Triblock Conjugates: Identification of a Highly Potent Antiinflammatory

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Journal of Medicinal Chemistry

Tri-block Conjugates: Identification of a Highly Potent Anti-inflammatory Agent Palwinder Singh*,†, Jagroop Kaur†, Gurjit Singh‡, Rajbir Bhatti‡ †

‡

Department of Chemistry, Guru Nanak Dev University, Amritsar-143005. India

Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005. India

ABSTRACT: Rationally designed conjugates of chrysin, indole and barbituric acid were synthesized and screened for their anti-inflammatory activities through in-vitro and in-vivo experiments. Improved over the previously reported chrysin-indole-pyrazole conjugates and also in comparison to the chrysin, indole and barbituric acid based COX-2 inhibitors; the new compounds have displayed significantly better IC50 for COX-2 and some of them also exhibited inhibition of 5-LOX enzyme. For one of the test compounds, IC50 for COX-2 and 5-LOX 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 anti-inflammatory 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 COX-2 and 5-LOX. Overall, a highly promising anti-inflammatory agent was identified.

ACS Paragon Plus Environment

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INTRODUCTION 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 anti-inflammatory drugs.5 Out of the two isoforms of cyclooxygenase viz. COX-1 and COX-2; COX-1 is a constitutive enzyme, also known for its housekeeping functions whereas COX-2 is active only under inducible/inflammatory conditions. Therefore, the anti-inflammatory drugs need to selectively target COX-2, without disturbing the functioning of COX-1.5,6 Navigating the journey of anti-inflammatory drugs, starting from aspirin (non-selective COX-1/2 inhibitor) to coxibs (selective COX-2 inhibitor), these drugs are still associated with various side effects,7 thereby making a dire need for the development of new chemical entities. 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 anti-inflammatory 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 2-propanol moiety, respectively (Chart 1). The biological screening of these compounds revealed that they are better COX-2 and 5-LOX inhibitors as well as anti-inflammatory agents in comparison to their analogues 1 and 2. Chart 1. N N

Cl

Cl N

O O

N

Cl

N ( )n O

( )n O

O

OH

O

O OH

1a, n = 2, IC50 (COX-2) = 0.7 µM 1b, n = 3, IC50 (COX-2) = 6.2 µM O

O

2, n = 2, IC50 (COX-2) = 7.3 µM

R N

O

R N

O O

O

N R

N R

O

N

N

( )n O

OH O O OH

O

O OH

3, n = 2 - 4, R = H, CH3

O

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

anti-seizures,13 anticancer,14,10d anti-inflammatory,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 2-propanol moiety between indole and chrysin and thereby compounds 4 were designed. It was observed that the OH group in compound (S)-4a makes H-bond interaction with Arg and Tyr residue and hence may prove better anti-inflammatory agent. Second enantiomer of compound 4a ((R)-4a) and its racemic mixture were also prepared for making comparative studies. N-alkylation of 3-formyl indole with equivalent amount of 1,3-dibromopropane/1,4dibromobutane/1,5-dibromopentane 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) was heated with barbituric acid (1 mmol) at 160-180 oC for 15-20 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 1,3-dimethyl barbituric acid, respectively (Scheme 1). Treatment of 3-formylindole with (rac)-epichlorohydrin in the presence of NaH in acetonitrile at 25 oC gave compound (rac)-7. Similarly, compound (R)-7 and (S)-7 were prepared by the reaction of 3-formylindole with (R)-epichlorohydrin and (S)-epichlorohydrin, respectively. Reaction of compounds 7 with chrysin in the presence of K2CO3 in acetone at 60 oC 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 2-propanol moiety (present between indole and chrysin in compound 4) on the COX2/5-LOX inhibitory activity of the compounds, it was planned to replace the alkyl linker of compound 1a with 2-propanol fragment and hence compound (S)-10 was obtained by the reaction of compound (S)-9 with 1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one (Scheme 2).


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Scheme 1. Synthesis of compounds 3 and 4. CHO CHO

CHO ( )n

Br i

N H

N

Br

Cl O NaH, ACN, 0 oC-rt, 2-3h

( )n N

N ( )n Br

rac/R/S-

CHO

5a, n=2 (70%) 5b, n=3 (65%) 5c, n=4 (68%)

6a, n=2 (20%) 6b, n=3 (27%) 6c, n=4 (18%)

CHO HO N

Ph

O

ii O OH O chrysin

rac-7, R-7, S-7 (45%) CHO chrysin iii N

OHC

( )n

N

O

O

HO O

O

OH

OH

O

8a, n = 2, (65%) 8b, n = 3, (68%) 8c, n = 4, (60%)

O

rac-9, R-9, S-9 (46%) O

O N

160-180 oC O

O

R

O

O

N R R N

N

R O

O

N

N R

O

R

N OH

O

N R

R N

O O

O

N

160-180 oC

( )n O

OH O rac-4a, R-4a, S-4a; R = H (65 %) rac-4b, R-4b, S-4b; R = CH3 (60 %) Reaction conditions: i) NaH, DMF, 60 oC, 3h ii) K2CO3, Acetone, 60 oC, 5-6h, N2 atm iii) K2CO3, DMF, 60 oC, 5-6h, N2 atm

O

O

OH O 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 %)


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Scheme 2. Synthesis of compound 10. (Z)

N N

(Z)

CHO

N O

N

Cl

O

N

N Cl

(S)

OH

(S)

OH

160-180 oC O

O

O

O

(Z)

OH

O

(Z)

OH

S-9

O

S-10 (70 %)

COX-1/2 and 5-LOX inhibition screening assays. Compounds 3a – 3f, all isomers of 4a – b and (S)-10 were screened for the inhibition of COX-1, COX-2 and 5-LOX enzymes. All the enzyme immunoassays were performed as per the kit manufacturer’s protocol.18 Compounds were tested at 10-5, 10-6, 10-7 and 10-8 M concentration for both COX-1/2 and 5-LOX 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 COX-2 with IC50 (50% inhibitory concentration) 2.0 nM while its IC50 for COX-1 was 0.5 µM. However, enantiomerically pure compound (S)-4a exhibited still better inhibition of COX-2 having IC50 1.0 nM. Interestingly, compound (R)-4a showed very poor inhibition of both COX-2 and COX-1 indicating that the COX-2 inhibition by (rac)-4a might be due to the (S)- enantiomer only. The selectivity index of (rac)-4a and (S)-4a for COX-2 over COX-1 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 µM and 0.007 µM, respectively whereas these compounds have IC50 0.5 µM and 0.35 µM for COX-1. However, (R)-4b showed poor but equal inhibition of COX-1 and COX-2.


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Table 1. IC50 (µM) of compounds 1, 2, 3, 4 and 10 against COX-1, COX-2 and 5-LOX enzymes. Compound 1a

*IC

50

IC50 (µM) COX-2 COX-1 0.7 118

Selectivity index* 168.5

IC50 (µM) 5-LOX ---

1b

6.2

90

14.5

---

2

7.3

115

15.7

---

3a

---

---

---

15.5

3b

0.4

1

2.5

7.2

3c

---

---

---

---

3d

0.07

10

142.85

0.8

3e

10

---

---

---

3f

9

0.06

0.0066

---

(rac)-4a

0.002

0.5

250

0.0025

(S)-4a

0.001

0.34

340

0.0015

(R)-4a

7

54

7.7

---

(rac)-4b

0.01

0.5

50

5.46

(S)-4b

0.007

0.35

50

3.78

(R)-4b

60

66

1

---

(rac)-10

0.2

---

---

---

(S)-10

0.13

---

---

---

Indomethacin

0.96

0.08

0.08

---

Diclofenac

0.02

0.07

3.5

---

Celecoxib

0.04

15

375

---

Chrysin

25.5

39.9

1.5

---

Zileuton

---

---

---

0.3

Barbituric acid

12

4

0.33

---

(COX-1)/ IC

50

(COX-2).


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Compound 3b also showed inhibition of COX-2 with IC50 0.4 µM while its IC50 for COX-1 was 1 µM. Compound 3d exhibited IC50 0.07 µM for COX-2 and showed desirable selectivity for COX-2 over COX-1. Compounds 3a, 3c and 3e did not inhibit COX-1/2 at the four tested concentrations (except 3e which have IC50 10 µM for COX-2) whereas compound 3f showed IC50 0.06 µM for COX-1. In comparison to compound 1a, the modified compound (S)-10 exhibited better biological profile with IC50 0.15 µM for COX-2. Enantiomeric effect on biological activity was also visible in the case of compound 10. When compared with the standard COX-2 inhibitors, compound (rac)-4a, (S)-4a, (rac)-4b, (S)-4b and 3d were as potent as diclofenac and celecoxib but their selectivity for COX-2 over COX-1 was better than that of diclofenac. Interestingly, the COX-2 inhibitory activity of the compounds (S)-4a, (S)-4b and 3d was much improved over those observed with compounds 1 and 2 (Table 1). In parallel to the results of COX-2 screening assay, the compounds (rac)-4a and (S)-4a showed appreciable inhibition of 5-lipoxygenase with IC50 2.5 nM and 1.5 nM, respectively whereas compound (R)-4a did not inhibit 5-LOX. Compound 3d, 3b and (S)-4b exhibited IC50 0.8, 7.2 and 3.78 µM, respectively against 5-LOX (Table 1). It is worth to note that the 5-LOX used in the assay kit was from soybeans but its amino acid sequence identity to the mammalian 5-LOX 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 2-propanol moiety present between indole and chrysin of compound 4a and the stereochemistry at the asymmetric carbon significantly contribute for the inhibition of COX-2 and 5-LOX. Comparison of the COX-2 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 COX-2 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 COX-2 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,20-23 the compounds under present investigation (3 and 4) are not the frequent hitters. Remarkably, the compounds show selectivity between COX-1 and COX-2 which also indicates their nonpromiscuous nature. Moreover, it is not only the barbiturate moiety in compound 4 which has enhanced its COX-2 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. Further to rule out the possibility of aggregation induced non-specific inhibition of COX-2 and 5-LOX by the compounds under current investigation, the enzyme inhibition assays were performed in the presence of Triton (a non-ionic 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 COX-2 and 5-LOX 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 COX-1, COX-2 and 5-LOX enzymes in the presence of Triton. Compound

IC50 (µM) COX-1 115 ---

1a 3a

COX-2 0.67 ---

5-LOX --16

3b

0.42

1

7.0

3c

---

---

---

3d

0.072

9

0.81

3e

10

---

---

3f

20

1.5

---

(rac)-4a

0.0019

0.48

0.0027

(S)-4a

0.0011

0.33

0.0016

(R)-4a

6.5

59

---

(rac)-4b

0.009

0.51

5.46

(S)-4b

0.006

0.31

4.0

(R)-4b

58

62

---

(rac)-10

0.2

---

---

(S)-10

0.14

---

---

Celecoxib

0.041

14.5

---

Chrysin

25.0

40

---

Zileuton

---

---

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 capsaicin-induced 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 Kg-1. In other groups, the compounds were tested at doses of 5 and 10 mg Kg-1. 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 Kg-1 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 Kg-1. 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 Kg-1 dose and the observed effect increases with the increase in dose from 5 to 10 mg Kg-1 (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.

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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Anti-inflammatory Activity. Compounds 3b, 3d, 4a, 4b and 10 were also evaluated for antiinflammatory activity. Swiss albino mice (25-35 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 kg-1. 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 anti-inflammatory effect similar to

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 kg-1 to 10 mg kg-1. However, in comparison to the standard drug diclofenac, a significant increase in the anti-inflammatory activity was observed with compound (S)-4a. Moreover, compound (S)-4a also showed remarkable dose dependence effect. The antiinflammatory activity of compound (S)-10 was also increased in comparison to (rac)-10 and an instant dose response effect was also visible (Figure 2). 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 Kg-1 doses. But the animals treated with 2000 mg Kg-1 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 anti-inflammatory activity of the compounds. With few modifications, method described by Sakurada et al26 was used to assess the antinociceptive activity. Briefly, capsaicin (1.6 µg 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 Kg-1 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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A

B

C

D

E

F

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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 Kg-1. injection. Pre-treatment 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 COX-2 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 pre-treatment on analgesic effect of compound S-4a. All values are expressed as mean ± SEM. *p < 0.05 vs control group. Studies on direct binding of the compounds to the enzymes: UV-vis and isothermal calorimetric experiments In order to support the results of enzyme immunoassays and for checking the extent of interactions between the compounds and COX-2/5-LOX 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 (∆H) and entropy changes (∆S). The solution of compound 3 and 4 was titrated into the sample cell containing the enzyme in Tris-HCl 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 ∆HV0

(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 ∆H is the molar heat of ligand binding


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The enthalpy change for the ith injection ∆H(i) for an injection volume dVi is defined by equation 2.

∆H(i) = Q(i) + dVi/V0 [Q(i) – Q(i-1)/2] – Q(i-1)

(2)

The various parameters determined from ITC experiments for the titration of compound 3 and 4 against 5-LOX are given in table 3 (Figure 5). Same parameters during the interaction of compound (S)-4a with COX-2 were also determined. Table 3. Isothermal calorimetric data of compounds 3 and 4 for 5-LOX and COX-2. Compd

Ka (M-1)

∆H (KJ/mol)

T∆S (KJ/mol)

∆G (KJ/mol)

3b

(6.48±1.81)x104

-287.11

-259.34

-27.76

(S)-4a

(3.16±0.49)x105

-248.86

-216.94

-31.91

(rac)-4a

(2.36±1.09)x104

-1619.21

-1595.94

-23.26

(S)-4a (for

(6.34±0.89)x105

-253.72

-212.65

-41.07

COX-2)

Figure 5. ITC data of compound 3b,(S)-4a and (rac)-4a for 5-LOX. 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 5-LOX and COX-2. 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 5-LOX and (S)-4a for COX-2 were calculated with the help of UV-vis spectral studies31 using Equation 3.32 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 5-LOX having Ka 2.01×105 M-1. Similarly, compounds (rac)-4a and 3a also exhibited Ka 4.7×104 and 6.45×104 M-1, respectively. Ki of compound were calculated using Equation 4 (Table 2). Ki = IC50/1+ [L]/Kd

(4)

where, Kd = 1/Ka

∆G for the enzyme - compound complex was calculated from Equation 5 ∆G = -RT ln Ka

(5)

Table 4. Ka, Ki and ∆G values of compounds 3 and 4 for 5-LOX enzyme. Compd

Ka(M-1)

Ki (µM)

∆G (KJ mol-1K-1)

3a

6.45 x 104

3.66

-27.44

3b

4.25 x 104

3.23

-26.40

3d

1.81 x 104

0.796

-24.29

(S)-4a

2.01 x 105

0.225x10-3

-30.26

(rac)-4a

4.7 x 104

2.23

-26.65

(S)-4a (for COX-2)

5.84 x 105

0.09x10-3

-38.42

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 non-competitive inhibition. 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 S-4a in the active site of the enzymes and their comparison with the docking results of other molecules are discussed here. The H-bond and hydrophobic interactions of the compound with the active site amino acid residues were analyzed. The crystal co-ordinates of COX-2 in complex with arachidonic acid (AA) (pdb Id 1CVU)33 showed H-bonds 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 COX-2 (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 H-bond. H-bonding 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). For making comparison, compound (R)-4a and the drugs based on individual fragments of compound (S)-4a such as indomethacin, chrysin and barbituric acid (1,3-dicyclohexyl-5-methyl barbituric acid)15 were docked in the active site of COX-2. Compound (R)-4a did not show any H- bond interaction with the amino acid residues (Figure S3). Indomethacin, chrysin and 1,3dicyclohexyl-5-methyl barbituric acid also exhibit less number of H-bond interactions with the


18

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Figure 6. Compound S-4a docked in the active site of COX-2 (pdb ID 1CVU). Red lines indicate H-bonds (H′s are omitted for clarity).

active site residues than that shown by compound (S)-4a (Figure S4-S9). Justifying its selectivity for COX-2 over COX-1, compound (S)-4a does not enter into the active site of COX-1 (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 COX-2 (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 COX-2 (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 5-LOX (pdb Id 3V99)36 also showed its appreciable interactions with the amino acid residues. H-Bond 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 5LOX and the OH group of propanol moiety is rather engaged in H-bonding with H2O (Figure S14). Similarly, the comparison between the docking of compound (S)-4b and (R)-4b in the active site of 5-LOX (Figure S15, S16) clearly demonstrates the difference in the 5-LOX 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 COX-2.

Figure 7. Compound (S)-4a docked in the active site of 5-LOX (pdb ID 3V99). Red lines indicate H-bonds (H′s are omitted for clarity). Rationale for the increase in COX-2 inhibitory and anti-inflammatory 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


20

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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. Table 5. Lipinski’s values for compounds 1-4 and 10. Compound

ClogP

TPSA (Å2)

nON

1a

7.37

99.5

8

1

1b

7.64

99.5

8

1

2

9.03

86.6

7

1

3a

3.94

147.4

10

3

3b

4.2

147.4

10

3

3c

4.72

147.4

10

3

3d

4.08

125.6

10

1

3e

4.35

125.6

10

1

3f

4.8

125.6

10

1

4a

3.03

167.6

11

4

4b

3.16

145.9

11

2

10

6.45

119.7

9

2

nOHNH

CONCLUSION Linkage of chrysin, indole and barbiturate units through appropriate spacer groups provided highly potent molecules for the inhibition of COX-2 and 5-LOX enzymes. Compound (S)-4a showed promising inhibition of COX-2 and desirably negligible inhibition of COX-1. This compound also exhibited appreciable inhibition of 5-LOX. Introduced on the basis of the results of molecular docking studies, the presence of 2-propanol linker between indole and chrysin in


21


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compound 4a, 4b and 10 and the stereochemistry at the asymmetric carbon seems to be responsible for the enhanced COX-2 inhibition, analgesic as well as anti-inflammatory 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 COX-2 over COX-1 and significant role of chiral propanol fragment on the enzyme inhibition are pointing towards the non-promiscuous 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 anti-inflammatory agent. EXPERIMENTAL SECTION General. Melting points were determined in capillaries and are uncorrected. 1H and

13

C NMR

spectra were recorded on Bruker 500 MHz and 125 MHz NMR spectrometer, respectively using CDCl3 and DMSO-d6 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 GF-254. Column Chromatography was performed with 60-120 mesh silica. The purity of the tested compounds was checked by qHNMR (quantitative 1H 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: Non-spinning

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%.


22

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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 compounds- barbituric acid/1, 3-dimethyl barbituric acid/ 1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one (1 mmol) was heated at 160-180 oC for 1020 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 8a- 8c. Indole-3-carboxaldehyde was treated with 1,3-dibromopropane/1,4-dibromobutane/1,5-dibromopentane 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. 5-{1-[3-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3-ylmethylene}pyrimidine-2,4,6-trione (3a). Compound 3a was synthesized by the general procedure given above using 8a and barbituric acid. Yellow solid; yield 70%; mp 280-283 oC. IR (KBr): 3453, 3048, 1738, 1692, 1655, 1613 cm-1. 1H NMR (500 MHz, DMSO-d6) δ: 2.34-2.37 (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), 7.36-7.38 (m, 2H), 7.58-7.64 (m, 3H), 7.75-7.77 (m, 1H), 7.91-7.92 (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, DMSO-d6) 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. 5-{1-[4-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-butyl]-1H-indol-3-ylmethylene}pyrimidine-2,4,6-trione (3b). Compound 3b was synthesized by the general procedure given


23


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Page 24 of 44

above using 8b and barbituric acid. Yellow solid; yield 73%; mp 295-298 oC. 1H NMR (500 MHz, DMSO-d6) δ: 1.75-1.81 (m, 2H), 1.97-2.03 (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), 7.35-3.39 (m, 2H), 7.55-7.61 (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), 8.64 (s, 1H), 9.55 (s, 1H, =CH), 11.02 (s, 1H, NH), 11.09 (s, 1H, NH), 12.76 (s, 1H, OH).

13

C

NMR (125 MHz, DMSO-d6) 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. 5-{1-[5-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-pentyl]-1H-indol-3-ylmethylene}pyrimidine-2,4,6-trione (3c). Compound 3c was synthesized by the general procedure given above using 8c and barbituric acid. Yellow solid; yield 65%; mp 290- 295 oC. 1H NMR (500 MHz, DMSO-d6) δ: 1.47-1.48 (m, 2H), 1.79-1.81 (m, 2H), 1.91-1.96 (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), 7.37-7.38 (m, 2H), 7.58-7.63 (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). 13

C NMR (125 MHz, DMSO-d6) 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. 5-{1-[3-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3-ylmethylene}1,3-dimethyl-pyrimidine-2,4,6-trione (3d). Compound 3d was synthesized by the general procedure given above using 8a and 1,3 dimethyl barbituric acid. Yellow solid; yield 76%; mp


24

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288-290 oC. 1H NMR (500 MHz, CDCl3+TFA) δ: 2.51-2.55 (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), 7.43-7.49 (m, 2H), 7.52 (d, J = 7.0 Hz, 1H), 7.56-7.59 (m, 1H), 7.62-7.64 (m, 1H), 7.92-7.94 (m, 2H), 8.03 (d, J = 7.5 Hz, 1H), 9.05 (s, 1H, =CH), 9.70 (s, 1H). 13

C 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. 5-{1-[4-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-butyl]-1H-indol-3-ylmethylene}1,3-dimethyl-pyrimidine-2,4,6-trione (3e). Compound 3e was synthesized by the general procedure given above using 8b and 1,3-dimethyl barbituric acid. Yellow solid; yield 85%; mp 265 oC. 1H NMR (500 MHz, CDCl3+TFA) δ: 1.93-1.98 (m, 2H), 2.20-2.26 (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), 7.43-7.45 (m, 2H), 7.49-7.51 (m, 1H), 7.55-7.61 (m, 3H), 7.92 (d, J = 7.5 Hz, 2H), 8.008.02 (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. 5-{1-[5-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-pentyl]-1H-indol-3-ylmethylene}1,3-dimethyl-pyrimidine-2,4,6-trione (3f). Compound 3f was synthesized by the general procedure given above using 8c and 1,3-dimethyl barbituric acid. Yellow solid; yield 63%; mp 233-235 oC. IR (KBr): 3445, 1716, 1655, 1610 cm-1. 1H NMR (500 MHz, CDCl3+TFA) δ: 1.601.66 (m, 2H), 1.89-1.95 (m, 2H), 2.07-2.13 (m, 2H), 3.45 (s, 3H), 3.46 (s, 3H), 4.07 (t, J = 6.0


25


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Page 26 of 44

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), 7.44-7.47 (m, 2H), 7.48-7.51 (m, 1H), 7.56 (t, J = 8 Hz, 2H), 7.60-7.63 (m, 1H), 7.93 (d, J = 2H), 8.03 (d, J = 1.5 Hz, 1H), 9.03 (s, 1H, =CH), 9.67 (s, 1H). 13C NMR (125 MHz, CDCl3+TFA) δ: 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. 1-Oxiranylmethyl-3a,7a-dihydro-1H-indole-3-carboxaldehyde (7). A mixture of indole-3carboxaldehyde (1 mmol) and rac/R/S- epichlorohydrin (1.5 mmol) was stirred in acetonitrile in the presence of NaH (0 oC to room temp) for 2-3 h to give rac/R/S- N-substituted 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 85-88 o

C. IR (KBr): 2804, 2759, 1648, 1389, 1333 cm-1. 1H NMR (500 MHz, CDCl3) δ: 2.50-2.51 (dd,

J = 2.5 Hz, J = 4.6 Hz, 1H), 2.88 (t, J = 4.5 Hz, 1H), 3.34-3.37 (m, 1H), 4.15-4.19 (dd. J = 5.8 Hz, J = 15.2 Hz, 1H), 4.56-4.59 (dd, J = 2.6 Hz, J = 15.2 Hz, 1H), 7.33-7.39 (m, 2H), 7.43-7.44 (m, 1H), 7.78 (s, 1H), 8.33-8.34 (m, 1H), 10.02 (s, 1H, CHO); 13C 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 for C12H11NO2 [M+H]+ calcd. 202.0862, found 202.0838. [α]D (25 oC) = 22o ((S)-7) and -17o ((R)-7) (1, DMSO). 1-[2-Hydroxy-3-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indole-3carbaldehyde (9). A mixture of rac/R/S- 7 (1 mmol) and chrysin (1 mmol) was heated in dimethyl formamide in the presence of potassium carbonate at 60 oC for 5-6 h under nitrogen atmosphere to get respective compound 9. The crude product was purified by column


26

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chromatography using ethyl acetate: hexane (3:7) as eluent. Light yellow solid; yield 46%; mp 185-190 oC. IR (KBr): 3302, 3069, 2825, 2770, 1655, 1625 cm-1. 1H NMR (500 MHz, DMSOd6) δ: 4.12-4.14 (m, 2H), 4.26 (s, 1H), 4.33-4.37 (m, 1H), 4.53-4.57 (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), 7.24-7.32 (m, 2H), 7.58-7.70 (m, 4H), 8.10-8.13 (m, 3H), 8.31 (s, 1H), 9.94 (s, 1H, CHO), 12.82 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) 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. 5-{1-[2-Hydroxy-3-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3ylmethylene}-pyrimidine-2,4,6-trione (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 280-285 oC. IR (KBr): 3409, 3175, 3043, 1730, 1689, 1654, 1612 cm-1. 1H NMR (500 MHz, DMSO-d6) δ: 4.22-4.27 (m, 2H), 4.36 (s, 1H), 4.51-4.56 (m, 1H), 4.74-4.77 (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), 7.68-7.73 (m, 3H), 7.86-7.88 (m, 1H), 7.99-8.01 (m, 1H), 8.21 (d, J = 8 Hz, 2H), 8.79 (s, 1H, =CH), 9.70 (s, 1H), 11.14 (s, 1H, NH), 11.22 (s, 1H, NH), 12.91 (s, 1H, OH);

13

C

NMR (125 MHz, DMSO-d6) 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). 5-{1-[2-Hydroxy-3-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3ylmethylene}-1,3-dimethyl-pyrimidine-2,4,6-trione (4b). Compound rac/R/S– 4b was synthesized by the general procedure given above using rac/R/S– 9 and 1,3-dimethyl barbituric acid. Yellow


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solid; yield 60%; mp 262-265 oC. IR (KBr): 3410, 3148, 3058, 1716, 1661, 1612 cm-1. 1H NMR (500 MHz, CDCl3 + TFA) δ: 3.27 (s, 3H), 3.33 (s, 3H), 4.23 (s, 2H), 4.54-4.64 (m, 3H), 6.45 (s, 1H), 6.59 (s, 1H), 6.98 (s, 1H), 7.38 (s, 2H), 7.44-7.60 (m, 4H), 7.88 (d, J = 7.0 Hz, 3H), 8.82 (s, 1H, =CH), 9.63 (s, 1H). 13C 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). 2-(3-Chloro-phenyl)-4-{1-[2-hydroxy-3-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)propyl]-1H-indol-3-ylmethylene}-5-methyl-2,4-dihydro-pyrazol-3-one (10). Compound (S)-10 was synthesized from (S)-9 and 1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one by the general procedure given above. Yellow solid; yield 70%, mp 175-178 oC. IR (KBr): 3396, 3110, 3048, 1659, 1613 cm-1. 1H NMR (500 MHz, DMSO-d6) δ: 2.41 (s, 3H), 4.18-4.19 (m, 2H), 4.30 (s, 1H), 4.46-4.51 (m, 1H), 4.67-4.69 (m, 1H), 5.70-5.71 (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), 7.37-7.43 (m, 3H), 7.57-7.64 (m, 3H), 7.78 (d, J = 5 Hz, 1H), 7.93-7.94 (d, J = 5 Hz, 1H), 8.07-8.12 (m, 4H), 8.22 (s, 1H, =CH), 9.86 (s, 1H), 12.80 (s, 1H).

13

C NMR (125 MHz, DMSO-d6) 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 oC) = 16o (1, DMSO). Biological studies. For evaluating in-vivo analgesic, anti-inflammatory and mechanistic studies, swiss-albino 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 Analgesic Activity. The capsaicin-induced 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 Kg-1. In group III and IV animals were injected compound 3b at doses of 5 and 10 mg Kg-1, respectively and in group V and VI animals were injected with compound 3d at doses of 5 and 10 mg Kg-1, respectively intraperitoneally. Similarly, compounds 10, 4a and 4b were studied in groups VII to XII at doses of 5 and 10 mg Kg-1. The decrease in number of paw licking and twitching was recorded as an indicator of analgesic effect of drug diclofenac and synthesized compounds. Anti-inflammatory 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 kg-1, respectively while group V and VI were


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administered 1b at doses of 5 and 10 mg kg-1 respectively; groups VII and VIII were treated with compound 10 using 5 and 10 mg kg-1 dose and groups IX and X were given respectively 5 and 10 mg kg-1 of compound 3b. Similarly, compounds 3d, 4a and 4b were studied in groups XI to XIV at doses of 5 and 10 mg Kg-1. 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 anti-inflammatory effect. 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 Kg-1, third group was treated with (S)-4a at a dose of 300 mg Kg-1 and the fourth treated with 2000 mg Kg-1. 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 heamatoxylin- 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 Kg-1 followed 30


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min later by intraplanter capsaicin injection in group. The number of paw lickings was observed for 10 min after capsaicin injection as a measure of nociception. Lipoxygenase inhibitory activities For 5-LOX inhibition studies, solutions of compounds at 0.01 µM, 0.1 µM, 1 µM, 10 µM and 100 µM concentrations were prepared in DMSO. 10 µL of each compound from the above concentrations was added to 90 µL solution (in assay buffer) of 5- LOX enzyme (Soybean lipoxygenase) taken in the wells of a 96-well plate. Each compound was tested in duplicate and the average of two values with deviation

Triblock Conjugates: Identification of a Highly Potent Antiinflammatory

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