Febuxostat-based amides and some derived heterocycles targeting xanthine oxidase and COX inhibition. Synthesis, in vitro and in vivo biological evaluation, molecular modeling and in silico ADMET studies

Bioorganic Chemistry 113 (2021) 104948

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Febuxostat-based amides and some derived heterocycles targeting xanthine

oxidase and COX inhibition. Synthesis, in vitro and in vivo biological

evaluation, molecular modeling and in silico ADMET studies

a

a,

b

a

c

Aya Y. Rashad , Shaymaa E. Kassab , Hoda G. Daabees , Ahmed E. Abdel Moneim , Sherif A.

F. Rostom

d,*

a

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Damanhur University, Damanhur, El-Buheria 22516, Egypt

Medicinal Chemistry Department, Faculty of Pharmacy, King Salman International University, Ras Sedr, South Sinai 46612, Egypt

Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo 11795, Egypt

b

c

d

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords:

Febuxostat

Amides

Various febuxostat derivatives comprising carboxamide functionalities and different substituted heterocycles

were synthesized and evaluated for their biological activities as xanthine oxidase (XO) and cyclooxygenase

(

COX) inhibitors. All the tested compounds exhibited variable in vitro XO inhibitory activities (IC50 values

.009–0.077 µM), among which the analog 17 has emerged as the most potent derivative (IC50 0.009 µM),

Heterocycles

Xanthine oxidase

COX

0

representing nearly 3-times the potency of febuxostat (IC50 0.026 µM). The same analogs were further investi-

gated for their in vitro COX-1 and COX-2 inhibitory activity, where ﬁfteen analogs demonstrated recognizable

COX-2 inhibitory potential (IC50 values range 0.04 – 0.1 µM), when correlated with celecoxib (IC50 0.05 µM),

together with appreciable selectivity indices. Compounds 5a, 14b, 17, 19c, 19e and 21b that showed signiﬁcant

in vitro XO and/ or COX inhibitory potentials were further investigated for their in vivo hypouricemic as well as

anti-inﬂammatory activities. Interestingly, the in vivo results were concordant with the collected in vitro data.

Docking of compounds 5a, 14b, 17, 19c, 19e and 21b with the active sites of XO and COX-2 isozymes

demonstrated superior binding proﬁle compared with the reported ligands (febuxostat and celecoxib, respec-

tively). Their docking scores were reasonable and cohering to a great extent with their corresponding in vitro IC50

values. Moreover, in silico computation of the predicted pharmacokinetic and toxicity properties (ADMET),

together with the ligand efﬁciency (LE) of the same six compounds suggesting their liability to act as new orally

active drug candidates with a predicted high safety proﬁle.

Docking

ADMET

1

. Introduction

that catalyzes the oxidation of hypoxanthine and xanthine to uric acid

[

6]. This process is associated with the production of reactive oxygen

Hyperuricemia is a common metabolic disorder originated from

species (ROS) that is involved in many pathological conditions such as

inﬂammation, cancer and metabolic disorders [7,8]. Therefore, XO in-

hibition constitutes the most signiﬁcant target for treating gout and

other pathological conditions caused by ROS [9,10].

elevated serum uric acid (the ﬁnal product of purine catabolism), as a

result of the overproduction or under excretion of urates [1,2]. This case

is considered as the main cause of gout owing to the deposition of

monosodium urate crystals in the joints, beside acting as pro-

inﬂammatory stimuli in some tissues resulting in acute inﬂammatory

gouty arthritis [1,3]. Hyperuricemia and gout are important risk factors

of several chronic disorders including metabolic syndrome, chronic

kidney disease (CKD) and cardiovascular troubles [4,5]. Xanthine oxi-

dase (XO) is an interconvertible form of xanthine dehydrogenase (XDH)

According to their structure relevance to the naturally occurring

purine derivatives (xanthines), XO inhibitors (XOIs) are classiﬁed into

two main groups namely; purine-like and non-purine inhibitors [11].

Allopurinol (an analog of hypoxanthine) is the prototype of purine-like

inhibitors, and was the cornerstone in treating gout for decades [12,13].

Due to the structural similarity of purine-like XOIs with the natural

*

Corresponding author.

E-mail address: sherif.rostom@alexu.edu.eg (S.A.F. Rostom).

https://doi.org/10.1016/j.bioorg.2021.104948

Received 12 January 2021; Received in revised form 6 April 2021; Accepted 22 April 2021

Available online 27 April 2021

0

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

bases, they could inhibit several purine and pyrimidine metabolic en-

zymes leading to obvious side effects among which Steven Johnson

syndrome [14]. On the other hand, febuxostat (1; Fig. 1) was the ﬁrst

non-purine selective non-competitive XOI approved in Europe in 2008,

then in the USA in 2009 [15], being more potent than allopurinol and

with fewer undesirable side effects [16,17]. However, in February 2019,

the FDA has stated a black box warning for febuxostat regarding its

cardiovascular mortality, being higher than that of allopurinol [18,19].

Consequently, there is an imperative need to discover novel non-purine

XOIs for the treatment of hyperuricemia and gout, with better efﬁcacy

and minimum side effects. Literature survey pointed out that several

non-purine XOIs belonging to various chemical entities have been

developed and exhibited excellent potency including pyrazoles [20],

isoxazoles [21], imidazoles [22], thiazoles [23,24], 1,2,3-triazoles

favorable for the stabilization of the ligand-enzyme complex and thus

the XOI activity [35]. Inspiringly, the motif of febuxostat structure

modiﬁcation is focused essentially on the 5-carboxylic acid function-

ality, which was either derivatized into various carboxamide derivatives

as H-bond forming centers or converted to a substituted heterocycle (A

and B; Fig. 1). In this context, small-sized amides e.g. the 2ry and 3ry

carboxamido, hydroxamic and acid hydrazide groups were taken into

account being H-bond donors and/or acceptors. Furthermore, size

enlargement of the acid hydrazide function with various atomic spacers

to the corresponding N-formyl, N-acetyl, sulfonamido, semicarbazide,

thiosemicarbazide and N-acylhydrazone derivatives were considered

hoping to assist the interaction of the target molecules with the desired

enzymatic targets through various intermolecular forces. In addition,

variation in the nature and size of the substituents was also selected so as

to impart different electronic, lipophilic and steric environment that

would inﬂuence the targeted biological activity. For instance, benzyl or

phenyl groups substituted with halo as well as methyl or methoxy

functions were introduced representing E-withdrawing and donating

entities, respectively. On the other hand, based on the contribution of

substituted 1,2,4-triazoles, 1,3,4-oxa(thia)diazoles and pyrazoles in a

variety of biological activities including the anti-inﬂammatory potential

[36–39], it was planned to integrate the inherited XO inhibitory activity

of febuxostat and the biologically-enhancing heterocycles in one struc-

ture entity hoping to yield novel lead compounds with improved XO

and/or COX inhibition activities. It is noteworthy mentioning that, most

of the newly synthesized heterocycle-substituted analogs are structur-

ally relevant to the vicinal diarylheterocycles class of COX-2 inhibitors

[40,41]. Docking studies were performed in order to deﬁne the plausible

binding pattern and afﬁnity of the newly synthesized compounds in the

active site of targeted enzymes (XO and COX-2) from a molecular

viewpoint. Moreover, in silico computation of the compliance to the

Lipinski’s rule of ﬁve (RO5), physicochemical parameters and drug

likeness score of the biologically active compounds were performed in

order to predict their pharmacokinetic and toxicity (ADMET) proﬁles,

and to assess their suitability to serve as possible orally-active dug

candidates. Finally, ligand efﬁciency metrics such as ligand efﬁciency

(LE) and lipophilic ligand efﬁciency (LLE) were applied to normalize the

in vitro biological activity with respect to the physicochemical properties

of the molecule, and they also measure how effectively the molecule

uses its structural features in binding to the target.

[

13,25], 1,2,4-triazoles [26,27], 1H-tetrazoles [28]. Although XOIs

can signiﬁcantly reduce serum uric acid levels in chronic gouty patients,

yet they failed in treating acute gout attacks [29]. Hence, the use of non-

steroidal anti-inﬂammatory drugs (NSAIDs) in the management of acute

gouty pain attacks imposes itself. NSAIDs exert their anti-inﬂammatory

effects through inhibiting the constitutive cyclooxygenase 1 (COX-1)

and the inducible COX-2 isozymes, which are responsible for the

biosynthesis of several inﬂammatory mediators including prostaglan-

dins (PGs) [30]. Consequently, a monotherapy with a drug with dual

hypouricemic/anti-inﬂammatory activity would be prioritized from

both pharmaco-economic and patient compliance perspectives. This

premise of combining the afore-mentioned two activities in one entity

has been previously discussed and reported [24,31]. Careful literature

survey revealed that limited endeavors have been conducted aiming to

modify the structure of febuxostat in order to ameliorate its selectivity

towards the XO active site and hence, the corresponding hypouricemic

efﬁciency. In particular, few reports have discussed the derivatization of

the 5-carboxylic acid group into 2ry or 3ry carboxamide functionalities

[

32].

In view of the afore-mentioned facts, this work aims at the devel-

opment of multi-target designed ligands (MTDLs) possessing dual XO

and/or COX inhibitory activities. The targeted compounds were

designed utilizing pharmacophoric hybridization strategy, by

combining different pharmacophores believed to be responsible for the

desired activities in one framework. Febuxostat 1 was relied upon as the

main scaffold to be derivatized being a well-known XOI, beside its re-

ported anti-inﬂammatory activities resulting from the ability to inhibit

both the XO-induced oxidative stress, as well as the COX-2 isozyme

2. Results and discussion

[

33,34]. According to the reported structure activity relationship (SAR)

study, 3D-QSAR models, docking results and key structure features

affecting the XOI activity of some febuxostat analogs, introduction of H-

bond donor and acceptor groups instead of the 5-carboxylic acid group is

2.1. Chemistry

The synthetic routes employed to obtain the target XOIs are outlined

Fig. 1. Febuxostat [1], the general structures of the targeted substituted amides [A], and derived heterocyclic ring systems [B].

2

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

in Schemes 1–3. As shown in Scheme 1, febuxostat 1 was converted to

the ethyl ester 2 following a reported method [42]. The resulting ester 2

was treated with hydroxylamine hydrochloride neutralized with potas-

sium hydroxide to afford the corresponding hydroxamic acid 3.

Furthermore, the reported acid hydrazide 4 [43] was prepared via a

modiﬁed method involving the treatment of the ethanolic solution of the

ethyl ester 2 with excess hydrazine hydrate. Reﬂuxing the acid hydra-

zide 4 with equimolar amounts of alkyl or aryl isothiocyanates in

ethanol gave the corresponding thiosemicarbazides 5a-f. However, the

semicarbazide 8 and thiosemicarbazide 9 were prepared by one-pot

reaction of febuxostat 1, 1H-benzotriazole and thionyl chloride in

DCM, where the resulting N-acylbenzotriazole was treated with the

semicarbazide hydrochloride or thiosemicarbazide, respectively. The

obtained low yields from this method have obliged the shift to an

alternative synthetic pathway for the preparation of amides utilizing

acid chlorides [44,45]. In this study, the reported acyl chloride 7 [32,46]

was prepared by heating febuxostat 1 with thionyl chloride, then

reﬂuxed with semicarbazide hydrochloride or thiosemicarbazide in THF

containing few drops of triethylamine (TEA) to yield the corresponding

analogs 8 and 9, respectively. Moreover, the febuxostat amide de-

rivatives 10a-e were achieved in good yields upon reacting the acyl

chloride 7 with variable amines in THF containing few drops of TEA.

Here, it should be mentioned that lower yields of the amides were ob-

tained by utilizing the one-pot reaction of thionyl chloride, febuxostat

and the respective amines in the presence of a catalytic amount of TEA.

presence of catalytic amounts of glacial acetic acid afforded the corre-

sponding N-acylhydrazones 14a,b in good yields. Moreover, condensa-

tion of the acid hydrazide

4 with acetylacetone afforded the

corresponding 3,5-dimethyl-1H-pyrazole 15, while fusing 4 with ethyl

ethoxymethylenecyanoacetate at 170 ⁰C resulted in the targeted pyr-

azole 16. On the other hand, treating 4 with carbon disulﬁde or with an

equimolar amount of benzoic acid in the presence of phosphorus oxy-

chloride yielded the corresponding 1,3,4-oxadiazol-2-thione 17 or the 2-

aryl oxadiazole 18, respectively.

In Scheme 3, alkaline intramolecular cyclization of the substituted

thiosemicarbazides 5a-f afforded the corresponding 1,2,4-triazolin-5-

thiones 19a-f. Whereas, heating the N-phenyl derivative 5a with phos-

phorus oxychloride yielded the corresponding 1,3,4-thiadiazole 20.

Furthermore, the 1,3,4-oxadiazoles 21b,d were prepared by treating the

thiosemicarbazides 5b,d with iodine and potassium iodide in aqueous

sodium hydroxide solution. The structures of the newly synthesized

compounds were conﬁrmed using IR, ¹H NMR, C NMR, Mass Spec-

trometry and elementary microanalyses, and are detailed in the exper-

imental section.

13

2

.2. Biological evaluation

.2.1. In vitro Xanthine Oxidase (XO) inhibitory activity

All of the synthesized target compounds were investigated for their in

vitro ability to inhibit bovine XO enzyme using spectrophotometric assay

based on the measurement of uric acid production at λ 295 nm [48,49].

The IC50 values (the concentration (µM) causing 50% enzyme inhibi-

tion) were determined each in triplicate, and the results were recorded

in Table 1. Febuxostat was employed as the positive control.

Interestingly, the analog 10a (R = CH

2 6 5

-C H ) was previously reported as

anticancer [46] and antibacterial [47] agent.

Referring to Scheme 2, treatment of the acid hydrazide 4 with either

formic acid or acetic anhydride gave rise to the corresponding N-formyl

1

1 or N-acetyl 12 derivatives, respectively in good yields. Meanwhile,

The results presented in Table 1 revealed that all the tested com-

pounds exhibited variable XO inhibitory activities, with IC50 values

ranging between 0.009 and 0.077 µM as compared to febuxostat (IC50

the 4-toluenesulfonamide derivative 13 was successfully prepared by

reacting the acid hydrazide 4 with tosyl chloride in dry pyridine.

Furthermore, condensing 4 with benzaldehyde or anisaldehyde in the

0

.026 µM). Compounds 8, 10b and 19d were proven to be equipotent

Scheme 1. Reagents and conditions. i: H

reﬂux, 1–4 h; v: 1H-benzotriazole, SOCl , DCM, RT, 2 h; vi: NH

NH CONHNH .HCl (or NH CSNHNH ), TEA, THF, reﬂux, 2 h; ix: SOCl

2

SO

4

, EtOH, reﬂux, 9 h; ii: NH

CONHNH

, Et

2

OH.HCl, KOH, EtOH, RT,1h; iii: NH

2

NH

2

⋅H

2

O 99%, EtOH, reﬂux,12 h; iv: RNCS, EtOH,

◦

2

.HCl, NaHCO (or NH CSNHNH

3

2

), DMF, 60 C, 4 h; vii: SOCl

2

, reﬂux, 45 min; viii:

2

3

N, DCM, amines, RT, 5 min; x: amines, TEA, THF, reﬂux, 2 h.

3

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Scheme 2. Reagents and conditions. i: HCOOH, reﬂux, 3 h; ii: Ac

2

O, AcONa, RT, 2 h; iii: Tosyl chloride, pyridine, RT, 24 h; iv: RCHO, EtOH, reﬂux, 1–3 h; v:

(

CH

3

CO)

2

CH

2

, DMF, reﬂux, 5 h; vi: EtOCH = C(COOEt)CN, 170 ^◦C, 1 h; vii: CS

2 6 5 3

/KOH, EtOH, reﬂux, 6 h; viii: C H COOH, POCl , reﬂux, 8 h.

Scheme 3. Reagents and conditions. i: 5a-e: 2 N NaOH, cetrimide, reﬂux, 2–6 h and 5f: TEA, dioxane, reﬂux, 5 h; ii: POCl

3 2

, reﬂux, 11 h; iii: I /KI, NaOH, RT, 15 min.

with febuxostat as XOIs with IC50 value of 0.026 µM. Interestingly, the

analog 17 has emerged as the most potent XOI (IC50 0.009 µM), repre-

senting nearly 3-times the potency of febuxostat. Whereas, compounds

febuxostat.

2.2.2. In vitro COX-1 and COX-2 inhibitory activities

5

a, 10c, 14b, 15, 18, 19a,c,e, 20 and 21b exhibited distinctive inhib-

All of the synthesized target compounds (together with febuxostat 1,

the reference standard XOI in this study) were investigated for their in

vitro inhibitory activity towards COX-1 and COX-2 isozymes utilizing an

ovine COX-1 / human recombinant COX-2 assay kit (Catalog no.

560131; Cayman Chemicals Inc. Ann Arbor, MI, USA) according to the

manufacturer’s instructions and a literature procedure [50,51]. The ef-

ﬁcacies of the tested compounds were calculated as the concentration

that brings about 50% enzyme inhibition (IC50; µM), and the COX-2

selectivity index (SI value) was calculated as IC50 (COX-1)/IC50 (COX-

2). The results were determined each in triplicate and were recorded in

Table 1. Diclofenac sodium and celecoxib were employed as positive

itory afﬁnities towards the XO enzyme recording IC50 values lying be-

tween 0.011 and 0.020 µM (2.4–1.3 times more active than febuxostat).

Furthermore, the analogs 4, 10a, 11, 12, 14a, 19b, and 21d showed

nearly equipotent XOI activity with febuxostat, being slightly more

potent (IC50 values 0.021–0.024 µM). Meanwhile, compounds 9, 10e

and 16 were also nearly equipotent to febuxostat, yet with a relatively

lower activity (IC50 0.032, 0.028 and 0.033 µM, respectively). On the

contrary, the synthesized derivatives 3, 10d, 13 and 19f (IC50 values

0

.077, 0.063, 0.068 and 0.047 µM, respectively) were considered as the

least active XOIs in this study, showing nearly 33–55% of the activity of

4

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Table 1

XOI in this study) displayed moderate COX-1 inhibitory potential when

compared with diclofenac sodium (IC50 5.37 vs 3.8 µM, respectively).

However, it showed weak afﬁnity (IC50 0.34 µM) and selectivity (SI

value 15.79) to the COX-2 isozyme when compared with celecoxib (IC50

0.05 µM and SI value 294).

It is noteworthy to mention that the in vitro XO and COX-1/COX-2

enzymatic inhibitory activities of the newly synthesized compounds

were also expressed as mean ± conﬁdence level (95%), and are provided

in addition to some representative graphs of IC50 calculations in the

supplementary section.

In vitro xanthine oxidase (XO) and COX-1/COX-2 enzymatic inhibitory activities

of the newly synthesized compounds.

Cpd. No.

XO

COX-1

COX-2

a

b

c

d

IC50 (µM)

SI

e

Celecoxib

NT

14.7 ± 0.0577

3.80 ± 0.0067

5.37 ± 0.0333

6.53 ± 0.0882

4.97 ± 0.0667

12.57 ± 0.0667

5.90 ± 0.0577

7.73 ± 0.0667

9.03 ± 0.0667

8.70 ± 0.1155

8.37 ± 0.0667

6.70 ± 0.1155

5.87 ± 0.0333

7.13 ± 0.0667

6.53 ± 0.0333

12.73 ± 0.0882

9.33 ± 0.0667

9.63 ± 0.0667

6.87 ± 0.0333

11.63 ± 0.0667

7.37 ± 0.0333

8.37 ± 0.0333

9.77 ± 0.0882

12.97 ± 0.0667

12.27 ± 0.3180

10.73 ± 0.0667

12.77 ± 0.0882

11.87 ± 0.0333

10.63 ± 0.0667

13.47 ± 0.0333

10.67 ± 0.0667

0.05 ± 0.0006

0.84 ± 0.0590

0.34 ± 0.0067

0.27 ± 0.0033

0.37 ± 0.0033

0.04 ± 0.0003

0.28 ± 0.0067

0.14 ± 0.0033

0.11 ± 0.0033

0.12 ± 0.0033

0.11 ± 0.0033

0.16 ± 0.0033

0.22 ± 0.0033

0.26 ± 0.0033

0.12 ± 0.0067

0.04 ± 0.0003

0.10 ± 0.0033

0.07 ± 0.0007

0.29 ± 0.0088

0.07 ± 0.0003

0.14 ± 0.0033

0.10 ± 0.0033

0.09 ± 0.0007

0.04 ± 0.0003

0.05 ± 0.0007

0.08 ± 0.0003

0.04 ± 0.0003

0.05 ± 0.0003

0.07 ± 0.0006

0.04 ± 0.0007

0.08 ± 0.0003

294

Diclofenac Na

NT

4.52

1

3

4

5

8

9

1

2

(Febuxostat)

0.026 ± 0.0012

0.077 ± 0.0014

0.021 ± 0.0015

0.017 ± 0.0006

0.026 ± 0.0019

0.032 ± 0.0032

0.021 ± 0.0021

0.026 ± 0.0026

0.017 ± 0.0023

0.063 ± 0.0029

0.028 ± 0.0015

0.021 ± 0.0018

0.023 ± 0.0026

0.068 ± 0.0039

0.024 ± 0.0024

0.011 ± 0.0008

0.015 ± 0.0020

0.033 ± 0.0018

0.009 ± 0.0010

0.018 ± 0.0009

0.016 ± 0.0009

0.023 ± 0.0023

0.018 ± 0.0016

0.026 ± 0.0027

0.019 ± 0.0021

0.047 ± 0.0039

0.020 ± 0.0019

0.018 ± 0.0014

0.024 ± 0.0018

15.79

24.19

13.43

314.25

21.07

55.21

82.09

72.50

76.09

41.88

26.68

27.42

54.42

318.25

93.30

137.57

23.69

166.14

52.64

83.70

108.56

324.25

245.40

134.13

319.25

237.40

151.86

336.75

133.38

a

2.2.3. In vivo hypouricemic effect

0a

0b

0c

0d

0e

1

Compounds 5a, 14b, 17, 19c, 19e and 21b that showed signiﬁcant

in vitro XOI potentials were further investigated for their in vivo hypo-

uricemic activities in potassium oxonate-induced acute hyperuricemia

mouse model using febuxostat as a positive control [20,52].

As presented in Table 2 and illustrated in Fig. 2, all the six tested

compounds exhibited an observable and continuous reduction in serum

uric acid (SUA) levels along the speciﬁed time intervals (1, 2, 4, 6 and 8

h). Taking the intermediate interval (4 h) as a check point, the investi-

gated compounds showed a signiﬁcant reduction in SUA levels (P <

2

3

4a

4b

5

6

0

.0001) with a range of 52.53–59.60 %, which was slightly better than

7

febuxostat (48.48%). Meanwhile, the area under the curve (AUC Uric acid,

1-8h) was measured to determine the % cumulative reduction in SUA

along the whole bioassay period (1–8 h) after drug administration. The

obtained results showed a noticeable reduction of 43.60–50.25% in the

AUC during the whole period compared with the hyperuricemic model

8

9a

9b

9c

9d

9e

9f

0

(

P < 0.0001). In particular, the AUC (uric acid, 1–8 h) of compounds

(5a, 14b and 17) and the analogs (19c and 21b) revealed more potent

hypouricemic effects than that of febuxostat (p values > 0.0001 and >

1b

1d

0

.01, respectively). Interestingly, the obtained in vivo results were, to a

a

Compounds’ concentration causing 50% in vitro (XO) enzyme inhibition.

great extent, concordant with the corresponding in vitro XOI activities.

However at 4 h time interval, although the 4-methoxytriazole derivative

19e showed more potent in vivo hypouricemic effect when compared

with febuxostat (p > 0.001), yet it showed a cumulative reduction in

SUA approximately like that of febuxostat with non-signiﬁcant differ-

ence. The reasonable in vivo activity of compounds 5a, 14b, 17, 19c,

b,c

Compounds’ concentration causing 50% inhibition of cyclooxygenase 1 and 2

COX-1 and COX-2) enzymatic activity, respectively.

(

a,b

All values are expressed as Mean ± SEM of triplicate determinations.

COX-2 selectivity index: (COX-1 IC50 / COX-2 IC50).

NT: not tested.

d

e

1

9e and 21b would reﬂect their good pharmacokinetic and systemic

control anti-inﬂammatory agents against COX-1 and COX-2 enzymes,

respectively.

bioavailability proﬁles, therefore presenting hopeful candidates for the

treatment of hyperuricemia.

As presented in Table 1, all the investigated compounds revealed

mild COX-1 inhibitory effects (IC50 range 4.97–13.47 µM) as compared

with the standard non-selective COX inhibitor; diclofenac sodium (IC50

2

.2.4. In vivo anti-inﬂammatory screening

To assess the in vivo anti-inﬂammatory activity (AI) of the same six

3

.8 µM). Among these, seventeen compounds (3, 4, 8–12, 14a,b, 15,

7, 18 and 19a) displayed an acceptable COX-1 inhibition proﬁle (IC50

compounds (5a, 14b, 17, 19c, 19e and 21b) with acceptable in vitro

COX-2 selectivity indices together with febuxostat, a carrageenan-

induced mouse paw edema bioassay was employed using celecoxib as

a reference standard [53,54]. The ability of the tested compounds to

decrease edema thickness (mm) in mouse paw along the speciﬁed time

intervals (2, 4 and 8 h) after carrageenan injection, and the percentage

edema inhibition (anti-inﬂammatory activity; AI%) were recorded in

Table 3 and depicted in Fig. 3. The results revealed that the investigated

six compounds demonstrated considerable AI activities manifested as

prominent reduction in paw thickness when compared with the

1

range 4.97–9.77 µM). Meanwhile, ﬁfteen analogs (5a, 13, 14a,b, 16,

8, 19a-f, 20 and 21b,d) demonstrated an appreciable COX-2 inhibitory

1

activity lying in the nanomolar to sub-micromolar level (IC50 values

range 0.04–0.1 µM), when correlated with celecoxib (IC50 0.05 µM), the

standard selective COX-2 inhibitor. In particular, compounds 5a, 13,

1

9b,e and 21b exhibited COX-2 inhibition proﬁle superior to that of

celecoxib (IC50 0.04 vs 0.05 µM, respectively), whereas the analogs 19c

and 19f (IC50 0.05 µM) proved to be equipotent with celecoxib. Mean-

while, the rest of the active compounds displayed nearly 50 – 70% of the

COX-2 inhibitory efﬁciency of celecoxib. Regarding the selectivity to

COX-2 enzymatic inhibition, ﬁve compounds namely (5a, 13, 19b,e and

“carrageenan only” group at the speciﬁed time intervals with P values <

0.0001. Comparing the AI% of the test compounds with that of the

reference drug celecoxib after 2 h indicated that compound 19e was the

2

1b) demonstrated an outstanding COX-2 selectivity index values (SI)

most active member as shown from its potent and quick onset of action

lying between 314.25 and 336.75, which were superior to that of cele-

coxib (SI 294). Additionally, the triazoles 19c and 19f revealed a

considerable selectivity towards the same isozyme (SI values 245.4 and

(

73.33%) which was superior to celecoxib (72.57%). Meanwhile, reli-

able AI% values were displayed by the analogs 5a, 14b, 19c and 21b,

comparable with celecoxib (AI% 62.86 – 68.76 vs 72.57%, respectively),

whereas the analog 17 proved to be the least active member with AI% of

2

37.4, respectively) comparable with that of celecoxib. Meanwhile, the

analogs 14b, 16, 19a,d, 20 and 21d were found to be moderately se-

lective COX-2 inhibitors (SI values range 108.56–166.14), whereas the

rest of the tested compounds showed mild to weak selectivity indices

lying between 13.43 and 93.3. Here, it should be highlighted that under

the same experimental conditions, febuxostat 1 (the reference standard

5

4.29. Interestingly, the same paw edema inhibitory proﬁle of the tested

six compounds was maintained after 4 h time interval. Nevertheless

after 8 h, compounds 19e and 21b proved to be more active than cel-

ecoxib (86.9 and 89.29 vs 83.57%, respectively), while the analog 5a

was nearly equipotent with the reference drug (81.67 vs 83.57%,

5

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Table 2

In vivo hypouricemic effects of compounds 5a, 14b, 17, 19c, 19e, 21b and febuxostat in potassium oxonate-induced acute hyperuricemia mouse model.*

a

b,c

e

Cpd. No.

Serum uric acid (SUA; mmol/L)

AUC (Uric acid, 1-8h) (% cumulative SUA reduction)

1

h

2 h

4 h

6 h

8 h

Model

1.23 ± 0.005

1.04 ± 0.004

0.77 ± 0.006

(25.96)

0.99 ± 0.003

0.51 ± 0.005

(48.48)

0.95 ± 0.005

0.39 ± 0.004

(58.95)

0.91 ± 0.007

0.24 ± 0.005

(73.63)

4.06 ± 0.015

c

Febuxostat

1.13 ± 0.004

2.34 ± 0.015

d

(

8.13)

(42.36)

c,f

5

1

2

a

1.11 ± 0.006

9.76)

1.09 ± 0.004

11.38)

1.08 ± 0.004

12.20)

1.13 ± 0.002

8.13)

1.12 ± 0.003

8.94)

1.13 ± 0.005

8.13)

0.74 ± 0.003

(28.85)

0.45 ± 0.004

(54.55)

0.34 ± 0.003

(64.21)

0.22 ± 0.003

(75.82)

2.19 ± 0.012

(

(46.06)

c,f

4b

7

0.72 ± 0.002

(30.77)

0.43 ± 0.008

(56.57)

0.33 ± 0.003

(65.26)

0.21 ± 0.003

(76.92)

2.12 ± 0.015

(

(47.78)

c,f

0.71 ± 0.004

(31.73)

0.40 ± 0.005

(59.60)

0.31 ± 0.004

(67.37)

0.14 ± 0.003

(84.62)

2.02 ± 0.014

(

(50.25)

c,g

9c

9e

1b

0.75 ± 0.003

(27.88)

0.45 ± 0.002

(54.55)

0.37 ± 0.003

(61.05)

0.23 ± 0.003

(74.73)

2.25 ± 0.009

(

(44.58)

c

0.77 ± 0.005

(25.96)

0.47 ± 0.004

(52.53)

0.38 ± 0.004

(60.00)

0.22 ± 0.003

(75.82)

2.29 ± 0.012

(

(43.60)

c,g

0.75 ± 0.003

(27.88)

0.47 ± 0.004

(52.53)

0.35 ± 0.005

(63.16)

0.25 ± 0.004

(72.53)

2.26 ± 0.013

(

(44.33)

*

a

b

c

d

e

f

Data were analyzed by one-way ANOVA followed by Tukey’s Karmer post hoc test for multiple comparisons.

Dose level for all compounds: 5 mg/kg b.wt. intragastrically.

values are expressed as Mean ± SEM (number of animals n = 5 mice).

Means are signiﬁcantly different from the hyperuricemic model group at p < 0.0001.

Values between parentheses (% reduction in SUA).

Area under the curve; AUC (Uric acid, 1-8h) (mmol/L. h).

Means are signiﬁcantly different from febuxostat at p < 0.0001.

Means are signiﬁcantly different from febuxostat at p < 0.01.

g

Fig. 2. Hypouricemic effects of compounds 5a,

1

4b, 17, 19c, 19e and 21b in comparison with

febuxostat. (a) Time-dependent changes of serum

uric acid levels after 1–8 h of the treatment. (b)

Serum uric acid levels after 4 h of the treatment.

(

c) Areas under the curve (AUC) of serum uric

acid after 1–8 h of the treatment. The data are

analyzed by one-way ANOVA followed by Tukey’

s Karmer post hoc test for multiple comparisons

and expressed as the Mean ± SEM (n = 5). **** P

<

0.0001 compared to the hyperuricemic model.

#

### P < 0.0001, ### P < 0.001, ## P < 0.01,

ns: non-signiﬁcant compared to febuxostat.

respectively). It is to be noted that although compound 17 has displayed

a weak in vitro COX-2 inhibitory potency (IC50 0.14 µM), it showed an

acceptable in vivo AI activity at the three time intervals compared with

celecoxib, yet still the least potent member among the tested com-

pounds. On the contrary, febuxostat exhibited inﬂammatory or very

weak anti-inﬂammatory activity under the same experimental condi-

tions, which matches with its in vitro COX-2 inhibitory activity. On the

other hand, the area under the curve (AUC paw edema thickness, 0-8h) was

measured to determine the % cumulative edema inhibition along the

whole bioassay period (0–8 h) after carrageenan administration. The

results showed that compounds 5a, 14b, 19c, 19e and 21b displayed

nearly equipotent AI proﬁle with celecoxib (17.88 – 20.01 vs 19.97%,

respectively), while the analog 17 is still acting as the least active

member.

2.2.5. Structure-activity correlation

A careful inspection of the structures of the tested compounds

revealed that they belong to two series namely, carboxamides with

variable substitutions and sizes (Schemes 1 and 2), and analogs sup-

ported with substituted heterocycles (Schemes 2 and 3). Concerning the

newly synthesized small-sized amide derivatives, the synthesis of the

hydroxamic acid 3 led to 3-fold reduction in the XOI activity, when

compared with febuxostat (IC50 0.077 vs 0.026 µM, respectively).

Contrariwise, derivatization into the corresponding acid hydrazide 4

6

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Table 3

the same isozyme (IC50 0.04 vs 0.05 µM and SI value 314.25 vs 294).

Furthermore, the development of the N-formyl 11 and N-acetyl 12 de-

rivatives led to a limited or no reﬁnement in the XOI activity (0.021 and

In vivo anti-inﬂammatory activity (AI) of compounds 5a, 14b, 17, 19c, 19e, 21b

and febuxostat in carrageenan-induced mouse paw edema bioassay (acute in-

ﬂammatory model).*

0

.023 µM, respectively), when correlated to either the parent hydrazide

a

b

Cpd. No.

Thickness of edema (mm)

2 h 4 h

AUC (Paw edema thickness,

f

or febuxostat. It is to be noted that the target compounds 8–12

demonstrated weak to marginal COX-1/2 potentials. However, intro-

ducing of a benzenesulfonyl moiety as in compound 13 led to 2.6 times

fall in the XOI activity (IC50 0.068 µM), though with a remarkable COX-2

inhibitory potency and selectivity relative to celecoxib (IC50 0.04 vs

0

-8h) (% cumulative

0

h

8 h

edema inhibition)

Control

2.56 ± 3.61 ±

3.73 ±

3.40 ±

10.32 ± 0.084

(

carrageenan) 0.019

0.037

0.025

0.035

0

.05 µM and SI value 318.25 vs 294). In addition, the development of the

N-acylhydrazones 14a,b produced a noticeable amelioration of the XOI

activity (0.024 and 0.011 µM, respectively), together with a moderate

COX-2 inhibitory efﬁcacy and selectivity. Notably, better XO and COX-2

enzymatic inhibition was linked to the presence of the electron-donating

methoxy group as revealed by the analog 14b.

2

.55 ±

c,ns

Celecoxib

0.022

2.84 ±

2.80 ±

2.69 ±

8.259 ± 0.071

c

0

.017

0.033

0.029

(19.97)

d

(

72.57)

(78.46)

(83.57)

c,ns

5

1

a

2.53 ± 2.86 ±

2.79 ±

2.68 ±

8.255 ± 0.079

c

0

.020

0.038

68.76)

0.029

0.009

(20.01)

On the other hand, replacing the carboxylic acid group of febuxostat

1 with a heterocyclic ring, either directly or through a carbonyl bridge

has remarkably modulated the XOI activity of the target compounds. In

this context, although the carbonyl-bridged 3,5-dimethylpyrazole de-

rivative 15 was proven to be nearly 2-times as potent as febuxostat (IC50

(

(77.78)

(81.67)

c,ns

4b

7

2.54 ± 2.93 ±

2.83 ±

2.73 ±

8.401 ± 0.206

c

0

.024

0.108

62.86)

0.063

0.047

(18.59)

(

(74.87)

(76.90)

0

.015 vs 0.026 µM, respectively), yet its COX-1/2 inhibition capability

c,ns

2.55 ± 3.03 ±

2.99 ±

2.78 ±

8.685 ± 0.097

was marginal. Meanwhile, the more polar pyrazole congener 16 showed

a noticeable lowering in the XOI activity (IC50 0.033 µM), though the

COX-2 inhibition was moderately enhanced (IC50 0.07 µM). Regarding

the 2-substituted-1,3,4-oxadiazole structure variants (17, 18 and 21b,

d), their XOI activity appeared to be closely joined with the 2-substitu-

ent of the oxadiazole counterpart. Intriguingly, the 2-thioxo function has

emerged as the most favorable substituent, where the analog 17 proved

to be the most active XOI in this study. This analog has displayed a

unique IC50 value lying in the nanomolar range (0.009 µM), being 3-

times as active as febuxostat (IC50 0.026 µM), nevertheless with weak

COX-2 activity and selectivity. Replacing the 2-thioxo function with an

aromatic moiety as in 18 (IC50 0.018 µM) led to 1.4-fold improvement in

the activity of febuxostat, yet with insigniﬁcant effect on the COX1/2

inhibitory potential. Contrariwise, introducing an NH spacer between

the aryl moiety and the oxadiazole counterpart as in 21b,d resulted in a

reasonable enhancement of both the XO and COX-2 inhibitory activities,

especially in the presence of the electron-withdrawing substituent (21b;

c

0

.016

0.046

54.29)

.55 ± 2.94 ±

0.036

0.019

(15.84)

(

(62.22)

(73.10)

2

2.90 ±

2.72 ±

c

c,ns

1

2

9c

9e

1b

0.022

0.050

0.036

0.037

8.475 ± 0.110

(

62.86)

(70.09)

(79.76)

(17.88)

2

.59 ± 2.87 ±

2.82 ±

2.70 ±

c

c,ns

0.012

0.014

0.021

0.016

8.333 ± 0.047

(

73.33)

(80.00)

(86.90)

(19.25)

2

.55 ± 2.89 ±

2.83 ±

2.64 ±

c

c,ns

0.022

0.061

0.034

0.015

8.315 ± 0.114

(

67.62)

(76.07)

(89.29)

(19.43)

3

.67 ±

3.75 ±

3.33 ±

e

Febuxostat

2.54 ± 0.029

.019 (-7.62)

0.037

0.036

10.36 ± 0.087

0

(-3.42)

(5.95)

(- 0.39)

ns: non-signiﬁcant compared to celecoxib.

*

Data were analyzed by one-way ANOVA followed by Tukey’ s Karmer post

hoc test for multiple comparisons.

a

Dose level for all compounds, po: 5 mg/kg b.wt.

6 4

R = 4-Cl-C H ), showing higher selectivity than celecoxib towards the

b

values are expressed as Mean ± SEM (number of animals n = 5 mice).

COX-2 isozyme (SI value 336.75 vs 294, respectively). Here, it should be

mentioned that bioisosteric replacement of the 1,3,4-oxadiazole with

the 1,3,4-thiadiazole ring as in 20 did not provide any signiﬁcant

advantage to the inhibition of both enzymatic activities, being compa-

rable with both febuxostat and celecoxib reference standards. On the

other hand, direct attachment of a 4-substituted-1,2,4-triazolin-5-thione

moiety to febuxostat’s thiazole ring appeared to noticeably affect both

the XO and COX-2 inhibitory activities. In this context, compound 19a

c

Means are signiﬁcantly different from the control group at p < 0.0001.

d

Values between parentheses: (percentage anti-inﬂammatory activity (AI%).

e

Means are signiﬁcantly different from the celecoxib group at p < 0.0001.

f

Area under the curve; AUC (Paw edema thickness, 0-8h) (mm. h).

showed slight improvement in the XO IC50 value (0.021 µM), yet both

derivatives 3 and 4 displayed low COX-1 and 2 inhibition potential.

Shifting to the 2ry and 3ry carboxamides 10a-e, the results revealed that

the 2ry amides 10a-c showed better XOI activity with IC50 values

comparable with- or even better than febuxostat (0.017–0.026 µM),

when compared with their 3ry congeners 10d,e (0.063 and 0.028 µM,

respectively). The descending order of the carboxamides’ activity was

(

R = C

.016 µM; 1.6-times > febuxostat), with moderate COX-2 inhibition

IC50 0.09 µM; 45% < celecoxib). Whereas, introducing an electron-

withdrawing (19b; R = Cl and 19c; R = F), or electron-donating

) groups resulted in a limited

6 5

H ) proved to be the most potent XOI among this series (IC50

0

(

19d; R = CH

3

and 19e; R = OCH

3

1

0c < 10a < 10b < 10e < 10d, where the electron-donating substituent

10c; R = 2-CH -C -NH) showed the highest potency. Meanwhile,

these amides 10a-e showed moderate COX-1 inhibition (IC50 range

.87–9.03 µM) compared with diclofenac sodium (IC50 3.8 µM), how-

impact on XOI inhibition (IC50 range 0.018–0.026 µM), yet still equi-

potent with- or even better than febuxostat. On the contrary, replace-

ment of the triazole-4-aryl moiety with an aralkyl one (19f: R =

(

3

6 4

H

5

CH

C

2 6

H5) led to the least active member in the series (IC50 0.047 µM),

ever with very weak action on COX-2. Moreover, size enlargement of the

acid hydrazide function with various atomic spacers to the corre-

sponding semicarbazide 8 did not offer any advantage to the XOI ac-

tivity being equipotent to febuxostat (IC50 0.026 µM), whereas its

bioisosteric thiosemicarbazide analog 9 showed 20% decrease in the

potency of febuxostat (IC50 0.032 µM). Nevertheless, introducing a

showing about 50% of febuxostat’s XO inhibitory activity. With regard

to the activity against COX-2 isozyme, the analogs 19b (R = 4-Cl-C

and 19e (R = 4-OCH -C ) were proven to be potentially active (IC50

.04 µM) and selective (SI values 324.25 and 319.25, respectively),

when compared with celecoxib. Meanwhile, the analogs 19c (R = 4-F-

) and 19f (R = CH ) were equiactive with celecoxib as COX-2

inhibitors (IC50 0.05 µM), yet with slight lower selectivity (SI value

45.4 and 237.4, respectively). Finally, the tolyl derivative 19d (R = 4-

CH -C ) was considered as the least active and selective member

within this series (IC50 0.08 µM and SI value 134.13). Finally, it is

6 4

H )

3

6 4

H

0

C

6

H

4

2 6 5

C H

phenyl moiety to the thiosemicarbazide as in 5a (R = C

6 5

H ) resulted in

1

.5 times improvement in the XOI potential (IC50 0.017 µM) as

2

compared with febuxostat. At the same time, 5a was proven to be more

3

6 4

H

active than celecoxib as COX-2 inhibitor, with higher selectivity towards

7

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Fig. 3. In vivo anti-inﬂammatory effects of compounds 5a, 14b, 17, 19c, 19e, 21b and febuxostat in comparison with celecoxib. (a) Time-dependent changes of paw

edema thickness after 0–8 h of the treatment. (b) paw edema thickness after 8 h of the treatment. (c) Areas under the curve (AUC) of paw edema thickness after 0–8 h

of the treatment. The data are analyzed by one-way ANOVA followed by Tukey’ s Karmer post hoc test for multiple comparisons and expressed as the Mean ± SEM (n

=

5). **** indicates P < 0.0001, compared to control. #### indicates p < 0.0001 compared to celecoxib.

worthwhile to mention that the in vivo results were matching with the

collected in vitro data.

compounds recorded approximately similar binding poses with excel-

lent docking scores (-10.0 to ꢀ 9.06 kcal/mol), which were obviously

superior to that of febuxostat (-7.70 kcal/mol) (Table 4). Interestingly, it

could be clearly noticed that the docking scores are in harmony with the

results obtained in vitro enzymatic assay. In particular, the highest en-

ergy scores achieved by compounds 17, 14b and 19c (-10.0, ꢀ 9.90 and

ꢀ 9.79 kcal/mol, respectively), and their motivating binding proﬁle to

the 1N5X target, would reﬂect their higher afﬁnity and potential in the

in vitro enzymatic inhibitory activity (IC50 0.009, 0.011 and 0.018 µM,

respectively), as compared to febuxostat (Table 4). Docking scores,

protein–ligand interactions, and binding modes in the docking simula-

tion for the most active XOIs 17, 14b and 19c, together with the

structurally relevant XDH ligand febuxostat are summarized in Table 5

and presented in Figs. 4–6, while those for compounds 5a, 19e and 21b

are presented in the supplementary section (Table SM2 and Figs. SM2-

SM4).

2

.3. Molecular docking studies

.3.1. Molecular docking study on xanthine oxidase

Inspired by the promising in vitro and in vivo XO inhibitory activities

displayed by compounds 5a, 14b, 17, 19c, 19e and 21b, an in silico

molecular docking simulation was conducted in order to estimate their

binding modes and molecular interactions with the active site of the

enzyme. Since the structure of the human XO has not yet been fully

resolved, and owing to its close sequence identity with the bovine iso-

form (ca. 90%), the X-ray structure of the bovine milk xanthine dehy-

drogenase (XDH) was selected for this study [55]. This isozyme was

retrieved from the RCSB Protein Data Bank (PDB ID: 1N5X) [56,57] and

the X-ray crystal structure of the bovine milk XDH-TEI-6720 (febuxo-

stat) complex was utilized and manipulated using the Molecular Oper-

ating Environment software (MOE, 2016. 0802). The docking poses

were selected according to the top-scored conformations along with

favorable binding interactions. Binding afﬁnities to XDH enzyme were

estimated based on docking scores, H-bonds, and the relative positioning

of the docked compounds with respect to the co-crystallized ligand

The 2D and 3D docking poses of febuxostat with the active site of

IN5X showed the capability of binding with the protein through six H-

bonds, and one electrostatic interaction (See the Supplementary section,

Fig. SM1). In detail, the carboxylate group acted as a H-bond acceptor to

the Arg880 and Thr1010, while the thiazole-N and the carbonitrile

group were involved as acceptors in H-bonding with Glu802, Asn768

and Lys771, respectively. Such mode of binding resulted in directing the

carboxylate function into the innermost of the active site of 1N5X, with

subsequent protrusion of the isobutoxyphenyl moiety outwards. More-

(

febuxostat). The molecular docking protocol was validated by

extracting the co-crystallized ligand and re-docking it into the XDH

binding site. The original docking pose (generated by 1N5X from PDB)

was retrieved with a small root mean square deviation (RMSD) of 0.54 Å

-

over, the carboxylate O formed a salt bridge with the guanidinium

(

between the docked pose and the co-crystallized ligand), and with

group of Arg880, whereas the benzonitrile fragment was involved in

binding energy score of ꢀ 7.70 kcal/mol.

π

-hydrophobic interactions with Leu648 and Leu1014. Finally, the

thiazole ring was sandwiched between both Phe914 and Phe1009 via

face to-face and face-to-edge -stacking interactions, respectively [57].

Molecular modeling analysis revealed that the six investigated

compounds were well penetrated and positioned onto the binding site of

XDH and were stabilized by a number of polar and hydrophobic mo-

lecular interactions. The binding site residues and overall binding

pattern of the above-mentioned compounds were found to be nearly

similar to those observed with the reported ligand (febuxostat) [57],

with some differences in their binding fashions. All the investigated six

π

Under the same experimental environment, the 2D and 3D docking

poses of the most active XOI and top-scoring oxadiazole derivative 17

exhibited a distinctive binding mode in the deepest part of the binding

site of 1N5X. Compound 17 could occupy almost the same spatial space

and arrangement of febuxostat, though with participation in eight H-

8

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Table 4

Summary of the in vitro xanthine oxidase (XO) and COX-2 enzymatic inhibition proﬁle and the corresponding docking scores for the most active compounds 5a, 14b,

1

7, 19c, 19e and 21b.

b

Cpd. No.

XO

XO Docking score (Kcal/mol)

COX-2

COX-2 Docking score (Kcal/mol)

a

c

d

IC50 (µM)

SI

e

Celecoxib

NT

0.05 ± 0.0006

0.34 ± 0.0067

0.04 ± 0.0003

0.07 ± 0.0007

0.14 ± 0.0033

0.05 ± 0.0007

0.04 ± 0.0003

0.04 ± 0.0007

294

ꢀ 7.99

ꢀ 5.13

ꢀ 7.52

ꢀ 7.32

ꢀ 7.15

ꢀ 8.28

ꢀ 7.73

ꢀ 7.56

1

5

1

2

(Febuxostat)

0.026 ± 0.0012

0.017 ± 0.0006

0.011 ± 0.0008

0.009 ± 0.0010

0.018 ± 0.0016

0.019 ± 0.0021

0.018 ± 0.0014

ꢀ 7.70

ꢀ 9.80

ꢀ 9.90

ꢀ 10.00

ꢀ 9.79

ꢀ 9.06

ꢀ 9.73

15.79

314.25

137.57

52.64

245.40

319.25

336.75

a

4b

7

9c

9e

1b

a

b

c

Compounds’ concentration causing 50% in vitro (XO) enzyme inhibition.

Docking scores were obtained utilizing MOE, 2016. 0802 software.

Compounds’ concentration causing 50% inhibition of cyclooxygenase 2 (COX-2) enzymatic activity.

COX-2 selectivity index: (COX-1 IC50 / COX-2 IC50).

NT: not tested.

d

e

Table 5

Molecular docking scores and binding modes of compounds 14b, 17, 19c and the reported ligand (febuxostat, 1) in XDH enzyme (PDB ID: 1N5X) using MOE, 2016.

0

802.

Cpd.

No.

Structure

Docking score (Kcal/mol) Amino acid residue

Interacting fragment

Type of molecular interactions

1

ꢀ 7.70

Glu802

Thiazole N

C≡N

H-bond (acceptor)

2H-bonds (acceptor)

H-bond (acceptor) and ionic

bond

Feb.

Asn768; Lys771

Arg880; Thr1010

Arg880; Arg880

Phe914; Phe1009

Leu648; Leu1014

C = O

OH

Thiazole ring

Benzonitril ring

π

-

π

stacking and

interactions

-H interactions

π

-H

π

1

4b

ꢀ 9.90

Asn768; Lys771

Ser876

C≡N

2H-bonds (acceptor)

H-bond (acceptor)

2H-bonds (acceptor)

H-bond (acceptor)

Thiazole sulfur

Thiazole N

Glu802

Arg880; Thr1010

Met1038

C = O

P-methoxy gp

N-acylhydrazone N

N-acylhydrazone H

benzonitrile ring

Thiazole ring

P-methoxyphenyl

ring

Arg880

Glu1261

Val1011

Phe1009

Ala1078

Phe649

π

-H interaction

Isobutoxy tail

1

7

ꢀ 10.00

Asn768; Lys771

Thr1010; Ser876

Ala1079; Arg880

Thr1010; Arg880;

Arg880

C≡N

2H-bonds (acceptor)

H-bond (acceptor) and ionic

bond

Thiazole sulfur

oxadiazole 2-thioxo

gp

Oxadiazole NH

2H-bonds (acceptor) and ionic

bond

Thr1010

Leu648

Oxadiazole N

4

Phe914, Phe1009;

Arg880

Benzonitril ring

Oxadiazole ring

H-bond (acceptor)

π

-H interaction

stacking, -H and -cation

-

π

interact.

1

9c

ꢀ 9.79

Asn768; Lys771

Glu802

C≡N

2H-bonds (acceptor)

H-bond (acceptor)

Thiazole N

Triazole NH

Arg880 and Arg880

Thr1010

H-bond (acceptor) and ionic

bond

Triazole N

2

Phe914; Phe1005;

Arg880

Triazole 5-thioxo gp

H-bond (acceptor)

2H-bonds (acceptor) and ionic

bond

Glu1261 and Ala1078

Ser876

P-ﬂuorophenyl ring

Benzonitril ring

Triazole ring

Arg880

H-bond (acceptor) and

interact.

π

-H

H-bond (acceptor)

π

-cation interaction

9

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Fig. 4. Docking and binding pattern of compound 14b (in pink) into XDH active site (PDB 1N5X) and its overlay over febuxostat (in cyan) in 3D (left panel). Ligand

interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 5. Docking and binding pattern of compound 17 (in pink) into XDH active site (PDB 1N5X) and its overlay over febuxostat (in cyan) in 3D (left panel). Ligand

interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 6. Docking and binding pattern of compound 19c (in pink) into XDH active site (PDB 1N5X) and its overlay over febuxostat (in cyan) in 3D (left panel). Ligand

interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

bonds (Fig. 5). Replacement of the carboxylic acid group with an oxa-

diazole counterpart led to the formation of a network of four H-bonds (as

acceptor). For instance, the 2-thioxo group acted as H-bond acceptor

with Ala1079 in the active site. Meanwhile, the NH was involved in two

H-bond interactions with both the Arg880 and Thr1010, whereas the

oxadiazole-N4 participated in one H-bond with Thr1010. In addition,

two ionic bonds with the guanidinium group of Arg880 were developed

between the oxadiazole 2-thioxo and the oxadiazole NH fragments.

kcal/mol, respectively). Both compounds exhibited the same fashion

and type of binding presented by febuxostat toward the amino acid

residues Arg880, Thr1010, Asn768, Lys771 and Glu802 as H-bond ac-

ceptors, though with observable extra-binding capacities. In detail, the

N-acylhydrazone 14b was involved in a network of nine H-bonds, where

the presence of the 4-methoxybenzylidene hydrazide counterpart acted

as an anchor in a deep pocket in the active site of 1N5X, through initi-

ating three H-bonds (as acceptor) between the 4-methoxy group, N-

acylhydrazone-N and the N-acylhydrazone-H with Met1038, Arg880

and Glu1261, respectively. Moreover, in addition to the existing six H-

bonds with Asn768, Lys771, Ser876, Glu802, Arg880 and Thr1010,

some arene-H interactions were formed with Val1011, Phe1009,

Ala1078 and Phe649 (Fig. 4). Shifting to the triazole derivative 19c, it

was perfectly accommodated in the active site of 1N5X by inducing a set

Moreover, some

π-

π

stacking,

π

-H and

π-cation interactions were

developed between the oxadiazole moiety and Phe914, Phe1009 and

Arg880. Furthermore, two H-bonds existed essentially between the

carbonitrile group and Asn768 and Lys771, whereas another two H-

bonds were initiated between the thiazole-S and Thr1010 and Ser876.

Such binding proﬁle could intensify the ﬁtting of 17 into the active site

of 1N5X and would justify the obtained high docking score (-10.0 Kcal/

mol), and therefore the corresponding distinctive in vitro enzymatic

inhibitory activity (IC50 0.009 µM) (Table 4).

of nine H-bonds, as well as some ionic and -hydrophobic interactions.

π

In particular, the substituted triazole moiety occupied the same space

and binding pattern of the carboxylic group of febuxostat by forming

On the other hand, compounds 14b and 19c were proven to be well-

overlayed and almost superimposable with the original ligand

2

two H-bonds between the triazole NH, N and the same amino acids

Arg880 and Thr1010, respectively, whereas the triazole 5-thioxo group

could contribute into two bifurcated H-bonds with Phe914 and

(

febuxostat) recording high afﬁnity binding scores (-9.90 and ꢀ 9.79

1

0

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Phe1005. Here it should be noticed that the 4-ﬂuorophenyl substituent

served as an anchor which augmented compound’s binding in the

innermost part of the active pocket of 1N5X. This ﬂuorinated fragment

was involved in an extra H-bond with Glu1261 as an acceptor, together

energy score of ꢀ 7.99 kcal/mol. A synopsis of the COX-2 enzymatic

inhibition proﬁle together with the corresponding docking scores of the

six active compounds is recorded in Table 4. Docking scores, pro-

tein–ligand interactions, and binding modes in the docking simulation

for febuxostat 1 and the most active COX-2 inhibitors 5a, 19c and 21b,

together with celecoxib ligand are summarized in Table 6 and depicted

in Figs. 7–10, while those for compounds 14b, 17 and 19e are presented

in the supplementary section (Table SM3 and Figs. SM6-SM8).

with a

ionic bonds took place between the triazole NH, 5-thioxo group and the

guanidinium group of Arg880, while a -cation interaction was observed

π-H interaction with Ala 1078 were also observed. Finally, two

π

between the triazole ring and Arg880 (Fig. 6). Collectively, the molec-

ular modeling study veriﬁed the objective of the design of the target

compounds at the molecular level, where the derivatization or

replacement of febuxostat’s carboxylic functionality retains or even in-

creases the afﬁnity for the XO enzyme. Additionally, it would also

explain the excellent in vitro and in vivo inhibitory potential displayed by

the afore-mentioned active compounds.

The results revealed that, although febuxostat 1 has occupied the

same binding site of COX-2 (Fig. 7), yet it showed remarkably low

docking score (-5.13 Kcal/mol) as compared with the ligand celecoxib

(-7.99 Kcal/mol). Interestingly, the newly synthesized febuxostat de-

rivatives 5a, 14b, 17, 19c, 19e and 21b have obviously penetrated and

occupied the same binding site of 3LN1, with excellent binding energy

range comparable with that of celecoxib (ꢀ 8.28 to ꢀ 7.15 Kcal/mol).

These compounds were engaged in several polar as well as hydrophobic

molecular interactions, with binding patterns approximately similar to

those displayed by celecoxib, though with little differences. It is worth

mentioning that, the docking scores of the investigated compounds were

reasonable and cohering to some extent with their IC50 and selectivity

index values obtained from the biological results.

2

.3.2. Molecular docking study on COX-2 isozyme

In a trial to investigate the possible binding proﬁle of the parent

febuxostat 1 and the most active XOIs (5a, 14b, 17, 19c, 19e and 21b)

with the active site of COX-2 isozyme, an in silico molecular docking

study was carried out using Molecular Operating Environment (MOE)

version 2016.0802. The COX-2 isozyme co-crystallized with celecoxib

was retrieved from the PDB (ID: 3LN1) [39], and re-docking of celecoxib

in the vicinity of the COX-2 active site validated the docking protocol

with root mean square deviation (RMSD) of 0.09 Å and with binding

The differences between the binding sites of COX-1 and COX-2 iso-

zymes obtained from the X-ray crystal structures are well-documented.

In particular, the COX-2 binding site possessed an additional 2-pocket

Table 6

Molecular docking scores and binding modes of febuxostat 1, compounds 5a, 19c and 21b and the reported ligand (celecoxib) in COX-2 isozyme (PDB ID: 3LN1) using

MOE, 2016. 0802.

Cpd. No.

Structure

Docking score (Kcal/

mol)

Amino acid residue

Interacting fragment

Type of molecular interactions

Celecoxib

ꢀ 7.99

Gln178; Leu338; Ser339

Arg499; Phe504

His75

NH

Oxygens of SO

2

3H-bonds (donor)

2H-bonds (acceptor)

H-bond (acceptor)

2

Benzenesulfonamide

ring

Ala513

π-H interaction

-H interaction

Tyr371

P-methylphenyl ring

π

CH

3

1

ꢀ 5.13

ꢀ 7.52

ꢀ 8.28

Arg106

Leu338

Glu510

Arg499

Ser339

C≡N

H-bond (acceptor)

Ionic bond

Feb.

Thiazole N

Thiazole sulfur

4-methylthiazole gp.

C = O

OH

thiazole ring

π-H interaction

5

a

Gly340

Thiosemicab. NH

C≡N

1

H-bond (donor)

Arg106

H-bond (acceptor)

2H-bonds (acceptor)

H-bond (acceptor)

2H-bonds (acceptor)

H-bond (acceptor)

Gln178; Asp501

His75

C = S

Carbonyl (C=O)

Thiazole sulfur

Thiazole N

Benzonitril ring

thiazole ring

C≡N

Ser339; Leu338

Arg499

His75

π

-H interaction

stacking interaction

His75

π

-

π

1

9c

Trp373; Gly512

Arg106

2H-bonds (acceptor)

H-bond (acceptor)

2H-bonds (acceptor)

H-bond (acceptor) and ionic

bond

Triazole N

Thiazole sulfur

2

Glu510; Val509

Arg499; Arg499

Ser339; Gln178; Leu338;

Ser339

Triazole 5-thioxo gp

P-ﬂuorophenyl ring

2H-bonds (acceptor) and

interactions.

π-H

Arg499

Triazole NH

Leu338

Benzonitril ring

Triazole ring

Ionic bond

Val509; Arg499

π

-H interaction

-H and -cation interactions

π

2

1b

ꢀ 7.56

Pro500

NH

H-bond (donor)

Arg499

Oxadiazole N3,

C≡N

N

4

2H-bonds (acceptor)

H-bond (acceptor)

Met508; Trp373

Gln178

Thiazole sulfur

Asp501

P-chlorophenyl ring

Thiazole ring

π

-H interaction

-H interactions

stacking interaction

Leu338; Ser339

His75

2

π

Oxadiazole ring

π-π

1

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Fig. 7. Docking and binding pattern of Febuxostat 1 (in orange) into COX-2 active site (PDB 3LN1) and its overlay over celecoxib (in turquoise) in 3D (left panel).

Ligand interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 8. Docking and binding pattern of compound 5a (in orange) into COX-2 active site (PDB 3LN1) and its overlay over celecoxib (in turquoise) in 3D (left panel).

Ligand interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 9. Docking and binding pattern of compound 19c (in orange) into COX-2 active site (PDB 3LN1) and its overlay over celecoxib (in turquoise) in 3D (left panel).

Ligand interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 10. Docking and binding pattern of compound 21b (in orange) into COX-2 active site (PDB 3LN1) and its overlay over celecoxib (in turquoise) in 3D (left panel).

Ligand interactions in 2D (right panel). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

where His90, Gln192 and Tyr355 control the entrance of ligands to this

pocket. Additionally, the replacement of His513 in COX-1 by Arg513 in

COX-2 plays a key role in forming H-bonds with the ligands in the COX-2

binding site [58,59]. In this view, the 2D and 3D docking poses of cel-

ecoxib (the selective COX-2 inhibitor) in the active site of COX-2

revealed a distinctive binding pattern with the COX-2 polar side

pocket by forming many interactions especially by the aminosulfonyl

group. Three H-bonds were formed with Gln178, Ser339 and Leu338 by

involved in two H-bonds with Phe504 and Arg499 amino acids as H-

bond acceptors. Furthermore, some hydrophobic interactions with

Ala513 And Tyr371 were also observed [60] (Supplementary section,

Fig. SM5).

Referring to the docking results of 5a in the active site of 3LN1, it

showed high afﬁnity binding energy (ꢀ 7.52 kcal/mol) being involved in

a network of eight H-bonds. This compound displayed a noticeable

binding proﬁle comparable with celecoxib via forming the same type of

interaction with the amino acid residues Arg499, His95, Leu338, Ser339

2

the NH group as H-bond donor, while the sulfonamide oxygens were

1

2

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

and Gln178 as H-bond acceptors. Moreover, 5a was involved in a

2.4. In silico prediction of the physicochemical properties, drug likeness

score, pharmacokinetics, toxicity proﬁle and ligand efﬁciency metrics

network of two extra H-bonds with Arg106 and Asp501 as H-bond

acceptor, meanwhile the NH

Gly340 as H-bond donor. Additionally, some

between His75 and both benzonitril and thiazole rings were detected

Fig. 8). On the other hand, compared with celecoxib, the top-scoring

1

group participated in one H-bond with

π

hydrophobic interactions

Early computation and prediction of the physicochemical properties

and the pharmacokinetic proﬁle of new drug candidates is a corner stone

in the lead optimization step and drug development process, to save

time, effort and cost [61]. In this context, the term “drug-likeness”

indicated a combined balance between structure features and variable

molecular characteristics of a compound, in order to be comparable with

previously recognized drugs. Lipinski’s rule of 5 (RO5) [62] and Veber’s

criteria [63] are among the widespread principles employed to evaluate

the drug-like characteristics including the hydrophobicity, ﬂexibility

(number of rotatable bonds; nROTB), electronic distribution, H-bonding

capacity, and molecular size. These parameters would manipulate

molecule’s behavior in a living system including bioavailability, mem-

brane transport properties, afﬁnity to proteins, reactivity, toxicity and

metabolic stability. Therefore, the in silico physicochemical properties,

drug likeness score, pharmacokinetic parameters, toxicity proﬁle and

ligand efﬁciency metrics of the most active compounds 5a, 14b, 17,

19c, 19e and 21b were investigated using web-based applications

including Molinspiration [64], Pre-ADMET [65] and Osiris property

explorer [66] softwares (Table 7).

(

compound 19c (ꢀ 8.28 Kcal/mol) showed a remarkable binding mode

in 3LN1 active site via acquiring the same vicinal diaryl conﬁguration

required for maximum ﬁtting in the COX-2 isozyme. In this context, the

binding efﬁciency of 19c was strengthened by participating in eight H-

bonds with Trp373, Gly512, Arg106, Glu510, Val509, Arg499, Ser339

and Gln178, in addition to the

π

hydrophobic interactions with Leu338,

Val509, Ser339 and Arg499 amino acids. Besides, two ionic bonds were

seen between triazole NH and triazole 5-thioxo group together with

Arg499 (Fig. 9). Finally, docking data of compound 21b revealed a

remarkably high binding afﬁnity (-7.56 Kcal/mol) and was well

accommodated in the active site of 3LN1 through a set of H-bond in-

teractions with Arg499, Met508, Trp373 and Gln178 as H-bond accep-

tors. The NH group formed one H-bond with Pro500 as H-bond donor, in

addition to some

π

hydrophobic interactions with Asp501, Leu338,

Ser339 and His75 amino acid residues (Fig. 10).

The data presented in table 7 revealed that all the tested compounds

comply with Lipinski’s rule of 5, where MW range 372–477 (≤500),

HBA range 5–7 (≤10) and HBD range 1–3 (≤5), whereas only three

Table 7

In silico prediction of the physicochemical properties, drug likeness, pharmacokinetics and ligand efﬁciency metrics of compounds 5a, 14b, 17, 19c, 19e and 21b.

5

a

14b

17

19c

19e

21b

Febuxostat

Physicochemical parameters and drug likeness

a

LogP

4.31

465.60

7

5.33

448.55

7

3.65

372.48

6

5.06

465.58

6

4.95

477.62

7

6.66

465.97

7

3.68

316.38

5

b

M.Wt

c

HBA

d

HBD

3

1

e

Lipinski’s violation

0

1

0

1

0

1

0

f

NROTB

9

8

5

6

7

5

g

TPSA

99.07

74.82

406.62

0.01

0.17

96.61

75.67

402.76

0.04

0.37

87.74

78.73

312.83

0.36

0.40

79.53

81.56

329.97

0.02

0.07

88.77

78.37

413.58

0.02

0.08

96.87

75.58

393.18

0.001

0.07

83.22

80.29

278.17

12.63

0.48

h

%

ABS

i

Volume

j

S

Drug likeness score

Pharmacokinetics (ADME)

k

Caco-2

12.10

0.05

20.14

0.05

22.68

0.06

96.08

0.19

100

49.83

0.04

48.13

0.04

34.46

0.05

9.10

l

MDCK

0.33

m

HIA

94.18

0.10

98.12

0.61

99.00

0.11

99.25

0.26

98.30

0.03

93.02

0.43

n

BBB

o

PPB

93.05

89.98

94.22

92.00

91.60

97.47

Ligand efﬁciency metrics

p

LE (XO)

0.34

3.46

0.32

3.09

0.34

2.63

0.31

1.82

0.44

4.40

0.38

3.20

0.33

2.68

0.31

2.24

0.32

2.77

0.31

2.41

0.33

1.08

0.32

0.74

0.47

3.91

0.40

2.79

q

LLE

LE (COX-2)

LLE

a

n-Octanol and water partition coefﬁcient.

Molecular weight.

b

c

d

e

f

Number of H-bond acceptors.

Number of H-bond donors.

Lipinski’s rule of 5 violations (log P ≤ 5, M.Wt. ≤ 500, HBA ≤ 10 and HBD ≤ 5).

Number of rotatable bonds (Veber’s criteria ≤ 10).

g

h

i

2

Topological polar surface area (Veber’s criteria ≤ 140 Å ).

Percentage of absorption = 109 – (0.345*TPSA).

Molecular volume.

j

Solubility in mg/L.

k

l

Permeability through human colon adenocarcinoma cells.

Permeability through Madin-Darby canine kidney cells.

Human intestinal absorption percentage.

Blood brain barrier penetration coefﬁcient.

Plasma protein binding.

m

n

o

p

q

Ligand efﬁciency = (pIC50*1.37) / non-hydrogen atoms.

Lipophilic ligand efﬁciency = pIC50-logP.

1

3

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

(

14b, 19c and 21b) violated the Lipinski’s rule showing logP values > 5.

2.41 and 0.74, respectively). Collectively, compounds 5a and 17 com-

plied with the optimum cut-off value for lead compounds for both XO

and COX-2 inhibitory activities (LLE ≥ 3), though the analogs 14b, 19c,

19e and 21b were not accepted to be lead compounds or drug

candidates.

Moreover, all of the tested compounds obeyed Veber’s criteria display-

ing nROTB values of 5–9 (≤10) and topographical polar surface area

(

TPSA; a sum of polar atoms’ surfaces: a descriptor for drug absorption,

2 2

penetrability and bioavailability) range 79.53–99.07 Å (≤140 Å ).

These results indicate acceptable molecular ﬂexibility with consequent

expected good permeability and oral bioavailability. Additionally, the

percentage of absorption (%ABS; calculated as %ABS = 109–0.345 ×

TPSA) [67] and the solubility were also determined. Furthermore, all the

tested compounds exhibited % ABS range 74.82–81.56%, which is an

indicator of a considerable bioavailability upon oral administration. It is

to be noted that TPSA is inversely proportional to %ABS e.g. the analog

Finally, prediction of the expected toxicity risks pointed out that

none of the tested compounds revealed any irritant effect, whereas only

compound 21b possessed high tumorigenic tendency. However, high

mutagenic probability was linked to compounds 19c, 19e and 21b,

whereas the analogs 5a, 19c and19e possessed high liability of repro-

ductive toxicity.

1

9c possesses the maximum absorption (81.56%), whereas its corre-

3. Conclusion

2

sponding TPSA was least among the series (79.53 Å ). Their solubility

values lied between 0.01 and 0.001 mg/L, satisfying the solubility

requirement (>0.0001 mg/L) [68,69], so they are foreseen to possess

good oral absorption, bioavailability and transport characteristics.

Regarding the drug-likeness score values [23], the results revealed that

all the evaluated compounds gave positive values lying between 0.07

and 0.40. Prominently, the analogs 14b and 17 showing the highest XOI

activity displayed the highest values (0.37 and 0.40, respectively),

which were comparable with febuxostat (0.48).

The prime aim of the present study was to develop multi-target

designed ligands (MTDLs) derived from febuxostat to be evaluated for

their expected XO and COX inhibitory activities. The targeted com-

pounds were designed utilizing pharmacophoric hybridization strategy

through structure modiﬁcation of febuxostat’s 5-carboxylic acid func-

tionality into different carboxamides or various substituted heterocy-

cles. The obtained in vitro XO inhibitory data revealed that all the tested

compounds exhibited variable XOI activities (IC₅₀values 0.009–0.077

µM). Among these, compound 17 has emerged as the most potent XOI

(IC 0.009 µM), representing nearly 3-times the potency of febuxostat

In another context, ADME properties were also calculated using Pre-

ADMET software [65] and the value of each of the following parameters

was calculated and compared with their optimal values: Caco-2 cell

permeability coefﬁcient (Permeability through cells derived from

human colon adenocarcinoma), Madin-Darby canine kidney (MDCK)

cell permeability coefﬁcient, human intestinal absorption (HIA), blood

brain barrier (BBB) coefﬁcient, and plasma protein binding (PPB). As

depicted in Table 7, the results indicated that all the investigated com-

pounds showed moderate cell permeability in Caco-2 cell model

5

0

(IC₅₀0.026 µM). Moreover, the analogs 5a, 10c, 14b, 15, 18, 19a,c,e,

20 and 21b proved to be 2.4–1.3 times more active than febuxostat,

whereas 8, 10b and 19d exhibited equipotency with febuxostat.

On the other hand, the same analogs were further investigated for

their in vitro COX-1 & 2 inhibitory potential, where ﬁfteen analogs (5a,

13, 14a,b, 16, 18, 19a-f, 20 and 21b,d) demonstrated an appreciable

COX-2 inhibitory activity lying in the nanomolar to sub-micromolar

level (IC₅₀values 0.04 – 0.1 µM), when correlated with celecoxib (IC

0.05 µM). For instance, compounds 5a, 13, 19b,e and 21b demonstrated

an outstanding COX-2 selectivity index values (SI) lying between 314.25

and 336.75, being superior to that of celecoxib (SI value 294). Addi-

tionally, the triazoles 19c and 19f revealed a considerable selectivity

towards the same isozyme comparable with that of celecoxib (SI values

245.4 and 237.4, respectively). Compounds 5a, 14b, 17, 19c, 19e and

21b that exhibited signiﬁcant in vitro XO and/ or COX inhibitory po-

tentials were further investigated for their in vivo hypouricemic as well

as anti-inﬂammatory activities. Remarkably, the in vivo results were in

harmony with the collected in vitro data. Furthermore, in silico molecular

docking simulation of the most active compounds (5a, 14b, 17, 19c,

19e and 21b) with the active site of XDH enzyme (PDB ID: 1N5X)

revealed similar binding poses with excellent docking scores (ꢀ 10.00 to

ꢀ 9.06 kcal/mol), which were obviously superior to that of febuxostat

(ꢀ 7.70 kcal/mol). Interestingly, the docking scores go in hand with the

results obtained in the in vitro enzymatic assay. Meanwhile, docking of

the same six active compounds and febuxostat in the active site of the

COX-2 isozyme (PDB ID: 3LN1) showed obvious occupation the same

binding site, with excellent binding energy range (ꢀ 8.28 to ꢀ 7.15 Kcal/

mol) comparable with that of celecoxib (ꢀ 7.99 Kcal/mol). The docking

scores of the investigated compounds were reasonable, cohering to a

great extent with their IC₅₀and selectivity indices obtained from the in

vitro results. Moreover, in silico computation of the predicted pharma-

cokinetic and toxicity properties (ADMET) of the same six active com-

pounds showed limited violations of Lipinski’s RO5 and none for Veber’s

criteria, suggesting their liability to act as new orally-active drug can-

didates with a predicted high safety proﬁle. Furthermore, LE values of

the six tested compounds regarding their XO and COX-2 inhibitory ac-

tivities were concordant with the acceptable LE value for drug candi-

dates (>0.3). Collectively, the overall XO and COX-2 inhibitory proﬁles

exhibited by the newly synthesized febuxostat derivatives 5a, 14b, 17,

19c, 19e and 21b would stimulate further structure development and

optimization in order to deﬁne the scope and limitation of the bio-

activities of such scaffold.

(

12.10–49.83 nm/s) as compared with the acceptable range (4–70 nm/

50

s). Nevertheless, all of them displayed low permeability in MDCK cell

model (0.04–0.06 nm/s) in relation to the recommended range (25–500

nm/s). Concerning the HIA, the tested compounds were well absorbed,

exhibiting high HIA values (94.18–99.25%). Meanwhile, moderate BBB

permeability coefﬁcient (reported 0.1–2) was displayed by compounds

5

2

a, 14b, 17, 19c and 19e in the range of 0.1–0.61, whereas the analog

1b showed low permeability (0.03; <0.1). Furthermore, all the tested

compounds were found to be strongly bound to plasma proteins (>90%)

ranging between 89.98% and 100% [50,69].

On the other hand, ligand efﬁciency metrics are used to normalize

the in vitro biological activity with respect to the physicochemical

properties of the molecule, and they also measure how effectively the

molecule uses its structural features in binding to the target [70]. Among

the commonly used metrics are the ligand efﬁciency (LE) and lipophilic

ligand efﬁciency (LLE). LE is deﬁned as the binding energy of the ligand

per atom and it evaluates the potency with respect to its molecular size,

where the acceptable LE should be around 0.3 for lead-likeness and >

0

.3 for drug-likeness. Meanwhile, LEE indicates if lipophilicity is

responsible for the speciﬁcity of a molecule in binding to the target and

the potency. The recommended LLE value for lead compound should be

≥

3, and ≥ 5 for a drug like candidate [71,72]. In the present study, both

LE and LLE values of the six tested compounds regarding their XO and

COX-2 inhibitory activities were calculated according to the reported

equations [72], and compared to the reference drug febuxostat. The

results revealed that all the tested compounds were concordant with the

acceptable LE value for drug candidates (>0.3) regarding both XOI ac-

tivity (0.32 to 0.44) and COX-2 inhibitory action (0.31 to 0.38).

Shifting to the obtained LLE values for the XOI activity, compounds

5

a and 17 (3.46 and 4.40, respectively) complied with the recom-

mended LEE value (≥3) as lead compounds. Whereas, the analogs 14b,

9c, 19e and 21b showed lower LLE values of 2.63, 2.68, 2.77 and 1.08,

1

respectively. Regarding the COX-2 inhibitory activity, compounds 5a

and 17 had LLE values of 3.09 and 3.2, respectively; meanwhile the

analogs 14b, 19c, 19e and 21b showed lower LLE values (1.82, 2.24,

1

4

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

4

. Experimental

S, 9.70. Found: C, 58.29; H, 5.10; N, 16.72; S, 9.66.

4

.1. Chemistry

4.1.3. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-N-

substituted hydrazine-1-carbothioamides (5a-f)

Melting points were determined in open-glass capillaries using Stuart

To a solution of the acid hydrazide 4 (0.66 g, 2 mmol) in absolute

ethanol (25 mL), the appropriate isothiocyanate (2 mmol) was added.

The reaction mixture was heated under reﬂux for 1–4 h then left to cool

to room temperature. The solid obtained was ﬁltered, washed with cold

ethanol and petroleum ether, dried and recrystallized from ethanol and

few drops of DMF.

Scientiﬁc melting point apparatus (SMP10) and are all uncorrected.

Reactions progress was followed up by using thin-layer chromatography

(

TLC) on silica gel-precoated aluminum sheets (Type 60 GF254; Merck;

Germany). The spots were visualized by exposure to UV-lamp at λ 254

nm or iodine vapors. Infrared spectra (IR) were recorded on Perkin-

Elmer 1430 IR spectrophotometer using KBr discs and on Schimadzu

FT-IR Afﬁnity-1 spectrophotometer. ¹H NMR spectra were scanned on

4.1.3.1. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

Bruker spectrophotometer (400 MHz) using DMSO‑d

6

or CDCl

3

as sol-

N-phenylhydrazine-1-carbothioamide (5a). Yellow ﬁbrous crystals, yield:

0

ꢀ 1

vents and D O to detect exchangeable protons. Signal splitting were

2

91%, m.p.: 199–201 C. IR (KBr, cm ): 3306, 3237 (NH), 2229 (CN),

1605 (CO), 1375 (CS). ¹H NMR (δ ppm, DMSO‑d ): 10.34 (s, 1H,

designated as follows: s: singlet; d: doublet; t: triplet; q: quartet; dd:

doublet of doublet and m: multiplet. ¹C NMR proton decoupled spectra

were recorded on Bruker 100 MHz spectrophotometer and were

6

3

CSNHPh, D O exchangeable), 9.80 (s, 2H, CONHNH, D O exchange-

2

able), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H),

7.46–7.32 (m, 5H, Ar-H), 7.17 (t, J = 7.6 Hz, 1H, Ar-H), 4.00 (d, J = 6.4

Hz, 2H, OCH ), 2.68 (s, 3H, CH ), 2.14–2.04 (m, 1H, CH), 1.02 (d, J =

measured in δ scale using either DMSO‑d

6 3

or CDCl as solvents. Micro-

analyses (C, H, N and S) were conducted using FLASH 2000 CHNS/O

analyzer, Thermo Scientiﬁc at the Regional Center for Mycology and

Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The found

values were within ± 0.4% of the calculated values. Electron impact

mass spectra (EI-MS) were carried out using direct inlet unit (DI-50) in

the gas chromatograph/mass spectrometer Shimadzu QP-5050. Com-

pound 2 was synthesized as previously reported [42].

2

3

1

3

6.8 Hz, 6H, (CH ) ). C NMR (δ ppm, DMSO‑d ): 180.97, 164.61,

161.94, 161.08, 144.09, 139.20, 132.94, 131.31, 128.13, 125.94,

125.39, 125.10, 123.84, 115.43, 114.05, 101.62, 75.13, 27.60, 18.74,

17.43. Anal. Calcd (%) for C₂₃H₂₃N O S (465.59): C, 59.33; H, 4.98; N,

3

2

6

5

2 2

15.04; S, 13.77. Found: C, 59.58; H, 4.61; N, 15.47; S, 13.63.

4

.1.3.2. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

4

.1.1. 2-(3-Cyano-4-isobutoxyphenyl)-N-hydroxy-4-methylthiazole-5-

N-(4-chlorophenyl) hydrazine-1-carbothioamide (5b). Yellow ﬁbrous

0

ꢀ 1

carboxamide (3)

crystals, yield 77%, m.p.: 206–208 C. IR (KBr, cm ): 3430, 3311 (NH),

1

A solution of hydroxylamine hydrochloride (1.39 g, 20 mmol) in

absolute ethanol (10 mL) was neutralized with KOH (1.12 gm, 20 mmol)

then ﬁltered after stirring for 0.5 h at room temperature. Ethyl 2-(3-

cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate 2 [42] (0.34

g, 1 mmol) was then added to the hydroxylamine ﬁltrate followed by

addition of KOH (1.12 g, 20 mmol) portion wise. The reaction mixture

was stirred at room temperature for 1 h, then treated with cold water

2

1

8

7

232 (CN), 1614 (CO), 1390 (CS). H NMR (δ ppm, DMSO‑d

H, CSNHPh, D O exchangeable), 9.90 (s, 2H, 2NH, D O exchangeable),

.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H),

.50–7.38 (m, 5H, Ar-H), 4.01 (d, J = 6.4 Hz, 2H, OCH ), 2.68 (s, 3H,

), 2.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

δ ppm, DMSO‑d ): 180.94, 164.69, 161.99, 161.17, 157.36, 138.20,

32.99, 131.35, 129.23, 127.98, 127.54, 125.38, 123.66, 115.46,

14.07, 101.63, 75.15, 27.63, 18.77, 17.48. Anal. Calcd (%) for

22ClN S (500.03): C, 55.25; H, 4.43; N, 14.01; S, 12.82. Found:

C, 55.67; H, 4.09; N, 14.22; S, 12.66.

6

): 10.35 (s,

2

1

3

CH

3

3 2

) ). C NMR

(

6

1

(

10 mL), and the pH was adjusted to 7 by adding glacial acetic acid. The

precipitate obtained was ﬁltered, washed with water and ethanol (1:1),

dried and recrystallized from acetonitrile as beige macrocrystals.

C

23

H

5 2

O

0

ꢀ 1

Yield: 65%, m.p.: 122–124 C. IR (KBr, cm ): 3181 (broad band OH

1

and NH), 2227 (CN), 1657 (CO). H NMR (δ ppm, DMSO‑d

1

8

6

): 11.05 (br s,

4

.1.3.3. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

H, OH, D O exchangeable), 9.29 (br s, 1H, NH, D O exchangeable),

2 2

N-(4-ﬂuorophenyl) hydrazine-1-carbothioamide (5c). Yellow macro-

.23 (d, J = 2.4 Hz, 1H, Ar-H), 8.17 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 7.36

crystals, yield: 65%, m.p.: 200–202 ⁰C. IR (KBr, cm ): 3306, 3163

ꢀ 1

(

d, J = 8.8 Hz, 1H, Ar-H), 3.99 (d, J = 6.4 Hz, 2H, OCH

2

), 2.57 (s, 3H,

_N_H₎_,₂₂₂₉₍_C_N₎_,₁₆₁₀₍_C_O₎_,₁₃₅₈₍_C_S₎_.1H NMR (δ ppm, DMSO‑d

(

6

):

1

3

CH

3

), 2.15–2.03 (m, 1H, CH), 1.01 (d, J = 6.8 Hz, 6H, (CH

3 2

) ). C NMR

1

0.34 (s, 1H, CSNHPh, D

exchangeable), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz,

H, Ar-H), 7.43–7.38 (m, 3H, Ar-H), 7.21–7.16 (m, 2H, Ar-H), 4.01 (d, J

6.4 Hz, 2H, OCH ), 2.68 (s, 3H, CH

), 2.14–2.04 (m, 1H, CH), 1.02 (d,

): 181.28, 164.67,

2 2

O exchangeable), 9.84 (s, 2H, 2NH, D O

(

δ ppm, DMSO‑d

6

): 163.92, 161.81, 159.80, 154.71, 132.85, 131.26,

1

25.47, 123.60, 115.46, 113.93, 101.53, 75.10, 27.62, 18.76, 16.97.

S (331.39): C, 57.99; H, 5.17; N, 12.68;

S, 9.67. Found: C, 57.76; H, 5.04; N, 12.50; S, 9.89.

1

17 3 3

Anal. Calcd (%) for C16H N O

=

2

3

13

J = 6.8 Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

1

61.99, 161.22, 160.70, 158.47, 135.51, 132.99, 131.35, 128.26,

25.40, 123.69, 115.48, 114.65, 114.08, 101.64, 75.16, 27.64, 18.78,

4

.1.2. 2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbohydrazide

(

4)

5 2 2

7.49. Anal. Calcd (%) for C23H22FN O S (483.58): C, 57.13; H, 4.59;

Hydrazine hydrate 99% (1.50 g, 1.46 mL, 30 mmol) was added to a

N, 14.48; S, 13.26. Found: C, 56.92; H, 4.69; N, 14.25; S, 13.60.

solution of the ethyl ester 2 (1.72 g, 5 mmol) in absolute ethanol (30

mL). The reaction mixture was heated under reﬂux for 24 h, and then

cooled to room temperature. The precipitate formed was ﬁltered,

washed with cold ethanol and diethyl ether, dried and recrystallized

from ethanol as yellow macrocrystals.

4

.1.3.4. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

N-(4-tolyl)hydrazine-1-carbothioamide (5d). Yellow ﬁbrous crystals,

0

ꢀ 1

yield: 82%, m.p.: 205–207 C. IR (KBr, cm ): 3315, 3201 (NH), 2230

1

(

CN), 1624 (CO), 1311 (CS). H NMR (δ ppm, DMSO‑d

CSNHPh, D O exchangeable), 9.78 (S, 1H, NH, D O exchangeable), 9.73

S, 1H, NH, D

O exchangeable), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd,

J = 8.8, 2.4 Hz, 1H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.31 (d, J =

.0 Hz, 2H, Ar-H), 7.14 (d, J = 8.0 Hz, 2H, Ar-H), 4.01 (d, J = 6.4 Hz, 2H,

OCH ), 2.68 (s, 3H, CH ), 2.28 (s, 3H, 4-CH

-Ph), 2.14–2.04 (m, 1H,

). C NMR (δ ppm, DMSO‑d ):

78.04, 166.74, 164.57, 161.93, 160.94, 139.71, 136.57, 134.25,

32.92, 131.28, 128.55, 125.41, 123.84, 115.41, 114.05, 101.63, 75.15,

6

): 10.32 (s, 1H,

0

ꢀ 1

Yield: 72%, m.p.: 166–168 C. IR (KBr, cm ): 3344, 3300 (NH

2

),

): 9.58 (s,

O exchangeable), 8.22 (d, J = 2.4 Hz, 1H, Ar-H), 8.15 (dd, J

8.8, 2.4 Hz, 1H, Ar-H), 7.36 (d, J = 8.8 Hz, 1H, Ar-H), 4.56 (s, 2H, NH2,

O exchangeable), 3.99 (d, J = 6.4 Hz, 2H, OCH ), 2.57 (s, 3H, CH ),

.13–2.03 (m, 1H, CH), 1.01 (d, J = 6.8 Hz, 6H, (CH ). C NMR (δ

ppm, DMSO‑d ): 163.74, 161.76, 160.97, 154.61, 132.79, 131.17,

25.51, 124.90, 115.44, 113.91, 101.52, 75.09, 27.60, 18.72, 16.94.

Anal. Calcd (%) for C16 S (330.41): C, 58.16; H, 5.49; N, 16.96;

2

1

3

1

211 (NH), 2231 (CN), 1618 (CO). H NMR (δ ppm, DMSO‑d

H, NH, D

6

(

2

=

8

D

2

3

2

3

1

3

2

3

)

2

13

CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

3

)

2

6

1

18 4 2

H N O

1

5

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

0

2

7.59, 20.56, 18.72, 17.39. Anal. Calcd (%) for C24

H

25

N

5

O

2

S

2

(479.62):

mmol) in DMF (10 mL) was stirred for 4 h at 60 C. The reaction mixture

C, 60.10; H, 5.25; N, 14.60; S, 13.37. Found: C, 60.39; H, 5.09; N, 14.24;

S, 13.46.

was cooled to room temperature, poured onto crushed ice and neutral-

ized with 2 N HCl. The precipitate obtained was ﬁltered, washed with

water, dried and recrystallized from ethanol as buff macrocrystals,

4

.1.3.5. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

N-(4-methoxyphenyl) hydrazine-1-carbothioamide (5e). Off white ﬁbrous

Method B. A suspension of the acid chloride 7 (0.34 g, 1 mmol) and

semicarbazide HCl (0.11 g, 1 mmol) in tetrahydrofuran (5 mL) con-

taining catalytic amount of TEA was heated up under reﬂux for 2 h. The

reaction mixture was cooled to room temperature and neutralized by 2 N

HCl. The precipitate obtained was separated and treated as mentioned

under Method A.

0

ꢀ 1

crystals, yield: 92%, m.p.: 196–198 C. IR (KBr, cm ): 3225 (NH), 2227

1

(

CN), 1676 (CO), 1373 (CS). H NMR (δ ppm, DMSO‑d

CSNHPh, D O exchangeable), 9.74 (s, 1H, NH, D O exchangeable), 9.69

s, 1H, NH, D

6

): 10.31 (s, 1H,

2

(

2

O exchangeable), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd,

J = 8.8, 2.4 Hz, 1H, Ar-H), 7.40 (d, J = 8.8 Hz, 1H, benzyl Ar-H), 7.28 (d,

J = 8.0 Hz, 2H, Ar-H), 6.90 (d, J = 9.2 Hz, 2H, Ar-H), 4.01 (d, J = 6.4 Hz,

Yield: method A (26%) and method B (96%), m.p.: 233–235 ⁰C. IR

ꢀ

1

2

1

5

H, OCH

2

), 3.74 (s, 3H, 4-CH

3

O-Ph), 2.68 (s, 3H, CH

3

), 2.14–2.04 (m,

):

(KBr, cm ): 3430 (NH ), 3295 (NH), 2231 (CN), 1671 (CONHNH),

2

1

3

1

H, CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

1643 (CONH

exchangeable), 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 8.18 (dd, J = 8.8, 2.4 Hz,

1H, Ar-H), 8.01 (s, 1H, NH, D

O exchangeable), 7.39 (d, J = 8.8 Hz, 1H,

Ar-H), 6.04 (s, 2H, NH , D

O exchangeable), 4.00 (d, J = 6.4 Hz, 2H,

OCH ), 2.62 (s, 3H, CH

), 2.12–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz,

6H, (CH ): 164.35, 161.91, 161.34,

). C NMR (δ ppm, DMSO‑d

58.75, 156.17, 132.95, 131.32, 125.42, 124.06, 115.47, 114.02,

101.58, 75.14, 27.62, 18.76, 17.21. Anal. Calcd (%) for C17

2

). H NMR (δ ppm, DMSO‑d

6 2

): 9.92 (s, 1H, NH, D O

81.17, 164.59, 161.95, 160.96, 156.81, 151.63, 132.95, 132.01,

31.31, 127.51, 125.42, 123.86, 115.45, 114.06, 113.31, 101.63, 75.15,

2

5.25, 27.62, 18.76, 17.44. Anal. Calcd (%) for C24

H

25

N

5

O

3

S

2

(495.62):

2

C, 58.16; H, 5.08; N, 14.13; S, 12.94. Found: C, 58.40; H, 5.22; N, 14.36;

S, 12.53.

2

3

1

3

)

2

6

1

4

.1.3.6. N-Benzyl-2-(2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-

19 5 3

H N O S

carbonyl)hydrazine-1-carbothioamide (5f). Off white powder, yield:

(373.43): C, 54.68; H, 5.13; N, 18.75; S, 8.59. Found: C, 54.82; H, 5.46;

N, 18.41; S, 8.23.

0

ꢀ 1

5

0%, m.p.: 164–166 C. IR (KBr, cm ): 3332 (NH), 2225 (CN), 1657

): 10.22 (s, 1H, CSNHPh,

O exchangeable), 8.70 (s, 1H,

O exchangeable), 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 8.19 (dd, J

8.8, 2.4 Hz, 1H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.31–7.21 (m,

(

CO), 1382 (CS). ¹H NMR (δ ppm, DMSO‑d

6

D

2

O exchangeable), 9.55 (s, 1H, NH, D

, D

2

4.1.7. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)

hydrazine-1-carbothioamide (9)

NHCH

2

=

5

H, Ar-H), 4.74 (d, J = 6.0 Hz, 2H, PhCH

2

NH), 4.00 (d, J = 6.4 Hz, 2H,

), 2.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz,

): 178.34, 163.80, 161.81,

Method A. A mixture of the N-acyl benzotriazole 6 (0.21 g, 0.5 mmol)

OCH

2

), 2.65 (s, 3H, CH

3

and thiosemicarbazide (0.09 g, 1 mmol) in DMF (5 mL) was stirred at 60

1

3

0

6

1

H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

C for 4 h. The reaction mixture was cooled to room temperature, poured

61.04, 154.64, 132.87, 131.25, 128.09, 127.10, 126.67, 125.53,

25.40, 124.96, 115.51, 113.97, 101.53, 75.12, 27.64, 18.78, 17.47,

onto crushed ice and neutralized with 2 N HCl. The precipitate obtained

was ﬁltered, washed with water, dried and recrystallized from ethanol

as buff macrocrystals.

25 5 2 2

7.00. Anal. Calcd (%) for C24H N O S (479.62): C, 60.10; H, 5.25; N,

4.60; S, 13.37. Found: C, 60.29; H, 5.11; N, 14.98; S, 13.43.

Method B. A suspension of the acid chloride 7 (0.34 g, 1 mmol) and

thiosemicarbazide (0.09 g, 1 mmol) in tetrahydrofuran (5 mL) con-

taining catalytic amount of TEA was heated under reﬂux for 2 h. The

reaction mixture was cooled to room temperature, neutralized by 2 N

HCl. The precipitate obtained was ﬁltered and treated as mentioned

under method A.

4

.1.4. 5-(5-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)-4-methylthiazol-2-

yl)-2-isobutoxybenzonitrile (6)

To a stirred solution of 1H-benzotriazole (0.95 g, 8 mmol) in

dichloromethane (10 mL), thionyl chloride (0.24 g, 145 µL, 2 mmol) was

added at room temperature. After 0.5 h stirring, febuxostat 1 (0.63 g, 2

mmol) was added and the stirring was continued for further 2 h. A white

precipitate was separated, ﬁltered and washed with dichloromethane

Yield: method A (36%) and method B (70%), m.p.: 221–223 ⁰C. IR

ꢀ 1

1

(KBr, cm ): 3264 (NH), 2231 (CN), 1650, (CONH), 1370 (CS). H NMR

(

three times with 10 mL for each) and the combined organic ﬁltrate was

(δ ppm, DMSO‑d ): 10.17 (s, 1H, NH, D O exchangeable), 9.42 (s, 1H,

6

2

washed with 2 N NaOH (three times with 10 mL for each). The organic

layer was separated, washed with water and brine, dried over anhydrous

NH, D O exchangeable), 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 8.18 (dd, J =

2

8.8, 2.4 Hz, 1H, Ar-H), 7.94 and 7.68 (2 br s, 2H, NH , D O exchange-

2

Na

2

SO

4

and ﬁltered. The solvent was evaporated under reduced pressure

able), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 4.01 (d, J = 6.4 Hz, 2H, OCH ),

2

to afford the corresponding N-acyl benzotriazole. The resulting precip-

itate was dried and submitted for the next step without further

puriﬁcation.

2.64 (s, 3H, CH ), 2.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H,

3

1

3

(CH ) ). C NMR (δ ppm, DMSO‑d ): 181.95, 164.52, 161.93, 160.98,

3

2

6

156.99, 132.93, 131.27, 125.41, 123.69, 115.44, 114.04, 101.61, 75.14,

7.62, 18.75, 17.39. Anal. Calcd (%) for C17 (389.49): C,

2

19 5 2 2

H N O S

4

.1.5. 2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl

chloride (7)

To an ice-cold dry solution of thionyl chloride (2 mL), febuxostat acid

(0.32 g, 1 mmol) was added. The reaction mixture was then heated

52.42; H, 4.92; N, 17.98; S, 16.46. Found: C, 52.77; H, 4.80; N, 17.53; S,

16.10.

1

4.1.8. 2-Isobutoxy-5-(4-methyl-5-(N-substituted acetamide)thiazol-2-yl)

benzonitriles (10a-e)

under reﬂux for 45 min. After cooling down the reaction mixture, excess

of the thionyl chloride was removed by distillation giving the expected

acid chloride product as a brownish solid which was then used without

further puriﬁcation in the next step for the preparation of the target

amides 8 and 9.

Method A. A mixture of febuxostat 1 (0.36 g, 1 mmol), the appropriate

amine (1 mmol), TEA (0.30 g, 420 µL, 3 mmol) and thionyl chloride

(

0.12 g, 73 µL, 1 mmol) in dichloromethane (10 mL) was stirred for

5

–20 min at room temperature. The reaction mixture was washed ﬁrst

with 2 N HCl (10 mL), then with 10% K CO (10 mL). The organic layer

was separated, washed with water and brine and dried over anhydrous

Na SO . The solvent was evaporated to dryness under reduced pressure

4

.1.6. 2-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)

2

3

hydrazine-1-carboxamide (8)

2

4

Method A. A mixture of the N-acyl benzotriazole 6 (0.21 g, 0.5 mmol),

to afford the respective carboxamide residues. The amide derivatives

semicarbazide HCl (0.11 g, 1 mmol) and sodium bicarbonate (0.08 g, 1

10a-d were recrystallized from ethanol, while the amide derivative 10e

1

6

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

was recrystallized from acetone.

2-Isobutoxy-5-(4-methyl-5-(morpholine-4-carbonyl)thiazol-2-yl)benzoni-

trile (10e). White powder, yield: method A (25%) and method B (73%),

0

ꢀ 1

1

Method B. A suspension of the acid chloride 7 (0.34 g, 1 mmol) and the

appropriate amine (1 mmol) in THF (5 mL) containing catalytic amount

of TEA was heated under reﬂux for 2 h. The reaction mixture was cooled

to room temperature and neutralized by using 2 N HCl. The precipitate

of the respective amides obtained was ﬁltered, washed with water, dried

and recrystallized as mentioned under Method B.

m.p.: 164–166 C. IR (KBr, cm ): 2227 (CN), 1629 (CO). H NMR (δ

ppm, CDCl

): 8.11 (d, J = 2.4 Hz, 1H, Ar-H), 8.04 (dd, J = 8.8, 2.4 Hz,

1H, Ar-H), 7.00 (d, J = 8.8 Hz, 1H, Ar-H), 3.89 (d, J = 6.4 Hz, 2H, OCH ),

3.75–3.67 (m, 8H, morpholinyl-H), 2.49 (s, 3H, CH ), 2.25–2.15 (m, 1H,

). C NMR (δ ppm, CDCl ): 165.44,

162.59, 162.35, 153.23, 132.47, 131.96, 126.05, 124.03, 115.56,

12.78, 103.07, 75.80, 66.97, 50.93, 28.28, 19.17, 16.68. Anal. Calcd

%) for C20 S (385.48): C, 62.32; H, 6.01; N, 10.90; S, 8.32.

Found: C, 62.16; H, 5.78; N, 10.65; S, 8.59.

3

2

3

1

3

CH), 1.08 (d, J = 6.8 Hz, 6H, (CH

3

)

2

3

1

(

23 3 3

H N O

N-Benzyl-2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carboxamide

(

10a). Beige microcrystals, yield: method A (23%) and method B (79%),

0

ꢀ 1

1

m.p.: 158–160 C. IR (KBr, cm ): 3448 (NH), 2224 (CN), 1633 (CO). H

NMR (δ ppm, CDCl

2

1

5

2

4

.1.9. 2-(3-Cyano-4-isobutoxyphenyl)-N’-formyl-4-methylthiazole-5-

carbohydrazide (11)

3

): 8.12 (d, J = 2.4 Hz, 1H, Ar-H), 8.04 (dd, J = 8.8,

.4 Hz, 1H, Ar-H), 7.38–7.33 (m, 5H, benzyl Ar-H), 7.00 (d, J = 8.8 Hz,

H, Ar-H), 6.11 (t, J = 6.4 Hz, 1H, NH, D O exchangeable), 4.62 (d, J =

.6 Hz, 2H, PhCH ), 2.73 (s, 3H, CH ),

), 3.89 (d, J = 6.4 Hz, 2H, OCH

.25–2.15 (m, 1H, CH), 1.08 (d, J = 6.8 Hz, 6H, (CH ). C NMR (δ

): 164.70, 162.48, 161.63, 156.49, 137.81, 132.59, 132.06,

29.02, 127.99, 127.94, 126.04, 125.77, 115.54, 112.77, 103.05, 75.83,

4.40, 28.28, 19.17, 17.59. Anal. Calcd (%) for C23 S (405.52):

A solution of the acid hydrazide 4 (0.17 g, 0.5 mmol) in formic acid

2

(

3 mL) was heated under reﬂux for 3 h. After cooling to room temper-

2

3

1

3

ature, the product was precipitated after addition of 6 mL of distilled

water and sodium bicarbonate solution. The precipitate formed was

ﬁltered, washed with water and sodium bicarbonate solution, dried and

recrystallized from ethanol as yellow ﬁbrous crystals.

3 2

)

ppm, CDCl

3

1

4

23 3 2

H N O

0

ꢀ 1

Yield: 93%, m.p.: 236–238 C. IR (KBr, cm ): 3279 (NH), 2230

C, 68.12; H, 5.72; N, 10.36; S, 7.91. Found: C, 68.34; H, 5.60; N, 10.72;

S, 7.68.

1

(

CN), 1695 (CHO), 1667 (CONHNH). H NMR (δ ppm, DMSO‑d

6

): 10.31

(

s, 1H, CONH, D O exchangeable), 10.15 (s, 1H, NHCHO, D O

2

exchangeable), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz,

H, Ar-H), 8.13 (s, 1H, CHO), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 4.01 (d, J =

6.4 Hz, 2H, OCH ), 2.62 (s, 3H, CH ), 2.14–2.04 (m, 1H, CH), 1.02 (d, J

2

-(3-Cyano-4-isobutoxyphenyl)-N-(4-methoxyphenyl)-4-methylthiazole-5-

1

carboxamide (10b). Light gray microcrystals, yield: method A (34%)

0

ꢀ 1

2

3

and method B (93%), m.p.: 181–183 C. IR (KBr, cm ): 3366 (NH),

1

3

1

= 6.8 Hz, 6H, (CH ) ). C NMR (δ ppm, DMSO‑d ): 164.64, 161.94,

3

2

6

2

228 (CN), 1632 (CO). H NMR (δ ppm, CDCl

Ar-H), 8.15 (d, J = 2.4 Hz, 1H, Ar-H), 8.02 (s, 1H, NH, D

able), 7.49 (d, J = 8.8 Hz, 2H, Ar-H), 7.03 (d, J = 8.8 Hz, 1H, Ar-H), 6.89

d, J = 8.8 Hz, 2H, Ar-H), 3.89 (d, J = 6.4 Hz, 2H, OCH ), 3.80 (s, 3H, 4-

CH O-Ph), 2.79 (s, 3H, CH

), 2.63–2.51 (m, 1H, CH), 1.07 (d, J = 6.8 Hz,

): 164.85, 162.52, 159.88, 157.10,

3

): 8.19 (d, J = 8.8 Hz, 1H,

1

60.19, 159.88, 156.45, 132.97, 131.39, 125.32, 123.39, 115.41,

13.96, 101.57, 75.13, 27.60, 18.73, 17.15. Anal. Calcd (%) for

2

O exchange-

C

17

H

18

N

4

O

3

S (358.42): C, 56.97; H, 5.06; N, 15.63; S, 8.94. Found: C,

(

2

+

5

7.21; H, 5.35; N, 15.84; S, 9.08. EI-MS: [M 358.60].

3

1

3

6

1

H, (CH

3

)

2

). C NMR (δ ppm, CDCl

3

4

.1.10. N’-Acetyl-2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-

56.86, 132.63, 132.04, 130.44, 126.01, 125.94, 122.56, 115.59,

14.41, 112.77, 102.99, 75.82, 55.64 28.27, 19.17, 17.60. Anal. Calcd

carbohydrazide (12)

A solution of the acid hydrazide 4 (0.17 g, 0.5 mmol) and anhydrous

sodium acetate (0.04 g, 0.5 mmol) in acetic anhydride (3 mL) was stirred

for 2 h at room temperature. The precipitate obtained was ﬁltered,

washed with water, sodium bicarbonate solution and petroleum ether,

dried and recrystallized from ethanol as white powder.

(

%) for C23

H

23

N

3

O

3

S (421.52): C, 65.54; H, 5.50; N, 9.97; S, 7.61.

+

Found: C, 65.17; H, 5.86; N, 9.71; S, 7.82. EI-MS: [M 421.53].

2

-(3-Cyano-4-isobutoxyphenyl)-4-methyl-N-(2-tolyl)thiazole-5-carbox-

amide (10c). White powder, yield: method A (20%) and method B

0

ꢀ 1

0

ꢀ 1

Yield: 80%, m.p.: 237–239 C. IR (KBr, cm ): 3263, 3027 (NH),

(

67%), m.p.: 160–162 C. IR (KBr, cm ): 3450 (NH), 2230 (CN), 1641

1

2235 (CN), 1693 (CH

0.17 (br s, 1H, NH, D

3

CO), 1670 (CONH). H NMR (δ ppm, DMSO‑d

6

):

CO). H NMR (δ ppm, CDCl ): 8.16 (d, J = 2.4 Hz, 1H, Ar-H), 8.09 (dd,

3

1

2

O exchangeable), 9.98 (br s, 1H, CH CONH, D O

3

2

J = 8.8, 2.4 Hz, 1H, Ar-H), 7.89 (d, J = 8.0 Hz, 1H, Ar-H), 7.40 (s, 1H, Ar-

H), 7.24 (s, 1H, Ar-H), 7.16–7.12 (m, 1H, Ar-H), 7.02 (d, J = 8.8 Hz, 1H,

exchangeable), 8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz,

1

H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 4.00 (d, J = 6.4 Hz, 2H, OCH

.62 (s, 3H, CH ), 1.02 (d,

), 2.14–2.02 (m, 1H, CH), 1.91 (s, 3H, COCH

): 168.50, 164.52,

61.94, 160.48, 156.24, 132.99, 131.39, 125.37, 123.67, 115.44,

13.98, 101.58, 75.14, 27.62, 20.58, 18.75, 17.12. Anal. Calcd (%) for

S (372.44): C, 58.05; H, 5.41; N, 15.04; S, 8.61. Found: C,

2

),

Ar-H), 5.30 (s, 1H, NH, D

OCH ), 2.81 (s, 3H, CH

2

O exchangeable), 3.90 (d, J = 6.4 Hz, 2H,

2

3

2

3

), 2.34 (s, 3H, 2-CH

3

-Ph), 2.26–2.16 (m, 1H,

). C NMR (δ ppm, CDCl ): 164.77,

62.26, 159.55, 156.48, 135.14, 132.33, 131.81, 130.48, 129.01,

26.77, 126.04, 125.57, 122.98, 115.19, 112.49, 102.81, 99.78, 75.55,

7.98, 18.86, 17.73, 17.41. Anal. Calcd (%) for C23 S (405.52):

1

3

1

3

J = 6.8 Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

CH), 1.09 (d, J = 6.8 Hz, 6H, (CH

3

)

2

3

1

2

18 20 4 3

C H N O

23 3 2

H N O

5

8.34; H, 5.68; N, 15.19; S, 8.20.

C, 68.12; H, 5.72; N, 10.36; S, 7.91. Found: C, 68.39; H, 5.86; N, 10.54;

+

S, 7.85. EI-MS: [M 405.19].

4

.1.11. N’-(2-(3-Cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)-

-methylbenzene-sulfonohydrazide (13)

2

-Isobutoxy-5-(4-methyl-5-(piperidine-1-carbonyl)thiazol-2-yl)benzonitrile

A mixture of the acid hydrazide 4 (0.17 g, 0.5 mmol) and tosyl

(

10d). White powder, yield: method A (37%) and method B (91%), m.

0

ꢀ 1

1

chloride (0.10 g, 0.5 mmol) in pyridine (5 mL) was stirred at room

temperature for 24 h. The reaction mixture was poured onto crushed ice

that is acidiﬁed to pH 3–4 using 2 N HCl. The precipitate obtained was

ﬁltered, washed with water, dried and recrystallized from ethanol as

light brown macrocrystals.

p.: 151–153 C. IR (KBr, cm ): 2225 (CN), 1615 (CO). H NMR (δ ppm,

CDCl

): 8.11 (d, J = 2.4 Hz, 1H, Ar-H), 8.02 (dd, J = 8.8, 2.4 Hz, 1H, Ar-

), 2.46 (s,

), 2.24–2.14 (m, 1H, CH), 1.72–1.63 (m, 10H, piperidinyl-H),

3

H), 6.99 (d, J = 8.8 Hz, 1H, Ar-H), 3.88 (d, J = 6.4 Hz, 2H, CH

2

3

1

H, CH

3

1

3

.08 (d, J = 6.8 Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, CDCl

3

): 164.80,

0

ꢀ 1

Yield: 83%, m.p.: 230–232 C. IR (KBr, cm ): 3295 (NH), 2227

62.18, 161.26, 152.44, 132.42, 131.86, 126.24, 125.09, 115.65,

12.70, 102.94, 75.72, 29.81, 28.27, 26.18, 24.56, 19.18, 16.54. Anal.

1

(

CN), 1654 (CO), 1368, 1341 (SO

2

). H NMR (δ ppm, DMSO‑d

6

): 10.60

br s, 1H, CONH, D O exchangeable), 10.09 (br s, 1H, SO

2

NH, D O

2

Calcd (%) for C21

25 3 2

H N O S (383.51): C, 65.77; H, 6.57; N, 10.96; S,

exchangeable), 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 8.17 (dd, J = 8.8, 2.4 Hz,

8

.36. Found: C, 65.56; H, 6.18; N, 10.70; S, 8.73.

1

4

2

H, Ar-H), 7.74 (d, J = 7.6 Hz, 2H, Ar-H), 7.37 (d, J = 8.0 Hz, 3H, Ar-H),

.00 (d, J = 6.4 Hz, 2H, OCH ), 2.48 (s, 3H, CH ), 2.37 (s, 3H, 4-CH -Ph),

.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H, (CH ). C NMR (δ

2

3

1

3

3 2

)

1

7

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

ppm, DMSO‑d

6

): 164.89, 161.95, 160.28, 156.13, 143.49, 135.89,

ether and the precipitate obtained was ﬁltered, washed with diethyl

ether, dried and recrystallized from ethanol as yellow ﬁbrous crystals.

1

32.96, 131.43, 129.42, 127.76, 125.25, 123.31, 115.40, 113.95,

01.56, 75.11, 27.60, 21.07, 18.73, 16.91. Anal. Calcd (%) for

(484.59): C, 57.01; H, 4.99; N, 11.56; S, 13.23. Found: C,

0

ꢀ 1

Yield: 80%, m.p.: 228–230 C. IR (KBr, cm ): 3326 (NH), 2228

1

C

23

H

24

4

N O

4

S

2

(CN), 1697 (COOC

(d, J = 2.4 Hz, 1H, Ar-H), 8.22 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 7.82 (s,

H, pyrazole-C -H), 7.54 (br s, 2H, NH , D O exchangeable), 7.33 (d, J

= 8.8 Hz, 1H, Ar-H), 4.23 (q, J = 7.2 Hz, 2H, CH CH ), 3.98 (d, J = 6.4

Hz, 2H, OCH ), 2.78 (s, 3H, CH

), 2.14–2.05 (m, 1H, CH), 1.28 (t, J =

7.2 Hz, 3H, CH CH ). Anal. Calcd (%)

), 1.02 (d, J = 6.8 Hz, 6H, (CH

for C22 S (453.52): C, 58.27; H, 5.11; N, 15.44; S, 7.07. Found: C,

2

H

5

), 1670 (CON). H NMR (δ ppm, DMSO‑d

6

): 8.28

5

7.27; H, 4.64; N, 11.66; S, 13.61.

1

3

2

4

.1.12. N’-(4-Substituted benzylidene)-2-(3-cyano-4-isobutoxyphenyl)-4-

2

3

methylthiazole-5-carbohydrazides (14a,b)

2

3

A solution of the acid hydrazide 4 (0.17 gm. 0.5 mmol) and the

appropriate aldehyde (0.5 mmol) in absolute ethanol (7 mL) and 2 drops

glacial acetic acid was reﬂuxed for 1–3 h and left to cool to room tem-

perature. The solid obtained was ﬁltered, washed with cold ethanol,

dried and recrystallized from ethanol.

2

3

3 2

)

23 5 4

H N O

58.61; H, 4.85; N, 15.19; S, 7.28.

4.1.15. 2-Isobutoxy-5-(4-methyl-5-(2-thioxo-2,3-dihydro-1,3,4-

oxadiazol-5-yl)thiazol-2-yl) benzonitrile (17)

4

.1.12.1. N’-Benzylidene-2-(3-cyano-4-isobutoxyphenyl)-4-methyl-

The acid hydrazide 4 (0.17 g, 0.5 mmol) was dissolved in a solution

of KOH (0.03 g, 0.55 mmol) in water (1 mL) and ethanol (7 mL). Carbon

disulphide (1 mL) was then gradually added while stirring and the re-

action mixture was heated under reﬂux for 6 h. The solvents were

evaporated under reduced pressure, the residue was suspended in water

(30 mL) and acidiﬁed to pH 5 using glacial acetic acid. The precipitate

obtained was ﬁltered, washed with water, dried and recrystallized from

ethyl acetate: hexane (3:2) as yellow macrocrystals.

thiazole-5-carbohydrazide (14a). Off white powder, yield: 92%, m.p.:

0

ꢀ 1

1

2

78–280 C. IR (KBr, cm ): 3164 (NH), 2229 (CN), 1658 (CO). H NMR

): 11.90 (s, 1H, NH, D O exchangeable), 8.30 (s, 1H, N

CH), 8.24 (d, J = 8.8 Hz, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 7.78 (d, J = 7.6

(

δ ppm, DMSO‑d

6

2

=

Hz, 2H, Ar-H), 7.51–7.43 (m, 3H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H),

4

.01 (d, J = 6.4 Hz, 2H, OCH

2

), 2.75 (s, 3H, CH

3

), 2.15–2.05 (m, 1H,

). C NMR (δ ppm, DMSO‑d ):

61.93, 161.33, 160.06, 154.59, 153.30, 133.95, 133.05, 131.36,

30.11, 129.72, 129.06, 127.39, 124.15, 115.48, 114.02, 101.59, 75.12,

7.60, 18.74, 17.24. Anal. Calcd (%) for C23 S (418.52): C,

1

3

CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

3

)

2

6

0

ꢀ 1

1

2

6

7

Yield: 97%, m.p.: 220–222 C. IR (KBr, cm ): 3141 (NH), 2244

1

(CN), 1336 (CS). H NMR (δ ppm, DMSO‑d

H), 8.23 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 7.39 (s, 1H, NH, D

), 2.66

).

): 176.69, 165.79, 162.14, 156.19, 133.04,

6

): 8.32 (d, J = 2.4 Hz, 1H, Ar-

H

22

4

N O

2

O

6.01; H, 5.30; N, 13.39; S, 7.66. Found: C, 66.43; H, 5.08; N, 12.92; S,

.47.

exchangeable), 7.36 (s, 1H, Ar-H), 4.00 (d, J = 6.4 Hz, 2H, OCH

2

(s, 3H, CH

3

), 2.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

3 2

)

1

3

C NMR (δ ppm, DMSO‑d

6

1

4

31.60, 128.33, 124.79, 115.31, 113.89, 113.43, 101.60, 75.15, 27.60,

4

.1.12.2. N’-(4-Methoxybenzylidene)-2-(3-cyano-4-isobutoxyphenyl)-4-

16 4 2 2

8.73, 17.13. Anal. Calcd (%) for C17H N O S (372.46): C, 54.82; H,

methylthiazole-5-carbohydrazide (14b). Yellow powder, yield: 94%, m.

0

ꢀ 1

1

.33; N, 15.04; S, 17.22. Found: C, 55.17; H, 4.51; N, 15.28; S, 17.14. EI-

p.: 269–271 C. IR (KBr, cm ): 3038 (NH), 2226 (CN), 1658 (CO). H

NMR (δ ppm, DMSO‑d ): 11.77 (s, 1H, NH, D O exchangeable), 8.31 (s,

H, N = CH), 8.24 (d, J = 8.8 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.72 (d,

J = 8.4 Hz, 2H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.05 (d, J = 8.4 Hz,

H, Ar-H), 4.01 (d, J = 6.4 Hz, 2H, OCH ), 3.81 (s, 3H, 4-CH O-Ph), 2.75

s, 3H, CH ).

), 2.15–2.05 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H, (CH

): 161.92, 161.20, 159.70, 156.43, 152.90,

+

MS: [M 372.35].

6

2

1

4

.1.16. 2-Isobutoxy-5-(4-methyl-5-(2-phenyl-1,3,4-oxadiazol-5-yl)

thiazol-2-yl)benzonitrile (18)

2

3

To a cooled mixture of equimolar amounts of the acid hydrazide 4

(

1

3

3 2

)

3

(

0.17 g, 0.5 mmol) and benzoic acid (0.21 g, 0.5 mmol), phosphorous

C NMR (δ ppm, DMSO‑d

6

oxychloride (3 mL) was added while stirring. The reaction mixture was

then heated under reﬂux for 8 h, left to cool to room temperature and

poured onto ice-cold water. The precipitate formed was ﬁltered, washed

with water and 10% sodium bicarbonate solution, dried and recrystal-

lized from ethanol and few drops of DMF as yellow macrocrystals.

1

34.20, 133.11, 131.34, 129.89, 129.06, 126.52, 125.03, 123.82,

14.56, 114.03, 101.60, 75.13, 55.37, 27.61, 18.75, 16.88. Anal. Calcd

(

%) for C24

H

24

N

4

O

3

S (448.54): C, 64.27; H, 5.39; N, 12.49; S, 7.15.

+

Found: C, 64.54; H, 5.62; N, 12.72; S, 7.29. EI-MS: [M 448.86].

0

ꢀ 1

1

Yield: 84%, m.p.: 254–256 C. IR (KBr, cm ): 2228 (CN). H NMR (δ

ppm, CDCl

): 8.21 (d, J = 2.4 Hz, 1H, Ar-H), 8.11 (d, J = 6.4 Hz, 3H, Ar-

H), 7.60–7.53 (m, 3H, Ar-H), 7.04 (d, J = 8.8 Hz, 1H, Ar-H), 3.91 (d, J =

.4 Hz, 2H, OCH ), 2.91 (s, 3H, CH

), 2.25–2.18 (m, 1H, CH), 1.10 (d, J

6.8 Hz, 6H, (CH ): 166.54, 164.30,

4

.1.13. 5-(5-(3,5-Dimethyl-1H-pyrazole-1-carbonyl)-4-methylthiazol-2-

yl)-2-isobutoxybenzonitrile (15)

3

A mixture of the acid hydrazide 4 (0.17 g, 0.5 mmol) and acetyla-

cetone (0.10 g, 103 µL, 1 mmol) in DMF (5 mL) was heated under reﬂux

for 5 h. After cooling to room temperature, the precipitate formed was

ﬁltered, washed with ethanol, dried and recrystallized from ethanol as

yellow microcrystals.

6

2

3

1

3

=

3

)

2

). C NMR (δ ppm, CDCl

62.64, 159.71, 157.21, 132.67, 132.17, 132.14, 129.33, 127.09,

25.85, 123.53, 115.51, 114.62, 112.83, 103.20, 75.86, 29.84, 19.20,

3

1

0

ꢀ 1

20 4 2

7.75. Anal. Calcd (%) for C23H N O S (416.50): C, 66.33; H, 4.84; N,

Yield: 69%, m.p.: 200–202 C. IR (KBr, cm ): 2227 (CN), 1667 (CO).

H NMR (δ ppm, CDCl ): 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J =

1

3.45; S, 7.70. Found: C, 66.06; H, 4.41; N, 13.59; S, 7.93.

3

8

.8, 2.4 Hz, 1H, Ar-H), 7.02 (d, J = 8.8 Hz, 1H, Ar-H), 6.06 (s, 1H,

4

.1.17. 5-(5-(4-Substituted-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-4-

pyrazole-C

4

-H), 3.90 (d, J = 6.4 Hz, 2H, OCH

-pyrazole), 2.33 (s, 3H, 3-CH

-pyrazole), 2.26–2.16 (m, 1H,

). C NMR (δ ppm, CDCl ): 171.30,

65.88, 162.60, 161.02, 151.85, 144.97, 132.72, 132.31, 126.29,

20.15, 115.69, 112.68, 111.84, 102.97, 75.77, 28.30, 19.77, 19.21,

4.81, 13.96. Anal. Calcd (%) for C21 S (394.49): C, 63.94; H,

2 3

), 2.87 (s, 3H, CH ), 2.64

methylthiazol-2-yl)-2-isobutoxybenzonitriles (19a-f)

(

s, 3H, 5-CH

3

1

3

CH), 1.09 (d, J = 6.8 Hz, 6H, (CH

3

)

2

3

1

5

4.1.17.1. 5-(5-(4-(4-Substituted phenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-

triazol-3-yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitriles

(19a-e). To

H

22

N

4

O

2

the appropriate thiosemicarbazide derivative (5a-e) (0.2 mmol) and

cetrimide (0.05 g), 2 N NaOH (10 mL) was added. The reaction mixture

was stirred under reﬂux for 2–6 h, left to cool to room temperature and

acidiﬁed to pH 5–6 with 10% HCl. The solid product thus separated was

ﬁltered, washed with water, dried and recrystallized from ethanol.

4.1.17.1.1. 5-(5-(4-Phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-

yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitrile (19a). Yellow powder,

.62; N, 14.20; S, 8.13. Found: C, 63.61; H, 5.47; N, 14.05; S, 8.55.

4

.1.14. Ethyl 5-amino-1-(2-(3-cyano-4-isobutoxyphenyl)-4-

methylthiazole-5-carbonyl)-1H-pyrazole-4-carboxylate (16)

A mixture of the acid hydrazide 4 (0.17 g, 0.5 mmol) and ethyl

0

ethoxymethylenecyanoacetate (0.17 g, 1 mmol) was heated at 170 C in

0

ꢀ 1

an oil bath for 1 h. The reaction mixture was triturated with diethyl

yield: 97%, m.p.: 284–286 C. IR (KBr, cm ): 3142 (NH), 2248 (CN),

1

8

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

1

359 (CS). ¹H NMR (δ ppm, DMSO‑d

exchangeable), 8.06 (d, J = 2.4 Hz, 1H, Ar-H), 7.96 (dd, J = 8.8, 2.4 Hz,

H, Ar-H), 7.55–7.51 (m, 3H, Ar-H), 7.45–7.41 (m, 2H, Ar-H), 7.29 (d, J

8.8 Hz, 1H, Ar-H), 3.94 (d, J = 6.4 Hz, 2H, OCH ), 2.40 (s, 3H, CH ),

.10–2.01 (m, 1H, CH), 0.99 (d, J = 6.8 Hz, 6H, (CH ). C NMR (δ

ppm, DMSO‑d ): 168.28, 165.47, 161.87, 155.85, 144.37, 133.60,

32.71, 131.12, 130.06, 129.53, 128.99, 125.00, 115.38, 115.10,

13.95, 101.55, 75.09, 27.61, 18.75, 16.87. Anal. Calcd (%) for

OS (447.58): C, 61.72; H, 4.73; N, 15.65; S, 14.33. Found: C,

1.98; H, 4.81; N, 15.39; S, 14.07.

.1.17.1.2. 5-(5-(4-(4-Chlorophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-

6

): 14.35 (s, 1H, NH, D

2

O

benzyl-2-(2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carbonyl)

hydrazine-1-carbothioamide 5f (0.24 gm, 0.5 mmol) in dioxane (3 mL),

TEA (1 mL) was added drop wise while stirring. The reaction mixture

was stirred under reﬂux for 5 h, left to cool to room temperature and

acidiﬁed to pH 5–6 with 10% HCl. The formed precipitate was ﬁltered,

washed with water, dried and recrystallized from ethanol as golden

yellow microcrystals.

1

=

2

3

1

3

2

3 2

)

6

1

Yield: 97%, m.p.: 189–191 ⁰C. IR (KBr, cm ): 3411 (NH), 2231

ꢀ 1

C

23

H

21

N

5

2

(CN), 1356 (CS). ¹H NMR (δ ppm, DMSO‑d

6 2

): 14.36 (s, 1H, NH, D O

6

exchangeable), 8.21 (d, J = 2.4 Hz, 1H, Ar-H), 8.12 (dd, J = 8.8, 2.4 Hz,

1H, Ar-H), 7.37 (d, J = 8.8 Hz, 1H, Ar-H), 7.28–7.23 (m, 3H, Ar-H), 7.04

4

triazol-3-yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitrile (19b). Yellow

(dd, J = 7.6, 2.4 Hz, 2H, Ar-H), 5.24 (s, 2H, PhCH ), 4.00 (d, J = 6.4 Hz,

2

macrocrystals, yield: 91%, m.p.: 247–249 ⁰C. IR (KBr, cm ): 3138

ꢀ 1

2H, OCH

), 2.13 (s, 3H, CH

), 2.10–2.04 (m, 1H, CH), 1.01 (d, J = 6.8

): 168.12, 166.15, 161.95,

2

3

1

13

(

NH), 2242 (CN), 1333 (CS). H NMR (δ ppm, DMSO‑d

6

): 14.36 (s, 1H,

Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

NH, D

.8, 2.4 Hz, 1H, Ar-H), 7.62 (d, J = 8.8 Hz, 2H, Ar-H), 7.51 (d, J = 8.8

Hz, 2H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 3.95 (d, J = 6.4 Hz, 2H,

OCH ), 2.42 (s, 3H, CH

2

O exchangeable), 8.15 (d, J = 2.4 Hz, 1H, Ar-H), 7.86 (dd, J =

156.21, 143.86, 135.36, 132.86, 131.36, 128.58, 127.76, 126.80,

125.14, 115.38, 114.09, 113.99, 101.57, 75.13, 46.74, 27.59, 18.73,

8

23 5 2

15.87. Anal. Calcd (%) for C24H N OS (461.60): C, 62.45; H, 5.02; N,

2

3

), 2.14–2.06 (m, 1H, CH), 0.99 (d, J = 6.8 Hz,

): 176.83, 165.74, 162.17,

15.17; S, 13.89. Found: C, 62.61; H, 5.26; N, 15.22; S, 13.65.

1

3

6

1

H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

56.10, 155.78, 147.49, 133.08, 131.63, 128.54, 128.37, 124.85,

15.37, 115.31, 113.93, 113.57, 101.62, 75.18, 27.64, 18.77, 17.16.

4.1.18. 2-Isobutoxy-5-(4-methyl-5-(2-(phenylamino)-1,3,4-thiadiazol-5-

yl)thiazol-2-yl)benzonitrile (20)

Anal. Calcd (%) for C23

H

20ClN

5

OS

2

(482.02): C, 57.31; H, 4.18; N,

A mixture of phosphorous oxychloride (2 mL) and the thio-

semicarbazide derivative 5a (0.1 g, 0.22 mmol) was stirred under reﬂux

for 11 h. The reaction mixture was cooled and poured onto ice-cold

water. The precipitate obtained was ﬁltered, washed with water, dried

and recrystallized from dioxane: acetone (5:1) as yellow macrocrystals.

+

1

4

4.53; S, 13.30. Found: C, 57.60; H, 4.34; N, 14.41; S, 13.43. EI-MS: [M

+

81.33, (M +2) 483.30].

4

.1.17.1.3. 5-(5-(4-(4-Fluorophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-

triazol-3-yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitrile (19c). Yellow

0

ꢀ 1

0

ꢀ 1

powder, yield: 85%, m.p.: 289–291 C. IR (KBr, cm ): 3220 (NH), 2242

CN), 1368 (CS). ¹H NMR (δ ppm, DMSO‑d

): 14.35 (s, 1H, NH, D

exchangeable), 8.12 (d, J = 2.4 Hz, 1H, Ar-H), 8.02 (dd, J = 8.8, 2.4 Hz,

H, Ar-H), 7.54–7.51 (m, 2H, Ar-H), 7.41–7.36 (m, 2H, Ar-H), 7.32 (d, J

8.8 Hz, 1H, Ar-H), 3.96 (d, J = 6.4 Hz, 2H, OCH ), 2.43 (s, 3H, CH ),

.12–2.02 (m, 1H, CH), 1.00 (d, J = 6.8 Hz, 6H, (CH ). C NMR (δ

ppm, DMSO‑d ): 168.39, 165.49, 161.86, 161.25, 155.90, 144.41,

32.75, 131.48, 131.17, 129.88, 125.00, 116.39, 115.35, 114.98,

13.92, 101.55, 75.08, 27.57, 18.71, 16.88. Anal. Calcd (%) for

20FN OS (465.57): C, 59.34; H, 4.33; N, 15.04; S, 13.77. Found: C,

9.09; H, 4.58; N, 15.16; S, 13.44.

.1.17.1.4. 5-(5-(4-(4-Tolyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-

-yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitrile (19d). Yellow pow-

Yield: 96%, m.p.: 210–212 C. IR (KBr, cm ): 3250 (NH), 2265

1

(

6

2

O

(CN). H NMR (δ ppm, DMSO‑d

6 2

): 10.62 (s, 1H, NH, D O exchangeable),

8.27 (d, J = 2.4 Hz, 1H, Ar-H), 8.20 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 7.63

(d, J = 8.0 Hz, 2H, Ar-H), 7.39–7.34 (m, 3H, Ar-H), 7.03 (t, J = 7.6 Hz,

1

=

2

3

1H, Ar-H), 3.97 (d, J = 6.4 Hz, 2H, OCH

2

), 2.63 (s, 3H, CH

3

), 2.13–2.03

). C NMR (δ ppm,

): 164.45, 163.67, 161.86, 151.70, 148.62, 140.30, 132.90,

131.35, 129.26, 125.40, 122.85, 122.35, 117.63, 115.50, 113.96,

101.57, 75.14, 27.65, 18.78, 17.26. Anal. Calcd (%) for C23 OS

1

3

13

2

3

)

2

(m, 1H, CH), 1.01 (d, J = 6.8 Hz, 6H, (CH

DMSO‑d

3 2

)

6

1

H

21

N

5

2

C

23

H

5

2

(447.58): C, 61.72; H, 4.73; N, 15.65; S, 14.33. Found: C, 61.86; H, 4.49;

N, 15.50; S, 14.09.

5

4

3

4.1.19. 5-(5-(2-((4-Substituted phenyl)amino)-1,3,4-oxadiazol-5-yl)-4-

methylthiazol-2-yl)-2-isobutoxybenzonitriles (21b,d)

0

ꢀ 1

der, yield: 89%, m.p.: 291–293 C. IR (KBr, cm ): 3225 (NH), 2242

CN), 1332 (CS). ¹H NMR (δ ppm, DMSO‑d

): 14.31 (s, 1H, NH, D

exchangeable), 8.10 (d, J = 2.4 Hz, 1H, Ar-H), 7.99 (dd, J = 8.8, 2.4 Hz,

), 2.39

-Ph), 2.09–2.01 (m, 1H, CH), 0.99 (d, J =

): 168.38, 165.47,

(

6

2

O

To a suspension of the appropriate thiosemicarbazide derivative (5b,

d) (0.3 mmol) in 95% ethanol (10 mL) and 4 N NaOH (2 mL), a solution

of iodine in KI (aqueous 5%) was added dropwise while stirring at room

temperature until the color of iodine persisted for 15 min. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated to a small volume

and left overnight at room temperature. The precipitate formed was

ﬁltered, washed with cold ethanol, dried and recrystallized from

ethanol.

1

H, Ar-H), 7.33–7.28 (m, 5H, Ar-H), 3.96 (d, J = 6.4 Hz, 2H, OCH

2

(

s, 3H, CH

3

), 2.36 (s, 3H, 4-CH

3

1

3

6

1

5

.8 Hz, 6H, (CH

3

)

2

). C NMR (δ ppm, DMSO‑d

6

61.84, 155.77, 144.36, 139.64, 132.73, 131.14, 130.97, 129.94,

28.60, 125.02, 115.34, 115.10, 113.93, 101.54, 75.08, 27.57, 20.86,

8.71, 16.80. Anal. Calcd (%) for C24

.02; N, 15.17; S, 13.89. Found: C, 62.11; H, 4.87; N, 15.30; S, 13.74.

.1.17.1.5. 5-(5-(4-(4-Methoxyphenyl)-5-thioxo-4,5-dihydro-1H-

,2,4-triazol-3-yl)-4-methylthiazol-2-yl)-2-isobutoxybenzonitrile (19e).

23 5 2

H N OS (461.60): C, 62.45; H,

4

4.1.19.1. 5-(5-(2-((4-Chlorophenyl)amino)-1,3,4-oxadiazol-5-yl)-4-

1

methylthiazol-2-yl)-2- isobutoxy benzonitrile (21b). Yellow powder, yield:

0

ꢀ 1

0

ꢀ 1

1

Yellow powder, yield: 96%, m.p.< :300 C. IR (KBr, cm ): 3390 (NH),

88%, m.p.: 248–250 C. IR (KBr, cm ): 3413 (NH), 2237 (CN). H NMR

(δ ppm, DMSO‑d ): 10.94 (s, 1H, NH, D O exchangeable), 8.31 (d, J =

1

2

231 (CN), 1334 (CS). H NMR (δ ppm, CDCl

exchangeable), 7.97–7.93 (m, 2H, Ar-H), 7.24 (s, 2H, Ar-H), 7.04 (d, J =

.8 Hz, 2H, Ar-H), 6.96 (d, J = 8.8 Hz, 1H, Ar-H), 3.89 (s, 3H, 4-CH O-

Ph), 3.87 (d, J = 6.4 Hz, 2H, OCH ), 2.61 (s, 3H, CH ), 2.22–2.15 (m, 1H,

). C NMR (δ ppm, CDCl ): 172.54,

66.22, 162.48, 161.16, 157.41, 153.82, 144.33, 135.05, 132.51,

32.03, 130.01, 125.80, 115.54, 115.32, 112.75, 103.02, 75.85, 55.76,

8.29, 19.17, 17.70. Anal. Calcd (%) for C24 (477.60): C,

3 2

): 11.42 (s, 1H, NH, D O

6

2

2.4 Hz, 1H, Ar-H), 8.24 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 7.62 (d, J = 9.0

8

3

Hz, 2H, Ar-H), 7.44–7.37 (m, 3H, Ar-H), 4.00 (d, J = 6.4 Hz, 2H, OCH ),

2

3

2.72 (s, 3H, CH ), 2.14–2.04 (m, 1H, CH), 1.02 (d, J = 6.8 Hz, 6H,

3

1

3

13

CH), 1.07 (d, J = 6.8 Hz, 6H, (CH

3

)

2

3

(CH ) ). C NMR (δ ppm, DMSO‑d ): 161.99, 154.39, 152.88, 146.08,

3

2

6

1

2

6

1

133.01, 131.42, 129.01, 125.66, 125.11, 118.71, 115.36, 114.71,

114.00, 101.52, 99.59, 99.08, 75.14, 27.59, 18.73, 17.02. Anal. Calcd

(%) for C₂₃H₂₀ClN O S (465.96): C, 59.29; H, 4.33; N, 15.03; S, 6.88.

23 5 2 2

H N O S

5

2

+

0.36; H, 4.85; N, 14.66; S, 13.43. Found: C, 60.47; H, 4.72; N, 14.36; S,

3.50.

Found: C, 59.47; H, 4.20; N, 15.44; S, 6.94. EI-MS: [M 465.46, (M +2)

467.28].

4

.1.17.2. 5-(5-(4-Benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-4-

4.1.19.2. 5-(5-(2-(4-Tolylamino)-1,3,4-oxadiazol-5-yl)-4-methylthiazol-

methylthiazol-2-yl)-2-isobutoxybenzonitrile (19f). To a solution of N-

2-yl)-2-isobutoxybenzonitrile (21d). Yellow macrocrystals, yield: 95%,

1

9

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

m.p.: 233–235 ⁰C. IR (KBr, cm ): 3311 (NH), 2230 (CN). ¹H NMR (δ

ppm, DMSO‑d ): 10.63 (s, 1H, NH, D O exchangeable), 8.28 (s, 1H, Ar-

H), 8.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.47 (d, J = 8.0 Hz, 2H, Ar-H), 7.36

d, J = 8.8 Hz, 1H, Ar-H), 7.16 (d, J = 8.0 Hz, 2H, Ar-H), 3.98 (d, J = 6.4

Hz, 2H, OCH ), 2.71 (s, 3H, CH ), 2.26 (s, 3H, 4-CH

-Ph), 2.11–2.04 (m,

). C NMR (δ ppm, DMSO‑d ):

ꢀ 1

the concentration causing 50% enzyme inhibition (IC50) was performed

by using ﬁve different concentration of each compound according to

their molecular weight and measuring percentage of enzyme inhibition

either for COX-1 or COX-2 enzymes at each concentration then it was

calculated from the concentration-inhibition response curve (three

determinations).

6

2

(

2

3

1

3

1

2

H, CH), 1.01 (d, J = 6.8 Hz, 6H, (CH

3

)

2

6

64.31, 161.94, 159.55, 154.12, 152.56, 135.88, 132.96, 131.43,

30.95, 129.49, 125.13, 117.16, 115.40, 114.71, 113.97, 101.55, 75.12,

4.2.3. In vivo hypouricemic effect

7.58, 20.35, 18.72, 17.00. Anal. Calcd (%) for C24

H

23

N

5

O

2

S (445.54):

Male Swiss overnight-fasted mice (20–25 g) were purchased from

EgyVac (Giza, Egypt) and housed under standard conditions of humidity

C, 64.70; H, 5.20; N, 15.72; S, 7.20. Found: C, 64.61; H, 5.54; N, 15.90;

S, 7.62.

0

(50 ± 10%) and temperature (24–26 C) in a 12 h light/dark cycle with

free access to food and water for 1 week before the beginning of ex-

periments for adaptation. Also, they were handled according to the

National Institutes of Health Guidelines for the Care and Use of Labo-

ratory Animals. All the experimental procedures were performed ac-

cording to the ethical principles of the Institutional Animal Care and Use

Committee (IACUC) of faculty of science, Helwan University, Egypt

4

.2. Biological evaluation

.2.1. In vitro Xanthine Oxidase (XO) inhibitory activity

The in vitro bovine xanthine oxidase inhibitory effect was measured

spectrophotometrically at 295 nm under aerobic condition, following

the reported method [48,49] with some modiﬁcations. The reaction

(

approval no. HU2021/Z/AEA121-02). Mice were divided into nine

groups (ﬁve mice per group) and all except the blank group were

intraperitoneally administered with potassium oxonate (300 mg/kg) to

induce acute hyperuricemia according to a modiﬁed previously reported

procedure [20,52]. One hour after potassium oxonate administration,

compounds 5a, 14b, 17, 19c, 19e, 21b (5 mg/kg) and the positive

control febuxostat (5 mg/kg) were intragastrically administered to the

mice and they were dissolved in 0.5% CMC-Na, whereas the blank and

model groups were treated with 0.5% CMC-Na only. Blood samples were

gathered from the mice via orbital vein bleeding at 1, 2, 4, 6 and 8 h after

the administration of the drugs. After coagulation of the blood samples

at room temperature for 2 h, they were centrifugated at 3500 g for 10

min at 4 ⁰C. Serum uric acid levels were determined using a uric acid

assay kit (Biodiagnostic, Giza, Egypt) according to the manufacturer’s

instructions. In addition, the cumulative XOI activity during the entire

period of the experiment was measured by calculating AUC.

mixture consisted of 300

00 L of the tested compounds which were initially dissolved in DMSO

to yield a 10 µM solution and was then further diluted with phosphate

buffer to obtain the required concentrations, 100 L of freshly prepared

XO enzyme solution (0.2 units /mL of XO in phosphate buffer, Sigma,

X4875) and 100 L of distilled water.

μ

L of 50 mM sodium phosphate buffer (pH 7.5),

1

μ

◦

The assay mixture was pre-incubated at 37 C for 15 min. Then, 200

μ

L of substrate solution (0.15 mM of xanthine, Sigma, X7375) was added

◦

to the mixture which was incubated at 37 C for 30 min. Next, the re-

action was stopped by the addition of 200 L of 0.5 M HCl. The absor-

μ

bance was measured using UV/VIS spectrophotometer (8500P Double-

Beam Spectrophotometer) against a blank prepared in the same way

but the enzyme solution was replaced with the phosphate buffer.

Another reaction mixture was prepared (-ve control) containing 100 L

μ

of DMSO instead of the test sample in order to have maximum uric acid

formation and the ﬁnal concentration of DMSO in the reaction mixture

was less than (0.01% v/v) and this ratio does not interfere with the

activity. Febuxostat was used as positive control and all the experiments

were performed in triplicates and results were expressed as means of

three experiments. The XO inhibitory activity of the compounds was

calculated in terms of IC50 value by measuring the reduction in uric acid

production and comparing it to the product formed in absence of in-

hibitor. IC50 values were calculated based on a non-linear regression

analysis.

4

.2.4. In vivo anti-inﬂammatory screening

The anti-inﬂammatory activity of compounds 5a, 14b, 17, 19c, 19e,

2

1b was screened by carrageenan-induced mouse paw edema model

[

53,54]. Preparation and adaptation of the animal model used in this

bioassay was carried out as mentioned under the in vivo hypouricemic

test. Male Swiss mice (20–25 g) were divided into nine groups of ﬁve

mice each; the ﬁrst group served as a negative control that was

administered with the vehicle saline while the second group received

celecoxib (5 mg/kg) as a positive control. The tested compounds and

febuxostat were administered orally in a dose of 5 mg/kg in the

remaining groups. One hour after administration of these compounds,

edema in the right-hand paw was induced by subcutaneous injecting 0.1

mL of 1% w/v carrageenan (Sigma-Aldrich, USA) suspended in saline at

the sub-planter surface of the right-hand paw of each mouse in all the

groups. A Vernier caliper (LETICA Scientiﬁc Instruments, Barcelona,

Spain) was used for measuring the paw edema thickness (mm) before (0

h) and after the carrageenan injection at 2, 4 and 8 h and these thick-

nesses were compared to that of the negative control group to assess the

anti-inﬂammatory activity. The percentage of edema inhibition (anti-

inﬂammatory activity; AI%) of each group were calculated according to

the following equation:

The equation reported by D. Kumar et al. [48] was used to estimate

the degree of XO inhibitory activity, in which Ac is the absorbance at

2

95 nm without the test compound and As is the absorbance at 295 nm

with the test compound.

%XO inhibition = [(Ac ꢀ As)/Ac]x100

4

.2.2. In vitro COX-1 and COX-2 inhibitory activities

Cayman colorimetric COX (ovine) inhibitor screening assay kit

(

Catalog No. 560131) supplied by Cayman chemicals, Ann Arbor, MI,

USA, was used to detect the in vitro inhibitory activity of COX-1/COX-2

for the standard drugs and tested compounds. All test solutions and re-

agents as well as experimental procedures were carried out in agreement

with both the manufacturer’s instructions in the assay kits and in

accordance to a previously reported method [50,51]. Brieﬂy, to a series

of supplied reaction buffer solutions (960 µL, 0.1 M Tris-HCl, pH 8.0

containing 5 mM EDTA and 2 mM phenol) with either COX-1 or COX-2

(C

t

ꢀ C

0

) control ꢀ (C

ꢀ C

t

ꢀ C

0

) tested com pound

(AI%) =

*100

(

C

t

0

) control

where C

t

= the average right paw edema thickness at speciﬁc time in-

(

10 µL) enzyme in the presence of heme (10 µL) were added 1µ mL of

terval after carrageenan treatment and C

0

= the average right paw

various concentrations of test drug solutions. Tested compounds were

edema thickness at zero-time interval before carrageenan treatment.

◦

incubated with the enzyme for 5 min at 25 C after which 10 µL of

Arachidonic Acid (100 µM) solution were added and the COX reaction

was stopped by the addition of 50 µL of 1 M HCl. Moreover, 100 µL of

saturated stannous chloride solution was added then plate reader was

used to measure the absorbance spectrophotometrically. Determining

4.3. Molecular docking studies

Molecular docking studies were performed with Molecular

2

0

A.Y. Rashad et al.

Bioorganic Chemistry 113 (2021) 104948

Operating Environment (MOE 2016.0802) software, Chemical

Computing Group, Montreal, Canada. The crystal structure of bovine

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Declaration of Competing Interest

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inhibitors, Eur. J. Med. Chem. 186 (2020), 111883 .

The authors declare that they have no known competing ﬁnancial

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the work reported in this paper.

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Acknowledgements

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Sincere gratitude is conveyed to Global NAPI Pharmaceuticals

company for the generous support with febuxostat during this work.

Extended thanks are due to Dr. Waleed A.M. Ali, Biochemistry Depart-

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Febuxostat-based amides and some derived heterocycles targeting xanthine oxidase and COX inhibition. Synthesis, in vitro and in vivo biological evaluation, molecular modeling and in silico ADMET studies

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