223.55

7e 3.79 313 278 270 0.0015 408.126 0.004 231.329 0.0042 165

7f 2.03 590 276 250 0.0023 279.853 0.011 64.230 0.0046 150.65

7g 2.01 535 277 260 0.017 41.257 0.044 83.634 0.0041 169.02

7h 2.06 560 267 220 0.0021 312.182 0.012 63.241 0.0035 198

7i 2.16 481 282 180 0.0034 363.153 0.005 143.201 0.0040 173.25

7j 3.15 302 246 210 0.0013 275.156 0.003 101.387 0.0043 161.16

Indomethacin 3.5 223 318 6.29 0.0012 264.18 0.002 88.261 0.0039 156.24

Med Chem Res

123

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ab

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70

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a(6

8.6

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3±

0.0

19

a(6

2.9

2)

7b

15

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0.2

8±

0.0

23

ac

(67

.25

)0

.32

±0

.01

9ac

(63

.22

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b1

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6.4

70

.31

±0

.02

6a

(63

.95

)0

.34

±0

.02

5a

(61

.36

)

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16

.24

0.1

6±

0.0

18

ab

(81

.18

)0

.20

±0

.02

1ab

(77

.01

)1

?1

c1

0±

7.2

90

.24

±0

.01

8a

(72

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)0

.29

±0

.02

2a

(67

.42

)

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18

.71

0.2

9±

0.0

26

ac

(65

.88

)0

.32

±0

.01

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(63

.21

)–

––

–

7e

17

.76

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1±

0.0

27

ac

(63

.53

)0

.35

±0

.02

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(59

.77

)1

?1

e1

0±

8.8

20

.33

±0

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(61

.63

)0

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±0

.01

8a

(60

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18

0.2

0±

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17

ab

(76

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(74

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(69

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(66

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(78

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(73

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(65

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(65

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3.6

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(64

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1a

(57

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Med Chem Res

123

release the parent drug molecules, similar to that of indo-

methacin, a reference drug considered in this study. The

rapid rate of hydrolysis observed in plasma and more sta-

bility in the absence of plasma under similar conditions in

buffers (pH 1.2 and 7.4) is important and implies that

enzymatic reactivity of test compounds is independent of

their intrinsic ester reactivity.

In vivo anti-inflammatory activity

All the synthesized compounds (7a–7j) were screened for

in vivo anti-inflammatory activity with carrageenan-

induced rat paw edema using indomethacin as reference

drug. Carrageenan (1 % w/v) was used to produce paw

edema. The paw edema induced by subplantar injection

of carrageenan was more prominent in control group

where indomethacin exerted 82 % anti-inflammatory

effect after 2 h. Among the tested compounds (Table 3),

7a, 7c, 7f, and 7h exhibited more than 70 % edema

inhibition and decreased the difference in paw thickness

comparable to that of indomethacin (p \ 0.05). The

comparable anti-inflammatory activity of these com-

pounds may be attributed to the combined effect of

improved physicochemical properties of esters and con-

tribution by their corresponding promoieties. Further-

more, equimolar physical mixtures of test compounds

(1a–1j) were also studied for their anti-inflammatory

activity (Table 3). However, these physical mixtures

showed lower anti-inflammatory activity as compared

with their corresponding test compounds.

Table 4 Effect of active test compounds and their physical mixtures on gastric mucosa

Ulcerogenic activity Antiulcer activity

Treatment Dose (mg/kg) Ulcer index* mean ± SEM Treatment Dose (mg/kg) Ulcer index* mean ± SEM

Control 0.5 % CMC 0.38 ± 0.19 Control 0.5 % CMC 0.33 ± 0.11

Indomethacin 48 5.98 ± 0.22 Pyloric ligated – 5.06 ± 0.17bc

7a 73.88 0.12 – 0.028 7a 18.47 1.19 ± 0.21ab

1 ? 1a – – 1 ? 1a 10 ? 9.53 2.7 ± 0.16abc

7c 64.92 0 7c 16.23 0.92 ± 0.19a

1 ? 1c – – 1 ? 1c 10 ? 7.29 2.4 ± 0.24abc

7f 61.64 0.1 ± 0.017 7f 15.41 1.15 ± 0.16ab

7h 68.24 0 7h 17.06 0.87 ± 0.14a

1 ? 1h – – 1 ? 1h 10 ? 8.12 2.2 ± 0.28abc

* Values are expressed as mean ± SEM (n = 6) and analyzed by one-way ANOVA followed by Dunnett’s testa Significant as compared to pyloric ligated group (p \ 0.05)b Significant as compared to control group (p \ 0.05)c Significant as compared to control group (p \ 0.05)

Table 5 Effect of test compounds and their physical mixtures on biomarkers of oxidative stress

Treatment LPO (nmol MDA/mg protein) GSH (lg of GSH/mg protein) SOD (U/mg protein) Catalase (U/mg protein)

Control 4.12 ± 0.38 32.45 ± 1.45 8.79 ± 0.26 13.28 ± 0.64a

Pyloric ligated 13.25 ± 0.67* 12.34 ± 0.86* 3.64 ± 0.31* 5.26 ± 0.34*

7a 6.24 ± 0.42a 23.42 ± 1.34a 6.72 ± 0.24a 10.13 ± 0.42a

1 ? 1a 10.96 ± 0.52b 19.25 ± 1.11c 6.18 ± 0.33bc 7.41 ± 0.5c

7c 6.11 ± 0.46a 26.24 ± 1.18a 7.38 ± 0.29a 10.78 ± 0.53a

1 ? 1c 10.89 ± 0.34b 19.34 ± 1.02bc 6.01 ± 0.19bc 7.28 ± 0.37c

7f 6.43 ± 0.31a 21.83 ± 1.06a 6.67 ± 0.22a 9.12 ± 0.26a

7h 5.98 ± 0.18a 27.34 ± 1.21a 7.94 ± 0.17a 11.53 ± 0.79a

1 ? 1h 10.94 ± 0.38b 19.39 ± 1.12bc 6.11 ± 0.25bc 7.71 ± 0.21bc

Data are expressed as mean ± SEM (n = 6)

* Significant as compared to control group (p \ 0.001)a Significant as compared to pyloric ligated group (p \ 0.001)b Significant as compared to pyloric ligated group (p \ 0.01)c Significant as compared to pyloric ligated group (p \ 0.05)

Med Chem Res

123

Ulcerogenic and antiulcer activity

Although the anti-inflammatory activity of many synthe-

sized compounds was comparable to the reference drug, the

gastric studies were only performed with most active

compounds (7a, 7c, 7f, and 7h). These test compounds and

indomethacin were given orally to rats and animals were

sacrificed after treatment for determining the ulcerogenic

index of the said compounds. None of the test compounds

showed any ulcerogenic effects (Table 4) in the gastric

mucosa in fasted rats. However, gastric ulcers were

observed in all the animals treated with indomethacin.

Further, the same test compounds along with their physical

mixtures were screened for their gastroprotective effects in

rats using pyloric ligation (PL) method, which produced a

significant increase in ulcer index (5.06 ± 0.17) as com-

pared to the control group (0.33 ± 0.11). All four com-

pounds (7a, 7c, 7f, and 7h) showed significantly reduced

gastric damage ([76 % protection) (Table 4). The reduc-

tion in ulcer index by the physical mixture of test com-

pounds was also less as compared to their test compounds

(Table 4). This may be due to the polar nature of their

antioxidant moieties resulting in poor bioavailability,

whereas reduction in ulcer index by the test compounds

was significant (p \ 0.05) which may be due to the

improved physicochemical properties and contribution by

the antioxidant promoieties after their cleavage. From the

results of these studies, it is evident that in all the groups

treated with test compounds, the structure of gastric

mucosa is quite normal.

Table 6 Calculation of various molecular properties of test compounds (7a–7j)

Compounds Mol. weight

(mol_MW)aDipoleb Total solvent

accessible

surface area

(SASA)c

Donor

HBdAcceptors

HBeQPlogPo/wf QPlogSg Human

oral

absorptionh

No. of

violations

of LRi

QPlogBBj PSAk

7a 262.218 1.837 463.321 4 5.5 0.442 -1.907 2 0 -1.362 102.697

7b 262.218 5.522 469.323 4 5.5 0.166 -1.991 2 0 -1.948 123.194

7c 276.245 4.713 496.844 3 5.5 0.624 -2.881 3 0 -1.362 102.697

7d 318.326 4.111 538.123 3 5.5 2 -3.266 3 0 -1.521 107.609

7e 302.326 3.012 555.783 3 4.75 2.984 -3.56 3 0 -1.354 91.653

7f 306.271 5.405 498.53 4 7.2 0.521 -1.473 2 0 -2.027 130.544

7g 306.271 2.123 526.147 4 7.2 0.365 -2.015 3 0 -1.964 125.708

7h 290.229 2.258 471.353 3 6.25 0.582 -1.983 3 0 -1.335 119.319

7i 314.251 3.403 530.353 3 7.25 0.304 -2.181 2 0 -2.161 140.127

7j 308.374 3.484 570.679 3 4.25 2.291 -3.914 3 0 -1.319 91.32

Indomethacin 357.793 4.935 595.039 1 5.75 4.269 -5.111 3 0 -0.651 82.039

Range 95 % of drugs: a (130.0–725.0), b (1.0–12.5), c (300.0–1000.0), d (0.0–6.0), e (2.0–20.0), f (-2.0 to 6.5), g (-6.5 to 0.5), h (1, 2, or 3 for

low, medium, or high), i (maximum 4), j (-3.0 to 1.2), k (7.0–200.0)a Molecular weight of the compounds in daltonb Computed dipole moment of the moleculec Total solvent accessible surface area in square angstroms using a probe with 1.4 A radiusd Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solutione Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solutionf Predicted octanol/water partition coefficientg Predicted aqueous solubilityh Predicted qualitative human oral absorptioni Number of violations of Lipinski’s rule of fivej Predicted brain/blood partition coefficientk Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms in an aqueous solution

Fig. 2 Correlation between calculated and predicted log P values of

test compounds (7a–7j)

Med Chem Res

123

Biochemical evaluation

Oxidative stress plays an important role in gastric mucosal

damage (Rastogi et al., 1998; Pal et al., 2010). In aerobic

cells, mitochondria are the major source of free radicals

and also a sensitive target for free radical-mediated damage

(Orrenius et al., 2007). Increased generation of free radi-

cals causes oxidative stress and is responsible for gastric

mucosal injury (Rastogi et al., 1998). To find out whether

the antiulcer activity of said compounds is mediated

through its antioxidant action, pyloric ligation-induced

mitochondrial oxidative stress was measured in gastric

mucosal cells in both the presence and absence of these test

compounds. Treatment with pyloric ligation increased

peroxidation of mitochondrial lipids and depleted the

mitochondrial total glutathione, superoxide dismutase, and

catalase contents (Table 5). Test compounds (7a, 7c, 7f,

and 7h) at their anti-inflammatory doses significantly pre-

vented mitochondrial lipid peroxidation, depletion of total

glutathione, superoxide dismutase, and catalase contents.

However, the effects of physical mixtures on antioxidant

enzymes were lesser compared with their test compounds

(Table 5). The results suggest that the inhibition of PL-

induced mitochondrial oxidative stress by test compounds

may result from the scavenging of free radicals, the for-

mation of which is augmented by pyloric ligation-induced

gastric mucosal injury.

In silico ADME profiling

Certain molecular properties which could influence the

metabolism, cell permeability, and bioavailability for the

synthesized compounds (7a–7j) were evaluated using

QikProp (version 3.4, Schrodinger, 2011). Some of the

parameters such as QPlogPo/w, QPlogS, polar surface area

(PSA), and molecular surface area such as solvent acces-

sible surface area (SASA) are recognized parameters for

prediction of drug transport properties. ADME prediction

methods were used to assess the bioavailability of the test

compounds (7a–7j). Herein, we calculated the compliance

of all the compounds to the Lipinski’s ‘‘rule of five’’ which

has been widely used as a filter for predicting the druggable

properties of any molecule (Lipinski et al., 2001).

According to this rule, poor absorption or permeation is

more likely when there are more than five H-bond donors,

ten H-bond acceptors, the molecular weight (MW) is

greater than 500, and the calculated log P (Clog P) is

greater than 5. Molecules violating more than one of these

rules may have problems with bioavailability. Further,

PSA, which is a measure of a molecule’s hydrogen bonding

capacity, is another key property that has been linked to

drug bioavailability. Passively absorbed molecules with a

PSA [200 are thought to have low-oral bioavailability

(Clark and Pickett, 2000). Predictions of ADME properties

for test compounds (7a–7j) are given in Table 6. Interest-

ingly, it has been observed that the results of practically

evaluated log P and log S values of test compounds are in

consonance with the theoretically predicted log P and

log S values as evident from their good correlation coef-

ficient of R2 = 0.973 and 0.962, respectively, as shown in

Figs. 2 and 3. This good correlation further reveals that

other predicted ADME properties of these compounds (7a–

7j) could be a reliable indicator supporting their druggable

properties. The results of predicted properties for all the

test compounds are in the ranges predicted by QikProp for

95 % of known oral drugs and also satisfies the Lipinski’s

rule of five to be considered as drug like. Moreover, the

standard drug indomethacin also does not show any vio-

lation. Theoretically, these compounds should present good

passive oral absorption and differences in their bioactivity

may not be attributed to this aspect.

Conclusion

In conclusion, a number of GA esters (7a–7j) have been

synthesized, characterized, and studied for physicochemi-

cal properties. All the compounds exhibited promising anti-

inflammatory activity. In particular, 7a, 7c, 7f, and 7h

emerged as the most active compounds in the series. The

results of gastric ulcerogenic studies and biochemical

evaluation suggest that these compounds are gastric safe.

Further, the predicted ADME properties of all the tested

compounds were found to be in the ranges as predicted by

QikProp for 95 % of known oral drugs and also satisfy the

Lipinski’s rule of five which signifies a good absorption

and hence, good bioavailability. The present investigation

suggests GA esters as potent new gastric safe anti-inflam-

matory agents, which can be further explored for other

therapeutic outcomes including anticancer activity.

Fig. 3 Correlation between calculated and predicted log S values of

test compounds (7a–7j)

Med Chem Res

123

Experimental

Materials

Chemicals and reagents viz: GA, nitroblue tetrazolium,

hydrogen peroxide, ethylenediaminetetracetic acid (EDTA),

thiobarbituric acid, and p-nitrosodimethyl aniline, and anti-

oxidant compounds were purchased from Sigma-Aldrich

chemicals. Indomethacin was procured from Ind-Swift lab-

oratories limited. All the solvents were of analytical grade

and distilled before use.

Methods

The melting points were recorded in open capillaries on

Casiaa Siamea (VMP-AM) melting point apparatus and are

uncorrected. Infrared (IR) spectra were recorded on a

Perkin-Elmer FT-IR 240-C spectrophotometer using KBr

disks. 1H NMR spectra were recorded on Bruker Avance II

400 MHz spectrometer in CDCl3/DMSO-d6 using tetra-

methylsilane (TMS) as internal standard. Mass spectra

were obtained with Waters Micromass Q-TOF Micro mass

spectrometer at 70 eV using electron ionization (EI) sour-

ces. The elemental analyses were performed using Thermo

EA 2110 series elemental analyzer. All the reactions were

monitored by thin layer chromatography (TLC) on pre-

coated silica gel 60 F254 plates (aluminum base) (E. Merck,

Mumbai) and spots were visualized under UV light

(254 nm). Silica gel from E. Merck, Mumbai (100–200

mesh) was used for column chromatography. UV–Vis

spectra were obtained on a Perkin-Elmer 554 double-beam

spectrophotometer and on a Hitachi U-2001 spectropho-

tometer. High-pressure liquid chromatography (HPLC)

separation was carried out using reverse phase HPLC C-18

column at isocratic mode. The Waters Associates machine

fitted with pump (Model-510), injector (Model 6UK),

detector (Lambda-Max Model 481 LC spectrophotometer),

and interfaced with Winchrom were used. The elution was

carried out at ambient temperature. Lichrosphere C-18

column (250 mm length 9 4.6 mm diameter, E. Merck,

India), was used for qualitative analysis. The samples were

prepared by filtering the appropriately diluted extract

through 0.4-lm filter before injection. Elution was carried

out using water:acetonitrile:methanol:acetic acid

(79.5:18:2:0.5) at a flow rate of 1.0 mL/min for the

detection of parent compound.

Synthesis of compounds (7a–7j)

GA esters (7a–7j) were synthesized by conjugating GA (1)

with potassium salts of various naturally occurring anti-

oxidant compounds (1a–1j, Fig. 1). These agents are

important components of human diet and their safety

profile is well known (Cotelle, 2001; Martin et al., 1998).

The sequence of steps involved in the synthesis of GA

esters is shown in Scheme 1.

Methyl 3,4,5-trihydroxybenzoate (2)

To a stirred solution of GA (1) (10 g, 58.8 mmol) in

methanol (60 mL), sulfuric acid (2 mL) was added. The

reaction mixture was heated under reflux for 2 h at 100 �C

and evaporated. The residual mass was taken in water

(70 mL) and extracted with ethyl acetate (3 9 125 mL).

The organic layer was washed with brine, dried over

sodium sulfate, filtered, and concentrated in vacuo to afford

compound 2 as white solid (Dodo et al., 2008) with 91 %

yield; mp 209–210 �C; IR (KBr) tmax: 3026, 2931, 2881,

1718, 1625, 1587, 1503, 1238, 1134, 1031 cm-1; 1H NMR

(DMSO-d6, 400 MHz): 9.25 (br s, 3H), 7.04 (s, 2H), 3.74

(s, 3H, OCH3); ESI-MS (m/z): 185 [M??1] (100).

Methyl 3,4,5-tribenzyloxybenzoate (3)

To a stirred solution of 2 (10 g, 54.35 mmol) and potas-

sium carbonate (22.5 g, 16.21 mmol) in DMF (5 mL),

benzyl chloride (20.54 g, 16.30 mmol) was added. The

reaction mixture was stirred at 80 �C under N2 atmosphere.

After 15 h, water was added, and the whole was extracted

with chloroform. The organic layer was washed with brine,

dried over sodium sulfate, filtered, and concentrated in

vacuo to afford 3 as white powder (Dodo et al., 2008) with

84 % yield; mp 104–105 �C; IR (KBr) tmax: 3031, 2947,

2877, 1715, 1586, 1498, 1430, 1214, 1109, 1027 cm-1; 1H

NMR (CDCl3, 400 MHz,): 7.45–7.25 (m, 17H), 5.13 (s,

4H, 3,5-CH2–O–Ar), 5.11 (s, 2H, 4-CH2–O–Ar), 3.89 (s,

3H, OCH3); ESI-MS (m/z): 455.2 (M??1), 477.2

[M??Na] (100).

3,4,5-Tribenzyloxybenzoic acid (4)

To a stirred solution of 3 (10 g, 22.03 mmol) in methanol

(50 mL), dioxane (100 mL), H2O (40 mL), and NaOH

(4 g, 0.1 mol) was added. The reaction mixture was heated

under reflux for 6 h at 120 �C, and evaporated. The

residual mass was taken in water (70 mL) and extracted

with chloroform (3 9 125 mL). The organic layer was

washed with brine, dried over sodium sulfate, filtered, and

concentrated in vacuo to afford 4 as white solid (Dodo

et al., 2008) with 72 % yield; mp 267 �C; IR (KBr) tmax:

3437, 2870, 1684, 1563, 1505, 1428, 1228, 1127,

1025 cm-1; 1H NMR (DMSO-d6, 400 MHz): 7.47 (s, 2H,

2,6-Ar–H), 7.35–7.33 (m, 4H, Ar–H), 7.29–7.21 (m, 8H,

Ar–H), 7.19–7.16 (m, 3H, Ar–H), 4.99 (s, 4H, 3,5-CH2–O–

Ar), 4.88 (s, 2H, 4-CH2–O–Ar); 13C NMR (DMSO-d6,

100 MHz): 169.7 (COOH), 151–108 (Ar–C), 74 (1C,

Med Chem Res

123

4-CH2–O–Ar), 70 (2C, 3,5-H2–O–Ar); ESI-MS (m/z):

463.2 [M??Na] (100).

3,4,5-Tribenzyloxybenzoylchloride (5)

To a stirred solution of 4 (10 g, 22.73 mmol) in DMF

(0.2 mL) and toluene (70 mL), oxalyl chloride (2.86 g,

22.7 mmol) was added slowly. The reaction mixture was

stirred at room temperature under N2 atmosphere, and

concentrated in vacuo. The residue was taken in toluene

(30 mL) and the solution was filtered. Cyclohexane (5 mL)

was added, and the mixture was cooled to room tempera-

ture to afford 5 as white solid (Dodo et al., 2008) with

61 % yield; mp 130–131 �C; IR (KBr) tmax: 2922, 2879,

1747, 1587, 1499, 1453, 1239, 1126, 1034 cm-1; 1H NMR

(CDCl3, 400 MHz): 7.42–7.23 (m, 17H, Ar–H), 5.17 (s,

2H, 4-CH2–O–Ar), 5.13 (s, 4H, 3,5-CH2–O–Ar); 13C NMR

(100 MHz, CDCl3, d ppm): 167 (C=O, acid chloride),

152–111 (Ar–C), 75 (1C, 4-CH2–O–Ar), 71 (2C, 3,5-CH2–

O–Ar).

General procedure for the synthesis of compounds

(6a–6j)

To a stirred solution of compound 5 (4.58 g, 0.01 mol), a

solution of potassium salt of different phytophenols (1a–1j;

0.01 mol) in THF (15 mL) was added dropwise over a

period of 1 h at 0 �C. The reaction mixture was stirred at

room temperature overnight and concentrated in vacuo.

The residue was taken in ethyl acetate and organic layer

was washed with 10 % sodium bicarbonate, brine, dried

over sodium sulfate, filtered, and concentrated in vacuo.

The residue was purified by recrystallization using hexane,

ethyl acetate to afford the desired products (6a–6j) as white

solid in the range 51–69 %.

20-Hydroxyphenyl-3,4,5-tribenzyloxybenzoate (6a)

White solid; yield 56 %; mp 149–150 �C; UV (MeOH)

kmax 268 nm; IR (KBr) tmax: 3070, 2940, 2836, 1727,

1593, 1509, 1459, 120, 1123, 1036 cm-1; 1H NMR

(CDCl3, 400 MHz): 7.52 (2H, s, Ar–H-2,6), 7.45–7.31

(13H, m, Ar–H), 7.30–7.25 (2H, m, Ar–H), 7.20–7.13 (2H,

m, Ar–H-40,60), 7.07–7.05 (1H, dd, J = 1.5, J = 8.5, Ar–

H-50), 6.99–6.95(1H, m, Ar–H-30), 5.48 (1H, br s, OH),

5.17 (6H, s, 3,4,5-CH2–O–Ar); ESI-MS (m/z): 555.2

[M??Na] (100).

30-Hydroxyphenyl-3,4,5-tribenzyloxybenzoate (6b)

White solid; yield 53 %; mp 111 �C; UV (MeOH) kmax

267 nm; IR (KBr) tmax: 3030, 2930, 2856, 1732, 1593,

1498, 1195, 1125, 1032 cm-1; 1H NMR (400 MHz,

CDCl3, d ppm): 7.45 (2H, m, Ar–H-2,6), 7.40–7.24 (12H,

m, Ar–H), 7.22–7.16 (5H, m, Ar–H), 7.07–6.89 (m, 1H,

Ar–H-40), 6.70–6.65 (1H, m, Ar–H-20), 5.09 (4H, s, 3,5-

CH2–O–Ar), 5.08 (2H, s, 4-CH2–O–Ar); ESI-MS (m/z):

555.2 [M??Na] (100), 533.2 [M??1] (27).

20-Methoxyphenyl-3,4,5-tribenzyloxybenzoate (6c)


269 nm; IR (KBr) tmax: 3063, 2928, 2871, 1727, 1586,

1499, 1196, 1102, 1025 cm-1; 1H NMR (CDCl3,

400 MHz): 7.56 (2H, s, Ar–H-2,6), 7.45–7.30 (12H, m,

Ar–H), 7.28–7.22 (4H, m, Ar–H), 7.14–7.11 (1H, m, Ar–H-

60), 7.02–6.96 (2H, m, Ar–H-30,50), 5.16 (4H, s, 3,5-CH2–

O–Ar), 5.14 (2H, s, 4-CH2–O–Ar), 3.81 (3H, s, 20-OCH3);13C NMR (CDCl3, 100 MHz): 164.3 (COOAr), 152.6 (Ar–

C), 151.4 (Ar–C), 142.9 (Ar–C), 137.4 (Ar–C), 136.6 (Ar–

C), 128.5 (Ar–C), 128.2 (Ar–C), 128 (Ar–C), 128 (Ar–C),

127.6 (Ar–C), 127 (Ar–C), 124.4 (Ar–C), 122.9 (Ar–C),

120.8 (Ar–C), 112.5 (Ar–C), 109.7 (Ar–C), 75.2 (–CH2–O–

Ar), 71.2 (–CH2–O–Ar), 55.9 (–OCH3); ESI-MS (m/z):

547.2 [M??1] (43).

20-Methoxy-40-(100-propenylphenyl)-3,4,5-

tribenzyloxybenzoate (6d)


260 nm; IR (KBr) tmax: 3031, 2920, 1734, 1591, 1505,

1188, 1114, 1027 cm-1; 1H NMR (CDCl3, 400 MHz): 7.48

(2H, s, Ar–H-2,6), 7.38–7.36 (4H, m, Ar–H), 7.34–7.30

(5H, m, Ar–H), 7.28–7.23 (2H, m, Ar–H), 7.21–7.17 (4H,

m, Ar–H), 6.97–6.95 (1H, m, Ar–H-40), 6.75–6.72 (2H, m,

Ar–H-30,60), 5.94–5.88 (1H, m, H-200), 5.09–5.02 [8H, m

(6H, 3,4,5-Ar–OCH2, 2H, H-100)], 3.73 (3H, s, 20-OCH3),

3.34–3.33 (2H, d, H-300, J = 6.68); 13C NMR (CDCl3,

100 MHz): 164.3 (COOAr), 152.6 (Ar–C), 151.1 (Ar–C),

142.8 (Ar–C), 139.1 (Ar–C), 138.2 (Ar–C), 137.4 (Ar–C),

137.1 (Ar–C), 136.6 (Ar–C), 128.5 (Ar–C), 128.2 (Ar–C),

128 (Ar–C), 127.9 (Ar–C), 127.6 (Ar–C), 124.5 (Ar–C),

122.6 (Ar–C), 120.7 (Ar–C), 116.1 (Ar–C), 112.9 (Ar–C),

109.7 (Ar–C), 75.2 (–CH2–O–Ar), 71.2 (–CH2–O–Ar),

55.9 (–OCH3), 40.1 (CH2–CH = CH2); ESI-MS (m/z):

587.3 [M??1] (3), 609.3 [M??Na] (100).

50-Methyl-20-isopropylphenyl-3,4,5-tribenzyloxybenzoate

(6e)

White solid; yield 69 %; mp 92–95 �C; UV (MeOH) kmax

271 nm; IR (KBr) tmax: 3033, 2921, 2865, 1729, 1590,

1489, 1198, 1116, 1009 cm-1; 1H NMR (CDCl3,

400 MHz): 7.53 (2H, s, Ar–H-2,6), 7.45–7.41 (6H, m, Ar–

H), 7.39–7.31 (6H, m, Ar–H), 7.30–7.27 (3H, m, Ar–H),

7.24–7.22 (1H, d, J = 7.96, Ar–H-30), 7.07–7.05 (1H,

Med Chem Res

123

J = 7.96, Ar–H-40), 6.91 (1H, rough singlet, Ar–H-60),5.17 (4H, s, 3,5-CH2–O–Ar), 5.16 (2H, s, 4-CH2–O–Ar),

2.99–2.95 (1H, septet, J = 6.8, H-70), 2.33 (3H, s, H-100),1.19–1.17 (6H, d, J = 6.8, H-80,90); 13C NMR (CDCl3,

100 MHz): 164.9 (COOAr), 152.6 (Ar–C), 148.1 (Ar–C),

142.9 (Ar–C), 137.1 (Ar–C), 136.5 (Ar–C), 128.5 (Ar–C),

128.2 (Ar–C), 128 (Ar–C), 128 (Ar–C), 127.5 (Ar–C)

127.2 (Ar–C), 126.5 (Ar–C), 124.5 (Ar–C), 122.8 (Ar–C),

109.5 (Ar–C), 75.2 (–CH2–O–Ar), 71.2 (–CH2–O–Ar),

27.2 (–CH3), 23 (CH(CH3)), 20.9 (CH(CH3)); ESI-MS (m/

z): 595.3 [M??Na] (100), 573.3 [M??1] (5).

40-Formyl-20-methoxyphenyl-3,4,5-tribenzyloxybenzoate

(6f)


263 nm; IR (KBr) tmax: 3033, 2922, 2860, 1728, 1701,

1592, 1503, 1459, 1199, 1124, 1025 cm-1; 1H NMR

(DMSO-d6, 400 MHz): 10.01 (1H, s, CHO), 7.64–7.61

(2H, m, Ar–H-30,50), 7.53 (2H, s, Ar–H-2,6), 7.49–7.44

(5H, m, Ar–H), 7.42–7.36 (8H, m, Ar–H), 7.29–7.25 (3H,

m, Ar–H), 5.22 (4H, s, 3,5-CH2–O–Ar), 5.11 (2H, s,

4-CH2–O–Ar), 3.87 (3H, s, 20-OCH3); 13C NMR (DMSO-

d6, 100 MHz): 191.6 (CHO), 162.9 (–COOAr), 152.2 (Ar–

C), 151.6 (Ar–C), 144.3 (Ar–C), 137.1 (Ar–C), 136.4 (Ar–

C), 135.1 (Ar–C), 128.3 (Ar–C), 128.2 (Ar–C), 128.1 (Ar–

C), 127.9 (Ar–C), 127.8 (Ar–C), 127.5 (Ar–C), 127.3 (Ar–

C), 123.6 (Ar–C), 123.6 (Ar–C), 123.2 (Ar–C), 111.7 (Ar–

C), 108.7 (Ar–C), 74.3 (–CH2–O–Ar), 70.3 (–CH2–O–Ar),

55.9 (–OCH3); ESI-MS (m/z): 597.3 [M??Na] (100),

575.3 [M??1] (4).

30-Formyl-60-methoxyphenyl-3,4,5-tribenzyloxybenzoate

(6g)


269 nm; IR (KBr) tmax: 3032, 2923, 2853, 1721, 1698,

1605, 1586, 1509, 1199, 1121, 1016 cm-1; 1H NMR

(CDCl3, 400 MHz): 9.76 (1H, s, CHO), 7.68–7.66 (1H,

dd, Ar–H-40, J = 1.96, J = 8.4), 7.58 (1H, d, Ar–H-20,J = 1.96), 7.45 (2H, s, Ar–H-2,6), 7.35–7.22 (12H, m,

Ar–H), 7.18–7.15 (3H, m, Ar–H), 6.99–6.97 (1H, d, Ar–

H-50, J = 8.4), 5.05 (6H, s, 3,4,5-CH2–O–Ar), 3.76 (3H,

s, 20-OCH3); 13C NMR (CDCl3, 100 MHz): 190.1 (CHO),

164 (COOAr), 156.6 (Ar–C), 152.7 (Ar–C), 143.1 (Ar–

C), 140.5 (Ar–C), 137.4 (Ar–C), 136.5 (Ar–C), 130.3

(Ar–C), 130 (Ar–C), 128.6 (Ar–C), 128.6 (Ar–C), 128.3

(Ar–C), 128.1 (Ar–C), 127.6 (Ar–C), 123.8 (Ar–C), 123.7

(Ar–C), 75.2 (–CH2–O–Ar), 71.2 (–CH2–O–Ar), 56

(–OCH3); ESI-MS (m/z): 597.3 [M??Na] (100), 575.3

[M??1] (5).

30,40-(Methylenedioxy)phenyl-3,4,5-tribenzyloxybenzoate

(6h)



1590, 1501, 1484, 1200, 1120, 1032 cm-1; 1H NMR

(DMSO-d6, 400 MHz): 7.49–7.45 (6H, m, Ar–H),

7.41–7.32 (8H, m, Ar–H), 7.27–7.25 (3H, m, Ar–H),

6.85–6.83 (1H, m, Ar–H-50), 6.74 (1H, s, Ar–H-60),6.64–6.62 (1H, m, Ar–H-20), 6.04 (2H, s, O–CH2–O), 5.18

(4H, s, 3,5-CH2–O–Ar), 5.09 (2H, s, 4-CH2–O–Ar); 13C

NMR (DMSO-d6, 100 MHz): 164.1 (COOAr), 152 (Ar–C),

147.5 (Ar–C), 144.9 (Ar–C), 144.7 (Ar–C), 142 (Ar–C),

136.8 (Ar–C), 136.1 (Ar–C), 128.1 (Ar–C), 128 (Ar–C),

127.7 (Ar–C), 127.7 (Ar–C), 127.6 (Ar–C), 127.2 (Ar–C),

123.92 (Ar–C), 113.6 (Ar–C), 108.7 (Ar–C), 107.5 (Ar–C),

103.4 (Ar–C), 101.4 (O–CH2–O), 74.4 (–CH2–O–Ar), 70.5

(–CH2–O–Ar); ESI-MS (m/z): 583.2 [M??Na] (100),

561.2 [M??1] (12).

20-Oxo-20H-chromene-70-yl-3,4,5-tribenzyloxybenzoate (6i)


275 nm; IR (KBr) tmax: 3062, 2923, 2856, 1726, 1620,

1591, 1501, 1196, 1127, 1026 cm-1; 1H NMR (DMSO-d6,

400 MHz): 8.06–8.04 (1H, d, Ar–H-40, J = 9.6), 7.80–7.77

(1H, m, Ar–H-50), 7.55 (2H, s, Ar–H-2,6), 7.49–7.47 (4H,

m, Ar–H), 7.42–7.32 (9H, m, Ar–H), 7.28–7.24 (4H, m,

Ar–H), 6.46–6.44 (1H, d, J = 9.6, Ar–H-30), 5.21 (4H, s,

3,5-CH2–O–Ar), 5.10 (2H, s, 4-CH2–O–Ar); 13C NMR

(DMSO-d6, 100 MHz): 163.4 (COOAr), 159.4 (–C=O–),

154.1 (Ar–C), 152.9 (Ar–C), 152.2 (Ar–C), 143.4 (Ar–C),

142.6 (Ar–C), 136.9 (Ar–C), 136.3 (Ar–C), 129 (Ar–C),

128.2 (Ar–C), 128.1 (Ar–C), 127.8 (Ar–C), 127.7 (Ar–C),

127.7 (Ar–C), 127.4 (Ar–C), 127.3 (Ar–C), 123.4 (Ar–C),

118.3 (Ar–C), 116.6 (Ar–C), 115.4 (Ar–C), 109.9 (Ar–C),

108.9 (Ar–C), 74.3 (–CH2–O–Ar), 70.5 (–CH2–O–Ar);

ESI-MS (m/z): 607.3 [M??Na] (100), 585.3 [M??1] (2).

(1a,2b,5a)-50-Methyl-20-isopropylcyclohexyl-3,4,5-

tribenzyloxybenzoate (6j)


260 nm; IR (KBr) tmax: 3072, 2937, 2845, 1718, 1610, 1568,

1453, 1247, 1137, 1057 cm-1; 1H NMR (CDCl3, 400 MHz):

7.43 (2H, s, Ar–H-2,6), 7.42–7.34 (14H, m, Ar–H),

7.28–7.26 (1H, m, Ar–H), 5.17 (2H, s, 4-CH2–O–Ar), 5.14

(4H, s, 3,5-CH2–O–Ar), 3.44–3.38 (1H, m, H-10), 2.19–2.14

(1H, m, H-70), 1.98–1.94 (1H, m, CH), 1.68–1.58 (2H, m,

CH), 1.43–1.40 (2H, m, CH), 1.14–1.07 (2H, m, CH2), 0.92

(6H, d, 80,90-H), 0.89 (1H, m, CH), 0.82 (3H, d, 100-H); ESI-

MS (m/z): 601.1 [M??Na] (100).

Med Chem Res

123

General procedure for the synthesis of compounds

(7a–7j)

To a stirred solution of compounds 6a–6j (1 mmol) in

ethyl acetate (15 mL), Pd/C (10 %, 1.40 mmol) was added.

The reaction mixture was stirred at room temperature under

H2 atmosphere for variably 3–96 h. The progress of reac-

tion was monitored by TLC. The residue was filtered and

concentrated in vacuo. The purification was carried out by

recrystallization using hexane/ethyl acetate to afford the

desired products (7a–7j) as white solid in the range

54–78 %.

20-Hydroxyphenyl-3,4,5-trihydroxybenzoate (7a)



1502, 1020 cm-1; 1H NMR (DMSO-d6, 400 MHz): 9.29

(1H, br s, OH), 9.10–9.01 (1H, br s, OH), 8.72 (2H, s, OH),

7.13 (2H, s, Ar–H-2,6), 7.10–7.02 (2H, m, Ar–H-40,60),6.97–6.95 (1H, m, Ar–H-50), 6.84–6.81 (1H, m, Ar–H-30);ESI-MS (m/z): 261 [M?-1] (100); Anal. Calcd. %:

(C13H10O6): C, 59.51; H, 3.81. Found (%): C, 59.55; H,

3.84.

30-Hydroxyphenyl-3,4,5-trihydroxybenzoate (7b)



1498, 1134 cm-1; 1H NMR (DMSO-d6, 400 MHz): 9.33

(3H, br s, OH), 9.10–9.01 (1H, br s, OH), 7.16–7.10 (1H,

m, Ar–H), 7.05–7.03 (2H, s, Ar–H), 6.75–6.73 (1H, m,

Ar–H), 6.64–6.62 (2H, m, Ar–H); ESI-MS (m/z): 261.1

[M?-1] (100); Anal. Calcd. %: (C13H10O6): C, 59.49; H,

3.80. Found (%): C, 59.55; H, 3.84.

20-Methoxyphenyl-3,4,5-trihydroxybenzoate (7c)


275 nm; IR (KBr) tmax: 3430, 2923, 2854, 1711, 1614,

1537, 1502, 1260, 1107, 1017 cm-1; 1H NMR (DMSO-d6,

400 MHz): 9.03 (3H, br s, OH), 7.24–7.20 (1H, m, Ar–H-

40), 7.17 (2H, m, Ar–H-2,6), 7.09–7.01 (2H, m, Ar–H-

30,60), 6.98–6.95 (1H, m, Ar–H-50), 3.79 (3H, s, 20-OCH3);13C NMR (DMSO-d6, 100 MHz): 164 (1C, COOAr), 150.9

(Ar–C-20), 145.25 (Ar–C-3,5), 139.6 (Ar–C-4), 138.6 (Ar–

C-10), 126.3 (Ar–C-40), 122.7 (Ar–C-1), 120.2 (Ar–C-60),118.4 (Ar–C-50), 112.1 (Ar–C-30), 109.3 (Ar–C-2,6), 55.3

(OCH3-20); ESI-MS (m/z): 299.1 [M??Na] (100), 277.1

[M??1] (1); Anal. Calcd. %: (C14H12O6): C, 60.84; H,

4.34. Found (%): C, 60.87; H, 4.38.

20-Methoxy-40-propylphenyl-3,4,5-trihydroxybenzoate (7d)



1502, 1449, 1264, 1197, 1079 cm-1; 1H NMR (CDCl3,

400 MHz): 7.34 (2H, s, Ar–H-2,6), 6.99–6.97 (1H, m, Ar–

H-60), 6.83–6.74 (2H, m, Ar–H-30,50), 6.39 (3H, br s, OH),

3.76 (3H, s, 20-OCH3), 2.57–2.49 (2H, m, CH2CH2CH3),

1.67–1.57 (2H, m, CH2CH2CH3), 0.96–0.92 (3H, m,

CH2CH2CH3); 13C NMR (CDCl3, 100 MHz): 165.6 (CO-

OAr), 150.7 (Ar–C-20), 143.6 (Ar–C-3,5), 141.8 (Ar–C-4),

137.6 (Ar–C-10), 137.2 (Ar–C-40), 122.5 (Ar–C-1), 120.7

(Ar–C-60), 120.5 (Ar–C-50), 112.8 (Ar–C-30), 110.6 (Ar–C-

2,6), 55.8 (OCH3-20), 37.9 (CH2CH2CH3), 24.5

(CH2CH2CH3), 13.8 (CH2CH2CH3); ESI-MS (m/z): 341.1

[M??Na] (100); Anal. Calcd. %: (C17H18O6): C, 64.11; H,

5.68. Found (%): C, 64.14; H, 5.70.

50-Methyl-20-isopropylphenyl-3,4,5-trihydroxybenzoate

(7e)


278 nm; IR (KBr) tmax: 3439, 1713, 1611, 1542, 1510,

1092 cm-1; 1H NMR (CDCl3, 400 MHz): 7.41 (2H, s, Ar–

H-2,6), 7.26–7.21 (1H, d, J = 7.8, Ar–H-30), 7.06–7.04

(1H, d, 40, J = 7.8, Ar–H,), 6.88 (1H, s, Ar–H-60), 6.27

(3H, br s, OH), 3.03–2.96 (1H, septet, J = 6.8, H-70,), 2.31

(3H, s, H-100), 1.18–1.16 (6H, d, J = 6.8, H-80,90); 13C

NMR (CDCl3, 100 MHz): 166 (COOAr), 148 (Ar–C-10),143.7 (Ar–C-3,5), 137.2 (Ar–C-20,4), 136.7 (Ar–C-50),127.2 (Ar–C-1), 126.5 (Ar–C-30), 122.8 (Ar–C-40), 120.7

(Ar–C-60), 110.4 (Ar–C-2,6), 27.2 (Ar–C-70), 23 (Ar–C-

80,90), 20.8 (Ar–C-100); ESI-MS (m/z): 325.1 [M??Na]

(100), 303.1 [M??1] (19); Anal. Calcd. %: (C17H18O5): C,

67.50; H, 5.97. Found (%): C, 67.54; H, 6.

40-Hydroxymethyl-20-methoxyphenyl-3,4,5-

trihydroxybenzoate (7f)


276 nm; IR (KBr) tmax: 3442, 1717, 1588, 1500, 1216, 1111,

1056 cm-1; 1H NMR (DMSO-d6, 400 MHz): 9.16 (2H, s,

OH), 8.91 (1H, s, OH) 7.13 (2H, s, Ar–H-2,6), 7.07 (1H, rough

singlet, Ar–H-30), 7.03–6.97 (d, 1H, 50-Ar–H, J = 8.0),

6.92–6.90 (d, 1H, 60-Ar–H, J = 8.0), 5.22 (br s, 1H, CH2OH),

4.55 (s, 2H, CH2OH), 3.78 (s, 3H, 20-OCH3);13C NMR

(DMSO-d6, 100 MHz): 164.1 (COOAr), 150.7 (Ar–C-20),145.2 (Ar–C-3,5), 141 (Ar–C-4), 138.7 (Ar–C-40), 138.2 (Ar–

C-10), 122.2 (Ar–C-1), 118.3 (Ar–C-60), 118.1 (Ar–C-50), 110.5

(Ar–C-30), 109.2 (Ar–C-2,6), 62.9 (CH2-40), 55.3 (OCH3-2

0);ESI-MS (m/z): 305.1 [M?-1] (25); Anal. Calcd. %:

(C15H14O7): C, 58.78; H, 4.57. Found (%): C, 58.82; H, 4.61.

Med Chem Res

123

50-Hydroxymehyl-20-methoxyphenyl-3,4,5-

trihydroxybenzoate (7g)


277 nm; IR (KBr) tmax: 3252, 2924, 2854, 1712, 1610,

1541, 1510, 1193, 1030, 1119 cm-1; 1H NMR (DMSO-

d6, 400 MHz): 9.10 (3H, br s, OH), 7.13 (2H, s, Ar–H-

2,6), 7.07 (1H, rough singlet, Ar–H-40), 7.03–6.98 (1H, m,

Ar–H-60), 6.92–6.90 (1H, m, Ar–H-30), 4.55 (2H, s,

CH2OH), 3.78 (3H, s, 20-OCH3); 13C NMR (DMSO-d6,

100 MHz): 164.2 (COOAr), 155.5 (Ar–C-20), 150.7 (Ar–

C-4), 145.3 (Ar–C-3,5), 138.6 (Ar–C-10), 138.4 (Ar–C-50),122.2 (Ar–C-1), 118.3 (Ar–C-40), 118.1 (Ar–C-60), 110.6

(Ar–C-30), 109.2 (Ar–C-2,6), 62.9 (CH2-50), 55.9 (OCH3-

20); ESI-MS (m/z): 329.1 [M??Na] (100); Anal. Calcd.

%: (C15H14O7): C, 58.80; H, 4.58. Found (%): C, 58.82;

H, 4.61.

30,40-Methylenedioxyphenyl-3,4,5-trihydroxybenzoate (7h)



1615, 1544, 1502, 1026 cm-1; 1H NMR (DMSO-d6,

400 MHz): 9.10–9.01 (1H, br s, OH), 8.73 (2H, br s,

OH), 7.08 (2H, s, Ar–H-2,6), 6.75–6.72 (1H, m, Ar–H),

6.60–6.58 (1H, m, Ar–H), 6.55–6.52 (1H, m, Ar–H),

5.92 (2H, s, O–CH2–O); 13C NMR (DMSO-d6,

100 MHz): 164.4 (COOAr), 149.2 (Ar–C-30), 145 (Ar–

C-3,5), 144.4 (Ar–C-40), 143.2 (Ar–C-10), 142.8 (Ar–C-

4), 122.3 (Ar–C-1) 113.1 (Ar–C-50), 112 (Ar–C-60),107.1 (Ar–C-2,6), 105.2 (Ar–C-20), 101.6 (O–CH2–O);

ESI-MS (m/z): 289 [M?-1] (100), Anal. Calcd. %:

(C14H10O7): C, 57.91; H, 3.45. Found (%): C, 57.94, H,

3.47.

20-Oxo-20H-chromene-70-yl-3,4,5-trihdroxybenzoate (7i)


282 nm; IR (KBr) tmax: 3395, 1705, 1680, 1604, 1542, 1510,

1218 1030 cm-1; 1H NMR (DMSO-d6, 400 MHz): 9.20

(2H, br s, OH), 8.96 (1H, br s, OH), 7.31–7.29 (1H, d,

J = 8.2, Ar–H-40), 7.17–7.08 (3H, m, Ar–H-2,6, Ar–H-50),6.98 (1H, s, Ar–H-80), 6.94–6.92 (1H, dd, J = 8.2, J = 2.2

Ar–H-60), 6.89–6.88 (1H, d, J = 2.2, Ar–H-30); 13C NMR

(DMSO-d6, 100 MHz): 165.2 (COOAr), 164.5 (–C=O–),

155.5 (Ar–C-70), 153.5 (Ar–C-80a), 149.7 (Ar–C-3,5), 138.8

(Ar–C-40), 130.7 (Ar–C-4), 128.6 (Ar–C-50), 124.2 (Ar–C-

1), 118.6 (Ar–C-40a), 118.3 (Ar–C-60), 116.1 (Ar–C-80),109.1 (Ar–C-30), 108.7 (Ar–C-2,6); ESI-MS (m/z): 337.1

[M??Na] (11); Anal. Calcd. %: (C16H10O7): C, 61.17; H,

3.23. Found (%): C, 61.15; H, 3.21.

(-)(1a,2b,5a)-50-Methyl-20-isopropylcyclohexyl-3,4,5-

trihydroxybenzoate (7j)

White solid; yield 67 %; mp 142–143 �C; [a]20D - 120 (c

0.006, EtOH); UV (MeOH) kmax 246 nm; IR (KBr) tmax:

3405, 2924, 2855, 1709, 1615, 1538, 1502, 1022 cm-1; 1H

NMR (DMSO-d6, 400 MHz): 9.20 (2H, br s, OH), 8.85

(1H, br s, OH), 6.91(2H, s, Ar–H), 3.17–3.12 (1H, m,

H-10), 2.20–2.15 (1H, m, CH), 1.83–1.80 (1H, m, CH),

1.79–1.57 (1H, m, CH), 1.52–1.48 (1H, m, CH2), 1.35–1.30

(1H, m, CH), 0.99–.092(2H, m, CH), 0.85–0.84(6H,

H-80,90), 0.79–0.77(1H, m, CH), 0.73–0.771 (3H, m,

H-100); ESI-MS (m/z): 331.1 [M??Na] (100); Anal. Calcd.

%: (C17H24O5): C, 66.19; H, 7.83. Found (%): C, 66.21; H,

7.84.

Physicochemical studies

Solubility studies

Solutions of GA (1) and synthesized compounds (7a–7j)

were prepared in methanol/buffer (pH 7.4). The kmax was

determined by scanning solutions containing 20–50 lg/mL

of GA between 200 and 400 nm using UV–visible spec-

trophotometer. The kmax of GA was found to be 258 nm.

Similarly, kmax of synthesized compounds (7a–7j) was

determined and shown in Table 2. The standard plot for

GA was constructed in methanol buffer. The stock solution

containing 1 mg/mL of GA was diluted to obtain solutions

of concentration in the range of 2–10 lg/mL. The spec-

trophotometric absorbances were recorded at 258 nm using

methanol/buffer as blank. Linear calibration curve was

obtained with slope as 0.281 and E1cm1 % 2810 (Table 2).

Similarly, calibration plots of synthesized compounds (7a–

7j) were established. The solubility studies of GA and

synthesized compounds were carried out in phosphate

buffer (pH 7.4) (Table 2). Excess amount of each com-

pound was added to 10 mL of buffer and shaken for 24 h at

37 ± 2 �C using water bath shaker. Solutions were filtered

and analyzed spectrophotometrically for determining the

amount of compounds.

Partition coefficient determination

Partition coefficients of GA and synthesized compounds

(7a–7j) were determined in octanol/phosphate buffer (pH

7.4) system using shake flask method (Table 2). Saturated

solutions of all the compounds were prepared in n-octanol

(5 mL) and equal volumes of phosphate buffer (pH 7.4)

were added to the solutions in conical flasks. The sealed

flasks were kept for shaking in a water bath shaker main-

tained at 37 ± 2 �C for 24 h and then allowed to stand for

Med Chem Res

123

30 min for both the phases to fully separate. Thereafter, the

respective phases were analyzed spectrophotometrically.

Chemical stability

Degradation rate of test compounds (7a–7j) in aqueous

solutions (containing 0.02 % w/v Tween 80) of pH 1.2

(nonenzymatic-simulated gastric fluid, SGF) and isotonic

phosphate buffer of pH 7.4 was determined at 37 �C. The

ionic strength of the buffer solutions was adjusted to 0.5 by

addition of a calculated amount of potassium chloride. The

reactions were initiated by adding 250 lL of a methanolic

solution (2 9 10-3 M) of the test compounds to 2.5 mL of

preheated buffer solutions in screw-capped test tubes and at

appropriate intervals, aliquots of 20 lL were withdrawn

and analyzed by HPLC for the residuals.

Enzymatic hydrolysis (in human plasma)

Enzymatic hydrolysis was carried out for test compounds

(7a–7j) on 80 % human plasma by the method described

by Mahfouz et al. (1998). Human plasma was obtained by

centrifugation of blood samples containing 0.3 % citric

acid at 3,0009g for 15–20 min. Human plasma fractions

(4 mL) were diluted with 1 mL of isotonic phosphate

buffer (pH 7.4) to give a final volume of 5 mL (80 %

plasma). Incubation was performed at 37 �C using shaking

water bath. The reactions were initiated by adding 100 mL

of stock solution of (1 mg/mL) to 5 mL of preheated

plasma. At appropriate time intervals, samples were taken

and diluted with phosphate buffer and analyzed spectro-

photometrically at 258 nm for the appearance of parent

compound GA. The value of rate constants (k) and half

lives (t�) for the hydrolysis of test compounds was cal-

culated from the linear portion of the plotted logarithm of

GA concentration versus time.

Pharmacological studies

Animals

Wistar rats (8–10 weeks old; 200–250 g) of both sexes

procured from central animal house, Panjab University,

Chandigarh, India were used. The animals were housed in

plastic cages under standard laboratory conditions and

maintained on rat chow and water until used and fasted

24 h prior to gastric ulcer studies. The experimental pro-

tocol was approved by the Institutional Animal Ethics

Committee (IAEC/98-112 dated 28.03.11) and conducted

according to the guidelines of Committee for the Purpose

of Control and Supervision of Experiments on Animals

(CPCSEA), New Delhi, India. Unless otherwise stated, the

standard conditions were adopted in all experiments.

Anti-inflammatory activity

Anti-inflammatory activity was determined using carra-

geenan-induced foot paw edema assay method in rats

(Winter et al., 1962). Indomethacin is one of the most

potent NSAIDs among non-selective COX inhibitor.

However, its use is restricted due to high incidences of

ulcerogenic side effects. In the present study, this potential

NSAID has been used as a standard drug. Rats were ran-

domly distributed into different groups (n = 6) of control,

standard, test. Separate groups of rats (n = 6) were used to

study the anti-inflammatory effects of physical mixtures

(1 ? 1a–1j).

The test compounds were suspended in 0.5 % car-

boxymethylcellulose (CMC) and administered orally at

molar equivalent doses of parent compound GA (12 mg/

kg, p.o.). Control animals were given the corresponding

amount of vehicle (0.5 %, CMC) and animals of standard

group received indomethacin (12 mg/kg, p.o.) suspended

in 0.5 % CMC. Acute edema was induced in the left hind

paw of rats by injecting freshly prepared solution of car-

rageenan (Type IV, 0.1 mL, 1 %) under plantar region of

the left hind paw. In the right paw, saline (1 mL, 0.9 %)

was injected, which served as control for comparison. The

increase in paw volume was measured by using plethys-

mometer (water displacement, UGO BASILE, Italy) at 2

and 4 h after carrageenan challenge. Percentage change in

paw volume was calculated and expressed as the amount of

inflammation.

Ulcerogenic and antiulcer Activity

Wistar rats of either sex were distributed at random in

different groups of six animals each. Animals were treated

with indomethacin (48 mg/kg, p.o.), equimolar doses of

test compounds to indomethacin once daily for four con-

secutive days. The animals were killed under deep ether

anesthesia and stomachs were removed. The abdomen of

each rat was opened through great curvature and examined

under dissecting microscope for lesions or bleedings. The

severity of the mucosal damage (ulcerogenic index) was

calculated by means of scores. The ulcers were scored as: 0

normal colored stomach, 0.5 red coloration, 1.0 spot ulcers,

1.5 hemorrhagic streaks, 2.0 ulcers [3 but \5, 3.0 ulcers

[5 (Milanino et al., 1988).

Separate groups of rats (n = 6) were used to see the

effect of test compounds and their physical mixtures on

pyloric ligation (PL)-induced gastric mucosal injury. Ani-

mals were divided into different groups: control, pyloric

ligated, PL-induced plus test compounds (7a, 7c, 7f, and

7h; separate group for each test compound), and PL-

induced plus physical mixture of same test compound

(1 ? 1a, 1 ? 1c, 1 ? 1f, and 1 ? 1h; separate group for

Med Chem Res

123

each physical mixture). Gastric ulcers were produced by

ligation of the pyloric end of the rat stomach (Shay et al.,

1945). The abdomen was opened under ether anesthesia

below the xiphoid process; the pyloric portion of the

stomach was slightly lifted and ligated avoiding any

damage to the adjacent blood vessels. Test compounds and

their physical mixtures were administered orally at their

anti-inflammatory doses 1 h prior to pyloric ligation.

Animals were sacrificed 8 h after pyloric ligation and the

stomachs were collected for ulcer scores and biochemical

studies.

Biochemical evaluation

Preparation of tissue homogenate

The glandular parts of excised stomachs from ulcer studies

were homogenized in ice cold phosphate buffer (pH 7.4)

with a Potter–Elvehjerr glass homogenizer for 30 s. The

homogenate was centrifuged at 8009g for 10 min. The

supernatant was again centrifuged at 12,0009g for 15 min

and the obtained postmitochondrial fraction (PMF) was

used for following estimations.

Estimation of lipid peroxidation

The malondialdehyde (MDA) content, a measure of lipid

peroxidation, was assayed in the form of thiobarbituric

acid-reactive substances by the method of Wills (1965). In

brief, 0.5 mL of postmitochondrial supernatant and 0.5 mL

of Tris–hydrochloric acid were incubated at 37 �C for 2 h.

After incubation, 1 mL of 10 % trichloroacetic acid was

added and centrifuged at 10009g for 10 min. To 1 mL of

supernatant, 1 mL of 0.67 % thiobarbituric acid was added

and the tubes were kept in boiling water for 10 min. After

cooling, 1 mL of double-distilled water was added and

absorbance was measured at 532 nm. Thiobarbituric acid-

reactive substances were quantified using an extinction

coefficient of 1.56 9 105 M-1/cm and expressed as nano-

mole of MDA per milligram of protein. Tissue protein was

estimated using the Biuret method, and the gastric MDA

content expressed as nanomole of MDA per milligram of

protein.

Estimation of reduced glutathione (GSH)

Reduced glutathione was assayed by the method of Jollow

et al. (1974). In brief, 1 mL of postmitochondrial super-

natant (10 %) was precipitated with 1 mL of sulfosalicylic

acid (4 %). The samples were kept at 4 �C for at least 1 h

and then subjected to centrifugation at 12009g for 15 min

at 4 �C. The assay mixture contained 0.1 mL supernatant,

2.7 mL phosphate buffer (0.1 M, pH 7.4), and 0.2 mL 5,5-

dithiobis-(2-nitro benzoic acid) (Ellman’s reagent,

0.1 mM, pH 8) in a total volume of 3.0 mL. The yellow

color developed was read immediately at 412 nm, and

GSH levels were calculated using molar extinction coeffi-

cient of 1.36 9 104 M-1 cm-1 and expressed as micro-

mole per milligram protein.

Estimation of superoxide dismutase activity

Cytosolic superoxide dismutase activity was assayed by the

method of Kono (1978). The assay system consisted of

0.1 mM ethylenediamine tetra-acetic acid, 50 mM sodium

carbonate, and 96 mM of nitro blue tetrazolium. In the

cuvette, 2 mL of above mixture was taken, and to it,

0.05 mL of postmitochondrial supernatant and 0.05 mL of

hydroxylamine hydrochloride (adjusted to pH 6 with

sodium hydroxide) were added. The auto-oxidation of

hydroxylamine was observed by measuring the change in

optical density at 560 nm for 2 min at 30-/60-s intervals.

The superoxide dismutase activity was expressed as units

per milligram protein.

Estimation of catalase

Catalase activity was assayed by the method of Claiborne

(1985). In brief, the assay mixture consisted of 1.95 mL

phosphate buffer (0.05 M, pH 7), 1 mL hydrogen peroxide

(0.019 M), and 0.05 mL postmitochondrial supernatant

(10 %) in a final volume of 3 mL. Changes in absorbance

were recorded at 240 nm. Catalase activity was quantified

using the millimolar extinction coefficient of hydrogen per-

oxide (0.07 mM) and expressed as micromoles of hydrogen

peroxide decomposed per minute per milligram protein.

In silico ADME profiling

Preparation of ligands

Structures of the ligands (7a–7j) were sketched using built

panel of Maestro (version 9.2, Schrodinger, 2011) and taken

in .mae format. LigPrep (version 2.5, Schrodinger, 2011) is a

utility of Schrodinger software suit that combines tools for

generating 3D structures from 1D (Smiles) and 2D (SDF)

representation, searching for tautomers, steric isomers and

perform a geometry minimization of the ligands. Molecular

Mechanics Force Fields (OPLS_2005) with default settings

were employed for the ligand minimization.

Calculation of QikProp descriptors for prediction

of ADME properties

QikProp is a quick, accurate, easy-to-use absorption, dis-

tribution, metabolism, and excretion (ADME) prediction

Med Chem Res

123

program design to produce certain descriptors related to

ADME. QikProp predicts physically significant descriptors

and pharmaceutically relevant properties of organic mole-

cules, either individually or in batches. QikProp has two

modes: normal mode and fast mode. In fast mode, certain

time-consuming calculations are omitted, some properties

are not predicted, and some have different values. In this

study, QikProp was run in normal processing mode with

default options (QikProp version 3.4, Schrodinger, 2011;

Ravindranathan et al., 2010). After preparing the ligands,

the program QikProp that generate the descriptors was run

with default options that were chosen to produce reason-

able descriptors. The selected properties or descriptors such

as molecular weight, computed dipole moment, total sol-

vent accessible surface area, donor hydrogen bonds,

acceptor hydrogen bonds, predicted octanol/water partition

coefficient (QPlogPo/w), predicted aqueous solubility

(QPlogS), human oral absorption, number of violations of

Lipinski’s rule of five, predicted blood/brain partition

coefficient (QPlogBB), and Van der Waals surface area

(PSA) that are known to influence metabolism, cell per-

meation, and bioavailability were calculated and compared

with the mean values described in the program for 95 % of

the drugs (Table 6).

Statistical analysis

Statistical analysis was carried out using one-way analysis

of variance (ANOVA). In all cases, post-hoc comparisons

of the means of individual groups were performed using

Dunnett’s test. A significance level of p \ 0.05 denoted the

significance in all cases.

Acknowledgments The research grant provided by the University

Grants Commission to MSD is duly acknowledged. Authors profusely

thank to Mr. Avtar Singh, SAIF (CIL), PU for carrying out the NMR

studies. Authors pay homage to Late Professor Pritam Dev Sharma,

one of the investigators. This work would have not been possible

without his guidance.

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