Triblock Conjugates: Identification of a Highly Potent Antiinflammatory Agent

Article abstract of DOI:10.1021/acs.jmedchem.5b00952

Rationally designed conjugates of chrysin, indole, and barbituric acid were synthesized and screened for their antiinflammatory activities through in vitro and in vivo experiments. Improved over the previously reported chrysin-indole-pyrazole conjugates a

Full text of DOI:10.1021/acs.jmedchem.5b00952

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Triꢀblock Conjugates: Identification of a Highly
Potent Antiꢀinflammatory Agent
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Palwinder Singh*,†, Jagroop Kaur†, Gurjit Singh‡, Rajbir Bhatti‡
†Department of Chemistry, Guru Nanak Dev University, Amritsarꢀ143005. India
‡Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsarꢀ143005. India
ABSTRACT: Rationally designed conjugates of chrysin, indole and barbituric acid were
synthesized and screened for their antiꢀinflammatory activities through inꢀvitro and inꢀvivo
experiments. Improved over the previously reported chrysinꢀindoleꢀpyrazole conjugates and also
in comparison to the chrysin, indole and barbituric acid based COXꢀ2 inhibitors; the new
compounds have displayed significantly better IC50 for COXꢀ2 and some of them also exhibited
inhibition of 5ꢀLOX enzyme. For one of the test compounds, IC50 for COXꢀ2 and 5ꢀLOX was 1
nM and 1.5 nM, respectively. Investigations on Swiss Albino mice through capsaicin induced
paw lickings and dextran induced inflammation showed that these compounds possess
appreciable analgesic and antiꢀinflammatory activities. Ki, Ka and ∆G for the enzyme –
compound interaction were calculated and found to be in agreement with the biological data. The
experimental results were supported by the molecular docking studies of the compounds in the
active site of COXꢀ2 and 5ꢀLOX. Overall, a highly promising antiꢀinflammatory agent was
identified.
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INTRODUCTION
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The implication of the chronic inflammation in the pathogeneses of arthritis, cancer,
cardiovascular, autoimmune as well as neurological diseases have made it a serious medical
issue.1 Biologically, the immune response of the body against harmful stimuli and pathogens2 –
the inflammation is associated with the alterations in arachidonic acid metabolism. The over
production of leukotrienes and prostanoids (prostaglandins and thromboxane) in arachidonic acid
pathway are basically responsible for the inflammatory diseases.3,4 The cyclooxygenase and
lipoxygenase are the real culprits and hence they are the primary targets of the antiꢀinflammatory
drugs.5 Out of the two isoforms of cyclooxygenase viz. COXꢀ1 and COXꢀ2; COXꢀ1 is a
constitutive enzyme, also known for its housekeeping functions whereas COXꢀ2 is active only
under inducible/inflammatory conditions. Therefore, the antiꢀinflammatory drugs need to
selectively target COXꢀ2, without disturbing the functioning of COXꢀ1.5,6 Navigating the
journey of antiꢀinflammatory drugs, starting from aspirin (nonꢀselective COXꢀ1/2 inhibitor) to
coxibs (selective COXꢀ2 inhibitor), these drugs are still associated with various side effects,7
thereby making a dire need for the development of new chemical entities.
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The combination of pharmacophores of two or more drugs in one molecule while retaining
their desired biological activity – the hybrid molecules are proving as highly beneficial to the
pharmaceutical industry.8,9 Recently, the hybrids of indole – pyrazole, indole – pyrimidine,
indole – chromone and indole – barbituric acid were identified with substantial anticancer and
antiꢀinflammatory activities.10 Expanding the horizon of this approach, the molecules obtained
by the combination of three heterocyclic components namely chrysin – indole – pyrazole and
chrysin – indole – indolinone (1 and 2, Chart 1)11 were found to exhibit better biological profile
than the conjugates of two heterocycles. For further modification of the molecules, it was
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planned to introduce barbiturate in place of pyrazole/indolinone and also increase the length of
the alkyl chain between indole and chrysin of compound 1/2 and thereby compound 3 was
designed. Likewise, compound 4 was planned by replacing pyrazole/indolinone and alkyl linker
of compound 1/2 with barbiturate and 2ꢀpropanol moiety, respectively (Chart 1). The biological
screening of these compounds revealed that they are better COXꢀ2 and 5ꢀLOX inhibitors as well
as antiꢀinflammatory agents in comparison to their analogues 1 and 2.
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Chart 1.
N
Cl
Cl
N
N
O
O
N
Cl
N
( )n
O
( )n
O
O
O
O
OH
OH
O
1a, n = 2, IC50 (COXꢀ2) = 0.7 µM
1b, n = 3, IC50 (COXꢀ2) = 6.2 µM
2, n = 2, IC50 (COXꢀ2) = 7.3 µM
O
R
O
R
N
N
O
O
N
N
O
R
O
R
N
N
O
( )n
O
OH
O
O
OH
O
OH
O
3, n = 2 ꢀ 4, R = H, CH3
4, a:R = H, b: R=CH3
RESULT AND DISCUSSION
Chemistry. Since barbituric acid is associated with a number of biological activities and do
make a part of anticonvulsant,12 antiꢀseizures,13 anticancer,14,10d antiꢀinflammatory,15
antioxidants,16 antidiabetic and antibacterial agents;17 here, we planned to replace pyrazole and
indolinone moieties of compounds 1 and 2 with barbituric acid for the design of compound 3.
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Further, the molecular docking studies instigated to introduce a 2ꢀpropanol moiety between
indole and chrysin and thereby compounds 4 were designed. It was observed that the OH group
in compound (S)ꢀ4a makes Hꢀbond interaction with Arg and Tyr residue and hence may prove
better antiꢀinflammatory agent. Second enantiomer of compound 4a ((R)ꢀ4a) and its racemic
mixture were also prepared for making comparative studies.
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Nꢀalkylation of 3ꢀformyl indole with equivalent amount of 1,3ꢀdibromopropane/1,4ꢀ
dibromobutane/1,5ꢀdibromopentane in the presence of NaH in dimethyl formamide gave the
products 5a – 5c (Scheme 1). During these reactions, compounds 6a – 6c were also obtained.
Reaction of compounds 5a – 5c with chrysin gave compounds 8a – 8c. Compound 8a (1 mmol)
o
was heated with barbituric acid (1 mmol) at 160ꢀ180 C for 15ꢀ20 min and compound 3a was
obtained. Using the same reaction conditions as for the synthesis of 3a; compounds 3b, 3c and
3d–3f were obtained by the reaction of compound 8a – 8c with barbituric acid and 1,3ꢀdimethyl
barbituric acid, respectively (Scheme 1).
Treatment of 3ꢀformylindole with (rac)ꢀepichlorohydrin in the presence of NaH in acetonitrile
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at 25 C gave compound (rac)ꢀ7. Similarly, compound (R)ꢀ7 and (S)ꢀ7 were prepared by the
reaction of 3ꢀformylindole with (R)ꢀepichlorohydrin and (S)ꢀepichlorohydrin, respectively.
o
Reaction of compounds 7 with chrysin in the presence of K2CO3 in acetone at 60 C resulted in
the formation of respective compounds 9. Further treatment of compound 9 with barbituric
acid/N, N′ꢀ dimethylbarbituric acid provided compounds 4a and 4b. In order to cross check the
effect of 2ꢀpropanol moiety (present between indole and chrysin in compound 4) on the COXꢀ
2/5ꢀLOX inhibitory activity of the compounds, it was planned to replace the alkyl linker of
compound 1a with 2ꢀpropanol fragment and hence compound (S)ꢀ10 was obtained by the
reaction of compound (S)ꢀ9 with 1ꢀ(3ꢀchlorophenyl)ꢀ3ꢀmethylꢀ2ꢀpyrazolinꢀ5ꢀone (Scheme 2).
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Scheme 1. Synthesis of compounds 3 and 4.
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6
CHO
7
8
9
CHO
CHO
N
Br
( )n
Br
( )n
N
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i
N
N
H
( )n
Br
Cl
rac/R/S-
CHO
O
5a, n=2 (70%)
5b, n=3 (65%)
5c, n=4 (68%)
NaH, ACN, 0 oCꢀrt, 2ꢀ3h
6a, n=2 (20%)
6b, n=3 (27%)
6c, n=4 (18%)
CHO
HO
O
Ph
N
ii
O
OH
O
chrysin
racꢀ7, Rꢀ7, Sꢀ7 (45%)
CHO
N
chrysin
iii
OHC
( )n
O
N
O
HO
O
O
O
OH
O
8a, n = 2, (65%)
8b, n = 3, (68%)
8c, n = 4, (60%)
OH
racꢀ9, Rꢀ9, Sꢀ9 (46%)
O
O
R
N
R
160ꢀ180 oC
N
160ꢀ180 oC
O
N
O
O
N
O
R
R
R
O
R
O
N
N
O
O
N
N
R
O
O
R
N
N
OH
( )n
O
O
O
O
O
OH
O
OH
3a, n = 2, R = H (70 %)
3b, n = 3, R = H (73 %)
3c, n = 4, R = H (65 %)
3d, n = 2, R = CH3 (76%)
3e, n = 3, R = CH3 (85 %)
3f, n = 4, R = CH3 (63 %)
racꢀ4a, Rꢀ4a, Sꢀ4a; R = H (65 %)
racꢀ4b, Rꢀ4b, Sꢀ4b; R = CH3 (60 %)
Reaction conditions:
i) NaH, DMF, 60 oC, 3h
ii) K2CO3, Acetone, 60 oC, 5ꢀ6h, N2 atm
iii) K2CO3, DMF, 60 oC, 5ꢀ6h, N2 atm
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Scheme 2. Synthesis of compound 10.
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(Z)
N
(Z)
CHO
N
N
Cl
N
O
O
9
N
N
O
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Cl
160ꢀ180 oC
(S)
OH
(S)
OH
O
O
O
O
O
(Z)
(Z)
OH
Sꢀ9
OH
Sꢀ10 (70 %)
COXꢀ1/2 and 5ꢀLOX inhibition screening assays. Compounds 3a – 3f, all isomers of 4a – b
and (S)ꢀ10 were screened for the inhibition of COXꢀ1, COXꢀ2 and 5ꢀLOX enzymes. All the
enzyme immunoassays were performed as per the kit manufacturer’s protocol.18 Compounds
were tested at 10ꢀ5, 10ꢀ6, 10ꢀ7 and 10ꢀ8 M concentration for both COXꢀ1/2 and 5ꢀLOX inhibitory
activities which were quantified by measuring the amount of prostaglandins produced by the
enzymes in the presence of different concentration of the compounds. In general, the amount of
prostaglandin produced by the enzyme in each well of the 96 well plate is inversely proportional
to the concentration of the inhibitor (compound) present therein. Compound (rac)ꢀ4a showed
excellent inhibition of COXꢀ2 with IC50 (50% inhibitory concentration) 2.0 nM while its IC50 for
COXꢀ1 was 0.5 µM. However, enantiomerically pure compound (S)ꢀ4a exhibited still better
inhibition of COXꢀ2 having IC50 1.0 nM. Interestingly, compound (R)ꢀ4a showed very poor
inhibition of both COXꢀ2 and COXꢀ1 indicating that the COXꢀ2 inhibition by (rac)ꢀ4a might be
due to the (S)ꢀ enantiomer only. The selectivity index of (rac)ꢀ4a and (S)ꢀ4a for COXꢀ2 over
COXꢀ1 was much higher than that of diclofenac and indomethacin and almost comparable with
celecoxib. Similar to the results of compound 4a, the IC50 of compound (rac)ꢀ4b and (S)ꢀ4b was
0.01 ꢁM and 0.007 ꢁM, respectively whereas these compounds have IC50 0.5 ꢁM and 0.35 ꢁM
for COXꢀ1. However, (R)ꢀ4b showed poor but equal inhibition of COXꢀ1 and COXꢀ2.
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Table 1. IC50 (µM) of compounds 1, 2, 3, 4 and 10 against COXꢀ1, COXꢀ2 and 5ꢀLOX enzymes.
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Compound
IC50 (µM)
Selectivity
index*
IC50 (µM)
5ꢀLOX
ꢀꢀꢀ
COXꢀ2
COXꢀ1
1a
1b
0.7
118
168.5
14.5
15.7
ꢀꢀꢀ
9
10
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6.2
90
115
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
7.3
2
ꢀꢀꢀ
15.5
7.2
ꢀꢀꢀ
3a
0.4
1
2.5
3b
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
3c
0.07
10
10
142.85
ꢀꢀꢀ
0.8
ꢀꢀꢀ
3d
ꢀꢀꢀ
3e
9
0.06
0.5
0.34
54
0.0066
250
340
7.7
ꢀꢀꢀ
3f
0.002
0.001
7
0.0025
0.0015
ꢀꢀꢀ
(rac)ꢀ4a
(S)ꢀ4a
(R)ꢀ4a
(rac)ꢀ4b
(S)ꢀ4b
(R)ꢀ4b
(rac)ꢀ10
(S)ꢀ10
Indomethacin
Diclofenac
Celecoxib
Chrysin
Zileuton
0.01
0.007
60
0.5
0.35
66
50
5.46
3.78
ꢀꢀꢀ
50
1
0.2
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.13
0.96
0.02
0.04
25.5
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.08
0.07
15
0.08
3.5
ꢀꢀꢀ
ꢀꢀꢀ
375
1.5
ꢀꢀꢀ
39.9
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.3
ꢀꢀꢀ
Barbituric
acid
12
4
0.33
*IC (COXꢀ1)/ IC (COXꢀ2).
50
50
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Compound 3b also showed inhibition of COXꢀ2 with IC50 0.4 µM while its IC50 for COXꢀ1 was
1 µM. Compound 3d exhibited IC50 0.07 ꢁM for COXꢀ2 and showed desirable selectivity for
COXꢀ2 over COXꢀ1. Compounds 3a, 3c and 3e did not inhibit COXꢀ1/2 at the four tested
concentrations (except 3e which have IC50 10 ꢁM for COXꢀ2) whereas compound 3f showed
IC50 0.06 µM for COXꢀ1. In comparison to compound 1a, the modified compound (S)ꢀ10
exhibited better biological profile with IC50 0.15 µM for COXꢀ2. Enantiomeric effect on
biological activity was also visible in the case of compound 10. When compared with the
standard COXꢀ2 inhibitors, compound (rac)ꢀ4a, (S)ꢀ4a, (rac)ꢀ4b, (S)ꢀ4b and 3d were as potent
as diclofenac and celecoxib but their selectivity for COXꢀ2 over COXꢀ1 was better than that of
diclofenac. Interestingly, the COXꢀ2 inhibitory activity of the compounds (S)ꢀ4a, (S)ꢀ4b and 3d
was much improved over those observed with compounds 1 and 2 (Table 1).
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In parallel to the results of COXꢀ2 screening assay, the compounds (rac)ꢀ4a and (S)ꢀ4a
showed appreciable inhibition of 5ꢀlipoxygenase with IC50 2.5 nM and 1.5 nM, respectively
whereas compound (R)ꢀ4a did not inhibit 5ꢀLOX. Compound 3d, 3b and (S)ꢀ4b exhibited IC50
0.8, 7.2 and 3.78 µM, respectively against 5ꢀLOX (Table 1). It is worth to note that the 5ꢀLOX
used in the assay kit was from soybeans but its amino acid sequence identity to the mammalian
5ꢀLOX is highest in the portion of catalytic domain near the iron atom.19 Therefore, it is apparent
from the results of enzyme immunoassays that the 2ꢀpropanol moiety present between indole and
chrysin of compound 4a and the stereochemistry at the asymmetric carbon significantly
contribute for the inhibition of COXꢀ2 and 5ꢀLOX.
Comparison of the COXꢀ2 inhibitory activity of compounds 3a and 4a indicated that it is not
only the replacement of pyrazole moiety of 1a with barbituric acid which is responsible for
improvement in the COXꢀ2 inhibitory activity of 4a but the propanol moiety between indole and
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chrysin and the stereochemistry at the asymmetric carbon also play significant role. Similarly,
the comparison of 3d and 4b for COXꢀ2 inhibitory activity also pointed towards the significance
of the propanol moiety. These observations indicated that in consistent with the literature reports
about the biological activities of compounds that structurally resemble with PAINS,20ꢀ23 the
compounds under present investigation (3 and 4) are not the frequent hitters. Remarkably, the
compounds show selectivity between COXꢀ1 and COXꢀ2 which also indicates their nonꢀ
promiscuous nature. Moreover, it is not only the barbiturate moiety in compound 4 which has
enhanced its COXꢀ2 inhibitory activity over that of compound 1 and 2 but additional structural
features of presence of OH group on the linker between indole and chrysin and the
stereochemistry at the asymmetric centre are also contributing. Hence, the issue of pan assay
interference (PAINS)20,24 for these compounds seems to be insignificant.
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Further to rule out the possibility of aggregation induced nonꢀspecific inhibition of COXꢀ2
and 5ꢀLOX by the compounds under current investigation, the enzyme inhibition assays were
performed in the presence of Triton (a nonꢀionic detergent).25 We observed no change in the
percentage inhibition and IC50 of the compounds for all the three enzymes (Table 2) except in the
case of compound 3f where a considerable loss of enzyme inhibitory activity was recorded.
Evidently, the most potent compounds of the present investigations exhibit specific inhibition of
COXꢀ2 and 5ꢀLOX and do not fall in the category of promiscuous compounds. On the basis of
the results of these immunoassays, compounds 3b, 3d, (S)ꢀ4a, (S)ꢀ4b and (S)ꢀ10 were pursued
for further investigations.
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Table 2. IC50 (µM) of compounds 1, 2, 3, 4 and 10 against COXꢀ1, COXꢀ2 and 5ꢀLOX enzymes
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in the presence of Triton.
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9
Compound
IC50 (µM)
COXꢀ1
115
COXꢀ2
0.67
ꢀꢀꢀ
5ꢀLOX
ꢀꢀꢀ
10
11
12
13
14
15
16
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18
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1a
3a
ꢀꢀꢀ
16
0.42
ꢀꢀꢀ
1
ꢀꢀꢀ
7.0
ꢀꢀꢀ
3b
3c
0.072
10
9
0.81
ꢀꢀꢀ
3d
ꢀꢀꢀ
3e
20
1.5
0.48
0.33
59
ꢀꢀꢀ
3f
0.0019
0.0011
6.5
0.0027
0.0016
ꢀꢀꢀ
(rac)ꢀ4a
(S)ꢀ4a
(R)ꢀ4a
(rac)ꢀ4b
(S)ꢀ4b
(R)ꢀ4b
(rac)ꢀ10
(S)ꢀ10
Celecoxib
Chrysin
Zileuton
0.009
0.006
58
0.51
0.31
62
5.46
4.0
ꢀꢀꢀ
0.2
ꢀꢀꢀ
ꢀꢀꢀ
0.14
0.041
25.0
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
14.5
40
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.3
Analgesic Activity. Swiss albino mice (25 – 35 g) of either sex were used for investigating the
analgesic activities of the compounds. The method used for capsaicinꢀinduced paw licking was
similar to that described by Sakurada et al26 with a few modifications. Briefly, 20 µL of
capsaicin was injected into the plantar surface of the right hind paw. Animals were observed
individually for 10 min after capsaicin administration and the time spent paw licking and
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twitching was recorded as an indicator of analgesia. Animals were divided into 12 groups of 5
each. All treatments were administered intraperitonealy 30 min before capsaicin injection. Group
I was control wherein the animals were injected with vehicle and in group II, animals were
injected with diclofenac at a dose of 25 mg Kgꢀ1. In other groups, the compounds were tested at
doses of 5 and 10 mg Kgꢀ1. The treatment with diclofenac was found to produce a marked
decrease in the number of paw licking and twitching after capsaicin injection. All the tested
compounds were also found to have desirable analgesic activity. At 5 mg Kgꢀ1 dose, compound
(S)ꢀ4a showed analgesic effect comparable with diclofenac but an increase in activity was
observed when the dose was increased to 10 mg Kgꢀ1. In comparison to its racemic mixture, the
analgesic activity of compound (S)ꢀ4a was much improved. Compound 10 also showed
significant analgesic effect at 5 mg Kgꢀ1 dose and the observed effect increases with the increase
in dose from 5 to 10 mg Kgꢀ1 (Figure 1). However, the difference in analgesic activity of (rac)ꢀ
10 and (S)ꢀ10 was not as significant as seen in the case of compound 4a.
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Figure 1. Effect of compounds 3b, 3d, 4a, 4b and 10 on capsaicin induced pain in mice. All
values are expressed as mean ± SEM (n= 5). Statistical significance has been calculated using
one way ANOVA followed by Tukey’s multiple range comparison test. *p < 0.001 vs control
group.
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Antiꢀinflammatory Activity. Compounds 3b, 3d, 4a, 4b and 10 were also evaluated for antiꢀ
inflammatory activity. Swiss albino mice (25ꢀ35 g) of either sex were made the subjects. The
method used for dextran induced inflammation was similar to that described by Maity et al27
with few modifications. Swiss albino mice of either sex were divided into 16 groups comprising
of five animals in each. Group I was treated with vehicle and served as control and group II was
treated with diclofenac (10 mg/kg). In other groups, the compounds were administered at doses 5
and 10 mg kgꢀ1. After 30 min of the treatment with diclofenac or the test compound, acute
inflammation was induced by subplantar injection of 0.2 ml freshly prepared 1% suspension of
dextran in 0.1% carboxymethyl cellulose. The paw thickness was measured using vernier calliper
and recorded every hour up to 6th h, and finally at 24 h (Figure 2). It was observed that
compound 3b, 3d, (rac)ꢀ4a, (rac)ꢀ4b and (rac)ꢀ10 have antiꢀinflammatory effect similar to
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Figure 2. Effect of compounds 3b, 3d, 4a, 4b and 10 on dextran induced inflammation in mice.
All values are expressed as mean ± SEM (n= 5). Statistical significance has been calculated
using one way ANOVA followed by Tukey’s multiple range comparison test. *p < 0.05 vs
control group.
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diclofenac. All these compounds showed a marked decrease in paw thickness at both dose
injections but there was no remarkable increase in the observed effect when dose was increased
from 5 mg kgꢀ1 to 10 mg kgꢀ1. However, in comparison to the standard drug diclofenac, a
significant increase in the antiꢀinflammatory activity was observed with compound (S)ꢀ4a.
Moreover, compound (S)ꢀ4a also showed remarkable dose dependence effect. The antiꢀ
inflammatory activity of compound (S)ꢀ10 was also increased in comparison to (rac)ꢀ10 and an
instant dose response effect was also visible (Figure 2).
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Acute toxicity studies. Acute toxicity studies with compound (S)ꢀ4a were performed on female
mice according to OECD guidelines.28 The animals showed no signs of any behavioral
alterations upon treatment with the most active compound (S)ꢀ4a at 50 and 300 mg Kgꢀ1 doses.
But the animals treated with 2000 mg Kgꢀ1 dose of (S)ꢀ4a showed some sniffing and scratching
movements occasionally in the first hour after treatment. However, these movements subsided
within the next two hours. Body weight of the animals showed variation in a narrow range of
±2g. Histological examination did not reveal any significant alterations in the liver, kidney or
myocardium when compared to vehicle treated control (Figure 3).
Support for the cyclooxygenase and lipoxygenase pathway inhibition for antiꢀinflammatory
activity of the compounds. With few modifications, method described by Sakurada et al26 was
used to assess the antinociceptive activity. Briefly, capsaicin (1.6 ꢁg in 20 µL of water) was
injected under the plantar surface of the right hind paw and the number of paw lickings was
observed for 10 min after capsaicin injection as a measure of nociception. In groups, the animals
were treated with substance P at a dose of 10 µg i.p. After 30 min, compound (S)ꢀ4a was injected
at a dose of 5 mg Kgꢀ1 followed 30 min later by intraplanter capsaicin injection. The treatment
with compound (S)ꢀ4a produced a marked decrease in the number of paw lickings after capsaicin
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C
D
E
F
Figure 3. (A) (C) (E): Histology of liver, kidney and myocardium of control (40x). (B) (D) (F):
Histology of liver, kidney and myocardium after treatment with compound (S)ꢀ4a (40x).
Compound (S)ꢀ4a was administered at a dose of 2000 mg Kgꢀ1.
injection. Preꢀtreatment with substance P decreased the analgesic effect of compound (S)ꢀ4a
(Figure 4). Substance P has been reported to increase the activity of lipoxygenase leading to
more production of LTB429 and also substance P is documented to increase the expression of
COXꢀ2 thereby increasing the production of PGE2.30 Thus, it may be concluded that compound
(S)ꢀ4a has analgesic effect in capsaicin induced algesia due to inhibition of both cyclooxygenase
and lipoxygenase pathways.
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Figure 4. Effect of Substance P preꢀtreatment on analgesic effect of compound Sꢀ4a. All values
are expressed as mean ± SEM. *p < 0.05 vs control group.
Studies on direct binding of the compounds to the enzymes: UVꢀvis and isothermal
calorimetric experiments
In order to support the results of enzyme immunoassays and for checking the extent of
interactions between the compounds and COXꢀ2/5ꢀLOX enzymes, the kinetic and
thermodynamics of the enzyme – ligand interactions were investigated. Out of all the other
techniques, ITC is the one which is capable of measuring not only the binding affinity but also
the enthalpy (ꢂH) and entropy changes (ꢂS). The solution of compound 3 and 4 was titrated into
the sample cell containing the enzyme in TrisꢀHCl buffer (pH 7.4). For each experiment, 19
consecutive injections of 2 µL of each compound (100 µM) were given to the sample cell after
regular time intervals of 120 s to ensure the equilibrium in each titration point. The total heat Q
produced/absorbed in the active cell, determined at fractional saturation Θ after the ith injection,
is given by equation 1
Q = n ΘMt ꢂHV0
(1)
Mt is the total concentration of the enzyme, V0 is the cell volume, n is the total number of
binding sites in the enzyme and ꢂH is the molar heat of ligand binding
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The enthalpy change for the ith injection ꢂH(i) for an injection volume dVi is defined by
3
4
5
6
equation 2.
ꢂH(i) = Q(i) + dVi/V0 [Q(i) – Q(iꢀ1)/2] – Q(iꢀ1)
(2)
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The various parameters determined from ITC experiments for the titration of compound 3 and 4
against 5ꢀLOX are given in table 3 (Figure 5). Same parameters during the interaction of
compound (S)ꢀ4a with COXꢀ2 were also determined.
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Table 3. Isothermal calorimetric data of compounds 3 and 4 for 5ꢀLOX and COXꢀ2.
Compd
3b
Ka (Mꢀ1)
ꢁH (KJ/mol)
ꢀ287.11
TꢁS (KJ/mol)
ꢀ259.34
ꢁG (KJ/mol)
ꢀ27.76
(6.48±1.81)x104
(3.16±0.49)x105
(2.36±1.09)x104
(6.34±0.89)x105
ꢀ248.86
ꢀ216.94
ꢀ31.91
(S)ꢀ4a
ꢀ1619.21
ꢀ253.72
ꢀ1595.94
ꢀ212.65
ꢀ23.26
(rac)ꢀ4a
(S)-4a (for
COXꢀ2)
ꢀ41.07
Figure 5. ITC data of compound 3b,(S)ꢀ4a and (rac)ꢀ4a for 5ꢀLOX. A1,B1: compound 3b, A2,B2:
compound (S)ꢀ4a, A3,B3: compound (rac)ꢀ4b. A. ITC raw data, B. Integrated heat pulse data.
Ka (Association constant) and Ki (Inhibitor constant) of compounds 3a, 3b, 3d, (S)ꢀ4a and
(rac)ꢀ4a for 5ꢀLOX and COXꢀ2. Ka quantifies the strength of the enzyme inhibitor complex
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whereas Ki is the concentration of the inhibitor which is required to decrease the maximum rate
of reaction by half. Small value of the Ki indicates that less concentration of the inhibitor is
required to decrease the enzymatic activity. Hence, Ka and Ki of compounds 3a, 3b, 3d, (S)ꢀ4a
and (rac)ꢀ4a for 5ꢀLOX and (S)ꢀ4a for COXꢀ2 were calculated with the help of UVꢀvis spectral
studies31 using Equation 3.32
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1/Af – Aobs = 1/Af – Afc + 1/ Ka [Af – Afc][L]
(3)
Where, Af = absorbance of free host, Aobs = observed absorbance, Afc = absorbance at saturation
and L = Ligand concentration
Compound (S)ꢀ4a showed significant interaction with 5ꢀLOX having Ka 2.01×105 Mꢀ1.
Similarly, compounds (rac)ꢀ4a and 3a also exhibited Ka 4.7×104 and 6.45×104 Mꢀ1, respectively.
Ki of compound were calculated using Equation 4 (Table 2).
Ki = IC50/1+ [L]/Kd
ꢂG for the enzyme ꢀ compound complex was calculated from Equation 5
ꢂG = ꢀRT ln Ka (5)
Table 4. Ka, Ki and ∆G values of compounds 3 and 4 for 5ꢀLOX enzyme.
(4)
where, Kd = 1/Ka
Ka(Mꢀ1)
6.45 x 104
4.25 x 104
1.81 x 104
Ki (ꢁM)
3.66
∆G (KJ molꢀ1Kꢀ1)
ꢀ27.44
Compd
3a
3.23
ꢀ26.40
3b
3d
0.796
ꢀ24.29
2.01 x 105 0.225x10ꢀ3
4.7 x 104
2.23
5.84 x 105 0.09x10ꢀ3
ꢀ30.26
(S)ꢀ4a
ꢀ26.65
(rac)ꢀ4a
ꢀ38.42
(S)-4a (for COXꢀ2)
It is evident from the data given in table 2 that the most potent compound (S)ꢀ4a exhibit
maximum favourable ∆G. A comparison of the IC50 and Ki values indicated that compound (S)ꢀ
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4a and (rac)ꢀ4a exhibit competitive inhibition of the enzymes (IC50 are different from Ki)
whereas compound 3d showed nonꢀcompetitive inhibition.
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Docking studies. The molecular docking studies helped in the design of the molecules. Further,
to visualize the interactions between the enzyme and the ligand and for rationalizing the trend of
enzyme inhibitory activity of the test compounds, the results of the docking of Sꢀ4a in the active
site of the enzymes and their comparison with the docking results of other molecules are
discussed here. The Hꢀbond and hydrophobic interactions of the compound with the active site
amino acid residues were analyzed. The crystal coꢀordinates of COXꢀ2 in complex with
arachidonic acid (AA) (pdb Id 1CVU)33 showed Hꢀbonds between the carboxylate group of AA
and Y385, S530. R120 also plays crucial role during the metabolic phase of the enzyme.34
Interaction of the compound with these amino acid residues may create its competition with the
natural substrate (AA) for binding to the enzyme and hence results into the higher efficacy of the
compound. Compound (S)ꢀ4a when docked in the active site of COXꢀ2 (pdb Id 1CVU) showed
docking score ꢀ10.2 kcal/mol and a number of interactions with the active site residues were
observed (Table S1, supporting information). The OH group of propanol linker interacts with
R120 (1.82 Å and 2.09 Å) and Y355 (1.69 Å) through Hꢀbond. Hꢀbonding between O of chrysin
unit and R120 (1.94 Å) is also visible (Figure 6, Figure S2). Chrysin unit of (S)ꢀ4a is favourably
placed in the hydrophobic pocket of the enzyme active site (Figure S2).
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For making comparison, compound (R)ꢀ4a and the drugs based on individual fragments of
compound (S)ꢀ4a such as indomethacin, chrysin and barbituric acid (1,3ꢀdicyclohexylꢀ5ꢀmethyl
barbituric acid)15 were docked in the active site of COXꢀ2. Compound (R)ꢀ4a did not show any
Hꢀ bond interaction with the amino acid residues (Figure S3). Indomethacin, chrysin and 1,3ꢀ
dicyclohexylꢀ5ꢀmethyl barbituric acid also exhibit less number of Hꢀbond interactions with the
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Figure 6. Compound Sꢀ4a docked in the active site of COXꢀ2 (pdb ID 1CVU). Red lines
indicate Hꢀbonds (H′s are omitted for clarity).
active site residues than that shown by compound (S)ꢀ4a (Figure S4ꢀS9). Justifying its selectivity
for COXꢀ2 over COXꢀ1, compound (S)ꢀ4a does not enter into the active site of COXꢀ1 (pdb ID
3KK6).35 Comparison of the results of molecular docking of compound 3a (Figure S10, Table
S1) and (S)ꢀ4a in the active site of COXꢀ2 (pdb Id 1CVU) supported the experimental results of
higher efficacy of (S)ꢀ4a than that of 3a. Similar to the biological data, the two enantiomers of
4b exhibited a large difference in their docking behaviour. Compound (S)ꢀ4b gets docked in the
active site of COXꢀ2 (Figure S11) whereas (R)ꢀ4b could not enter into the active site pocket of
the enzyme.
Molecular docking of (S)ꢀ4a in the active site of 5ꢀLOX (pdb Id 3V99)36 also showed its
appreciable interactions with the amino acid residues. HꢀBond interactions between the OH of
propanol linker and Q363 (1.72 Å) and between C=O of barbituric acid unit and N180 (2.17 Å)
were observed (Figure 7, Figure S12,S13). However, in parallel to the results of enzyme
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immunoassays, compound (R)ꢀ4a showed poor interactions with the active site residues of 5ꢀ
LOX and the OH group of propanol moiety is rather engaged in Hꢀbonding with H2O (Figure
S14). Similarly, the comparison between the docking of compound (S)ꢀ4b and (R)ꢀ4b in the
active site of 5ꢀLOX (Figure S15, S16) clearly demonstrates the difference in the 5ꢀLOX
inhibitory activity of these two isomers. Therefore, design of the molecules on the basis of
molecular docking studies and a comparative analysis of the molecular docking results identified
compound (S)ꢀ4a as the most potent candidate for selective inhibition of COXꢀ2.
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Figure 7. Compound (S)ꢀ4a docked in the active site of 5ꢀLOX (pdb ID 3V99). Red lines
indicate Hꢀbonds (H′s are omitted for clarity).
Rationale for the increase in COXꢀ2 inhibitory and antiꢀinflammatory activity of
compounds 3 and 4 over compounds 1 and 2 on the basis of Lipinski parameters. Taking
Lipinski parameters of the compounds into consideration (Table 5), it was observed that
compound 4a has desirable LogP and highest total polar surface area (TPSA) amongst the
compounds studied here. Moreover, compound 4a has largest number of OH/NH groups.
Therefore, the desirable polarity of compound 4a might be contributing for its higher activity
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than the other compounds investigated in the present studies. However, the stereochemistry at
the asymmetric carbon of compound 4a has also made significant contribution towards the
activity of the compound as compound (S)ꢀ4a was found to be more potent than its enantiomer
(R)ꢀ4a.
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Table 5. Lipinski’s values for compounds 1ꢀ4 and 10.
Compound
ClogP TPSA (Å2) nON nOHNH
1a
1b
2
7.37
7.64
9.03
3.94
4.2
99.5
99.5
8
1
1
1
3
3
3
1
1
1
4
2
2
8
86.6
7
3a
3b
3c
3d
3e
3f
147.4
147.4
147.4
125.6
125.6
125.6
167.6
145.9
119.7
10
10
10
10
10
10
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11
9
4.72
4.08
4.35
4.8
4a
4b
10
3.03
3.16
6.45
CONCLUSION
Linkage of chrysin, indole and barbiturate units through appropriate spacer groups provided
highly potent molecules for the inhibition of COXꢀ2 and 5ꢀLOX enzymes. Compound (S)ꢀ4a
showed promising inhibition of COXꢀ2 and desirably negligible inhibition of COXꢀ1. This
compound also exhibited appreciable inhibition of 5ꢀLOX. Introduced on the basis of the results
of molecular docking studies, the presence of 2ꢀpropanol linker between indole and chrysin in
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compound 4a, 4b and 10 and the stereochemistry at the asymmetric carbon seems to be
responsible for the enhanced COXꢀ2 inhibition, analgesic as well as antiꢀinflammatory activities
of these compounds. Irrespective of the presence of barbiturate alkylidene moiety in (S)ꢀ4a; no
loss in its enzyme inhibition activity in presence of Triton, its selectivity for COXꢀ2 over COXꢀ1
and significant role of chiral propanol fragment on the enzyme inhibition are pointing towards
the nonꢀpromiscuous nature of the compound. Overall, a systematic approach of preparing the
conjugate molecules by the selection of suitable heterocycles and appropriate linker between
them provided highly promising antiꢀinflammatory agent.
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EXPERIMENTAL SECTION
1
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General. Melting points were determined in capillaries and are uncorrected. H and C NMR
spectra were recorded on Bruker 500 MHz and 125 MHz NMR spectrometer, respectively using
CDCl3 and DMSOꢀd6 as solvents. Chemical shifts are given in ppm with TMS as internal
reference. J values are given in Hertz. Mass Spectra were recorded on Bruker micrOTOF QII
Mass Spectrometer. IR spectra were recorded on Varian 660 – IR Spectrometer. Reactions were
monitored by thin layer chromatography (TLC) on glass plates coated with silica gel GFꢀ254.
Column Chromatography was performed with 60ꢀ120 mesh silica. The purity of the tested
1
compounds was checked by qHNMR (quantitative H NMR)37 (Figure S99 and accompanying
calculation). Dimethylsulfone was used as internal calibrant (IC) and the purity of the compound
was calculated by using the following formula:
P [%] = nIC.Intt.MWt.mIC. PIC/nt.IntIC.MWIC.ms
Where Int is the integral, MW is the molecular weight, m is the mass, n is the number of protons,
P is the purity (in %), IC is the internal calibrant, s is the sample, and t is the target molecule.
For these experiments, 1H NMR spectra were recorded under the following conditions:
Pulse Program: zg with 90o pulse (Bruker) Spinning status: Nonꢀspinning
Sample temperature: 25 oC Acquired Data points: 64000, Dummy Scans: 4, Acquisition time: 4s
Spectral window: 20 ppm, O1P: 6 ppm, Number of Scans: 64
All the tested compounds were having purity >95%.
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General procedure for the synthesis of compounds 3, 4 and 10. A finely ground mixture of
aldehyde (1 mmol) and active methylene compoundsꢀ barbituric acid/1, 3ꢀdimethyl barbituric
acid/ 1ꢀ(3ꢀchlorophenyl)ꢀ3ꢀmethylꢀ2ꢀpyrazolinꢀ5ꢀone (1 mmol) was heated at 160ꢀ180 oC for 10ꢀ
20 min. The reaction was monitored with TLC. The crude reaction mass was then solidified by
washing with diethyl ether. The products were purified by washing with ethanol and hot water.
General procedure for the synthesis of compounds 8aꢀ 8c. Indoleꢀ3ꢀcarboxaldehyde was
treated with 1,3ꢀdibromopropane/1,4ꢀdibromobutane/1,5ꢀdibromopentane in the presence of NaH
in dimethyl formamide at 60 oC for 3h to get products 5a, 5b and 5c respectively. Further 5a, 5b
and 5c were treated with chrysin in the presence of K2CO3 in acetone (under nitrogen
atmosphere) at 60 oC for 6 – 7 h to give products 8a, 8b and 8c respectively.
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5ꢀ{1ꢀ[3ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpropyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
pyrimidineꢀ2,4,6ꢀtrione (3a). Compound 3a was synthesized by the general procedure given
o
above using 8a and barbituric acid. Yellow solid; yield 70%; mp 280ꢀ283 C. IR (KBr): 3453,
1
3048, 1738, 1692, 1655, 1613 cmꢀ1. H NMR (500 MHz, DMSOꢀd6) δ: 2.34ꢀ2.37 (m, 2H), 4.18
(t, J = 6.0 Hz, 2H), 4.60 (t, J = 6.0 Hz, 2H), 6.33 (d, J = 2 Hz, 1H), 6.72 (d, J = 2 Hz, 1H), 7.03
(s, 1H), 7.36ꢀ7.38 (m, 2H), 7.58ꢀ7.64 (m, 3H), 7.75ꢀ7.77 (m, 1H), 7.91ꢀ7.92 (m, 1H), 8.07 (d, J =
2 Hz, 2H), 8.69 (s, 1H, =CH), 9.57 (s, 1H), 11.02 (s, 1H, NH), 11.11 (s, 1H, NH), 12.79 (s, 1H,
OH). 13C NMR (125 MHz, DMSOꢀd6) δ: 29.1, 44.6, 66.2, 93.7, 99.0, 105.8, 109.1, 111.1, 112.2,
122.3, 118.4, 123.2, 124.3, 126.9, 129.6, 130.2, 131.0, 132.6, 136.9, 142.5, 143.3, 150.8, 157.8,
161.6, 163.6, 163.9, 164.7, 164.9, 182.5. HRMS (ESI) m/z for C31H23N3O7 [M+Na]+ calcd.
572.1428, found 572.1426.
5ꢀ{1ꢀ[4ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀbutyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
pyrimidineꢀ2,4,6ꢀtrione (3b). Compound 3b was synthesized by the general procedure given
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above using 8b and barbituric acid. Yellow solid; yield 73%; mp 295ꢀ298 C. H NMR (500
MHz, DMSOꢀd6) δ: 1.75ꢀ1.81 (m, 2H), 1.97ꢀ2.03 (m, 2H), 4.13 (t, J = 6.5 Hz, 2H), 4.49 (t, J =
7.0 Hz, 2H), 6.32 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 7.00 (s, 1H), 7.35ꢀ3.39 (m, 2H),
7.55ꢀ7.61 (m, 3H), 7.75 (d, J = 7.5 Hz, 1H), 7.87 (d, J = 7.0 Hz, 1H), 8.06 (d, J = 7.0 Hz, 2H),
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8.64 (s, 1H), 9.55 (s, 1H, =CH), 11.02 (s, 1H, NH), 11.09 (s, 1H, NH), 12.76 (s, 1H, OH). C
NMR (125 MHz, DMSOꢀd6) δ: 26.0, 26.3, 47.1, 68.3, 93.7, 98.9, 105.3, 105.8, 109.0, 111.0,
112.3, 118.4, 123.5, 124.2, 126.9, 129.6, 130.2, 131.0, 132.6, 136.9, 142.2, 143.3, 150.8, 157.7,
161.6, 163.6, 163.9, 164.9, 164.9, 182.5. HRMS (ESI) m/z for C32H25N3O7 [M+Na]+ calcd.
586.1584, found 586.1564.
5ꢀ{1ꢀ[5ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpentyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
pyrimidineꢀ2,4,6ꢀtrione (3c). Compound 3c was synthesized by the general procedure given
o
1
above using 8c and barbituric acid. Yellow solid; yield 65%; mp 290ꢀ 295 C. H NMR (500
MHz, DMSOꢀd6) δ: 1.47ꢀ1.48 (m, 2H), 1.79ꢀ1.81 (m, 2H), 1.91ꢀ1.96 (m, 2H), 4.09 (t, J = 5 Hz,
2H), 4.46 (t, J = 5 Hz, 2H), 6.34 (d, J = 2 Hz, 1H), 6.77 (d, J = 2 Hz, 1H), 7.03 (s, 1H), 7.37ꢀ7.38
(m, 2H), 7.58ꢀ7.63 (m, 3H), 7.75 (d, J = 5Hz, 1H), 7.90 (d, J = 5 Hz, 1H), 8.10 (d, J = 10 Hz,
2H), 8.68 (s, 1H, =CH), 9.56 (s, 1H), 11.03 (s, 1H, NH), 11.12 (s, 1H, NH), 12.78 (s, 1H, OH).
13C NMR (125 MHz, DMSOꢀd6) δ: 23.0, 28.3, 29.3, 47.3, 68.7, 39.6, 98.9, 105.3, 105.8, 108.9,
111.0, 112.3, 118.3, 123.5, 124.2, 126.9, 129.6, 130.2, 131.0, 132.6, 136.9,142.3, 143.3, 150.8,
157.8, 161.5, 163.6, 163.9, 164.9, 165.1, 182.5. HRMS (ESI) m/z for C33H27N3O7 [M+Na]+
calcd. 600.1741, found 600.1736.
5ꢀ{1ꢀ[3ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpropyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
1,3ꢀdimethylꢀpyrimidineꢀ2,4,6ꢀtrione (3d). Compound 3d was synthesized by the general
procedure given above using 8a and 1,3 dimethyl barbituric acid. Yellow solid; yield 76%; mp
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288ꢀ290 oC. 1H NMR (500 MHz, CDCl3+TFA) δ: 2.51ꢀ2.55 (m, 2H), 3.40 (s, 3H), 3.45 (s, 3H),
4.15 (t, J = 5.5 Hz, 2H), 4.63 (t, J = 7.0 Hz, 2H), 6.45 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 2.0 Hz,
1H), 7.08 (s, 1H), 7.28 (s, 1H), 7.43ꢀ7.49 (m, 2H), 7.52 (d, J = 7.0 Hz, 1H), 7.56ꢀ7.59 (m, 1H),
7.62ꢀ7.64 (m, 1H), 7.92ꢀ7.94 (m, 2H), 8.03 (d, J = 7.5 Hz, 1H), 9.05 (s, 1H, =CH), 9.70 (s, 1H).
13C NMR (125 MHz, CDCl3+TFA) δ: 28.6, 29.0, 29.3, 44.8, 65.2, 93.7, 99.6, 104.3, 105.1,
106.1, 110.9, 113.3, 113.5, 115.6, 119.1, 124.5, 125.1, 126.6, 129.3, 130.2, 132.9, 136.9, 144.5,
148.0, 151.9, 158.1, 161.3, 162.8, 165.2, 166.1, 182.8. HRMS (ESI) m/z for C33H27N3O7
[M+Na]+ calcd. 600.1741, found 600.1703.
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5ꢀ{1ꢀ[4ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀbutyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
1,3ꢀdimethylꢀpyrimidineꢀ2,4,6ꢀtrione (3e). Compound 3e was synthesized by the general
procedure given above using 8b and 1,3ꢀdimethyl barbituric acid. Yellow solid; yield 85%; mp
265 oC. 1H NMR (500 MHz, CDCl3+TFA) δ: 1.93ꢀ1.98 (m, 2H), 2.20ꢀ2.26 (m, 2H), 3.46 (s, 6H),
4.11 (t, J = 6.0 Hz, 2H), 4.44 (t, J = 7.0 Hz, 2H), 6.39 (d, J = 2.0 Hz, 1H), 7.01 (s, 1H), 7.28 (s,
1H), 7.43ꢀ7.45 (m, 2H), 7.49ꢀ7.51 (m, 1H), 7.55ꢀ7.61 (m, 3H), 7.92 (d, J = 7.5 Hz, 2H), 8.00ꢀ
8.02 (m, 1H), 9.01 (s, 1H, =CH), 9.68 (s, 1H); 13C NMR (125 MHz, CDCl3+TFA) δ: 26.1, 26.3,
28.4, 29.1, 47.7, 67.8, 93.4, 99.2, 104.7, 105.2, 107.1, 110.9, 112.7, 119.0, 123.9, 124.6, 126.5,
129.2, 130.5, 130.7, 132.5, 136.7, 143.2, 147.1, 151.7, 158.0, 161.6, 162.5, 164.6, 165.3, 165.4,
182.8. HRMS (ESI) m/z for C34H29N3O7 [M+Na]+ calcd. 614.1897, found 614.1837.
5ꢀ{1ꢀ[5ꢀ(5ꢀHydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpentyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ
1,3ꢀdimethylꢀpyrimidineꢀ2,4,6ꢀtrione (3f). Compound 3f was synthesized by the general
procedure given above using 8c and 1,3ꢀdimethyl barbituric acid. Yellow solid; yield 63%; mp
233ꢀ235 oC. IR (KBr): 3445, 1716, 1655, 1610 cmꢀ1. 1H NMR (500 MHz, CDCl3+TFA) δ: 1.60ꢀ
1.66 (m, 2H), 1.89ꢀ1.95 (m, 2H), 2.07ꢀ2.13 (m, 2H), 3.45 (s, 3H), 3.46 (s, 3H), 4.07 (t, J = 6.0
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Hz, 2H), 4.39 (t, J = 7.0 Hz, 2H), 6.40 (d, J = 2 Hz, 1H), 6.54 (d, J = 2 Hz, 1H), 7.04 (s, 1H),
7.44ꢀ7.47 (m, 2H), 7.48ꢀ7.51 (m, 1H), 7.56 (t, J = 8 Hz, 2H), 7.60ꢀ7.63 (m, 1H), 7.93 (d, J = 2H),
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8.03 (d, J = 1.5 Hz, 1H), 9.03 (s, 1H, =CH), 9.67 (s, 1H). C NMR (125 MHz, CDCl3+TFA) δ:
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23.2, 28.3, 28.4, 29.2, 29.2, 48.0, 68.2, 93.5, 99.4, 104.5, 104.9, 111.0, 113.4, 115.7, 119.0,
124.1, 124.7, 126.6, 129.3, 130.4, 130.7, 132.6, 136.8, 143.8, 147.6, 151.8, 158.1, 161.4, 162.7,
165.0, 165.7, 165.8, 182.8. HRMS (ESI) m/z for C35H31N3O7 [M+Na]+ calcd. 628.2054, found
628.2017.
1ꢀOxiranylmethylꢀ3a,7aꢀdihydroꢀ1Hꢀindoleꢀ3ꢀcarboxaldehyde (7). A mixture of indoleꢀ3ꢀ
carboxaldehyde (1 mmol) and rac/R/Sꢀ epichlorohydrin (1.5 mmol) was stirred in acetonitrile in
the presence of NaH (0 oC to room temp) for 2ꢀ3 h to give rac/R/Sꢀ Nꢀsubstituted product 7. The
completion of reaction was monitored with TLC. The crude product was purified by column
chromatography using ethyl acetate: hexane (2:8) as eluent. White solid; yield 45%; mp 85ꢀ88
oC. IR (KBr): 2804, 2759, 1648, 1389, 1333 cmꢀ1. 1H NMR (500 MHz, CDCl3) δ: 2.50ꢀ2.51 (dd,
J = 2.5 Hz, J = 4.6 Hz, 1H), 2.88 (t, J = 4.5 Hz, 1H), 3.34ꢀ3.37 (m, 1H), 4.15ꢀ4.19 (dd. J = 5.8
Hz, J = 15.2 Hz, 1H), 4.56ꢀ4.59 (dd, J = 2.6 Hz, J = 15.2 Hz, 1H), 7.33ꢀ7.39 (m, 2H), 7.43ꢀ7.44
13
(m, 1H), 7.78 (s, 1H), 8.33ꢀ8.34 (m, 1H), 10.02 (s, 1H, CHO); C NMR (125 MHz, CDCl3) δ:
45.1, 48.4, 50.2, 109.8, 118.7, 122.3, 123.1, 124.3, 125.2, 137.5, 138.8, 184.7. HRMS (ESI) m/z
o
for C12H11NO2 [M+H]+ calcd. 202.0862, found 202.0838. [α]D (25 C) = 22o ((S)ꢀ7) and ꢀ17o
((R)ꢀ7) (1, DMSO).
1ꢀ[2ꢀHydroxyꢀ3ꢀ(5ꢀhydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpropyl]ꢀ1Hꢀindoleꢀ3ꢀ
carbaldehyde (9). A mixture of rac/R/Sꢀ 7 (1 mmol) and chrysin (1 mmol) was heated in
o
dimethyl formamide in the presence of potassium carbonate at 60 C for 5ꢀ6 h under nitrogen
atmosphere to get respective compound 9. The crude product was purified by column
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chromatography using ethyl acetate: hexane (3:7) as eluent. Light yellow solid; yield 46%; mp
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185ꢀ190 C. IR (KBr): 3302, 3069, 2825, 2770, 1655, 1625 cmꢀ1. H NMR (500 MHz, DMSOꢀ
d6) δ: 4.12ꢀ4.14 (m, 2H), 4.26 (s, 1H), 4.33ꢀ4.37 (m, 1H), 4.53ꢀ4.57 (dd, J = 3 Hz, J = 6.6 Hz,
1H), 5.65 (d, J = 5.5 Hz, 1H, OH), 6.46 (s, 1H), 6.85 (s, 1H), 7.05 (s, 1H), 7.24ꢀ7.32 (m, 2H),
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7.58ꢀ7.70 (m, 4H), 8.10ꢀ8.13 (m, 3H), 8.31 (s, 1H), 9.94 (s, 1H, CHO), 12.82 (s, 1H, OH). C
NMR (125 MHz, DMSOꢀd6) δ: 49.8, 67.8, 70.8, 93.8, 99.1, 105.5, 105.8, 111.7, 117.6, 121.4,
122.68, 123.9, 125.0, 126.9, 129.6, 131.0, 132.6, 137.9, 142.3, 157.7, 161.6, 163.9, 164.9, 182.5,
185.5. HRMS (ESI) m/z for C27H21NO6 [M+H]+ calcd. 456.1441, found 456.1432.
5ꢀ{1ꢀ[2ꢀHydroxyꢀ3ꢀ(5ꢀhydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpropyl]ꢀ1Hꢀindolꢀ3ꢀ
ylmethylene}ꢀpyrimidineꢀ2,4,6ꢀtrione (4a). Compound rac/R/S– 4a was synthesized by the
general procedure given above using compound rac/R/S– 9 and barbituric acid. Yellow solid;
yield 65%; mp 280ꢀ285 oC. IR (KBr): 3409, 3175, 3043, 1730, 1689, 1654, 1612 cmꢀ1. 1H NMR
(500 MHz, DMSOꢀd6) δ: 4.22ꢀ4.27 (m, 2H), 4.36 (s, 1H), 4.51ꢀ4.56 (m, 1H), 4.74ꢀ4.77 (m, 1H),
5.78 (d, J = 6 Hz, 1H, OH), 6.55 (d, J = 2 Hz, 1H), 6.94 (d, J = 1 Hz, 1H), 7.14 (s, 1H), 7.45 (d, J
= 9 Hz, 2H), 7.68ꢀ7.73 (m, 3H), 7.86ꢀ7.88 (m, 1H), 7.99ꢀ8.01 (m, 1H), 8.21 (d, J = 8 Hz, 2H),
13
8.79 (s, 1H, =CH), 9.70 (s, 1H), 11.14 (s, 1H, NH), 11.22 (s, 1H, NH), 12.91 (s, 1H, OH); C
NMR (125 MHz, DMSOꢀd6) δ: 50.6, 67.8, 70.8, 93.9, 99.1, 105.5, 105.8, 109.0, 111.0, 112.4,
118.2, 123.4, 124.1, 126.9, 129.6, 130.2, 131.0, 132.6, 137.3, 143.4, 143.7, 150.9, 157.7, 161.6,
163.6, 163.9, 164.8, 165.0, 182.5. HRMS (ESI) m/z for C31H23N3O8 [M+K]+ calcd. 604.1116,
found 604.1114. [α]D (25 oC) = 21o ((S)ꢀ4a), ꢀ16o ((R)ꢀ4a) (1, DMSO).
5ꢀ{1ꢀ[2ꢀHydroxyꢀ3ꢀ(5ꢀhydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀpropyl]ꢀ1Hꢀindolꢀ3ꢀ
ylmethylene}ꢀ1,3ꢀdimethylꢀpyrimidineꢀ2,4,6ꢀtrione (4b). Compound rac/R/S– 4b was synthesized
by the general procedure given above using rac/R/S– 9 and 1,3ꢀdimethyl barbituric acid. Yellow
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solid; yield 60%; mp 262ꢀ265 oC. IR (KBr): 3410, 3148, 3058, 1716, 1661, 1612 cmꢀ1. 1H NMR
(500 MHz, CDCl3 + TFA) δ: 3.27 (s, 3H), 3.33 (s, 3H), 4.23 (s, 2H), 4.54ꢀ4.64 (m, 3H), 6.45 (s,
1H), 6.59 (s, 1H), 6.98 (s, 1H), 7.38 (s, 2H), 7.44ꢀ7.60 (m, 4H), 7.88 (d, J = 7.0 Hz, 3H), 8.82 (s,
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1H, =CH), 9.63 (s, 1H). C NMR (175 MHz, CDCl3+TFA) δ: 28.4, 29.1, 50.3, 68.8, 69.4, 93.6,
99.3, 104.7, 105.6, 107.2, 110.8, 113.0, 113.4, 115.6, 118.8, 124.1, 124.8, 126.5, 129.3, 130.3,
132.7, 137.0, 144.2, 147.3, 151.5, 158.0, 161.7, 162.6, 164.5, 165.8, 182.8. HRMS (ESI) m/z for
C33H27N3O8 [M+H]+ calcd. 594.1870, found 594.1846. [α]D (25 oC) = 20o ((S)ꢀ4b), ꢀ16o ((R)ꢀ4b)
(1, DMSO).
2ꢀ(3ꢀChloroꢀphenyl)ꢀ4ꢀ{1ꢀ[2ꢀhydroxyꢀ3ꢀ(5ꢀhydroxyꢀ4ꢀoxoꢀ2ꢀphenylꢀ4Hꢀchromenꢀ7ꢀyloxy)ꢀ
propyl]ꢀ1Hꢀindolꢀ3ꢀylmethylene}ꢀ5ꢀmethylꢀ2,4ꢀdihydroꢀpyrazolꢀ3ꢀone (10). Compound (S)ꢀ10
was synthesized from (S)ꢀ9 and 1ꢀ(3ꢀchlorophenyl)ꢀ3ꢀmethylꢀ2ꢀpyrazolinꢀ5ꢀone by the general
o
procedure given above. Yellow solid; yield 70%, mp 175ꢀ178 C. IR (KBr): 3396, 3110, 3048,
1659, 1613 cmꢀ1. H NMR (500 MHz, DMSOꢀd6) δ: 2.41 (s, 3H), 4.18ꢀ4.19 (m, 2H), 4.30 (s,
1
1H), 4.46ꢀ4.51 (m, 1H), 4.67ꢀ4.69 (m, 1H), 5.70ꢀ5.71 (d, J = 5 Hz, 1H, OH), 6.44 (s, 1H), 6.83
(s, 1H), 7.01 (s, 1H), 7.18 (d, J = 10 Hz, 1H), 7.37ꢀ7.43 (m, 3H), 7.57ꢀ7.64 (m, 3H), 7.78 (d, J =
5 Hz, 1H), 7.93ꢀ7.94 (d, J = 5 Hz, 1H), 8.07ꢀ8.12 (m, 4H), 8.22 (s, 1H, =CH), 9.86 (s, 1H), 12.80
(s, 1H). 13C NMR (125 MHz, DMSOꢀd6) δ: 13.5, 50.6, 67.8, 70.8, 93.9, 99.1, 105.5, 105.8,
112.1, 112.3, 116.3, 117.4, 118.2, 119.3, 123.0, 123.8, 124.1, 126.9, 129.2, 129.5, 130.9, 131.0,
132.6, 133.6, 137.4, 137.6, 140.5, 142.5, 152.1, 157.7, 161.6, 163.4, 163.9, 164.8, 182.5. HRMS
(ESI) m/z for C37H28N3O6Cl [M+H]+ calcd. 646.1739, found 646.1726. [α]D (25 C) = 16o (1,
o
DMSO).
Biological studies. For evaluating inꢀvivo analgesic, antiꢀinflammatory and mechanistic studies,
swissꢀalbino mice of either sex were used. The animals were housed at 25 ± 2 ºC under 12h
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light/ 12h dark cycle and free access to food and water in the central animal house at Guru Nanak
Dev University, Amritsar, Punjab. Animals were acclimatized to the laboratory before testing
and were used once throughout the experiments. All the protocols have been duly approved by
the Institutional Ethics Committee for the purpose of Control and Supervision of Experiments on
Animals (CPSCEA), Ministry of Environment and Forests, India
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Analgesic Activity. The capsaicinꢀinduced paw licking method was used which was as similar as
described by Sakurada et al26 with a few modifications. In this method, 20 µL of capsaicin was
injected into the plantar surface of the right hind paw. Animals were observed individually for 10
min after capsaicin administration and during this time number of paw licking and twitching
were recorded as an indicator of analgesia. Animals were divided into 12 groups of 5 each. All
treatments (diclofenac/ compounds) were administered intraperitonealy 30 min before capsaicin
injection. Group I was control wherein the animals were injected with vehicle; in group II,
animals were injected diclofenac at a dose of 25 mg Kgꢀ1. In group III and IV animals were
injected compound 3b at doses of 5 and 10 mg Kgꢀ1, respectively and in group V and VI animals
were injected with compound 3d at doses of 5 and 10 mg Kgꢀ1, respectively intraperitoneally.
Similarly, compounds 10, 4a and 4b were studied in groups VII to XII at doses of 5 and 10 mg
Kgꢀ1. The decrease in number of paw licking and twitching was recorded as an indicator of
analgesic effect of drug diclofenac and synthesized compounds.
Antiꢀinflammatory Activity. The method used for dextran induced inflammation was similar to
that described by Maity et al27 with few modifications. Here Swiss albino mice of either sex
were divided into 16 groups comprising of five animals in each. Group I was treated with vehicle
and served as control, group II was treated with diclofenac (10 mg/kg), groups III and IV were
administered 1a at doses of 5 and 10 mg kgꢀ1, respectively while group V and VI were
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administered 1b at doses of 5 and 10 mg kgꢀ1 respectively; groups VII and VIII were treated with
compound 10 using 5 and 10 mg kgꢀ1 dose and groups IX and X were given respectively 5 and
10 mg kgꢀ1 of compound 3b. Similarly, compounds 3d, 4a and 4b were studied in groups XI to
XIV at doses of 5 and 10 mg Kgꢀ1. 30 min after the respective treatments (diclofenac/ test
compounds), acute inflammation was induced by subplantar injection of 0.2 ml freshly prepared
1% suspension of dextran in 0.1% carboxymethyl cellulose. The paw thickness was measured
using vernier calipers and recorded every hour up to 6th hour and finally at 24 hours. In case of
diclofenac and test compounds the decrease in paw thickness was measured as an indicator of
antiꢀinflammatory effect.
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Acute toxicity studies
Acute toxicity studies were carried out in female mice using the most active compound (S)ꢀ4a
according to OECD guidelines. Briefly, animals were divided into four groups with 3 animals in
each group. Animals were fasted for 4 h prior to dosing and for further 2 h after dosing. The first
group was treated with vehicle, second group was treated with compound (S)ꢀ4a at a dose of 50
mg Kgꢀ1, third group was treated with (S)ꢀ4a at a dose of 300 mg Kgꢀ1 and the fourth treated with
2000 mg Kgꢀ1. All treatments were given orally in a single dose. The animals were observed
continuously for the first 4 h followed by periodic monitoring for 24 h. Thereafter the animals
were observed once daily for a period of 14 days. At the end of 14 days, the animals were
sacrificed; gross examination of liver, kidney and heart was carried out followed by histological
studies using heamatoxylinꢀ eosin staining.
Mechanistic Studies. The method described by Sakurada et al was used with few modifications
to assess the antinociceptive activity as given above. Here the animals are divided into three
groups, five animals each. In all the groups, the animals were treated with substance P at a dose
of 10 µg i.p. After 30 min the compounds 4a was injected at a dose of 5 mg Kgꢀ1 followed 30
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