Design, Synthesis, and Activity Evaluation of Stereoconfigured Tartarate Derivatives as Potential Anti-inflammatory Agents in Vitro and in Vivo

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Design, Synthesis, and Activity Evaluation of Stereoconﬁgured

Tartarate Derivatives as Potential Anti-inﬂammatory Agents In Vitro

and In Vivo

Priya Kumari, Palwinder Singh,* Jashanpreet Kaur, and Rajbir Bhatti

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sı Supporting Information

ABSTRACT: Preclinical and clinical data reveal that inﬂammation

is strongly correlated with the pathogenesis of a number of diseases

including those of cancer, Alzheimer, and diabetes. The

inﬂammatory cascade involves a multitude of cytokines ending

ultimately with the activation of COX-2/LOX for the production

of prostaglandins and leukotrienes. While the available inhibitors

for these enzymes suﬀer from nonoptimal selectivity, in particular

for COX-2, we present here the results of purposely designed

tartarate derivatives that exhibit favorable selectivity and signiﬁcant

eﬀectiveness against COX-2 and LOX. Integrated approaches of

molecular simulation, organic synthesis, and biochemical/physical

experiments identiﬁed 15 inhibiting COX-2 and LOX with

−

1

respective IC₅₀4 and 7 nM. At a dose of 5 mg kg to Swiss albino mice, 15 reversed algesia by 65% and inﬂammation by 33%

in 2−3 h. We ﬁnd good agreement between experiments and simulations and use the simulations to rationalize our observations.

INTRODUCTION

While a number of steroidal and nonsteroidal drugs

regulating a broad range of inﬂammatory diseases by targeting

COX-2 are identiﬁed, the parallel existence of house-keeping

■

Inﬂammation is the body’s ﬁrst alarm signal in response to

microbial invaders and pathogens whereas its continuing

prevalence (chronic) can lead to the development of several

diseases such as cardiovascular disease, cancer, Alzheimer’s

disease, asthma, rheumatoid arthritis, and diabetes.

Succeeding the discovery of cyclooxygenase and its two

17

COX-1 led to the emergence of selective COX-2 inhibitors

18−21

(

COXIBS).

However, the cardiovascular side eﬀects of

1

−6

these COX-2 inhibitory drugs were responsible for the

22,23

7

withdrawal of rofecoxib and valdecoxib

celecoxib

leaving only

24,25

8

−10

for the treatment of arthritis, acute pain, and

isoforms COX-1 and COX-2,

acid (AA) metabolism is extensively explored identifying PGE₂,

the cascade of arachidonic

menstrual pain. Since the blockage of the COX-2 channel

during the use of COXIBS shifts the AA gradient to the

PGD , PGF , and LT s′ (Figure 1) as the causative agents of

26

2

4

lipoxygenase and COX-1 channels, resulting in the higher

inﬂammation. Though LX has been reported to have an anti-

4

11,12

production of leukotrienes, the advantage of targeting both

inﬂammatory eﬀect,

the pronounced inﬂammatory con-

27−29

LOX and COX-2

motivated us to develop dual COX-2/

sequences of LTB , LTC , LTD , and LTE make LOX an

4

LOX inhibitors. As of now, besides the medicinal use of

attractive target for inﬂammation therapy. These metabolites

are generated by the inducible COX-2 and LOX under cell

signaling initiation by the activation of inﬂammatory

24,25

30

celecoxib

and zileuton, though these are also associated

31−35

with side eﬀects,

not many dual COX-2/LOX inhib-

29,36−42

itors

).

Although the heterocycles-templated biaryl substituted

are succeeded to the clinical trials/practice (Figure

1

0

cytokines. It is also thought that similar to the previous

2

16

corona viruses, such as SARS-CoV, the deaths induced by

SARS-CoV-2 (COVID-19) are due to the systemic inﬂamma-

tion caused by “cytokine storms” propelled by pro-inﬂamma-

tory cytokines including tumor necrosis factor-α (TNF-α),

interleukin-1/6 (IL-1/6), and interferon-γ (INF-γ). One such

small but potent signaling protein, IL-6, is a call-to-arms for the

macrophages which besides fueling inﬂammation also damages

normal cells. The ideal counter, then, would be the drugs that

block the activity of COX-2 and LOX- reducing the formation

of PGE , PGD , PGF , and the leukotrienes (enclosed in red

20,43

classical COX-2 inhibitors such as celecoxib (Figure 3A)

are found to occupy the AA binding pocket of COX-2, it

26

Received: May 16, 2021

Published: June 17, 2021

2

boxes in Figure 1).

©

2021 American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00880

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Figure 1. AA metabolism cascade. ³⁻¹⁵Inﬂammatory house-keeping prostaglandins/leukotrienes are respectively enclosed in red and blue boxes.

1

Colored arrows indicate COX-2/LOX targeting.

Figure 2. COX-2, 5-LOX, and COX-2/5-LOX dual inhibitors. 1−3 refer to numbering in the original publication.

would appear that it is relatively rare to have tartarate based

acyclic-templated COX-2/LOX inhibitors which can attain a

conformation similar to that of AA (Figure 3B) and hence can

better occupy the active site pocket of the enzymes. Thence,

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Figure 3. Overall design strategy for new COX-2, LOX inhibitors. (A) Energy minimized geometry of celecoxib. (B) U-shaped conformation of

arachidonic acid as attained in the active site pocket of the enzymes. (C) New molecules with conformation similar to that of AA showing H-

2

+

donor/acceptor sites. (D) Binol moiety. Energy minimized conformation of (E) (1R,2R)-15 and (F) (1S,2S)-15. (G) Interactions of 15 with Fe

of 5-LOX (from molecular docking studies).

a

Scheme 1. Synthesis of Key Intermediates 4 and 5

a

Reaction conditions: (i) NaH, THF, 18-crown-6, tetrabutylammonium bromide, 0−25 °C.

for creating inhibitors structurally similar to AA, tartarate was

chosen as one of the components of new molecules. The

biological acceptance, capability to undergo polar/nonpolar

interactions with the protein, and providing another asym-

metric carbon/s (except glycine) led us to choose amino acids

for coupling with tartarate. Making use of amino acid azides,

while creating an additional medicinally functional unit 1,2,3-

1S,2S and 1S/1R, 2R/2S conﬁgurations at the two carbons of

tartarate, the isomers with 1R, 2R stereochemistry exhibit

better interactions with 5-LOX (PDB ID 3V99) (5-LOX was

chosen because all the compounds dock in this enzyme)

(Table S1). Moreover, in addition to the 1R,2R stereo-

chemistry, the isomers with either both 10S, 15S or 10S/10R,

15R/15S (Figure 3C for numbering) were better interacting

with the enzyme. Out of the 10 stereoisomers 8a, 8b/8c, 8d,

8e, 8f/8n, 8g/8h, 8i, 8j/8k, 8l/8m, and 8o of compound 8 (the

pairs 8b/8c, 8f/8n, 8g/8h, 8j/8k, and 8l/8m were the same),

8a and 8g/8h with respective conﬁgurations 1R, 2R, 10S, 15S,

and 1R, 2R, 10S/10R, 15R/15S exhibited maximum inter-

actions with the active site residues of 5-LOX (Table S1). The

docking score of 8a and 8g/h was also signiﬁcantly better than

the other isomers. Similar results were obtained when the

stereoisomers of the rest of the compounds were docked in the

active sites of COX-2 and 5-LOX (Table S2, S3). As a result,

instead of synthesizing all isomers of the designed compounds,

we restricted to those with 1R, 2R, 10S, 15S conﬁgurations.

Only in the case of compound 8, the isomer 8g/h with 1R, 2R,

4

4,45

triazole,

structure) in the substituent chains may be achieved (Figure

C). It was, therefore, envisaged that the appropriate

the desired bending (so to achieve AA like

3

stereochemistry at the two asymmetric carbons of tartarate

and its suﬃcient backbone ﬂexibility may confer L-/extended-

U shaped geometry to the molecule enabling it to go over the

AA binding pocket of the enzyme. As a proof of concept, an

energy minimized closed conformation was observed for the

(

1R,2R)-tartarate based molecules (Figure 3E) whereas their

1S,2S)-isomer attained an open conformation (Figure 3F). A

pertinent role of the ﬂexible hydrophilic tartarate moiety was

warranted by replacing it with rigid hydrophobic binol (Figure

3

D). The designed molecules were also intended to

2

+

3+

orchestrate Fe /Fe of LOX as observed in the molecular

docking studies (Figure 3G). Based on this design, molecules

10S/10R, 15R/15S conﬁguration was synthesized for the

purpose of comparison. Some other implications of the

molecular docking studies including poor interactions of

binol derivatives in the active site pocket of COX-2 and better

interactions of the carboxylate compounds than their ester

analogues were also supported by the results of enzyme

immunoassays.

6

−23 (Figure S1, Schemes 1−5) were synthesized and

evaluated for in vitro COX-2/COX-1/LOX inhibitory activity

using enzyme immunoassays, examined for the in vivo

suppression of analgesia and inﬂammation, and checked for

acute toxicity, mode of action, pharmacokinetics, and

interactions with the enzymes as detailed below.

The molecular simulations with the stereoisomers of 6−23

were performed to make an initial assessment of their bioactive

conformations by evaluating the diﬀerences of their (stereo-

isomers) interactions in the active sites of COX-2 and 5-LOX.

It was observed that in comparison to the stereoisomers with

RESULTS AND DISCUSSION

■

Chemistry. The syntheses of the designed molecules are

depicted in Schemes 1−5. Reaction of (1R,2R)-diethyl

tartarate with propargyl bromide provided key intermediates

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a

Scheme 2. Synthesis of Compounds 6−10

a

(

i) EtOH:water (9:1), sodium ascorbate, CuSO .5H O, room temperature. For 8g/h: (i) (S)-tyrosine azide (1 equiv), (ii) (R)-tyrosine azide (1

4 2

equiv).

a

Scheme 3. Synthesis of Compounds 11−14

a

(

i) EtOH:water (9:1), sodium ascorbate, CuSO .5H O, room temperature.

4 2

4

(40%) and 5 (15%) (Scheme 1). For incorporating the

tryptophenyl azide at room temperature in the presence of

sodium ascorbate and copper sulfate, and compounds 6−10

were generated (Scheme 2). In a two step procedure,

treatment of 4 with (S)-tyrosine azide (1 equiv) and further

amino acid derived 1,2,3-triazole moiety in the molecule,

intermediate 4 was coupled with ethylbromoacetate azide/(S)-

serine azide/(S)-tyrosine azide/(S)-phenylalanine azide/(S)-

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reaction of monosubstituted product with another equivalent

of (R)-tyrosine azide gave compound 8g/h. Compound 8g/h

exhibited the same spectral data as compound 8a except a

diﬀerence in the optical rotation. Similar to the reactions of 4

with the azides, intermediate 5 was also made to react with

ethylbromoacetate azide, (S)-serine azide, (S)-tyrosine azide,

and (S)-phenylalanine azide to procure compounds 11−14

tartarate displayed lower potency than that of compounds 6−

10. While identifying a new inhibitor, it was observed that

compound 15 exhibited dramatically better COX-2 and LOX

inhibitory activity than all the other compounds including the

positive controls celecoxib and zileuton. Desirably, the

selectivity index 75 of compound 15 also fell between that of

selective (celecoxib) and nonselective (indomethacin) COX-2

inhibitors. In general, compounds 8, 9, and 10 with an

(

Scheme 3). In order to make comparison of the biological

activities, the ester moieties of 6 and 9 were transformed to

their corresponding acids in compounds 15 and 16,

respectively (Scheme 4).

aromatic side chain at C of the amino acid component of the

α

molecule exhibited lower potency for COX-2 but better for

LOX suggesting that the binding interface of LOX can

accommodate relatively bulky and lipophilic groups. This trend

in the inhibitory activity of the compounds is similar to the

results of molecular docking studies where the aromatic moiety

of the amino acid did not allow the molecule to ﬁt into the

active site of COX-2. Moreover, supporting the in-silico results,

compound 8g/h was found as potent as its stereoisomer 8a.

On the basis of the structure activity relationship studies, it was

a

Scheme 4. Synthesis of Compounds 15 and 16

inferred that either no or a small side chain at C of the amino

α

acid unit of these molecules seems to be optimal for the COX-

2

inhibitory activity. Overall, compounds 6, 7, 8a, 10, 15, 16,

and 21 were identiﬁed for further screening on animal models.

4

6,48

Human Whole Blood Assay.

Human whole blood

assay was performed to support the results of enzyme

immunoassays for the selectivity of compound 15 for COX-2

over COX-1. Here, the lipopolysaccharide (LPS) induced

expression of COX-2 was reversed (Figure 4A) whereas no

eﬀect on calcium ionophore induced expression of COX-1 was

observed (Figure 4B) (Table 2).

In Vivo Screening of the Compounds. The anti-

inﬂammatory and analgesic activities of the compounds were

checked on Swiss albino mice (25−35 g) of either sex.

Acetic Acid-Induced Writhing Test. Stretching of abdomen

a

(

i) Acetone:water (3:2), NaOH, room temperature.

49

Further, as it was envisaged that the acyclic tartarate moiety

and extension of hind-limbs were speciﬁed in acetic acid-

induced writhing test for evaluating the analgesic activities of

the compounds. The standard and the test groups of animals

may be better suited to provide an AA like structure to the

molecules, the hypothesis was checked by replacing it with

binol. The reactions of compound 18, obtained by the

propargylation of (S)-binol (17), with the same azides as used

in Schemes 2 and 3 gave products 19−23 (Scheme 5). All the

synthesized compounds were characterized by using NMR, IR,

and HRMS techniques (Experimental Section, SI).

−

1

were given indomethacin (10 mg kg ) and test compounds (5

−

1

mg kg ), respectively, 30 min before inducing the writhings

through 0.6% v/v acetic acid injection (including the control

group). The results recorded in terms of reduction in the

number of writhings are shown in Figure 5A. Compound 15

treated animals caused 65% inhibition of algesia that was

appreciably better than the 55% shown by the standard drug

indomethacin and comparable to the 62% analgesic eﬀect of

Biological Studies. In Vitro COX-1/2 and LOX Inhibitory

46,47

Assay. By performing enzyme immunoassays,

of compounds 6−16 and 19−23 against COX-1/2 and LOX

were determined at 10 −10 M concentrations in triplicate.

These were calculated by measuring the amount of

prostaglandins/leukotrienes produced in each enzymatic

reaction and quantiﬁed in terms of 50% inhibitory concen-

trations (IC ). Compounds 6 and 8a, 8g/8h exhibited

signiﬁcant inhibitory activity for COX-2 with respective IC₅₀

values 0.1 and 0.7 μM, but their selectivity for COX-2 over

COX-1 was either too high (600) or too low (∼14). Although

the IC of compound 7 was 0.4 μM, its selectivity for COX-2

over COX-1 was found between that of celecoxib (COX-2

selective) and indomethacin (COX-1/2 nonselective). Com-

pound 10 exhibited the IC value 0.5 μM for COX-2. Besides

their poor interactions with COX-2, the binol compounds were

included in the enzyme immunoassays so that the results of

molecular modeling studies get validated. It was noticed that

the replacement of the tartarate fragment of compounds 6−10

with the binol in compounds 19−23 lowered the potency of

the compounds against both COX-2 and LOX (Table 1).

Compounds 11−14 with substitution at only one OH group of

the activities

−

4

−8

−1

50

celecoxib at 5 mg kg dose. The analgesic activity of 6 and 7

was almost in parallel to their response in enzyme immuno-

assays whereas other compounds included in these experi-

ments were not very eﬀective in reducing the number of

writhings.

5

0

Carrageenan Induced-Paw Edema Test. It is an acute

51

inﬂammatory model in which the reduction in carrageenan-

induced thickness of hind paw in the presence of the test

compounds was taken as a measure of their anti-inﬂammatory

activity. The control, standard, and treatment groups of

5

0

−

1

animals were given indomethacin (10 mg kg ) and the test

−

1

compounds (5 mg kg ), respectively, 30 min prior to the 100

μL injection of freshly prepared 1% carrageenan solution. The

paw thickness (mm) was determined at 15, 30, 60, 120, 180,

240, 300, and 360 min time intervals. It was apparent from

50

−

1

Figure 5B that compound 15 (5 mg kg ) caused a 33%

decrease in the paw edema that was signiﬁcantly better than

the 30% decrease caused by indomethacin at 10 mg kg⁻and

comparable to the 33% reduction shown by celecoxib at 5 mg

1

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a

Scheme 5. Synthesis of Compounds 19−23

a

(

i) Acetone, anhydrous potassium carbonate, propargyl bromide, 60 °C, (ii) EtOH:water (9:1), sodium ascorbate, CuSO .5H O, room

4 2

temperature.

kg⁻dose. Moreover, the anti-inﬂammatory eﬀect of

compound 15 was observed after 2−3 h (Figure 5C) of

treatment against the similar eﬀect of indomethacin and

1

50

min before the treatment with compound 15. After 30 min,

each group of animals was treated with 0.6% v/v acetic acid,

and the number of abdominal writhings was noted for 30 min.

Pretreatment with Substance P (COX-2 and LOX stimulator)

made a signiﬁcant reversal in the eﬀect of compound 15,

delineating that 15 might induce its analgesic eﬀect by

inhibiting COX-2 and LOX pathways. L-arginine (nitric

oxide donor) treatment did not change the analgesic eﬀect

of 15. However, pretreatment with L-NIL, a nitric oxide

synthase inhibitor, potentiated the eﬀect of compound 15

(Figure 6). Hence, the results of these in vivo experiments

suggested nonimplication of the NO pathway in modulating

the analgesic eﬀect of compound 15.

50

celecoxib in 5−6 h and 3−4 h, respectively. This observation

points toward the fast-acting feature of 15 (Figure S109)

though more experiments are needed to prove it. The response

of compound 21, kept for positive comparison, was similar to

its poor analgesic eﬀect.

Further, to check the dose-dependent anti-inﬂammatory

−

1

eﬀect of compound 15; its 0.5, 2.5, 5, 10, and 20 mg kg doses

were given to 5 sets of animals (6 animals in each group) 30

min prior to carrageenan injection, and percentage decrease in

paw thickness (mm) was determined (Figure 5C, Table 3).

Characteristically, a dose-dependent response in terms of

decrease in paw thickness was observed and the eﬀect achieved

saturation between 5 and 10 mg kg⁻dose (Figure 5D).

Mechanistic Study. In addition to the enzyme immuno-

assay, in vivo studies were also performed to check if these

compounds target COX-2/LOX pathways. Substance P (10 μg

kg ) was used to study the involvement of COX-2 and LOX

pathways whereas the nitric oxide donor L-arginine (40 mg

kg ) and the nitric oxide synthase inhibitor N6-(1-

iminoethyl)-L-lysine dihydrochloride (L-NIL) (5 mg kg )

were utilized to study the participation of the nitric oxide

pathway. All these prescriptions were given intraperitoneally 30

Acute Toxicity Study. Acute toxicity studies were carried

out using Swiss albino mice. Brieﬂy, 2 groups were used with

three animals in each group (n = 3). The ﬁrst group was

normal control whereas in the second group, compound 15

1

−

1

was given orally a dose of 300 mg kg (due to quite low IC₅₀

−

1

value, only 300 mg kg dose was used for toxicity studies).

The study was performed after 4 h of the fasting period. Mice

were continuously monitored for a period of 4 h followed by

periodic monitoring for the next 14 days. Mice exhibited a

sedative eﬀect after 1 h of the treatment with slowed

movements, sluggish behavior, and sleep. The eﬀects subsided

within the next 1 h. No other gross behavioral changes were

−

1

−

1

−

1

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Table 1. IC₅₀(μM) Values of Compounds 6−23 against

COX-1, COX-2, and LOX

Table 2. Inhibition of TXB during Calcium Ionophore

2

Stimulation and PGE in LPS Stimulation of Human Whole

2

Blood by Compound 15

a

IC₅₀(μM)

b

PGE (ng/

Compounds

COX-1

COX-2

LOX

Selectivity index

2

mL)

TxB (ng/mL)

2

6

60

54

>10

90

>10

95

110

80

0.1

0.4

0.7

0.3

1.3

600

135

>14

−

calcium

+calcium

ionophore

7

8

9

1

2

−LPS +LPS

ionophore

a

g/h

0.15

0.14

0.09

0.45

6

14

12

8

0.007

0.5

11

6

0.6

3

control

indomethacin

0.18

2.0

0.20

1.80

0.9

1.6

0.65

0.36

1

5

0

1

2

3

4

5

6

9

0

1

2

3

0.5

55

40

35

32

0.004

1.0

23

30

1.5

54

>20

1.7

2.7

2.3

1.7

75

from mice (calculated w.r.t. C_m_a_x). The AUC trend in paw

thickness vs time graph at diﬀerent doses of the compound

w.r.t. the control and standard clearly showed that treatment

with 15 results in fast decrease of carrageenan-induced paw

edema (Figure S109). A comparison of the PK data for

celecoxib on rats at 5 mg kg i.p. dose, showing t₁_/₂5.2 h and

C_m_a_x1.2 μg/mL achieved in 0.28 h (T_m_a_x) with AUC₀_‑_t6.6 μg/

mL·h, indicates a better PK proﬁle of 15 though we know

celecoxib is an approved drug.

Plasma protein binding (PPB), water solubility, to be

substrate of CYP3A4, and the Marine−Darby canine kidney

(MDCK) parameters of 15 were also calculated and

compared with one of our previously reported potent molecule

2 and tepoxaline (Table 5). Due to the presence of four

carboxyl groups, 15 is highly soluble in water which may

decrease its rate of absorption from intestine into the blood.

But given other favorable factors, poor drug absorption can be

overcome by delivering the drug in the form of either

nanoparticles or surfactant emulsion or its ester form.

Advantageously, higher water solubility improved the perme-

ability of the drug 15 to 1.59 nm/s in the MDCK model in

comparison to 0.05 and 0.04 nm/s for 2 and tepoxaline,

respectively. Remarkably, small PPB of 15 in comparison to 2

and tepoxaline may enable it to be a fast acting drug requiring a

small dose. Higher PPB of the drug causes it to release slowly

and may make it diﬃcult to reach a therapeutic concentration,

thus requiring higher drug dose. Hence, compound 15 seems

relatively fast acting, and consequently, its small dose could be

suﬃcient if given in appropriate formulation (also evident from

the drug-response assay). Compound 2 being the substrate of

CYP3A4 (enzyme responsible for drug metabolism in the

liver) may get further reduced in concentration and also cause

drug−drug interactions whereas 15 (similar to tepoxaline) is a

weak substrate of CYP3A4. Overall, the pharmacokinetic

55

0.3

1.0

70

1

−

1

3.0

2.8

>7

2.7

32

0.08

3.5

375

86

52

>10

150

90

0.08

0.07

15

2.8

0.96

0.02

0.04

1.0

Indomethacin

Diclofenac

Celecoxib

Zileuton

5

3

0.3

b

a

Average of three values with deviation <3%. IC₅₀(COX-1)/(COX-

).

2

observed. Due to the ethical issues, the animals were not

sacriﬁced, and hence the images of the organs were not taken.

The toxicities of compounds 6, 8, 10, and 15, alone as well as

in combination with LPS, were also checked over the

microglial cells BV2. Though the results are not shown here,

all these compounds exhibited no toxicity to the cells (checked

up to 48 h) even at concentration >1 μM.

Pharmacokinetics of 15. A dose of 10 mg kg of 0.1%

CMC suspension of 15 was administered i.p. to the Swiss

albino mice. This compound exhibited t₁_/₂of 8.74 h and

attained a maximum concentration (C_m_a_x) of 26 μg/mL in 1 h

of its administering to the animal (Figure 7, Table 4). Even

after 24 h, the plasma drug concentration (1.98 μM) was above

the IC required for COX-2/LOX inhibition as determined in

−

1

5

0

the enzyme assays. The Cl value 0.0355 (μg/mL)/h indicates

that it takes ∼35h for the complete clearance of the compound

Figure 4. (A) PGE and (B) TXB inhibition in whole blood by indomethacin (1 μM) (abbreviated here as Indo) and compound 15 (1 μM). LPS

2

and calcium ionophore were used as stimulants of COX-2 and COX-1, respectively.

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Figure 5. (A) Eﬀect of compounds on acetic acid-induced writhings in Swiss albino mice. Data were analyzed by using one way ANOVA and

expressed as mean ± SEM of six animals per group followed by Tukey’s test. ***p < 0.0002 and ****p < 0.0001 compared to the control group.

(

B) Eﬀect of compounds 15 and 21 at 5 mg kg⁻¹dose and (C) 15 on various doses on carrageenan-induced hind paw edema in Swiss albino mice.

a

Data were analyzed by using one way ANOVA and expressed as mean ± SEM of six animals per group followed by Tukey’s test. p < 0.05

compared to control group. p < 0.05 compared to indomethacin group. p < 0.05 compared to 15 (5/0.5 mg/kg). p < 0.05 compared to 15 (2.5

mg/kg). p < 0.05 compared to 15 (5 mg/kg). (D) Dose-dependent reduction in paw thickness (%) on treatment of mice with 15 .

b

c

d

e

Table 3. Percentage Decrease in Paw Edema on Treatment

with Varied Doses of 15 (Dose-Response Assay)

S.

No.

Dose

percentage decrease in paw edema (anti-

(mg kg⁻¹)

inﬂammatory activity)

1

2

3

4

5

0.5

2.5

5

10

20

2.0

5.4

32.6

36.5

38.5

parameters of 15 are emerging superior to those of 2 and

tepoxaline. The low log P (−3.26) and high total polar surface

area (299.10) of 15 also comply with the high renal clearance

features of a drug.

Figure 6. Eﬀect of Sub-P, L-arginine, and L-NIL on the analgesic

activity of compound 15 in an acetic acid-induced writhings test. Data

were analyzed by using one way ANOVA and expressed as mean ±

Isothermal Titration Calorimetry (ITC) Experiments.

The binding interactions of the compounds with COX-1,

COX-2, 5-LOX, and HSA (human serum albumin) were

studied with the help of ITC experiments, and the data

supported the biological results. The values of the thermody-

namic parameters ΔH and ΔG (Table 6, Figure S110)

indicated the exothermic and spontaneous binding of

compound 15 with the enzymes. Apparently, compound 15

has better interactions with COX-2 in comparison to those

with COX-1 and LOX. Compound was also found interacting

with HSA- cordial for transportation and clearance in the

biological system though many factors and several pathways

are involved for the elimination of a compound.

a

SEM of six animals per group followed by Tukey’s test. p < 0.05

b

compared to control group, p < 0.05 compared to compound 15

group.

Molecular Modeling Studies. Docking calculations of the

compounds were performed to determine their binding

aﬃnities with COX-2 and 5-LOX. Figure 8A shows the most

potent compound 15 in the active site of COX-2. Three of the

four carboxyl groups of the inhibitor formed H-bonds with the

three very crucial amino acids of the COX-2 catalytic

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entry point and not in the interior of the cavity (Figure S112).

Compounds 8−14 and 19−23 as well as their acid

counterparts partially enter into the active site of the enzyme

and did not show H-bond interactions with any of the residues

there.

Since compound 15 also appeared potent for LOX, its

docking in 5-LOX showed that it ﬁts in the active site of the

2

+

enzyme and coordinates with Fe through the two carboxyl

groups (Figure 9A, B). Another carboxyl group was H-bonded

to Phe177. Additionally, the triazole rings of 15 were engaged

in π−π and cation−π interactions with Phe177 and Lys409,

respectively. It was observed that compound 15 exhibits more

2

+

number of interactions with Fe

and hence better

coordination than that shown by tepoxaline (Figure 9C, D).

Compounds 6−10 and 21−23 also exhibited appreciable

interactions with the active site amino acid residues of 5-LOX

Figure 7. PK Proﬁle for compound 15 in mice.

(

Figure S113−S118). Most of these compounds were

2+

coordinating with Fe that may be the reason for their LOX

inhibitory activities. As a result, we found a good correlation

between the experimental and calculated data suggesting the

stereoelectronic compliance of the drug/compound with the

target enzyme for its higher eﬃcacy. Only compound 15 in this

series showed unique interactions with both COX-2 and LOX,

even appreciably better than those exhibited by the reported

drug tepoxaline. It seems that the engaging of carboxyl groups

Table 4. PK Data of Compound 15

Parameter

Value

no. of animals

Dose level

t₁_/₂

T_m_a_x

C_m_a_x

30

−1

10 mg kg

8.74 h

1 h

26 μg/mL

241.27 μg/mL·h

0.0355 (μg/mL)/h

2

+

in H-bonding with COX-2 and coordinating with Fe in LOX

enable a 15 dual COX-2/LOX inhibitor.

AUC₀_‑_t

Cl_obs

Table 5. Predicted Pharmacokinetic Features of

Compounds 2, 15, and Tepoxaline

CONCLUSIONS

■

Chronic inﬂammation is a highly debilitating physiological

stage that is implicated in a number of diseases. Among the

various cellular targets of the anti-inﬂammatory agents, the

COX-2 and LOX inhibitors are the most extensively explored

but the side eﬀects associated with these compounds are the

major bottlenecks in their transformation to clinical drugs.

Even the clinically available drugs such as celecoxib (Celebrex)

(COX-2 inhibitor) and tepoxaline (Zubrin) (COX-2, LOX

inhibitor) suﬀer from side eﬀects. Since the interaction/

binding of the inhibitor with its cellular target is important for

the eﬃcacy of the compound, based on this concept, here, we

report a new series of COX-2, LOX dual inhibitors. Imparting

the ﬂexibility and stereochemical attributes to the molecules,

tartarate based acyclic compounds were developed as a

probable replacement of the usual heterocyclic-template

mono/biaryl substituted inhibitors of the AA pathway. As

shown by the combined results of molecular modeling studies

and the in vitro and in vivo experiments, compound 15 is a

promising candidate for anti-inﬂammatory drugs. This

compound exhibited the IC₅₀values 4 nM and 7 nM for

COX-2 and LOX, respectively. In contrast to the conventional

COX-2/LOX inhibitors, compound 15 does not carry a single

phenyl ring that may render its safe metabolism. Desirably, the

selectivity index of compound 15 for COX-2 over COX-1 was

less than that of highly selective celecoxib but more than

CYP3A4

substrate

MDCK

(nm/s)

pure water solubility

(mg/mL)

Compound PPB%

2

1

98

67

100

substrate

weakly

0.05

1.59

0.04

0.01

Freely

21.08

5

Tepoxaline

5

4−59

pocket,

i.e., Arg120, Tyr385, and Ser530 (Figure 8A, B).

These H-bonds must strengthen the enzyme−ligand inter-

actions and help in justifying the better COX-2 inhibitory

activity of 15. Apparently, the entrance as well as the interior

part of the active site pocket was occupied by compound 15,

and its H-bond distances with Ser530 and Tyr385 were shorter

and hence stronger than those shown by AA (Figure 8C).

Remarkably, compound 15 occupied the same pocket of COX-

2

where AA sits but with the additional advantage of H-

bonding, thus supporting the hypothesis made in the design of

the molecules. Compound 15 was certainly better ﬁt and more

interactive in the COX-2 active site in comparison to one of

our previous compound 2 (Figure 2, Figure 8D) as well as

COX-2/LOX inhibitor tepoxaline (Figure 2; Figure 8E, F)

though the IC value of 15 for COX-2 was slightly higher than

5

0

that of 2. Compound 6, the ester precursor of 15, exhibited

only π−π interactions with Tyr355 and Arg120 (Figure S111A,

B). Compound 7 interacted with Arg120 and Tyr115 at the

Table 6. ITC Data for the Binding of Compound 15 with the Enzymes

Physical parameters

COX-1

COX-2

5-LOX

HSA

K (M⁻¹)

(1.64 ± 0.49) × 10

3

(6.07 ± 0.63) × 10⁵

−43.73

−10.75

−32.98

(1.33 ± 0.12) × 10⁵

−41.81

−12.53

−29.28

(1.18 ± 0.66) × 10⁴

a

2

ΔH (kJ/mol)

TΔS(kJ/mol)

ΔG (kJ/mol)

−25.12 × 10

−24.91 × 10

−11.05 × 10

−10.81 × 10

2

−21.00

−23.26

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Figure 8. (A) 3D representation of compound 15 docked in the COX-2 (1CVU) active site. (B) 2D interaction diagram of compound 15 with

amino acid residues lining the active site of COX-2. (C) Compound 15 (in yellow-red) docked in COX-2 showing overlapping with AA (gray-red)

cocrystallized with the enzyme. (D) Compound 2 docked in COX-2. (E and F) Tepoxaline docked in the active site of COX-2 representing 3D and

2D views. Nonpolar H’s are removed for clarity.

nonselective indomethacin, that may help compound 15 to

overcome the COX-2/1 selectivity related limitations of the

reported anti-inﬂammatory drugs. Furthermore, compound 15

does not show in vivo toxicity up to 300 mg kg dose and

exhibited signiﬁcant reversal of inﬂammation and analgesia in

the treated Swiss albino mice. Our results suggest that the

novel tartarate−1,2,3-triazole carboxylate based COX-2/LOX

inhibitors deserve further investigations as potential anti-

inﬂammatory agents.

compound (dissolved in methanol) was injected to the column

Kromasil Silica, 5 μ, 250 mm × 4.6 mm, 60 Å) by autosampler of

(

Dionex Ultimate 3000 connected to a mass spectrometer. An isocratic

mobile phase consisting acetonitrile and water (composition

mentioned for each compound along with the LC-MS in SI) was

used with the ﬂow rate of 0.2 mL/min. The purity of the sample was

calculated with the following formula, and it is given along with the

LC-MS of each compound in the SI.

−

1

Area of Single Peak (summed EIC)

Area of All Integrated Peaks (TIC)

MS Sample Purity =

× 100

EXPERIMENTAL SECTION

General. Melting points were determined in capillaries using a

VEGGO digital melting point apparatus and are uncorrected. H

■

(1)

1

General Procedure for Synthesis of Compounds. Procedure

A. To a suspension of sodium hydride (0.77 g, 60% in mineral oil,

NMR spectra were recorded on Bruker 500 and JEOL 400 MHz

1

3

0

.019 mol) in THF (20 mL), a solution of L-diethyltartarate (2.09 g,

.01 mol) in THF (30 mL) was added dropwise over 30 min with

NMR spectrometers with the corresponding recording of C NMR

spectra respectively at 125 and 100 MHz. CDCl and DMSO-d were

3

6

used as the solvents with TMS as the internal reference. Chemical

stirring at 0 °C. After the mixture was stirred for 1 h at 0 °C,

tetrabutylammonium bromide (0.074 g, 0.002 mol) and 18-crown-6

(6 mg, 0.022 mmol) (catalytic amount) were added in one portion.

Propargyl bromide (2.37 g, 0.02 mol) was added dropwise over 30

min at 0 °C. The resulting mixture was stirred for 12 h at room

temperature, quenched with 1 N aqueous HCl, poured into water, and

extracted with three portions of ether. The combined organic layers

1

3

shifts are given in ppm and J values in hertz (Hz). For C and DEPT-

35 NMR spectra, positive signals correspond to CH₃and CH

1

carbons, and negative signals correspond to CH₂carbons. Mass

spectra were recorded on a Bruker micrOTOF QII mass spectrometer

for high resolution mass spectra (HRMS). IR spectra were recorded

on a Varian 660 IR spectrometer. Reactions were monitored by thin

layer chromatography (TLC) on glass plates coated with silica gel GF-

were washed with aqueous NaHCO and brine, dried over Na SO ,

3

2

4

2

54. Column chromatography was performed on 60−120 mesh silica.

and concentrated under vacuum to give the crude product which was

puriﬁed by column chromatography using ethyl acetate and hexane as

eluent to obtain compounds 4 and 5 in 40% and 15% yield,

respectively.

1

Percentage purity 98% was conﬁrmed with the help of a H NMR

spectrum (Figure S119) as reported earlier. The purity of the

compounds were also checked by LC-MS for which 2 μL of sample

60

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Figure 9. (A) 3D representation of 15 docked in the 5-LOX (3V99) active site. (B) 2D interaction diagram of compound 15 with amino acid

residues lining the active site of 5-LOX. Molecular docking of tepoxaline in the active site pocket of 5-LOX representing (C) 3D and (D) 2D views

of interactions. Nonpolar H’s are removed for clarity.

Procedure B. To a solution of S-binol (500 mg, 1.7 mmol) in

acetone, anhydrous potassium carbonate (720 mg, 5.20 mmol) and

propargyl bromide (620 mg, 5.21 mmol) were added. The reaction

mixture was allowed to stir at 60 °C for 16 h. After completion of the

reaction (TLC), the reaction mixture was ﬁltrated and the ﬁltrate was

evaporated under reduced pressure. The residue was puriﬁed by

recrystallization (petroleum ether/EtOAc) to give compound 18 in

ethanol were added in sequence. The reaction mixture was stirred at

room temperature and monitored with TLC. After the completion of

the reaction, it was diluted with distilled water and extracted with

ethyl acetate. Ethyl acetate was distilled oﬀ under reduced pressure to

give a thick oil/solid which was puriﬁed with column chromatography

using a mixture of ethyl acetate and hexane as eluent to isolate the

product. Compounds 6−10, 11−14, and 19−23 were procured with

70−90% yield. For compounds 11−14, 1.1 mmol of the azide was

used.

9

5% yield.

Procedure C. Preparation of Azides of Amino Acid. Ester of L-

amino acid (1 mmol) was dissolved in methanol followed by the

addition of anhydrous potassium carbonate (1.19 mmol), imidazole

sulphonyl azide hydrochloride (ISA.HCl) (1.19 mmol), and CuSO₄

Procedure F. Hydrolysis of Compounds 6 and 9. To the solution

of compound 6/9 (1 mmol) in acetone−water (3:2), 1 N NaOH (4

mmol) was added. The reaction mixture was allowed to stir at room

temperature until completion (TLC). After completion, acetone was

evaporated under vacuum and the pH of the water layer was adjusted

to acidic with 1 N HCl followed by extraction with ethyl acetate to

obtain desired product 16 after removing the ethyl acetate. However,

in the case of hydrolysis of 6, the desired compound 15 was isolated

from the water layer by washing with warm acetone.

(

1 mol %). The reaction mixture was stirred for 6 h at room

temperature. After the completion of the reaction, it was quenched

with water and extracted with ethyl acetate (3 × 25 mL). The

combined organic layers were separated, dried over Na SO , and

2

4

concentrated under vacuum to obtain the crude product which was

then puriﬁed by column chromatography using ethyl acetate/hexane

as eluent. All the amino acid azides were procured in 85−90% yield.

Procedure D. Preparation of Ethyl 2-Azidoacetate. To the

solution of ethyl bromoacetate (2.00 g, 12 mmol) in acetone (12

mL), sodium azide (2.73 g, 42 mmol) in water (12 mL) was added at

Diethyl (2R,3R)-2,3-Bis(prop-2-yn-1-yloxy)succinate (4). Com-

pound 4 was synthesized according to general procedure A using L-

diethyl tartarate and propargyl bromide and puriﬁed by column

chromatography using ethyl acetate−hexane (0.5:9.5 v/v) as eluent.

1

0

°C under vigorous agitation. This reaction mixture was then

Thick transparent oil, yield 40%. H NMR (400 MHz, CDCl ) δ 1.33

3

warmed to room temperature and heated at 60 °C overnight. The

aqueous phase was washed with dichloromethane (DCM) (3 × 15

mL). The organic layers were combined and washed with a solution

(t, J = 7.3 Hz, 6H, CH ), 2.45 (t, J = 2.5 Hz, 2H, CH ), 4.25−4.31

3

2

(m, 4H, CH ), 4.34−4.35 (m, 2H, CH ), 4.41−4.42 (m, 1H, CH of

2

1

3

CH ), 4.45−4.46 (m, 1H, CH of CH ), 4.77 (s, 2H, CH). C NMR

2

(10%) of sodium bicarbonate (20 mL) and water (20 mL). Then the

(100 MHz, CDCl ) δ 14.2 (+ve, CH ), 57.9 (−ve, CH ), 61.6 (−ve,

3

2

organic layer was dried on Na SO , and the solvent was evaporated

CH ), 75.7 (+ve, CH), 76.6 (+ve, CH), 78.32 (C), 168.3 (C O).

2

4

2

+

under vacuum to aﬀord ethyl 2-azidoacetate as a pale yellow liquid.

No further puriﬁcation was done of the crude product.

HRMS (microTOF-QII, MS, ESI): Calcd for C H O ([M + Na] )

1

4

18

6

305.0996, found 305.0994.

Procedure E. Compound 4/5/18 (1 mmol) was dissolved in

ethanol. To this solution, sodium ascorbate (5 mol %) and CuSO₄·

Diethyl (2R,3R)-2-Hydroxy-3-(prop-2-yn-1-yloxy)succinate (5).

Compound 5 was synthesized according to general procedure A

using L-diethyl tartarate and propargyl bromide. Sticky, transparent

5

H O (2 mol %) dissolved in water and the azide (2.1 mmol) taken in

2

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1

liquid, yield 15%. H NMR (500 MHz, CDCl ) δ 1.31−1.34 (m, 6H,

chromatography using ethyl acetate−hexane (6:4) as eluent. Thick

3

2

× CH ), 2.44 (t, J = 2.32 Hz, 1H, CH), 3.13 (d, J = 7.09 Hz, 1H,

oil, yield 74%. IR (ATR): 3138, 2952, 2124, 1744, 1453, 1371, 1267,

3

−

1

25

OH), 4.28−4.32 (m, 5H, 2CH + 1H of CH ), 4.40−4.44 (m, 1H,

1200, 1103, 1021, 812, 752, 506, 454 cm . [α]

= −32.5° (0.4,

2

D

1

3

1

H of CH ), 4.61−4.62 (m, 1H, CH), 4.63−4.64 (m, 1H, CH). C

CHCl ). H NMR (400 MHz, CDCl ) δ 1.19−1.27 (m, 6H, CH ),

2

3

NMR (125 MHz, CDCl ) δ 14.1 (+ve, CH ), 57.7 (−ve, CH ), 61.7

3.40−3.56 (m, 4H, CH ), 3.73 (s, 6H, OCH ), 4.07−4.19 (m, 4H,

3

2

3

(

−ve, CH ), 62.2 (−ve, CH ), 72.0 (+ve, CH), 75.6 (+ve, CH), 76.8

CH ), 4.51−4.52 (m, 2H, CH), 4.64−4.67 (m, 2H, CH ), 4.89−4.92

2

(

+ve, CH), 78.32 (C), 168.3 (CO).

(m, 2H, CH ), 5.49−5.53 (m, 2H, CH), 7.04−7.08 (m, 5H, ArH),

2

13

Diethyl (2R,3R)-2,3-Bis((1-(2-ethoxy-2-oxoethyl)-1H-1,2,3-triazol-

7.21−7.25 (m, 5H, ArH), 7.71 (s, 2H, ArH). C NMR (100 MHz,

CDCl ) δ 14.2 (+ve, CH ), 38.8 (−ve, CH ), 53.1 (+ve, OCH ), 61.5

5

-yl)methoxy)succinate (6). Compound 6 was synthesized according

3

2

3

to general procedure E using compound 4 and ethyl bromoacetate

azide and puriﬁed by column chromatography with ethyl acetate−

hexane (6:4). Thick oil, yield 73%. IR (ATR): 3145, 2981, 2094,

744, 1558, 1468, 1371, 1267, 1207, 1095, 1021, 872, 797, 708, 581,

47 cm . [α] = +40° (0.4, CHCl ). H NMR (400 MHz, CDCl )

(

−ve, CH ), 64.1 (+ve, CH), 65.0 (−ve, CH ), 78.8 (+ve, CH), 123.1

2

+ve, ArCH), 127.6 (+ve, ArCH), 128.9 (+ve, ArCH), 129.0 (+ve,

ArCH), 134.8 (ab, ArC), 144.2 (ab, ArC), 168.6 (CO), 168.8

CO). HRMS (microTOF-QII, MS, ESI): Calcd for C H O N

([M + H] ) 693.2879, found 693.2902.

1

4

(

3

4

40 10

6

−

1

25

D

1

+

3

δ 1.24 (t, J = 7.1 Hz, 6H, CH ), 1.30 (t, J = 7.3 Hz, 6H, CH ), 4.14−

3

Diethyl (2R,3R)-2,3-Bis((1-((S)-3-(1H-indol-3-yl)-1-methoxy-1-ox-

opropan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (10). Com-

pound 10 was synthesized according to general procedure E using

compound 4 and (S)-tryptophan methyl ester azide. Column

chromatography using ethyl acetate−hexane (6:4) was performed

for the puriﬁcation. Thick oil, yield 72%. IR (ATR): 3391, 3138,

4

.21 (m, 4H, CH ), 4.23−4.29 (m, 4H, CH ), 4.56 (s, 2H, CH),

2

.68−4.71 (m, 2H, CH ), 4.71−4.96 (m, 2H, CH ), 5.12−5−13 (m,

2

1

3

H, CH ), 7.73 (s, 2H, ArH). C NMR (100 MHz, CDCl ) δ 14.0

2

3

(

+ve, CH ), 14.1 (+ve, CH ), 50.8 (−ve, CH ), 61.5 (−ve, CH ),

3

2

6

2.4 (−ve, CH ), 64.9 (−ve, CH ), 78.8 (+ve, CH), 124.5 (+ve,

2

ArCH), 144.6 (ArC), 166.1 (CO), 168.8 (CO). HRMS

2

952, 2594, 2355, 2079, 1744, 1438, 1341, 1267, 1200, 1095, 1013,

+

−1

25

1

(

microTOF-QII, MS, ESI): Calcd for C H O N ([M + Na] )

2

32 10

6

857, 745, 603, 506 cm . [α] = −5.0° (0.4, CHCl ). H NMR (400

D

3

5

63.2072, found 563.2055.

Diethyl (2R,3R)-2,3-Bis((1-((S)-3-hydroxy-1-methoxy-1-oxopro-

pan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (7). Compound

was synthesized according to general procedure E using compound

and (S)-serine methyl ester azide. It was puriﬁed by column

chromatography using ethyl acetate−hexane (7:3) eluent. Thick oil,

yield 75%. IR (ATR): 3459, 3153, 2959, 2355, 2117, 1744, 1558,

446, 1371, 1274, 1207, 1095, 1021, 775, 700, 596, 492 cm . [α]

MHz, CDCl ) δ 1.16−1.28 (m, 6H, CH ), 3.52−3.66 (m, 4H, CH ),

3

2

3

.77 (s, 6H, OCH ), 3.96−4.04 (m, 2H, CH ), 4.08−4.16 (m, 2H,

3

2

CH ), 4.45−4.46 (m, 2H, CH), 4.57−4.60 (m, 2H, CH ), 4.84−4.88

2

7

4

(

m, 2H, CH ), 5.54−5.58 (m, 2H, CH), 6.52 (d, J = 2.3 Hz, 2H,

2

ArH), 7.10−7.18 (m, 4H, ArH), 7.31 (d, J = 7.8 Hz, 2H, ArH), 7.53−

7

13

.54 (m, 4H, ArH), 8.70 (br, 2H, NH). C NMR (100 MHz, CDCl )

3

δ 14.0 (+ve, CH ), 28.9 (−ve, CH ), 53.1 (+ve, OCH ), 61.7 (−ve,

−

1

25

3

2

3

1

=

D

CH ), 63.1 (+ve, CH), 64.5 (−ve, CH ), 78.8 (+ve, CH), 107.8 (ab,

1

2

+87.5° (0.4, CHCl ). H NMR (400 MHz, CDCl ). δ 1.26−1.32

3

ArC), 111.6 (+ve, ArCH), 117.8 (+ve, ArCH), 119.5 (+ve, ArCH),

22.0 (+ve, ArCH), 123.8 (+ve, ArCH), 124.2 (+ve, ArCH), 126.5

(

m, 6H, CH ), 3.80−3.82 (s, 6H, 2 × OCH ), 4.14−4.22 (m, 6H,

3

1

CH ), 4.32−4.37 (m, 2H, CH ), 4.43−4.46 (m, 2H, CH), 4.60−4.66

2

(

ab, ArC), 136.0 (ab, ArC), 143.5 (ab, ArC), 168.9 (CO), 169.03

(

7

m, 2H, CH ), 4.87−4.90 (m, 2H, CH ), 5.47−5.49 (m, 2H, CH),

2

CO). HRMS (microTOF-QII, MS, ESI): Calcd for C H O N

13

38 42 10

8

.94 (s, 2H, ArH). C NMR (100 MHz, CDCl ) δ 14.1 (+ve, CH ),

+

3

[M + Na] ) 793.2916, found 793.2925.

Diethyl (2R,3R)-2-((1-(2-Ethoxy-2-oxoethyl)-1H-1,2,3-triazol-5-

5

3.2 (+ve, OCH ), 62.1 (−ve, CH ), 62.8 (−ve, CH ), 64.5 (+ve,

3

2

CH), 64.6 (−ve, CH ), 78.3 (+ve, CH), 124.9 (+ve, ArCH), 143.5

2

yl)methoxy)-3-hydroxysuccinate (11). Compound 11 was synthe-

sized according to general procedure E using compound 5 and ethyl

bromo acetate azide. It was puriﬁed by column chromatography with

ethyl acetate−hexane (4:6). Thick oil, yield 76%. IR (ATR): 3503,

(

ArC), 167.4 (CO), 169.5 (CO). HRMS (microTOF-QII, MS,

+

ESI): Calcd for C H O N ([M + Na] ) 595.1970, found

2

32 12

6

595.1979.

Diethyl (2R,3R)-2,3-Bis((1-((S)-3-(4-hydroxyphenyl)-1-methoxy-1-

3

8

145, 2981, 2355, 2087, 1744, 1558, 1468, 1371, 1207, 1140, 1021,

oxopropan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (8a).

Compound 8a was synthesized according to general procedure E

using compound 4 and (S)-tyrosine methyl ester azide. Crude

product was puriﬁed by column chromatography with ethyl acetate−

hexane (7:3). Thick oil, yield 72%. IR (ATR): 3451, 3145, 3019,

−1 1

64, 797, 708, 581, 447 cm . H NMR (400 MHz, CDCl ) δ 1.24 (t,

3

J = 7.1 Hz, 3H, CH ), 1.29−1.34 (m, 6H, CH ), 4.11−4.20 (m, 1H,

3

CH of CH ), 4.21−4.32 (m, 5H, CH + CH ), 4.51 (d, J = 2.3 Hz, 1H,

2

CH), 4.62 (d, J = 2.3 Hz, 1H, CH), 4.66−4.69 (m, 1H, CH of CH ),

2

4

.92−4.95 (m, 1H, CH of CH ), 5.15 (s, 2H, CH ), 7.72 (s, 1H,

2

5

094, 1744, 1610, 1520, 1446, 1371, 1267, 1207, 1013, 827, 752, 596,

1

3

−

1

25

1

ArH). C NMR (100 MHz, CDCl

3

) δ 14.15 (+ve, CH ), 14.18 (+ve,

3

44 cm . [α]

= −7.50° (0.4, CHCl ). H NMR (400 MHz,

D

3

CH ), 14.2 (+ve, CH ), 50.9 (−ve, CH ), 61.8 (−ve, CH ), 62.3

3

2

CDCl ) δ 1.20−1.28 (m, 6H, CH ), 3.36−3.39 (m, 4H, CH ), 3.78

3

2

(

−ve, CH ), 62.5 (−ve, CH ), 64.5 (−ve, CH ), 72.3 (+ve, CH), 78.9

2

(

s, 6H, OCH ), 4.11−4.16 (m, 4H, CH ), 4.44 (s, 2H, CH), 4.57−

3

2

+ve, CH), 124.5 (+ve, ArCH), 144.6 (ArC), 166.1 (CO), 169.1

4

.60 (m, 2H, CH ), 4.81−4.84 (m, 2H, CH ), 5.47−5.51 (m, 2H,

2

CO), 171.1 (CO). HRMS (microTOF-QII, MS, ESI): Calcd

CH), 6.67 (d, J = 8.7 Hz, 4H, ArH), 6.75 (d, J = 8.7 Hz, 4H, ArH),

+

1

3

for C15

H

O

23

8

N

3

([M + Na] ) 396.1377, found 396.1394.

7

.55 (s, 2H, ArH). C NMR (100 MHz, CDCl ) δ 14.1 (+ve, CH ),

3

Diethyl (2R,3R)-2-Hydroxy-3-((1-((S)-3-hydroxy-1-methoxy-1-ox-

opropan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (12). Com-

pound 12 was synthesized according to general procedure E using

compound 5 and (S)-serine methyl ester azide. Crude product was

puriﬁed by column chromatography with ethyl acetate−hexane (4:6).

Thick oil, yield 73%. IR (ATR): 3481, 2959, 2087, 1736, 1446, 1371,

3

8.0 (−ve, CH ), 53.1 (+ve, OCH ), 61.6 (−ve, CH ), 64.3 (+ve,

2

3

2

CH), 64.5 (−ve, CH ), 78.9 (+ve, CH), 115.9 (+ve, ArCH), 124.1

2

(

+ve, ArCH), 125.5 (ab, ArC), 130.0 (+ve, ArCH), 143.7 (ab, ArC),

1

55.9, (ab, ArC), 168.5 (CO), 168.9 (CO). HRMS (microTOF-

+

QII, MS, ESI): Calcd for C H O N ([M + Na] ) 747.2592, found

3

4

40 12

6

747.2589.

−1 1

1

200, 1095, 1021, 857, 700, 596, 492 cm . H NMR (400 MHz,

Diethyl (2R,3R)-2-((1-((R)-3-(4-hydroxyphenyl)-1-methoxy-1-oxo-

CDCl ) δ 1.27 (t, J = 7.1 Hz, 3H, CH ), 1.33 (t, J = 7.1 Hz, 3H,

3

propan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)-3-((1-((S)-3-(4-hydrox-

yphenyl)-1-methoxy-1-oxopropan-2-yl)-1H-1,2,3-triazol-5-yl)-

methoxy)succinate (8g/h). Compound 8g/h was synthesized

according to general procedure E using compound 4 and (S)-tyrosine

methyl ester azide and (R)-tyrosine methyl ester azide. Thick oil, yield

CH ), 3.82 (s, 3H, OCH ), 4.14−4.21 (m, 3H, CH + CH of CH ),

3

2

4

=

.26−4.31 (m, 2H, CH ), 4.35−4.39 (m, 1H, CH of CH ), 4.47 (d, J

2

2.3 Hz, 1H, CH), 4.61 (d, J = 2.3 Hz, 1H, CH), 4.63−4.66 (m, 1H,

CH of CH ), 4.89−4.92 (m, 1H, CH of CH ), 5.47−5.50 (m, 1H,

CH), 7.96 (s, 1H, ArH). C NMR (100 MHz, CDCl

2

4%. [α]_D²⁵= −18° (0.4, CHCl ).

13

5

3

) δ 14.0 (+ve,

), 62.3

3

Diethyl (2R,3R)-2,3-Bis((1-((S)-1-methoxy-1-oxo-3-phenypropan-

CH ), 14.1 (+ve, CH ), 53.2 (+ve, OCH ), 61.8 (−ve, CH

3

2

-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (9). Compound 9 was

(−ve, CH ), 62.6 (−ve, CH ), 64.40 (−ve, CH ), 64.45 (+ve, CH),

2

synthesized according to general procedure E using compound 4 and

S)-phenylalanine methyl ester azide. It was puriﬁed by column

72.41 (+ve, CH), 78.68 (+ve, CH), 124.7 (ArCH), 143.6 (ArC),

(

167.4 (CO), 169.2 (CO), 171.2 (CO). HRMS (microTOF-

9

561

https://doi.org/10.1021/acs.jmedchem.1c00880

J. Med. Chem. 2021, 64, 9550−9566

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

+

1

QII, MS, ESI): Calcd for C H O N ([M + Na] ) 412.1327, found

4

H NMR (500 MHz, CDCl ) δ 2.37−2.38 (m, 2H, CH ), 4.56−4.63

1

5

23

9

3

2

12.1333.

Diethyl (2R,3R)-2-Hydroxy-3-((1-((S)-3-(4-hydroxyphenyl)-1-me-

thoxy-1-oxopropan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate

13). Compound 13 was synthesized according to general procedure

(m, 4H, 2CH ), 7.12−7.15 (m, 2H, ArH), 7.19−7.22 (m, 2H, ArH),

2

7.32−7.35 (m, 2H, ArH), 7.56−7.58 (m, 2H, ArH), 7.86−7.88 (d, J =

13

8

(

1

.25 Hz, 2H, ArH), 7.96−7.98 (d, J = 8.95 Hz, 2H, ArH). C NMR

(

125 MHz, CDCl ) δ 57.2 (−ve, CH ), 75.2 (C), 79.3 (+ve, CH),

3

2

E using compound 5 and (S)-tyrosine methyl ester azide and puriﬁed

by column chromatography with ethyl acetate−hexane (1:1). Thick

oil, yield 73%. IR (ATR): 3459, 3145, 2981, 2355, 2087, 1736, 1520,

16.0 (+ve, ArCH), 120.6 (ArC), 124.1 (+ve, ArCH), 125.6 (+ve,

ArCH), 126.4 (+ve, ArCH), 127.9 (+ve, ArCH), 129.4 (+ve, ArCH),

29.7 (ArC), 133.9 (ArC), 153.1 (ArC). HRMS (microTOF-QII,

1

−

1 1

1

446, 1371, 1200, 1095, 1013, 834, 700, 544 cm . H NMR (400

+

MS, ESI): Calcd for C H O ([M + Na] ) 305.0996, found

2

6

18

2

MHz, CDCl ) δ 1.21 (t, J = 7.1 Hz, 3H, CH ), 1.31 (t, J = 7.2 Hz, 3H,

3

05.0984.

Diethyl 2,2′-((([1,1′-Binaphthalene]-2,2′-diylbis(oxy))bis-

methylene))bis(1H-1,2,3-triazole-5,1-diyl))(S)-diacetate (19). Com-

CH ), 3.33−3.43 (m, 2H, CH ), 3.78 (s, 3H, OCH ), 4.05−4.13 (m,

3

2

3

1

H, CH of CH ), 4.15−4.22 (m, 1H, CH of CH ), 4.25−4.30 (m,

2

(

2

H, CH ), 4.46 (d, J = 2.3 Hz, 1H, CH), 4.57−4.62 (m, 2H, CH of

2

pound 19 was synthesized according to general procedure E using

compound 18 and ethylbromoacetate azide. It was puriﬁed by column

chromatography with ethyl acetate−hexane (1:1). Creamish solid,

yield 87%, mp 96 °C. IR (ATR): 3145, 3056, 2981, 1744, 1595, 1371,

CH + CH), 4.86−4.89 (m, 1H, CH of CH ), 5.49−5.52 (m, 1H,

2

CH), 6.70 (d, J = 8.2 Hz, 2H, ArH), 6.84 (d, J = 8.7 Hz, 2H, ArH),

1

3

7

.61 (s, 1H, ArH). C NMR (100 MHz, CDCl ) δ 14.10 (+ve, CH ),

3

1

4.15 (+ve, CH ), 38.0 (−ve, CH ), 53.1 (+ve, OCH ), 61.8 (−ve,

−1 1

3

2

3

1

207, 805, 581 cm . H NMR (400 MHz, CDCl ) δ 1.24 (t, J = 7.3

3

CH ), 62.3 (−ve, CH ), 64.3 (+ve, CH), 64.4 (−ve, CH ), 72.2 (+ve,

2

Hz, 6H, CH ), 4.19 (q, J = 7.0 Hz, 4H, 2CH ), 4.88 (s, 4H, 2CH ),

5

=

ArH), 7.51 (d, J = 9.1 Hz, 2H, ArH), 7.87 (d, J = 8.2 Hz, 2H, ArH),

7

(

1

3

2

CH), 79.0 (+ve, CH), 115.8 (ArCH), 123.3 (ArCH), 126.0 (ArC),

.12 (m, 2H, CH ), 5.25 (m, 2H, CH ), 6.79 (s, 2H, ArH), 7.13 (d, J

2

1

30.0 (ArCH), 143.8 (ArC), 155.5 (ArC), 168.5 (CO), 169.1

8.2 Hz, 2H, ArH), 7.19−7.24 (m, 2H, ArH), 7.32−7.36 (m, 2H,

(

CO), 171.1 (CO). HRMS (microTOF-QII, MS, ESI): Calcd

+

for C H O N ([M + Na] ) 488.1640, found 488.1635.

13

21

27

9

3

.94 (d, J = 8.7 Hz, 2H, ArH). C NMR (100 MHz, CDCl ) δ 14.1

3

Diethyl (2R,3R)-2-hydroxy-3-((1-((S)-1-methoxy-1-oxo-3-phenyl-

propan-2-yl)-1H-1,2,3-triazol-5-yl)methoxy)succinate (14). Com-

pound 14 was synthesized according to general procedure E using

compound 5 and (S)-phenyalanine methyl ester azide and puriﬁed by

column chromatography with ethyl acetate−hexane (3:7). Thick oil,

yield 75%. IR (ATR): 3145, 2952, 2355, 2117, 1744, 1625, 1558,

+ve, CH ), 50.7 (−ve, CH ), 62.4 (−ve, CH ), 63.7 (−ve, CH ),

3

2

15.9 (+ve, ArCH), 120.6 (ArC), 124.0 (+ve, ArCH), 124.1 (+ve,

ArCH), 125.5 (+ve, ArCH), 126.5 (+ve, ArCH), 128.0 (+ve, ArCH),

29.5 (+ve, ArCH), 134.0 (ArC), 145.0 (ArC), 153.6 (ArC), 166.25

CO). HRMS (microTOF-QII, MS, ESI): Calcd for C H O N

([M + Na] ) 643.2276, found 643.2276.

1

(

3

4

32

6

+

−

1

453, 1371, 1274, 1177, 820, 752, 566, 506, 462 cm . H NMR (400

MHz, CDCl ) δ 1.22 (t, J = 7.3 Hz, 3H, CH ), 1.32 (t, J = 7.1 Hz, 3H,

Dimethyl 2,2′-(((((S)-[1,1′-Binaphthalene]-2,2′-diyl)bis(oxy))bis-

3

(

methylene))bis(1H-1,2,3-triazole-5,1-diyl))(2S,2′S)-bis(3-hydroxy-

CH ), 3.42−3.56 (m, 2H, CH ), 3.75 (s, 3H, OCH ), 4.06−4.14 (m,

3

2

3

propanoate) (20). Compound 20 was synthesized according to

general procedure E using compound 18 and (S)-serine methylester

azide. Crude product was puriﬁed by column chromatography with

ethyl acetate−hexane (6:4). Creamish solid, yield 83%, mp 97 °C. IR

(ATR): 3280, 3153, 2952, 2117, 1900, 1744, 1595, 1505, 1431, 1326,

1

H, CH of CH ), 4.16−4.23 (m, 1H, CH of CH ), 4.25−4.31 (m,

2

H, CH ), 4.46 (d, J = 2.5 Hz, 1H, CH), 4.60−4.63 (m, 2H, CH of

2

CH + CH), 4.89 (m, 1H, CH of CH ), 5.52−5.55 (m, 1H, CH),

2

7

.03−7.06 (m, 2H, ArH), 7.22−7.29 (m, 3H, ArH), 7.66 (s, 1H,

13

ArH). C NMR (100 MHz, CDCl ) δ 14.12 (+ve, CH ), 14.17 (+ve,

3

−1 1

1

215, 1043, 805, 745, 544 cm . H NMR (500 MHz, CDCl ) δ 3.70

3

CH ), 38.7 (−ve, CH ), 53.2 (+ve, OCH ), 61.7 (−ve, CH ), 62.3

3

2

3

2

(

s, 3H, OCH ), 3.73 (s, 3H, OCH ), 4.11−4.12 (m, 2H, CH ), 4.21

3

2

(

−ve, CH ), 64.1 (+ve, CH), 64.6 (−ve, CH ), 72.3 (+ve, CH), 78.9

2

m, 1H, CH of CH ), 4.31−4.32 (m, 1H, CH of CH ), 4.83−4.92

2

+ve, CH), 123.0 (ArCH), 127.6 (ArCH), 128.9 (ArCH), 134.6

m, 2H, CH ), 5.03−5.05 (m, 1H, CH), 5.10−5.16 (m, 2H, 2CH),

2

ArC), 144.0 (ArC), 168.5 (CO), 169.0 (CO), 171.0 (CO).

5

.28−5.31 (m, 1H, CH), 6.96−7.05 (m, 2H, ArH), 7.13−7.18 (m,

2H, ArH), 7.23−7.24 (m, 2H, ArH), 7.34−7.35 (m, 2H, ArH), 7.48−

.51 (m, 1H, ArH), 7.54−7.59 (m, 1H, ArH), 7.88−7.89 (m, 2H,

HRMS (microTOF-QII, MS, ESI): Calcd for C H O N ([M +

2

1

27

8

3

+

Na] ) 472.1690, found 472.1664.

2R,3R)-2,3-Bis((1-(carboxymethyl)-1H-1,2,3-triazol-5-yl)-

7

(

1

3

ArH), 7.93−7.99 (m, 2H, ArH). C NMR (125 MHz, CDCl ) δ 53.1

(

methoxy)succinic acid (15). Compound 15 was synthesized

according to general procedure F using compound 6 and 1N

NaOH. Creamish solid, yield 78%. mp 132−34 °C. IR (ATR): 3384,

3

+ve, OCH ), 62.8 (−ve, CH ), 62.9 (−ve, CH ), 64.7 (+ve, CH),

3

2

1

15.9 (+ve, ArCH), 120.5 (ArC), 124.1 (+ve, ArCH), 124.2 (+ve,

−

1 1

ArCH), 125.5 (+ve, ArCH), 126.3 (+ve, ArCH), 128.0 (+ve, ArCH),

129.6 (+ve, ArCH), 129.7 (ArC), 134.0 (ArC), 153.7 (ArC), 167.2

3

145, 2892, 2363, 1722, 1625, 1222, 812, 641 cm . H NMR (500

MHz, DMSO-d ) δ 4.44 (s, 2H, 2xCH), 4.49−4.53 (m, 2H, CH ),

6

2

4

.75−4.77 (m, 2H, CH ), 5.25 (s, 4H, 2 × CH ), 7.98 (s, 2H, ArH).

(CO). HRMS (microTOF-QII, MS, ESI): Calcd for C34

H O N

32 8 6

2

1

3

+

C NMR (125 MHz, DMSO-d ) δ 50.9 (−ve, CH ), 64.2 (−ve,

([M + Na] ) 675.2174, found 675.2179.

6

2

Dimethyl 2,2′-(((((S)-[1,1′-Binaphthalene]-2,2′-diyl)bis(oxy))bis-

CH ), 78.6 (+ve, CH), 125.9 (+ve, ArCH), 143.8 (ArC), 169.1 (C

2

(

methylene))bis(1H-1,2,3-triazole-5,1-diyl))(2S,2’S)-bis(3-(4-

O), 170.7 (CO). HRMS (microTOF-QII, MS, ESI): Calcd for

−

hydroxyphenyl)propanoate) (21). Compound 21 was synthesized

according to general procedure E using compound 18 and (S)-

tyrosine methylester azide and puriﬁed by column chromatography

with ethyl acetate−hexane (7:3). Creamish solid, yield 86%, mp 86

C H O N ([M − H] ) 427.0844, found 427.0853.

14

16 10

6

(

2R,3R)-2,3-Bis((1-((S)-1-carboxy-2-phenylethyl)-1H-1,2,3-triazol-

5

-yl)methoxy)succinic acid (16). Compound 16 was synthesized

according to general procedure F using compound 9 and 1N NaOH.

Creamish solid, yield 80%. mp 150−153 °C. IR (ATR): 3429, 3145,

892, 1722, 1453, 1200, 820, 752, 506 cm . H NMR (500 MHz,

°

C. IR (ATR): 3145, 2952, 2370, 2117, 1893, 1744, 1617, 1513,

−

1 1

−

1 1

1

438, 1326, 1207, 1043, 805, 693, 544 cm . H NMR (500 MHz,

2

DMSO-d ) δ 3.09−3.14 (m, 2H, CH ), 3.32−3.35 (m, 2H, CH ),

3

2CH), 6.48 (d, J = 8.25 Hz, 4H, ArH), 6.72 (d, J = 8.28 Hz, 4H,

ArH), 6.92 (d, J = 8.49 Hz, 2H, ArH), 7.19 (t, J = 7.56 Hz, 2H, ArH),

7.35 (t, J = 7.22 Hz, 2H, ArH), 7.60 (s, 2H, ArH), 7.66 (d, J = 9.02

Hz, 2H, ArH), 7.96 (d, J = 8.17 Hz, 2H, ArH), 8.05 (d, J = 9.21 Hz,

2H, ArH), 9.24 (s, 2H, OH). C NMR (125 MHz, DMSO-d

(−ve, CH ), 53.2 (+ve, OCH ), 62.8 (−ve, CH ), 63.8 (+ve, CH),

115.5 (+ve, ArCH), 116.3 (+ve, ArCH), 119.7 (ArC), 124.1 (+ve,

ArCH), 124.2 (+ve, ArCH), 125.2 (+ve, ArCH), 125.8 (ArC), 126.8

(+ve, ArCH), 128.4 (+ve, ArCH), 129.4 (ArC), 129.7 (+ve, ArCH),

130.2 (+ve, ArCH), 133.7 (ArC), 143.6 (ArC), 153.9 (ArC), 156.6

DMSO-d ) δ 3.45−3.50 (m, 2H, CH ), 3.53−3.57 (m, 2H, CH ),

6

2

6

2

.62 (s, 6H, OCH ), 5.08−5.16 (m, 4H, 2CH ), 5.60−5.63 (m, 2H,

4

2

(

.36−4.40 (m, 2H, CH ), 4.47−4.50 (m, 2H, CH ), 4.70−4.73 (m,

3

2

H, CH ), 5.63−5.69 (m, 2H, 2CH), 7.12−7.21 (m, 10H, ArH), 8.04

2

1

3

s, 1H, ArH), 8.09 (s, 1H, ArH). C NMR (100 MHz, DMSO-d )

6

3

1

7.3 (−ve, CH ), 63.7 (+ve, CH), 64.1 (−ve, CH ), 78.4 (+ve, CH),

2

24.7 (+ve, ArCH), 127.2 (+ve, ArCH), 128.8 (+ve, ArCH), 129.3

1

3

(

+ve, ArCH), 136.7 (ArC), 143.5 (ArC), 170.2 (CO), 170.6 (C

6

) δ 36.5

O). HRMS (microTOF-QII, MS, ESI): Calcd for C H O N ([M

2

3

2

8

28 10

6

−

H] ) 607.1783, found 607.1775.

(

S)-2,2′-Bis(prop-2-yn-1-yloxy)-1,1′-binaphthalene (18). Com-

pound 18 was synthesized according to general procedure B using

S-binol and propargyl bromide. Creamish solid, yield 95%, mp 92 °C.

9

562

https://doi.org/10.1021/acs.jmedchem.1c00880

J. Med. Chem. 2021, 64, 9550−9566

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(

ArC), 169.0 (CO). HRMS (microTOF-QII, MS, ESI): Calcd for

0.09 mL = volume of the enzyme solution used.

Percent LOX inhibition = [{L.A. (P.C.) − L.A. (I.)}/L.A. (P.C.)]

× 100.

L.A. (P.C.) = lipoxygenase activity of positive control, L.A. (I.) =

lipoxygenase activity of inhibitor. IC₅₀values were determined from

the graph between percent inhibition versus concentration of

inhibitor.

+

C H O N ([M + Na] ) 827.2800, found 827.2817.

46

40

8

6

Dimethyl 2,2′-(((((S)-[1,1′-Binaphthalene]-2,2′-diyl)bis(oxy))bis-

(

methylene))bis(1H-1,2,3-triazole-5,1-diyl))(2S,2’S)-bis(3-phenyl-

propanoate) (22). Compound 22 was synthesized according to

general procedure E using compound 18 and (S)-phenylalanine

methylester azide. Compound was puriﬁed by column chromatog-

raphy using ethyl acetate−hexane (4:6) as eluent. Creamish solid,

yield 84%, mp 89−92 °C. IR (ATR): 3145, 3034, 2952, 2340, 2109,

COX-1 and COX-2 Inhibitory Immunoassay. The inhibitory

activities of all the synthesized compounds against COX-1 (ovine)

and COX-2 (human) were determined. Five diﬀerent concentrations

−

1 1

1

893, 1744, 1595, 1431, 1326, 1207, 1043, 909, 805, 574 cm . H

NMR (400 MHz, CDCl ) δ 3.00−3.17 (m, 2H, CH ), 3.29−3.34 (m,

2

−4

−8

3

2

(

10 to 10 M) of the compounds were prepared, and their enzyme-

H, CH ), 3.64−3.67 (m, 6H, 2OCH ), 5.12−5.22 (m, 4H, 2CH ),

2

3

2

inhibition assay was performed in triplicate as per the protocol

available with the COX Inhibitory Screening Assay Kit. The

background samples of both COX-1 and COX-2 were prepared by

putting 20 μL of each enzyme into separate test tubes and keeping

them in boiling water for 3 min. The background tubes for COX-1

and COX-2 were prepared by adding 160 μL of reaction buﬀer, 10 μL

of heme, and 10 μL of inactive COX-1 or COX-2. The initial activity

tubes of COX-1 and COX-2 were prepared by adding 160 μL of

reaction buﬀer, 10 μL of heme, and 10 μL of COX-1 and COX-2

enzyme to the respective tubes. The COX-1 and COX-2 inhibitor

tubes were prepared by adding 160 μL of reaction buﬀer, 10 μL of

heme, 10 μL of COX-1 or COX-2, and 10 μL of inhibitor solution.

After the incubation of the tubes at 37 °C for 10 min, 10 μL of

arachidonic acid was added to each of the tubes, which were

incubated at 37 °C for 2 min. The reaction was quenched by adding

5

.24−5.30 (m, 1H, CH), 5.32−5.38 (m, 1H, CH), 6.72−6.73 (m, 1H,

ArH), 6.76−6.83 (m, 5H, ArH), 7.03−7.13 (m, 6H, ArH), 7.14−7.18

(

7

4

m, 2H, ArH), 7.21−7.24 (m, 2H, ArH), 7.34−7.39 (m, 2H, ArH),

.46 (t, J = 3.6 Hz,1H, ArH), 7.48 (t, J = 3.5 Hz, 1H, ArH), 4.90−

13

.99 (m, 4H, ArH). C NMR (100 MHz, CDCl ) δ 38.3 (−ve,

3

CH ), 52.9 (+ve, OCH ), 63.6 (−ve, CH ), 64.0 (+ve, CH), 115.4

2

3

2

(

1

(

+ve, ArCH), 120.2 (ArC), 122.4 (+ve, ArCH), 123.9 (+ve, ArCH),

24.0 (+ve, ArCH),125.5 (+ve, ArCH), 126.5 (+ve, ArCH), 127.4

+ve, ArCH), 128.0 (+ve, ArCH), 128.6 (+ve, ArCH), 129.5 (+ve,

ArCH), 133.9 (ArC), 134.6 (ArC), 144.7 (ArC), 144.9 (ArC), 153.4

(

ArC), 168.3 (CO). HRMS (microTOF-QII, MS, ESI): Calcd for

+

C H O N ([M + Na] ) 795.2902, found 795.2899.

46

40

6

Dimethyl 2,2′-(((((S)-[1,1′-Binaphthalene]-2,2′-diyl)bis(oxy))bis-

methylene))bis(1H-1,2,3-triazole-5,1-diyl))(2S,2’S)-bis(3-(1H-indol-

-yl)propanoate) (23). Compound 23 was synthesized according to

(

3

the general procedure E using compound 18 and (S)-tryptophan

methylester azide and puriﬁed by column chromatography using ethyl

acetate−hexane (6:4) as eluent. Creamish solid, yield 86%, mp 98 °C.

IR (ATR): 3406, 3056, 2117, 1997, 1893, 1744, 1587, 1431, 1334,

30 μL of saturated SnCl to each reaction tube. As described in a

2

61

previous report, the prostaglandins produced in each well were

quantiﬁed using EIA.

Human Whole Blood Assay. The protocol for human whole blood

assay was approved by the Institutional Ethics Committee of Guru

Nanak Dev University, Amritsar, and the assay was performed as per

−

1 1

1

3

6

5

3

7

207, 1043, 805, 745, 581 cm . H NMR (500 MHz, CDCl ) δ

3

.02−3.14 (m, 2H, CH ), 3.29−3.36 (m, 2H, CH ), 3.64−3.67 (m,

2

61

H, 2OCH ), 5.11−5.21 (m, 4H, 2CH ), 5.25−5.28 (m, 1H, CH),

the procedure described in the previous report.

3

2

.29−5.37 (m, 1H, CH), 6.72−6.74 (m, 1H, ArH), 6.77−6.81 (m,

H, ArH), 7.04−7.11 (m, 6H, ArH), 7.13−7.18 (m, 2H, ArH), 7.21−

.23 (m, 2H, ArH), 7.34−7.38 (m, 2H, ArH), 7.46−7.49 (m, 2H,

Carrageenan Induced-Paw Edema Test. Swiss albino mice of

either sex weighing 25−35 g were used for the present studies. The

animals were kept under 12 h light/12 h dark cycle with free supply of

food and water and maintaining the temperature at 22 ± 2 °C. All the

protocols for animal studies were approved by the Institutional

Animal Ethical Committee (IAEC) of Guru Nanak Dev University,

Amritsar, Punjab, India. The animals were divided into diﬀerent

groups of six animals in each group. Group I, the control, comprised

of animals treated with vehicle (acetic acid/carrageenan), and group

II was treated with the standard reference drug indomethacin (10 mg

1

3

ArH), 7.89−7.91 (m, 2H, ArH), 7.94−7.98 (m, 2H, ArH). C NMR

(

100 MHz, CDCl ) δ 28.6 (−ve, CH ), 53.0 (+ve, OCH ), 63.1 (+ve,

3

2

3

CH), 63.1 (−ve, CH ), 107.7 (ArC), 111.6 (+ve, ArCH), 116.1 (+ve,

ArCH), 117.5 (+ve, ArCH), 119.5 (+ve, ArCH), 120.5 (ArC), 122.0

2

(

+ve, ArCH), 123.1 (+ve, ArCH), 124.1 (+ve, ArCH), 125.3 (+ve,

ArCH), 126.3 (ArC), 126.4 (+ve, ArCH), 127.8 (+ve, ArCH), 129.2

+ve, ArCH), 129.3 (ArC), 133.8 (ArC), 135.7 (ArC), 144.1 (ArC),

(

1

53.4 (ArC), 168.7 (CO). HRMS (microTOF-QII, MS, ESI):

−1

kg ). The other group/s of animals was/were administered test

+

Calcd for C H O N ([M + Na] ) 873.3120, found 873.3092.

−1

50

42

6

8

compounds at doses of 5 mg kg and variable doses in the case of

Lipoxygenase Inhibitory Activities. For LOX inhibition

compound 15 (for dose−response assay).

−

4

−5

−6

−7

−8

studies, solutions of compounds at 10 , 10 , 10 , 10 , and 10

100 μL of freshly prepared 1% carrageenan solution was injected

concentrations were prepared in DMSO. Ten microliters of each

compound from the above concentrations was added to 90 μL of

LOX (Soyabean lipoxygenase) solution in assay buﬀer taken in the

wells of a 96-well plate. Each compound was tested in triplicate, and

the average of three values with deviation <5% was taken for

calculation. Two wells were taken as blanks (assay buﬀer + AA), and

four wells were taken as positive controls (enzyme in assay buﬀer +

AA). The reaction was initiated by the addition of 10 μL of the

substrate (AA). After shaking the 96-well plate on a shaker for 5 min,

on the subplantar surface of the hind paw to induce paw edema.

Thirty min before carrageenan injection, standard and treatment

−1

groups received indomethacin (10 mg kg ) and test compounds (5

−1

mg kg ), respectively. Paw thickness (mm) was determined at

diﬀerent time intervals after carrageenan administration (15, 30, 60,

120, 180, 240, 300, and 360 min). A decrease in paw thickness (mm)

was taken as a measure of the anti-inﬂammatory potential of the

standard and the test compounds.

Acetic Acid-Induced Writhing Test. Acetic acid-induced

writhing is a screening model for assessing analgesic agents. Writhings

were indicated by stretching of the abdomen and extension of hind-

limbs. Writhings were induced by injecting freshly prepared 0.6% v/v

acetic acid solution intraperitonealy 30 min before acetic acid

injection; standard and treatment groups (six animals in each group)

1

00 μL of the chromogen (developing reagent) was added to each

well. The plate was again shaken for 5 min and read at 490 nm in a

microplate scanning spectrophotometer. Percent LOX inhibitory

activity was determined using the average of the two values for each

sample

−

1

received indomethacin (10 mg kg ) and test compounds (5 mg

−

1

[

A500/min /9.47 mM ] × [0.21 mL/0.09 mL]

sample dilution

−1

kg ), respectively. Thereafter, mice were placed in a transparent box

×

and the number of abdominal writhings was noted for 30 min.

Acute Toxicity Study. Acute toxicity studies were carried out

using Swiss albino mice. Brieﬂy, 2 groups were used with three

animals in each group (n = 3). The ﬁrst group was taken as normal

control while in the second group of animals, compound 15 was given

where:

A500/min = A500 (sample)/min − A500 (blank)/5 min,

−

1

9

0

.47 mM = extinction coeﬃcient of chromogen,

−

1

.21 mL = total volume of the solution in each well, and

orally at a dose of 300 mg kg . The study was performed after 4 h of

9

563

https://doi.org/10.1021/acs.jmedchem.1c00880

J. Med. Chem. 2021, 64, 9550−9566

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

the fasting period. Mice were continuously monitored for a period of

₁_H_a_n_d13C NMR spectra, mass spectra, and molecular

docking data (PDF )

4

h followed by periodic monitoring for the next 14 days.

Mechanistic Study. Three animal groups (n = 6) were utilized to

study the involvement of cyclooxygenase, lipoxygenase, and the nitric

oxide pathway in modulating the analgesic potential of the most active

Homology Models: NA Molecular formula strings of

compounds 6−23 (CSV)

PDB coordinates for computational models. Three-

dimensional models of target−ligand (compounds

docked in COX-2, 5-LOX) complexes (ZIP )

−

1

compound. Substance-P (10 μg kg ) was utilized to study the

involvement of COX-2 and LOX pathways. The nitric oxide donor L-

−

1

arginine (40 mg kg ) and nitric oxide synthase inhibitor N6-(1-

−

1

Iminoethyl)-L-lysine-dihydrochloride (L-NIL) (5 mg kg ) were

utilized to study the involvement of nitric oxide. Thirty minutes

before treatment with the test compound, the three groups of animals

received substance-P, L-arginine, and L-NIL, respectively. After 30

min, each group was treated with 0.6% v/v acetic-acid, and the

number of abdominal writhings was noted for 30 min.

AUTHOR INFORMATION

■

Corresponding Author

Palwinder Singh − Department of Chemistry, Guru Nanak

Dev University, Amritsar 143005, India; orcid.org/0000-

003-2332-5257 ; Email: palwinder_singh_2000@

yahoo.com

In Vivo Pharmacokinetic Studies. The in vivo PK studies were

evaluated using Swiss Albino mice (25−35 g) of either sex (10 groups

with three animals in each group). Ten mg kg⁻dose of compound 15

was suspended in 0.1% CMC and administered intraperitonealy. The

animals were anesthetized with ketamine (50 mg kg⁻i.p.). The blood

samples were withdrawn from the jugular vein at intervals of 30, 45,

and 60 min and 2, 3, 4, 6, 8, 11, and 24 h and collected in heparinised

tubes. Samples were withdrawn in triplicate (3 samples/time interval).

0

1

Authors

Priya Kumari − Department of Chemistry, Guru Nanak Dev

University, Amritsar 143005, India; orcid.org/0000-

0

002-1802-5412

100 μL of blood sample was withdrawn at each interval from one

Jashanpreet Kaur − Department of Pharmaceutical Sciences,

Guru Nanak Dev University, Amritsar 143005, India

Rajbir Bhatti − Department of Pharmaceutical Sciences, Guru

Nanak Dev University, Amritsar 143005, India;

orcid.org/0000-0002-5761-7074

animal. Further, the samples were prepared for LC-MS studies using

the procedure as described in the previous report.

Molecular Docking Studies. The molecular docking of the

62

compounds was performed in the active site pockets of COX-2 (pdb

6

3

64

ID 1CVU) and 5-LOX (pdb ID 3V99) using the procedure as

6

1

described in one of the previous reports.

ADME properties were calculated using online PreADMET

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00880

53

software.

Isothermal Titration Calorimetry Experiments. For ITC experi-

ments, solutions of compound 15 (100 μM) were prepared in HPLC

grade DMSO and 0.1 M Tris-Buﬀer (1:9) and enzyme (COX-1,

COX-2, 5-LOX) by dissolving 10 μL in 5 mL of Tris-buﬀer, and

solutions of HSA were prepared by dissolving 0.05 g of HSA in 1 mL

of Tris-Buﬀer. Enzyme solution in Tris-buﬀer was taken into the

sample cell and was titrated against the compound taken in a rotating

stirrer syringe (500 rpm) at 25 °C. Each experiment consists of 40

consecutive injections of 1 μL of the compound into the enzyme

solution, except in the case of COX-1, for which 20 injections were

there each of 2 μL, after regular time intervals of 120 s so that

equilibrium was attained at each titration point. The total heat, Q,

produced or absorbed by the active cell was determined at fractional

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Financial assistance from CSIR, New Delhi, is gratefully

acknowledged. P.K. thanks SERB-DST, New Delhi, for JRF.

ABBREVIATIONS USED

■

AA, arachidonic acid; COX-2, cyclooxygenase-2; COX-1,

cyclooxygenase-1; IC , 50% inhibitory concentration; IL-6,

5

0

th

saturation Θ after the i injection given by the equation

interleukin; i.p., intraperitoneally; L-NIL, N6-(1-iminoethyl)-L-

lysine-dihydrochloride; LOX, lipoxygenase; LPS, lipopolysac-

charide; LT4, leukotriene; NaH, sodium hydride; SEM,

standard error mean; THF, tetrahydrofuran

Q = nΘMtΔHV_o

where Mt is the total concentration of the enzyme, V is the cell

o

volume, n is the total number of binding sites in the enzyme, and ΔH

th

is the molar heat of ligand binding. The enthalpy change for the i

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https://doi.org/10.1021/acs.jmedchem.1c00880

J. Med. Chem. 2021, 64, 9550−9566

Article Doi

Design, Synthesis, and Activity Evaluation of Stereoconfigured Tartarate Derivatives as Potential Anti-inflammatory Agents in Vitro and in Vivo

DOI: 10.1021/acs.jmedchem.1c00880

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Full text of DOI:10.1021/acs.jmedchem.1c00880

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