Xanthine oxidase inhibitory activity of nicotino/isonicotinohydrazides: A systematic approach from in vitro, in silico to in vivo studies

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Xanthine oxidase inhibitory activity of nicotino/isonicotinohydrazides: A

systematic approach from in vitro, in silico to in vivo studies

b

c

a

Humaira Zafar ^a,, Muhammad Hayat , Sumayya Saied , Momin Khan , Uzma Salar , Rizwana Malik ,

⇑

M. Iqbal Choudhary ^a,d, Khalid Mohammed Khan ^a,

⇑

^aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

^bDepartment of Chemistry, University of Karachi, Karachi 75270, Pakistan

^cDepartment of Chemistry, Abdul Wali Khan University, Mardan 23200, Pakistan

^dDepartment of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah 21412, Saudi Arabia

a r t i c l e i n f o

a b s t r a c t

Article history:

Change in life style and eating habits has led to an increased prevalence of hyperuricemia worldwide. The

role of hyperuricemia is no more restricted to gout, but it has a central role in progression of CVD, hyper-

tension, metabolic syndrome, and arthritis. Among the different factors involved in regulation of serum

uric acid, xanthine oxidase (XO) is the best pharmacological target to control the levels of serum uric acid

as it catalyzes the ﬁnal steps in uric acid production. In the current study, a systemic search for the inhi-

bitors of xanthine oxidase, starting from synthesis to in vitro screening and leading to in vivo studies is

presented. Benzylidene nicotino/isonicotinohydrazides (1–54) were synthesized by treating nicotinic/

isonicotinic hydrazides with substituted aromatic aldehyde, and characterized by EI-MS and ¹H NMR.

Elemental analysis was also performed. All synthetic compounds were screened for xanthine oxidase

inhibitory activity initially using an in vitro spectroscopic XO inhibition assay. Among them twenty-

Received 22 December 2016

Revised 18 February 2017

Accepted 22 February 2017

Available online xxxx

Keywords:

Benzylidene

Hydrazides

Hyperuricemia

Gout

two derivatives were found to be active with IC₅₀values between 0.96 and 330.4

standard drug allopurinol IC₅₀= 2.00 0.01 M. Kinetic studies of ﬁve most active compounds (8, 35,

36, 39, and 45) with low IC₅₀values between 0.96 and 54.8 M showed a competitive mode of inhibition.

lM, as compared to

Xanthine oxidase inhibitors

In vitro

In silico, in vivo studies

l

Further in silico molecular docking was carried out to study the interactions of these inhibitors with cat-

alytically important amino acid residues in XO. Three compounds 8, 35, and 36 with IC₅₀values of 10,

12.4, and 0.96 lM, respectively, were also found to be non-cytotoxic, and thus selected for in vivo studies.

A simple and physiologically relevant animal model was used to analyze the in vivo XO inhibitory activity

of these compounds. Among these, two compounds 35, and 36 showed a signiﬁcant inhibition in male

Wistar rats, and identiﬁed as potential lead molecules for anti-hyperuricemic drug development.

1. Introduction

many diseases, including gout, hypertension, and renal dysfunc-

tion. The role of increased serum uric acid is still debatable in car-

Uric acid, a heterocyclic organic compound, is the end product

of purine metabolism in humans. The weak acidic pKa of uric acid

(5.8) limits its solubility at physiological pH, and it exist in salt

form i.e. urate ion. Therefore an elevated level of serum uric acid

increases the risk for monosodium urate crystal formation in con-

nective tissues, kidneys, and joints. This increase in serum uric acid

from the normal concentration (6.8 mg/dL) is referred as hyper-

uricemia.¹Hyperuricemia is involved in the pathophysiology of

diovascular diseases (CVD).²However, various researchers have

established a link between increased xanthine oxidase (XO) activ-

ity, and increased reactive oxygen species (ROS) production in the

progression of CVD due to serum uric acid.^2,3

The regulation of uric acid production and excretion are com-

plex processes involving various factors. Among these factors, XO

is responsible for the formation of uric acid in purine catabolism,

and is therefore involved in the regulation of uric acid levels in

serum. Consequently XO catalyzed reactions are the most relevant

pathway responsible for uric acid overproduction. Hence XO inhi-

bitors are excellent pharmacological agents for the treatment of

⇑

Corresponding authors.

E-mail addresses: hamramalik@gmail.com (H. Zafar), khalid.khan@iccs.edu,

drkhalidhej@gmail.com (K.M. Khan).

http://dx.doi.org/10.1016/j.bmc.2017.02.044

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2

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

O

CN

O

Me

O

N

R₁

N

R₂

O

N

H

N

H

NH

H

O

N

H

S

N

CN

N

H

N

1-27

HO

N

28-54

R₁= R₂= Aryl rings

N

H

Me

Allopurinol

Febuxostat

Topiroxostat

IC₅₀= 0.96- 330.4

M

µ

Standard = Allopurinol = IC₅₀= 2.00 0.01

M

µ

Fig. 1. Clinical drugs for XO inhibition.

Fig. 3. General representation of the synthetic library and their bioactivity.

O

N

OH

NH

Ar

O

4

NH₂

N

R₁

N

Glacial AcOH

EtOH, Reflux, 2 h

5

6

N

H

N

H

+

3

2

R₁

H

2-Arylquinazolin-4(3H )-ones 3-Pyridine carboxylic acid

H

N

IC₅₀= 518.23 2.23 µM

IC₅₀= 2.80- 108.50

M

µ

1

1-27

Fig. 2. General representation of our previous study.

Scheme 1. Synthesis of benzylidene nicotinohydrazides 1–27.

hyperuricemia, rather than uricosuric agents that increases excre-

tion of uric acid with severe adverse effects.^4,5

O

Allopurinol, febuxostat, and recently developed topiroxostat are

the clinical drugs for XO inhibition. However, these drugs have sev-

eral adverse effects, such as hypersensitivity drug reactions.

Adverse effects related to allopurinol includes interstitial nephritis,

peripheral neuritis, renal toxicity, and congenital malformations

when used during pregnancy. So, there is still a need to develop

new drugs with better pharmacological proﬁle and lesser side

effects.^6,7

5

NH₂

N

R₂

6

Glacial AcOH

EtOH, Reflux, 2 h

N

H

N

H

4

3

+

R₂

H

N

₁N

H

2

28-54

Scheme 2. Synthesis of benzylidene isonicotinohydrazides 28–54.

In current study, we used a simple and reliable in vivo assay that

measures the XO inhibition in animals through drug-drug interac-

tion.¹⁴6-Mercaptopurine (6-MP) is a pro-drug used in the treat-

ment of leukemia, and it is metabolized by three pathways.¹⁵

One of these pathways is catalyzed by XO that convert 6-MP into

6-thiouric acid which is also inactive product. The basic mecha-

nism of action of allopurinol is that it lowers the formation of 6-

thiouric acid by inhibiting the enzyme XO, and thus increase the

concentration of 6-MP.^16,17

Several classes of synthetic and natural compounds have been

reported as XO inhibitors. The synthetic inhibitors of XO are

broadly classiﬁed into purine and non-purine based inhibitors.

Non-purine based inhibitors have advantage over purine based

inhibitors as the former is not being metabolized by the enzymes

of purine metabolism. Although there are several studies reported

on XO inhibitors using in vitro assays, still very few of them

reached to the clinical trials. One reason behind this limited suc-

cess could be a poor correlation between in vitro and in vivo stud-

ies^8–10(See Fig. 1).

2. Results and discussion

Our group previously reported 3-pyridine carboxylic acid as an

inhibitor of XO.¹¹The compound was isolated from Pyrenacantha

staudtii Engl. and its IC₅₀value was found to be

2.1. Chemistry

518.23 2.23 lM. We have also reported nitrogen containing hete-

rocycles as potential XO inhibitors,^12,13such as 2-arylquinazolin-4

(3H)-ones (Fig. 2).¹²

Benzylidene nicotino/isonicotinohydrazides (1–54) were syn-

thesized from nicotinic and isonicotinic hydrazide by condensing

with different aromatic aldehydes in ethanol under reﬂux

(Schemes 1 and 2). Glacial acetic acid was used as a catalyst. The

structures of compounds 1–54 were elucidated by different spec-

troscopic techniques, such as ¹H NMR and EI-MS. Elemental analy-

sis was also performed and found satisfactory. Except compounds

7 and 16, all other derivatives were previously reported.¹⁸

It is worth noting that these synthetic derivatives, and the

recently developed drug topiroxostat have pyridine ring in the

structures. Therefore, in the light of our previous study and the

structural resemblance of the synthetic benzylidene nicotino/ison-

icotinohydrazides (1–54) (Fig. 3) with the drug topiroxostat, we

were encouraged to evaluate this library of compounds for their

XO inhibitory activity. We, herein, report the synthesis and XO

inhibitory activity in vitro, as well as in vivo.

2.2. In vitro XO inhibitory activity

The results of in vitro spectrophotometric inhibition assay

encouraged us to carry out in vivo studies. Signiﬁcant results were

obtained in in vivo studies. The mode of inhibition was determined

by kinetic studies and to predict the interaction of these com-

pounds with speciﬁc amino acids residues in the active site of

XO, in silico molecular docking studies were performed.

The most common in vivo model for XO inhibitors is based on

the use of potassium oxonate (uricase inhibitor) that increases

the amount of uric acid in experimental animals. The inhibitors

identiﬁed through this model still lack the clear indication that

the decrease in serum uric acid is due to inhibition of XO (i.e.,

decreased production) or test compounds have increased the

excretion of uric acid.¹¹

All the compounds 1–54 were evaluated for their XO inhibitory

activity. Compounds 1, 2, 5, 7, 8, 11, 12, 18–22, 24, 29, 35, 36, 38,

39, 45, 48, and 49 showed their inhibitory potential in the range

of IC₅₀= 0.96–330.4

lM, as compared to standard drug allopurinol

IC₅₀= 2.00 0.01 M (Table 1). The limited structure-activity rela-

l

tionship was rationalized by observing the effect of different sub-

stituted rings ‘R₁/R₂’ on inhibitory activity.

2.3. Structure-activity relationship of benzylidene nicotinohydrazides

The parent compound 1 has a phenyl ring with no substitution,

and showed an IC₅₀value of 210.7 1.2 lM. The better activity of

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

3

Table 1

XO inhibitory activity of benzylidene nicotino (1–27), and isonicotinohydrazides (28–54).

O

4

5

2

N

R₁

N

R₂

6

5

6

N

H

N

H

3

2

4

3

H

₁N

H

N

1

1-27

28-54

Comp.

R₁

IC₅₀SEM^a

210.7 1.2

(

lM)

Comp.

R₂

IC₅₀SEM^a

NA^b

(lM)

1

28

2

183.7 0.8

NA^b

29

173.56 2.1

NA^b

NO₂

3

30

O₂N

4

5

6

NA^b

31

32

33

NA^b

NO₂

183.7 0.4

NA^b

OH

7

161 4.5

34

NA^b

HO

OH

8

9

10 0.2

NA^b

35

36

12.4 0.1

0.9 0.01

OH

HO

OH

HO

C₂H₅O

10

NA^b

37

NA^b

C₂H₅O

11

12

242 6.3

264 4.5

38

39

330.4 2.1

54.8 1.2

OC₂H₅

HO

OC₂H₅

HO

OC₂H₅

13

14

15

16

NA^b

40

41

42

43

NA^b

H₃CO

OC₂H₅

OCH₃

H₃CO

OH

OCH₃

OC₂H₅

OCH₃

HO

17

NA^b

44

NA^b

OCH₃

HO

18

19

175.5 2.7

197.6 1.2

45

46

21.1 1.3

NA^b

HO

Br

OCH₃

HO

Cl

OCH₃

(continued on next page)

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4

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Table 1 (continued)

Comp.

R₁

IC₅₀SEM^a

173.8 1.7

(l

M)

Comp.

R₂

IC₅₀SEM^a

NA^b

(lM)

20

47

F

21

205.1 0.1

48

206 1.0

Cl

22

23

195 0.8

NA^b

49

50

179.3 0.3

NA^b

Cl

24

25

180.3 3.1

51

52

NA^b

H₃C

HO

NO₂

Cl

HO

NA^b

CH₃

SCH₃

26

27

NA^b

53

54

NA^b

SCH₃

CH₃

N

Standard^c= Allopurinol

2.00 0.01

SEM^a(Standard error of the mean); NA^b(Not active); Standard^c= Allopurinol (Inhibitor for Xanthine Oxidase activity).

compound 1, in comparison to 3-pyridine carboxylic acid

(IC₅₀= 518.23 2.23 M), might be due to its increased lipophilic-

ity. This is in consistent with the previous results where Biagi et al.

reported that the addition of lipophilic substituents (phenyl ring in

this case) on the unsubstituted heterocyclic ring system increases

the activity.¹⁹

Among the halobenzene containing derivatives, compound 19

with Br group at ortho position showed an IC₅₀of

197.6 2.1 M, while the presence of F at ortho position in com-

pound 20 increases the activity (IC₅₀= 173.8 1.7 M), as F is more

electronegative than Br and can better interact with the active site

via hydrogen bonding interactions or polar interactions. Among the

chlorobenzene derivatives, the presence of Cl at meta position

l

a

l

Among the nitrobenzene containing derivatives, only meta sub-

stituted compound 2 showed a weak inhibition with an IC₅₀value

(compound 21) showed IC₅₀value of 205.1 0.1

presence of Cl group at para position, as in compound 22, showed

an IC₅₀value of 195 0.8 M. Interestingly, the dichloro substitu-

lM, while the

of 183.7 0.8

lM, as compared to standard allopurinol

(IC₅₀= 2.00 0.01

lM). Whereas, ortho and para substituted

l

derivatives 3 and 4 were found to be inactive. It shows that nitro

group plays an important role in the activity, when it is present

at meta position. Might be other positional isomers 3 and 4

attained the conformation which is not ﬁtted well into the active

site of the enzyme.

tion at ortho and para positions, as in compound 23, made com-

pound inactive.

The presence of CH₃group at ortho position in compound 24

showed a weak activity with an IC₅₀value of 180.3 3.1 lM. The

presence of OH at ortho and CH₃at meta position in compound

25 was found to be inactive. Compounds 26, and 27 with dimethy-

lamine and thiomethyl at para positions, respectively, were also

found to be inactive.

Among the phenol containing compounds, hydroxyl group was

only favorable at para position, as in compound

value = 183.7 0.4 M). The substitution of two OH groups meta

to each other, increased the activity of compound

(IC₅₀= 161.0 4.5 M). Interestingly, compound 8 with two hydro-

xyl groups ortho to each other showed several folds increased

activity with IC₅₀of 10.0 0.2 M which is comparable to the stan-

dard inhibitor, allopurinol (IC₅₀= 2.0 0.01 M). Tri-hydroxy sub-

5 (IC₅₀

l

7

l

2.4. Structure-activity relationship of benzylidene

isonicotinohydrazides

l

Benzylidene isonicotinohydrazides were also evaluated for XO

inhibitory activity. The parent compound 28 with no substitution

on phenyl ring was found to be inactive. Might be the compound

attained a conformation which doesn’t ﬁt well in the active site.

Among the nitrobenzene containing compounds, compound 29

with a nitro group at meta position, showed a weak inhibition of

XO. Activity results of compounds 2 and 29 indicated that NO₂

group is only favorable for XO inhibitory activity when it is at meta

position.

stituted compound 9 was found to be inactive, indicating the

importance of number as well as position of OH groups on benzene

ring.

Amongst the ethylphenyl ether derivatives, compound 11

(IC₅₀= 242 6.3 lM) with OC₂H₅group at para position showed a

weak inhibitory activity. While compound 12 with OH and

OC₂H₅groups showed further decrease in the activity

(IC₅₀= 264.03 4.5 lM).

Compounds 13, 14, and 15 with mono, di, and tri-OCH₃groups

were found to be inactive. Among the OCH₃cum OH group deriva-

tives, only compound 18 with OH and OCH₃groups ortho to each

All the phenol containing derivatives (32, 33 and 52) were

found to be inactive. Compound 34 with OH groups at ortho and

para positions was also inactive. Interestingly, the presence of

two OH groups at ortho and one at para in compound 36 resulted

other, was weakly active (IC₅₀= 175.5 2.7 lM).

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

5

in a potent XO inhibitory activity (IC₅₀= 0.96 0.01

l

M). Its activ-

Among the ethylphenyl ether derivatives, 37 with ethoxy at

ortho position was found to be inactive, as was observed in case

of compound 10. Inactivity of the compounds 10 and 37 might

be due to the steric hindrance created by the ethoxy group in bind-

ing with the active site of enzyme. Compound 38 with OC₂H₅at

ity was compared with its positional isomer compound 9 with

three OH at ortho, meta, and para positions which was found to

be completely inactive. Might be compound 9 attained a conforma-

tion which is not ﬁtted properly into the active site of enzyme.

Compound 35 with two OH groups at meta and para positions also

para position was weakly active (IC₅₀= 330.4 2.1 lM). Activity

showed activity (IC₅₀= 12.4 0.1

compound 8 and almost similar in structure but only has a nicotine

ring, instead of isonicotine (Table 2).

l

M) which is comparable to

results of compounds 11, and 38 showed that ethoxy group is only

favorable when it is present at the para position. Compound 39

with OH and OC₂H₅(ortho and meta) was a moderate inhibitor

The XO inhibitory activity of compounds 35, and 36 indicated

that positions as well as number of OH substituent play an impor-

tant role in the activity of these compounds. This was in accor-

dance with earlier reports by Leigh et al., and by Pauf and Hille

that the OH group is involved in XO inhibitory activity of different

classes, such as hydrazide, thiosemicarbazides, curcumins, etc.^20,21

with an IC₅₀value of 54.8 1.2 lM, and showed almost ﬁve times

better inhibition as compared to structurally similar compound 12

which has a nicotine ring instead of isonicotine.

Compounds 41, 43, and 44 with OCH₃and OH cum OCH₃group

substitutions were found to be inactive. Only compound 45 with

OH and OCH₃at ortho (1⁰) and meta (2⁰) positions, respectively,

Table 2

Results of kinetics and molecular docking studies on selected inhibitors.

Compounds

IC₅₀SEM (

lM)

Ki SEM (

lM)

Type of Inhibition

Docking Score

DG_bind(kcal/mol)

8

10 0.26

0.96 0.01

12.4 0.1

54.8 1.2

21.1 1.3

7.27 0.021

Competitive

ꢀ7.4

ꢀ6.8

ꢀ5.9

ꢀ5.7

ꢀ5.3

ꢀ42

ꢀ40

ꢀ32

ꢀ16

ꢀ22

35

36

39

45

0.82 0.0789

15.46 0.0052

51.34 0.032

15.02 0.008

1.5

1

15

10

5

0.5

0

-5

-0.5

-1

-10

-15

-1.5

-20 -10

0

10

20

-30 -20 -10

0

10

20

30

1/Xanthine

Inhibitor (Compound 56)

Compound 8

(A)

(B)

20

10

0

-10

-20

-40

-20

0

20

40

Compound 8

Inhibitor (Compound 56)

(C)

Fig. 4. The inhibition of XO by compound 8, (A) Lineweaver-Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (xanthine) in the absence (-), and in

presence of 20 M (j), 10 M (h), 5 M (Δ), 2.5 M (▲), and 1.25 M (s) of compound 8. (B) Secondary replot of Lineweaver-Burk plot between the slopes of each line on

l

Lineweaver-Burk plot vs different concentrations of compound 8. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 8.

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

2

1.5

1

30

15

0

0.5

0

-0.5

-1

-15

-30

-1.5

-2

-30 -20 -10

0

10 20 30

-6 -4 -2

0

2

4

6

1/Xanthine

Inhibitor (Compound 82)

Compound 36

(A)

(B)

30

20

10

0

-10

-20

-30

-3

-2

-1

0

1

2

3

Inhibitor (Compound 82)

Compound 36

(C)

Fig. 5. The inhibition of XO by compound 36, (A) Lineweaver-Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (xanthine) in the absence (-), and

in presence of 16 M (s), 8 M (d), 4 M (h), 2 M (j), and 1 M (Δ) of compound 36. (B) Secondary replot of Lineweaver-Burk plot between the slopes of each line on

Lineweaver-Burk plot vs different concentrations of compound 36. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 36.

l

showed a signiﬁcant activity (IC₅₀= 21.1 1.3

l

M). Activity results

2.6. In silico studies

of compounds 18, and 45 indicated that the OH at ortho (1⁰) and

OCH₃at meta (2⁰) are contributing towards the activity of this ser-

ies of compounds.

To predict the interaction of these compounds with speciﬁc

amino acids residues in the active site of XO, in silico molecular

docking studies were performed. The in silico studies were per-

formed using bovine milk XDH with bound competitive inhibitor

oxypurinol (PDB ID: 3BDJ).

Oxypurinol is the hydroxylated product of allopurinol that is

formed by XO. This oxypurinol then interacts with the Mo atom

and catalytically important amino acids thereby inhibit the activity

of XO. The important amino acid residues that are involved in the

XO catalyzed reactions are Arg880, Phe914 and 1009, Glu1261,

Glu802, and Thr1010. Oxypurinol forms hydrogen bonds with

Halogen substitutions in benzylidene isonicotinohydrazides

were seemingly unable to contribute signiﬁcantly in inhibiting

XO as compounds 46, 47, and 50 with Cl, and F, were found to be

inactive. The presence of Cl group at meta position in compound

48 showed a weak activity with an IC₅₀value of 206 1.0

The presence of Cl group at para position in compound 49 lead to

a slightly better activity (IC₅₀= 179.3 0.3 M), in comparison to

lM.

l

compound 48. The same activity pattern was observed in case of

compounds 21, and 22.

Similarly, compounds 53, and 54 with dimethylamine and thio-

methyl at para positions, respectively, were found to be inactive, as

was observed in case of benzylidene nicotinohydrazides.

Arg880, Thr1010, and

p-p interactions with the two Phe residues

914 and 1009 (Fig. 7).^22,23

The phenyl ring in these inhibitors occupied the same position

as that of pyrimidine ring in oxypurinol structure (Fig. 8). There

were some common interactions observed in all the ﬁve inhibitors.

2.5. Kinetic studies

These involved p-p stacking of the benzene ring with two Phe resi-

dues (914 and 1009). While the change in substitutions on the ben-

zene ring, resulted in change in the hydrogen bonding pattern.

For instance, compounds 8 and 35 have similar structures with

only difference in the position of nitrogen in the pyridine ring.

Compound 8 has 3-pyridine, while 35 has a 4-pyridine ring which

resulted in some differences in interactions. Both OH groups (meta

and para) in compound 8 were involved in hydrogen bonding with

Five compounds 8, 35, 36, 39, and 45 were selected for kinetic

studies, based on their lower IC₅₀values (0.96 to 54.8 lM). The

mode of inhibition was analyzed from Lineweaver-Burk plot, and

was found to be competitive as Km values were increased in the

presence of each inhibitor without affecting the Vmax values

(Figs. 4–6). Ki values were calculated from secondary replots of

Lineweaver-Burk plot, and also from Dixon plot (Table 2).

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

7

1

15

10

5

0.5

0

-5

-0.5

-1

-10

-15

-20

-10

0

10

20

-50 -40 -30 -20 -10 0 10 20 30 40 50

1/Xanthine

Compound 45

(A)

(B)

20

10

0

-10

-20

-60 -40 -20

0

20

40

60

Inhibitor (Compound 86)

Compound 45

(C)

Fig. 6. The inhibition of XO by compound 45, (A) Lineweaver-Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (xanthine) in the absence (-), and

in presence of 96 M (h), 48 M (j), 24 M (Δ), 12 M (▲), and 6 M (s) of compound 45. (B) Secondary replot of Lineweaver-Burk plot between the slopes of each line on

Lineweaver-Burk plot vs different concentrations of compound 45. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 45.

l

the Arg880 (Fig. 9). However, the OH group at meta position in

compound 35 was able to interact with Thr1010 through hydrogen

production of formazan indicated the viability of cells and hence

the compound was referred as non-cytotoxic. All the ﬁve com-

pounds 8, 35, 36, 39, and 45 were found to be non-cytotoxic

against 3T3- cell lines. Three signiﬁcant inhibitors 8, 35, and 36

were selected among these ﬁve for in vivo studies.

6-MP, allopurinol, and test compounds 8, 35, and 36 were given

to test animals at a dose of 50 mg/kg through I.P. route. Rats were

sacriﬁced after 6 h of administration of drug and blood was col-

lected through heart puncture. Plasma samples were prepared

(materials and methods) for the analysis of 6-MP. In order to quan-

tify the plasma samples, calibration curve was plotted between

various amounts of 6-MP and related peak area. The standard

curve demonstrated a linear relationship between the peak areas

and concentrations for the pooled rat plasma, spiked with 6-MP

standards (Fig. 14).

bond. An additional

p-p stacking interaction was also observed

through the pyridine ring with Phe1013 (Fig. 10).

Compound 36 with three OH groups (ortho and para) showed

some additional interactions. Beside the hydrogen bond with

Thr1010, the OH group at para position also interacted with

Glu1261 through hydrogen bond. The ortho OH group was involved

in hydrogen bonding with the water molecule (Fig. 11).

Compound 45 with OH cum OCH₃groups at ortho and meta

positions, respectively, showed hydrogen bond between OCH₃

group and Ala1079, and also among NH and Ser876 (Fig. 12). How-

ever, the replacement of OCH₃group by OC₂H₅in compound 39

resulted in a loss of these hydrogen bonds (Fig. 13).

The blank group that was untreated showed no peak in the total

run time of 15 min that could otherwise overlap with the peak of

6-MP. The negative control group in which only 6-MP was injected

showed a non-quantiﬁable peak of 6-MP indicating that XO is

active, and has metabolized the drug (Fig. 15A-B).

The positive control group that was injected with 6-MP as well

as allopurinol showed a quantiﬁable peak of 6-MP at the same

retention time, indicating the inhibition of XO by allopurinol. The

2.7. In vivo studies of selected inhibitors

The signiﬁcant XO inhibitory activities of these compounds in

competitive manner motivated us to subject them to in vivo stud-

ies. The compounds were analyzed for their cytotoxic activity using

colorimetric MTT assay. This assay measures the MTT dye reduc-

tion (yellow color) to formazan (Purple color). The increase in the

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8

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Fig. 7. Ligplot and 3D view of docked pose of oxypurinol (ball and stick) interacting with active site amino acid residues via p-p stacking interaction stacking interactions

(blue dotted lines) with Phe914 and 1009, and form hydrogen bonds (yellow dotted lines) with Arg880, Thr1010, and also shows metal interaction with molybdenum

cofactor.

test group I that was injected with 6-MP as well as compound 8,

showed a non-quantiﬁable peak of 6-MP indicating that this com-

pound was not able to inhibit XO in in vivo conditions (Fig. 15C-D).

Test groups II and III that were injected with compounds 35,

and 36, respectively, in addition to 6-MP, showed a quantiﬁable

peak indicating the inhibition of XO. The peak areas of 6-MP in

the chromatogram of compounds 35 and 36 were found to be

47.4 and 73.5 mAU, respectively (Fig. 15E-F). Compounds 35 and

36 were thus able to inhibit the activity of XO up to 28%, and

44% in comparison to the 100% inhibition of the enzyme activity

by allopurinol. Although the percent inhibition was lower than

the standard inhibitor allopurinol, but it is still signiﬁcant (Fig. 16).

(35 and 36) were able to inhibit XO signiﬁcantly. These two com-

pounds can serve as potential leads for XO inhibition, but more

detailed in vivo studies are required.

4. Material and methods

XO (bovine milk, EC 1.17.3.2.), 6-MP, Dulbecco’s modiﬁed eagle

medium (DMEM), and cycloheximide were purchased from Sigma

Aldrich, USA. Xanthine was acquired from TCI, Japan. Allopurinol

and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bro-

mide (MTT) were obtained from MP Biomedicals, LLC, France. All

reagents were of analytical grade, and used without any puriﬁca-

tion. Dimethylsulfoxide (DMSO) was purchased from Fisher Scien-

tiﬁc UK. Potassium dihydrogen phosphate, dipotassium hydrogen

phosphate, and sodium nitroprusside were purchased from Merck,

Germany. Mouse ﬁbroblast cell line (3T3) was purchased from

American Type Culture Collection (ATCC), USA. 0.25%. Fetal bovine

serum (FBS) was purchased from A&E Scientiﬁc (PAA), USA. 0.4%

Trypan Blue solution was purchased from Amersco, USA. Sterile

heparinized capillary tubes were purchased from BD, USA.

3. Conclusion

We screened two series of synthetic benzylidene nicotine/

isonicotinohydrazides (1–54) using in vitro spectroscopic XO inhi-

bition assay. Five compounds with lower IC₅₀values (0.96 to

54.8 lM) were selected from the two series and were subjected

to mechanism based studies by evaluation of their kinetic param-

eters. All compounds showed a competitive pattern of inhibition

and were further explored by in silico molecular docking studies.

These inhibitors showed interactions with the catalytically impor-

tant amino acid residues, such as Phe (914, 1009), Arg880,

Thr1010, and Glu1261. These compounds were also found to be

non-cytotoxic against 3T3-cell line (Mouse ﬁbroblast cells), and

were evaluated for in vivo XO inhibition activity in vivo using

male Wistar rats. Three compounds (8, 35, and 36) were sub-

jected to the in vivo studies and among them two compounds

4.1. General procedure for the synthesis of benzylidene nicotino/

isonicotinohydrazides (1–54)

Nicotinic/isonicotinic hydrazides (1 mmol), substituted aro-

matic aldehyde (1 mmol) and glacial acetic acid (few drops) were

taken in ethanol (10 mL) and reﬂuxed for 3 h. Progress of the reac-

tion was monitored by TLC. After the completion of the reaction,

mixture was left for evaporation of solvent, and then crude product

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

9

Fig. 8. 3D view of docked pose of allopurinol (blue), compound 8 (green), and 35 (red) and interacting with active site amino acid residues through

p

-

p

stacking interactions

(blue dotted lines) and hydrogen bonds (yellow dotted lines).

washed with dichloromethane and hexane, and dried to afford

compounds 1–54.

ligands. The centroid of the co-crystallized ligand (oxypurinol)

was used to deﬁne a grid box (15 ꢁ 15 ꢁ 15 Å). Ligands were

docked using Glide extra precision (XP) module, and the best

docked poses were used for the interpretation of ﬁnal results.^26–28

4.2. Assay for XO inhibitory activity

The XO inhibitory activity was evaluated in 96-well plate ELISA

reader.^24,25Phosphate buffer (pH 7.4, 50 mM) was used to dissolve

XO (0.003 units/well). Test samples were dissolved in DMSO (90%).

4.5. In vitro cytotoxicity measurement assay

Mouse ﬁbroblast (3T3) cell line was grown in DMEM, provided

10

added in 96-well plate and incubated for 10 min at 30 °C in the

spectrophotometer. After incubation, 20 of xanthine

(0.15 mM) was added, and change in absorbance was monitored

for 15 min at 295 nm. Allopurinol drug was used as a positive con-

trol, and all experiments were performed in triplicate.

lL of test compounds, 20 lL of XO and 150 lL of buffer were

with 5% of FBS, 100 IU/mL of penicillin and 100 lg/mL of

streptomycin, and kept at 37 °C in 5% CO₂incubator. Cell culture

(5 ꢁ 10⁴cells/mL) was prepared, and poured into 96-well plates

and incubated overnight. After completion of incubation, fresh

medium was added (after removing the old medium) with test

compound of different concentrations (ranging from 0.1 M to

l

L

0.00016 M). After 48 h, 200

plate and further incubated for 4 h. Afterward, DMSO (100

l

L of MTT (0.5 mg/mL) was added to

L)

l

4.3. Protocol for kinetic studies

was added to each well. Reduction of MTT to formazan was

measured spectrophotometrically at 540 nm, using a micro plate

reader (Spectra Max plus, Molecular Devices, CA, USA). The exper-

iments were run in triplicate (n = 3). Cycloheximide was used as

Kinetic studies were performed in the same way as the inhibi-

tion studies were performed, by varying the inhibitor and substrate

concentrations. Four concentrations of xanthine were used

(0.0375, 0.075, 0.15, and 0.300 mM).

positive control (IC₅₀= 0.2 0.10 l

M).^29,30

4.4. Protocol for docking studies

4.6. In vivo measurement of XO inhibitory activity

Computational studies were performed using Glide 6.9 module

of Schrodinger suite of programs. Bovine milk xanthine dehydroge-

nase (PDB ID: 3BDJ) with bound oxypurinol was used to dock the

ligands. Protein was prepared, optimized and minimized using pro-

tein preparation wizard and the selected force ﬁeld was OPLS_2005.

Maestro Build and LIGPREP module were used to prepare the

6-Mercaptopurine (6-MP) is the structural analogue of hypox-

anthine and is being metabolized by XO. In this assay, 6-MP was

used as a substrate in order to analyze the activity of XO. 6-MP,

allopurinol, and our test samples were all administered to the

experimental animals by intra-peritoneal injections. 6-MP

(50 mg/kg) was injected in male Wistar rats via intra-peritoneal

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Fig. 9. Ligplot and 3D view of docked pose of compound 8 (ball and stick) interacting with active site amino acid residues via p-p stacking interaction with Phe914 and 1009,

and form hydrogen bond (yellow dotted lines) with Arg880.

route. Animals were sacriﬁced 6 h after the administration of the

allopurinol. Blood was collected by heart puncture, and plasma

samples were prepared. The amount of uncatalyzed 6-MP was

analyzed in the plasma samples by ultra-pressure liquid chro-

matography (UPLC) technique.

4.7. Animals welfare

Male Wistar rats (180–200 g) were acquired from the animal

house of Dr. Panjwani Center for Molecular Medicine and Drug

Research, International Center for Chemical and Biological

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

11

Sciences, University of Karachi, Pakistan. Before performing the

4.8. Grouping of animals

experiment, the ethical clearance (00010-2014) was obtained from

the Animal Use and Care Committee of International Center of

Chemical and Biological Sciences, University of Karachi. Animals

were then housed in standard conditions: 12 h light-dark cycle,

provision with food and water ad libitum.

Animals were dividing into six groups with 6 rats in each group.

Group I (Blank group): was untreated

Group II (Negative control group): Injected with 6-MP at a dose

of 50 mg/kg

Fig. 10. Ligplot and 3D view of docked pose of compound 35 (ball and stick) interacting with active site amino acid residues via p-p stacking interaction with Phe914, 1009,

1013, and form hydrogen bond (yellow dotted lines) with Thr1010.

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Fig. 11. Ligplot and 3D view of docked pose of compound 36 (ball and stick) interacting with active site amino acid residues via p-p stacking interactions (blue dotted lines)

with Phe914 and 1009, and forms hydrogen bonds (yellow dotted lines) with Thr1010, and Glu802.

Group III (Positive control group): Injected with 6-MP (50 mg/

kg), and allopurinol (50 mg/kg).

Group IV (Test group I): Injected with 6-MP (50 mg/kg), and

compound 8 (50 mg/kg).

Group V (Test group II): Injected with 6-MP (50 mg/kg), and

compound 35 (50 mg/kg).

vortexed for few sec, and then centrifuged at 14,000 rpm for

25 min, in order to make the solution clear. Clear supernatant was

transferred into the glass tubes for injection into UPLC chromato-

graphic system. In order to quantify the plasma samples, calibration

curve was plotted using various concentrations of 6-MP in the range

of 10 mg/kg to 50 mg/kg with 50 mg/kg of allopurinol.

Group VI (Test group III): Injected with 6-MP (50 mg/kg), and

compound 36 (50 mg/kg).

After 6 h of 6-MP administration, rats were sacriﬁced and blood

was collected by heart puncture in sterile heparinized capillary

tubes. Plasma was stored at ꢀ20 °C until further analysis (Table 3).

4.10. Determination of 6-MP in rat plasma by chromatographic

techniques

4.9. Preparation of plasma samples

The quantiﬁcation of 6-MP was carried out by slight modiﬁca-

tions in the previously reported method of Mohammad et al., with

slight modiﬁcations.¹⁶The method was then performed on UPLC

for better efﬁciency and quantiﬁcation.

Plasma samples were prepared by adding of dithiothreitol (DTT) -

methanol solvent (10 mg/dL) in 1:1 ratio. The samples were then

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

13

Fig. 12. Ligplot (left) and3D view (right) of docked pose of compound 45 (ball and stick) interacting (blue dotted lines) through

p-

p

stacking interaction with active site

amino acid residues Phe914 and 1009, and form hydrogen bonds (yellow dotted lines) with Glu802, Ala1079, and Ser876.

4.11. UPLC system and experimental conditions

4.12. Statistical analysis

UPLC (1260 series, Agilent technologies, USA) was used to cal-

culate the concentration of 6-MP in each plasma sample, ZORBAX

The results for in vitro XO inhibition studies were recorded using

microtitre plate reader (SpectraMax plus 384, Molecular Devices,

CA, USA). SoftMax Pro 4.8 software (Molecular Devices, CA, USA)

and Microsoft excel were used to process the results. Percent inhi-

bition was calculated by using the following formula:

Eclipse XDB-C18 (Rapid Resolution HT, 3.0 ꢁ 50 mm, 1.8-

lm, 600

Bar) column was used. Phosphoric acid: water: acetonitrile: DTT

in a ratio of 2.5 mL: 500 mL: 15 mL: 50 mg, was used as mobile

phase, and was ﬁltered and degassed under vacuum, before its

use. Samples were run for a total time of 15 min at a ﬂow rate of

0.08 mL/min, and wavelength of 322 nm.

Percent inhibition ¼ 100 ꢀ ðOD_{test compound}=OD_controlÞ ꢁ 100

Where OD is the optical density.

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Fig. 13. Ligplot (left) and3D view (right) of docked pose of compound 39 (ball and stick) interacting (blue dotted lines) through

p-p stacking with active site amino acid

residues Phe914 and 1009.

EZ-FIT, Enzyme kinetics software (Perrella Scientiﬁc, Inc., USA),

was used to measure the IC₅₀values and kinetic parameters,

respectively. One-way ANOVA and Statistica^Ò(Version 5.0) soft-

ware packages were used for statistical analysis of in vivo studies.

(dd, 1H, J_6,5= 4.8 Hz, J_6,2= 1.2 Hz, H-6), 8.44 (s, 1H, @CH), 8.25

(br.d, 1H, J_4,5= 8.0 Hz, H-4), 7.75 (m, 2H, H-2⁰, H-6⁰), 7.56 (m, 1H,

H-5), 7.45 (m, 3H, H-3⁰, H-4⁰, H-5⁰); EI-MS: m/z (rel. abund.%),

225 (M⁺, 6), 122 (92), 106 (100), 78 (70); Anal. Calcd for

C₁₃H₁₁N₃O (225.25): C, 69.32; H, 4.92; N, 18.66; Found: C, 69.30;

H, 4.91; N, 18.68.

4.13. Spectroscopic data of compounds 1–54

4.13.1. (E)-N’-Benzylidenenicotinohydrazide (1)^18a

4.13.2. (E)-N’-(3ʹ-Nitrobenzylidene) nicotinohydrazide (2)^18b

Yield: 89%; m.p. 196–198 °C (lit. 195–197 °C); ¹H NMR

(300 MHz, DMSO-d₆): d 12.27 (s, 1H, NH), 9.27 (s, 1H, H-2⁰), 8.76

Yield: 76%; m.p. 146–148 °C (lit. 148–149 °C); ¹H NMR

(400 MHz, DMSO-d₆): d 12.00 (s, 1H, NH), 9.09 (s, 1H, H-2), 8.76

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H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

15

4.13.7. (E)-N’-(3ʹ,5ʹ-Dihydroxybenzylidene) nicotinohydrazide (7)

Yield: 77%; m.p. 245–247 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.10 (s, 1H, NH), 10.23 (s, 1H, OH), 9.06 (s, 1H, OH), 8.96 (s, 1H,

H-2), 8.76 (d, 1H, J_6,5= 4.4 Hz, H-6), 8.57 (s, 1H, @CH), 8.25 (d,

0

1H, J_4,5= 4.4 Hz, H-4), 7.56 (m, 1H, H-5), 7.00 (d, 1H, J_{4 ,6 /4 ,2}

=

2.0 Hz, H-4⁰), 6.74 (m, 2H, H-2⁰, H-6⁰); EI-MS: m/z (rel. abund.%),

258 (M⁺, 18), 122 (78), 106 (100), 78 (61); HREI-MS calcd for

C₁₃H₁₁N₃O₃: m/z = 257.0800, found 257.0803; Anal. Calcd for

C₁₃H₁₁N₃O₃(257.24): C, 60.70; H, 4.31; N, 16.33; Found: C,

60.71; H, 4.33; N, 16.32.

4.13.8. (E)-N’-(3ʹ,4ʹ-Dihydroxybenzylidene) nicotinohydrazide (8)^18d

Yield: 70%; m.p. 198–200 °C; ¹H NMR (400 MHz, DMSO-d₆): d

9.07 (d, 1H, J_2,6= 1.2 Hz, H-2), 8.73 (d, 1H, J_4,5= 3.6 Hz, H-4), 8.23

(s, 1H, @CH), 8.11 (d, 1H, J_6,5= 8.5 Hz, H-6), 7.54 (m, 1H, H-5),

7.24 (s, 1H, H-2⁰), 6.92 (dd, 1H, J_{6 ,5}= 8.0 Hz, J_{6 ,2}= 2.0 Hz, H-6⁰),

0

6.77 (d, 1H, J_{5 ,6}= 8.0 Hz, H-5⁰); EI-MS: m/z (rel. abund.%), 257

(M⁺, 80), 135 (77), 123 (79), 106 (100); Anal. Calcd for

C₁₃H₁₁N₃O₃(257.24): C, 60.70; H, 4.31; N, 16.33; Found: C,

60.71; H, 4.32; N, 16.3O.

0

Fig. 14. Calibration curve between the amount of 6-MP and peak area.

(s, 1H, H-2), 8.55 (m, 2H, @CH, H-6), 8.26 (m, 3H, H-4, H-5, H-6⁰),

7.76 (m, 1H, H-5⁰), 7.56 (m, 1H, H-4⁰); EI-MS: m/z (rel. abund.%),

270 (M⁺, 5), 122 (35), 106 (100), 78 (79); Anal. Calcd for

4.13.9. (E)-N’-(2ʹ,3ʹ,4ʹ-Trihydroxybenzylidene) nicotinohydrazide

(9)^18e

Yield: 80%; m.p. 205–207 °C; ¹H NMR (300 MHz, DMSO-d₆): d

9.06 (s, 1H, OH), 8.74 (s, 1H, H-2), 8.74 (d, 1H, J_6,5= 4.8 Hz, H-6),

8.37 (s, 1H, @CH), 8.35 (d, 1H, J_4,5= 4.8 Hz, H-4), 7.61 (m, 1H, H-

C₁₃H₁₀N₄O₃(270.24): C, 57.78; H, 3.73; N, 20.73; Found: C,

57.76; H, 3.74; N, 20.74.

5), 6.78 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰), 6.44 (d, 1H, J_{5 ,6}= 8.5 Hz, H-

5⁰); EI-MS: m/z (rel. abund.%), 273 (M⁺, 50), 123 (31), 106 (100),

78 (51); Anal. Calcd for C₁₃H₁₁N₃O₄(273.24): C, 57.14; H, 4.06;

N, 15.38; Found: C, 57.12; H, 4.07; N, 15.39.

0

4.13.3. (E)-N’-(2ʹ-Nitrobenzylidene) nicotinohydrazide (3)^18c

Yield: 77%; m.p. 191–192 °C (lit. 190–192 °C); ¹H NMR

(400 MHz, DMSO-d₆): d 12.33 (s, 1H, NH), 9.08 (s, 1H, H-2), 8.86

(s, 1H, @CH), 8.77 (d, 1H, J_6,5= 4.2 Hz, H-6), 8.27 (d, 1H,

J_4,5= 8.0 Hz, H-4), 8.13 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰), 8.09 (d, 1H,

0

4.13.10. (E)-N’-(2ʹ-Ethoxybenzylidene) nicotinohydrazide (10) CAS #

292180-67-7

J_{3 ,4}= 8.5 Hz, H-3⁰), 7.83 (t, 1H, J_{4 ,3 /4 ,5}= 8.0 Hz, H-4⁰), 7.69 (t, 1H,

0

J_{5 ,4 /5 ,6}= 8.0 Hz, H-5⁰), 7.57 (m, 1H, H-5); EI-MS: m/z (rel. abund.

0

Yield: 78%; m.p. 186–188 °C; ¹H NMR (300 MHz, DMSO-d₆): d

12.00 (s, 1H, NH), 9.06 (s, 1H, H-2), 8.79 (s, 1H, @CH), 8.75 (d,

1H, J_6,5= 4.2 Hz, H-6), 8.25 (d, 1H, J_4,5= 8.0 Hz, H-4), 7.87 (d, 1H,

%), 270 (M⁺, 5), 122 (24), 106 (100), 78 (50); Anal. Calcd for

C₁₃H₁₀N₄O₃(270.24): C, 57.78; H, 3.73; N, 20.73; Found: C,

57.79; H, 3.74; N, 20.70.

J_{6 ,5}= 8.5 Hz, H-6⁰), 7.56 (m, 1H, H-5), 7.40 (t, 1H, J_{5 ,4 /5 ,6}= 8.0 Hz,

0

H-5⁰), 7.10 (d, 1H, J_{3 ,4}= 8.5 Hz, H-3⁰), 7.01 (t, 1H, J_{4 ,3 /4 ,5}= 8.0 Hz,

0

4.13.4. (E)-N’-(4ʹ-Nitrobenzylidene) nicotinohydrazide (4)^18c

H-4⁰), 4.12 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-1⁰⁰), 1.38 (t, 3H, J_{2 ,1}= 7.2 Hz,

CH₃-2⁰⁰); EI-MS: m/z (rel. abund.%), 269 (M⁺, 7), 147 (82), 119 (100),

78 (81); Anal. Calcd for C₁₅H₁₅N₃O₂(269.30): C, 66.90; H, 5.61; N,

15.60; Found: C, 66.89; H, 5.60; N, 15.62.

00 00

Yield: 81%; m.p. 257–259 °C (lit. 258–260 °C); ¹H NMR

(300 MHz, DMSO-d₆): d 12.29 (s, 1H, NH), 9.07 (s, 1H, H-2), 8.77

(d, 1H, J_6,5= 4.2 Hz, H-6), 8.54 (s, 1H, @CH), 8.29 (m, 3H, H-4, H-

2⁰, H-6⁰), 8.02 (d, 2H, J_{3 ,2 /5 ,6}= 8.0 Hz, H-3⁰, H-5⁰), 7.57 (m, 1H, H-

5); EI-MS: m/z (rel. abund.%), 270 (M⁺, 3), 122 (66), 106 (100), 78

(81); Anal. Calcd for C₁₃H₁₀N₄O₃(270.24): C, 57.78; H, 3.73; N,

20.73; Found: C, 57.77; H, 3.71; N, 20.75.

0

4.13.11. (E)-N’-(4ʹ-Ethoxybenzylidene)nicotinohydrazide (11) CAS #

324531-29-5

Yield: 81%; m.p. 155–157 °C; ¹H NMR (400 MHz, DMSO-d₆): d

11.85 (s, 1H, NH), 9.04 (d, 1H, J_2,6= 1.2 Hz, H-2), 8.75 (d, 1H,

J_6,5= 4.2 Hz, H-6), 8.37 (s, 1H, @CH), 8.23 (d, 1H, J_4,5= 8.0 Hz, H-

4.13.5. (E)-N’-(4ʹ-Hydroxybenzylidene) nicotinohydrazide (5)^18b

Yield: 70%; m.p. 230–232 °C (lit. 231–232 °C); ¹H NMR

(400 MHz, DMSO-d₆): d 11.78 (s, 1H, NH), 9.03 (s, 1H, H-2), 8.74

(d, 1H, J_6,5= 4.2 Hz, H-6), 8.42 (s, 1H, @CH), 8.27 (d, 1H,

4), 7.667(d, 2H, J_{2 ,3 /6 ,5}= 8.0 Hz, H-2⁰, H-6⁰), 7.57 (m, 1H, H-5),

0

7.01 (d, 2H, J_{3 ,2 /5 ,6}= 8.0 Hz, H-3⁰, H-5⁰), 4.10 (q, 2H, J_{1 ,2}= 7.2 Hz,

0

00 00

OCH₂-1⁰⁰), 1.35 (t, 3H, J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰); EI-MS: m/z (rel.

00 00

J_4,5= 8.0 Hz, H-4), 7.56 (m, 3H, H-5, H-2⁰, H-6⁰), 6.83 (d, 2H,

abund.%), 269 (M⁺, 37), 147 (100), 119 (74), 106 (53); Anal. Calcd

for C₁₅H₁₅N₃O₂(269.30): C, 66.90; H, 5.61; N, 15.60; Found: C,

66.89; H, 5.60; N, 15.61.

+

J_{3 ,2 /5 ,6}= 8.0 Hz, H-3⁰, H-5⁰); EI-MS: m/z (rel. abund.%), 241 (M ,

25), 123 (86), 106 (100), 78 (55); Anal. Calcd for C₁₃H₁₁N₃O₂

(241.25): C, 64.72; H, 4.60; N, 17.42; Found: C, 64.71; H, 4.62; N,

17.41.

0

4.13.12. (E)-N’-(3ʹ-Ethoxy-2ʹ-hydroxybenzylidene) nicotinohydrazide

(12) CAS # 292180-68-8

4.13.6. (E)-N’-(3ʹ-Hydroxybenzylidene) nicotinohydrazide (6)^18b

Yield: 74%; m.p. 203–205 °C (lit. 202–204 °C); ¹H NMR

(300 MHz, DMSO-d₆): d 11.94 (s, 1H, NH), 9.62 (s, 1H, H-2⁰), 9.05

(s, 1H, H-2), 8.77 (d, 1H, J_6,5= 4.2 Hz, H-6), 8.42 (s, 1H, @CH),

8.27 (d, 1H, J_4,5= 8.0 Hz, H-4), 7.70 (m, 1H, H-5), 7.55 (m, 1H, H-

Yield: 80%; m.p. 247–249 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.20 (s, 1H, NH), 10.78 (s, 1H, OH), 9.09 (d, 1H, J_2,6= 1.2 Hz, H-

2), 8.76 (dd, 1H, J_6,5= 4.8 Hz, J_6,2= 1.2 Hz, H-6), 8.64 (s, 1H, @CH),

0

8.27 (m, 1H, H-4), 7.57 (m, 1H, H-5), 7.17 (d, 1H, J_{6 ,5}= 8.5 Hz, H-

6⁰), 7.04 (d, 1H, J_{4 ,5}= 8.5 Hz, H-4⁰), 7.86 (t, 1H, J_{5 ,4 /5 ,6}= 8.5 Hz,

0

5⁰), 7.12 (d, 1H, J_{6 ,5}= 7.5 Hz, H-6⁰), 6.83 (d, 1H, J_{4 ,5}= 7.5 Hz, H-

4⁰); EI-MS: m/z (rel. abund.%), 241 (M⁺, 10), 123 (63), 106 (100),

78 (26); Anal. Calcd for C₁₁H₁₃N₃O₂(241.25): C, 64.72; H, 4.60;

N, 17.42; Found: C, 64.73; H, 4.61; N, 17.41.

H-5⁰), 4.06 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-1⁰⁰), 1.34 (t, 3H, J_{2 ,1}= 7.2 Hz,

CH₃-2⁰⁰); EI-MS: m/z (rel. abund.%), 285 (M⁺, 55), 163 (20), 106

(100), 78 (67); Anal. Calcd for C₁₅H₁₅N₃O₃(285.30): C, 63.15; H,

5.30; N, 14.73; Found: C, 63.16; H, 5.29; N, 14.74.

0

00 00

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16

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

4.13.13. (E)-N’-(2ʹ-Methoxybenzylidene) nicotinohydrazide (13)^18f

Yield: 94%; m.p. 157–159 °C; ¹H NMR (400 MHz, DMSO-d₆): d

11.99 (s, 1H, NH), 9.07 (d, 1H, J_2,6= 1.2 Hz, H-2), 8.79 (s, 1H,

@CH), 8.75 (dd, 1H, J_6,5= 6.5 Hz, J_6,2= 1.6 Hz, H-6), 8.26 (dt, 1H,

C₁₄H₁₃N₃O₂(255.27): C, 65.87; H, 5.13; N, 16.46; Found: C,

65.86; H, 5.11; N, 16.48.

4.13.14. (E)-N’-(3ʹ,4ʹ-Dimethoxybenzylidene) nicotinohydrazide

(14)^18g

0

J_{6 ,5}= 8.0, J_{6 ,4}= 1.6 Hz, H-6), 7.88 (dd, 1H, J_{3 ,4}= 6.5 Hz, J_{3 ,5}= 1.6

Hz, H-3⁰), 7.55 (m, 1H, H-4), 7.18 (td, 1H, J_{5 ,4 /5 ,6}= 8.0 Hz,

0

Yield: 84%; m.p. 166–168 °C (lit. 166–167 °C); ¹H NMR

J_{5 ,3}= 1.6 Hz, H-5⁰), 7.12 (t, 1H, J_{4 ,3 /4 ,5}= 8.0 Hz, H-4⁰), 7.03 (t, 1H,

J_5,4/5,6= 8.0 Hz, H-5), 3.86 (s, 3H, OCH₃); EI-MS: m/z (rel. abund.

%), 255 (M⁺, 32), 133 (100), 106 (100), 78 (89); Anal. Calcd for

0

(400 MHz, DMSO-d₆):

d 11.88 (s, 1H, NH), 9.04 (d, 1H,

J_2,6= 1.2 Hz, H-2), 8.75 (dd, 1H, J_6,2= 1.2 Hz, J_6,5= 4.8 Hz, H-6),

8.35 (s, 1H, @CH), 8.23 (td, 1H, J_4,5= 8.0 Hz, J_4(2,6)= 2.0 Hz, H-4),

Negative control group

Blank group

6-MP

(A)

(B)

Test group I

Positive control group

6-MP

(C)

(D)

Test group II

Test group III

6-MP

(E)

(F)

Fig. 15. UPLC chromatograms of (A) Blank group, (B) Negative control group, (C) Positive control group, (D) Test group I, (compound 8; 50 mg/kg), (E) Test group II,

(compound 35; 50 mg/kg), and (F) Test group III (compound 36; 50 mg/kg), showing the corresponding peaks of 6-MP.

Please cite this article in press as: Zafar H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.044

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

17

4.13.17. (E)-N’-(2ʹ-Hydroxy-5ʹ-methoxybenzylidene)

*P<0.01

**P<0.001

nicotinohydrazide (17)^18h

Yield: 92%; m.p. 242–244 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.20 (s, 1H, NH), 10.78 (s, 1H, OH), 9.07 (d, 1H, J_2,6= 1.2 Hz, H-

200

150

2), 8.77 (d, 1H, J_{6 ,4}= 3.6 Hz, H-6⁰), 8.65 (s, 1H, @CH), 8.26 (m, 1H,

0

H-6), 7.57 (m, 1H, H-4), 7.18 (d, 1H, J_{3 ,4}= 8.0 Hz, H-3⁰), 7.04 (d,

0

1H, J_{4 ,3}= 8.0 Hz, H-4⁰), 6.86 (t, 1H, J_5,4/5,6= 8.0 Hz, H-5), 3.81 (s,

3H, OCH₃); EI-MS: m/z (rel. abund.%), 271 (M⁺, 89), 123 (81), 106

(100), 51 (39); Anal. Calcd for C₁₄H₁₃N₃O₃(271.27): C, 61.99; H,

4.83; N, 15.49; Found: C, 61.98; H, 4.81; N, 15.51.

0

100

50

4.13.18. (E)-N’-(2ʹ-Hydroxy-3ʹ-methoxybenzylidene)

nicotinohydrazide (18)^18b

Yield: 73%; m.p. 197–199 °C (lit. 198–199 °C); ¹H NMR

(400 MHz, DMSO-d₆): d 12.20 (s, 1H, NH), 10.76 (s, 1H, OH), 9.07

(d, 1H, J_2,6= 1.2 Hz, H-2), 8.77 (dd, 1H, J_6,5= 4.8 Hz, J_6,4= 1.2 Hz,

H-6), 8.56 (s, 1H, @CH), 8.27 (td, 1H, J_4(2,6)= 2.0 Hz, J_4,5= 8.0 Hz,

0

Control Allopurinol

Compound 35 Compound 36

Fig. 16. Comparison of standard inhibitor allopurinol, compounds 35, and 36 with

the control group showing signiﬁcant peak areas of 6-mercaptopurine (6-MP) with

P values <0.01, and 0.001, respectively.

H-4), 7.57 (m, 1H, H-5), 7.17 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰), 7.05 (d,

0

1H, J_{4 ,5}= 8.5 Hz, H-4⁰), 7.86 (t, 1H, J_{5 (4 ,5 )}= 8.5 Hz, H-5⁰), 3.81 (s,

3H, OCH₃); EI-MS: m/z (rel. abund.%), 271 (M⁺, 50), 149 (86), 106

(100), 83 (68); Anal. Calcd for C₁₄H₁₃N₃O₃(271.27): C, 61.99; H,

4.83; N, 15.49; Found: C, 61.97; H, 4.84; N, 15.50.

0

Table 3

Cytotoxicity and in vivo XO inhibitory activity of selected inhibitors.

4.13.19. (E)-N’-(2ʹ-Bromobenzylidene) nicotinohydrazide (19)¹⁸ⁱ

Yield: 69%; m.p. 203–205 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.23 (s, 1H, NH), 9.02 (s, 1H, H-2), 8.81 (s, 1H, @CH), 8.76 (d,

1H, J_6,5= 4.8 Hz, H-6), 8.27 (d, 1H, J_4,5= 5.0 Hz, H-4), 8.01 (d, 1H,

S.

No.

Group

MTT

assay

Retention

Time

Peak Area Amount (6-

(mAU)

MP) (mg/mL)

1

2

Control group

Allopurinol

(50 mg/kg)

Compound 8

(50 mg/kg)

Compound 35

(50 mg/kg)

Compound 36

(50 mg/kg)

–

4.912

4.929

7.3

–

166.3

1.67 (100%)

J_{6 ,5}= 7.5 Hz, H-6⁰), 7.70 (d, 1H, J_{3 ,4}= 7.5 Hz, H-6⁰), 7.57 (m, 1H,

0

3

4

5

Non-

cytotoxic

Non-

cytotoxic

Non-

cytotoxic

4.901

4.946

4.918

0.001

47.4

73.5

–

H-5), 7.48 (t, 1H, J_{4 (3 ,5 )}= 8.0 Hz, H-4⁰), 7.48 (t, 1H, J₅

0

+

_{(4 ,6 )}= 8.0 Hz, H-6⁰); EI-MS: m/z (rel. abund.%), 303 (M , 3), 122

(70), 106 (100), 78 (68); Anal. Calcd for C₁₃H₁₀BrN₃O (304.14): C,

156.34; H, 3.31; N, 13.82; Found: C, 51.32; H, 3.32; N, 13.81.

0

0.475 (28%)

0.76 (44%)

4.13.20. (E)-N’-(2ʹ-Fluorobenzylidene) nicotinohydrazide (20)^18j

Yield: 90%; m.p. 149–151 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.12 (s, 1H, NH), 9.07 (d, 1H, J_2,6= 1.2 Hz, H-2), 8.79 (d, 1H,

J_6,5= 3.6 Hz, H-6), 8.68 (s, 1H, @CH), 8.27 (td, 1H, J_4,5= 8.0 Hz, J₄

7.57 (m, 1H, H-5), 7.34 (d, 1H, J_{2 ,6}= 2.0 Hz, H-2⁰), 7.74 (dd, 1H,

0

J_{6 ,5}= 8.0 Hz, J_{6 ,2}= 2.0 Hz, H-6⁰), 7.03 (d, 1H, J_{5 ,6}= 8.0 Hz, H-5⁰),

3.81 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃); EI-MS: m/z (rel. abund.%),

285 (M⁺, 73), 163 (100), 148 (40), 106 (44); Anal. Calcd For

0

_(2,6)= 2.0 Hz, H-4), 7.95 (t, 1H, J_{3 (4 ,F)}= 8.0 Hz, H-3⁰), 7.53 (m, 2H,

H-5, H-6⁰), 7.31 (m, 2H, H-4⁰, H-5⁰); EI-MS: m/z (rel. abund.%),

243 (M⁺, 15), 122 (97), 106 (100), 78 (85); Anal. Calcd for C₁₃H₁₀

F N₃O (243.24):C, 64.19; H, 4.14; N, 17.28; Found: C, 64.2O; H,

4.13; N, 17.27.

0

C

₁₅H₁₅N₃O₃(285.30): C, 63.15; H, 5.30; N, 14.73; Found: C,

63.17; H, 5.32; N, 14.70.

4.13.15. (E)-N’-(2ʹ,3ʹ,4ʹ-Trimethoxybenzylidene) nicotinohydrazide

(15) CAS # 556786-23-3

4.13.21. (E)-N’-(3ʹ-Chlorobenzylidene) nicotinohydrazide (21)^18k

Yield: 88%; m.p. 131–133 °C (lit. 130–132 °C); ¹H NMR

(300 MHz, DMSO-d₆): d 12.14 (s, 1H, NH), 9.05 (s, 1H, H-2), 8.77

(d, 1H, J_6,5= 4.2 Hz, H-6), 8.42 (s, 1H, @CH), 8.27 (d, 1H,

J_4,5= 8.0 Hz, H-4), 7.79 (s, 1H, H-2⁰), 7.70 (m, 1H, H-5), 7.55 (m,

3H, H-4⁰, H-5⁰, H-6⁰); EI-MS: m/z (rel. abund.%), 259 (M⁺, 3), 261

(M+2, 1.2), 122 (58), 106 (100), 78 (52); Anal. Calcd for C₁₃H₁₀ClN₃-

O (259.69): C, 60.12; H, 3.88; N, 16.18; Found: C, 60.13; H, 3.87; N,

16.19.

Yield: 76%; m.p. 182–184 °C; ¹H NMR (300 MHz, DMSO-d₆): d

11.90 (s, 1H, NH), 9.05 (s, 1H, H-2), 8.75 (d, 1H, J_6,5= 4.2 Hz, H-6),

8.24 (s, 1H, @CH), 7.65 (d, 1H, J_{6 ,5}= 8.0 Hz, H-6⁰), 7.70 (m, 1H, H-

0

5), 6.94 (d, 1H, J_{5 ,6}= 8.0 Hz, H-5⁰); EI-MS: m/z (rel. abund.%), 315

(M⁺, 19), 193 (100), 178 (48), 78 (26); Anal. Calcd for C₁₆H₁₇N₃O₄

(315.32): C, 60.94; H, 5.43; N, 13.33; Found: C, 60.95; H, 5.41; N,

13.34.

0

4.13.16. (E)-N’-(2ʹ-Hydroxy-6ʹ-methoxybenzylidene)

nicotinohydrazide (16)

4.13.22. (E)-N’-(4ʹ-Chlorobenzylidene) nicotinohydrazide (22)^18b

Yield: 85%; m.p. 226–228 °C (lit. 227–229 °C); ¹H NMR

Yield: 77%; m.p. 278–280 °C; ¹H NMR (400 MHz, DMSO-d₆): d

12.21 (s, 1H, NH), 10.55 (s, 1H, OH), 9.00 (d, 1H, J_2,6= 1.4 Hz, H-

2), 8.72 (dd, 1H, J_6,5= 5.0 Hz, J_6,4= 1.4 Hz, H-6), 8.60 (s, 1H, @CH),

(400 MHz, DMSO-d₆):

d 12.09 (s, 1H, NH), 9.05 (d, 1H,

J_2,6= 1.2 Hz, H-2), 8.76 (d, 1H, J_6,5= 4.2 Hz, H-6), 8.42 (s, 1H,

0

8.11 (m, 1H, H-4), 7.48 (m, 1H, H-5), 7.14 (d, 1H, J_{3 ,4}= 8.0 Hz, H-

3⁰), 7.05 (m, 2H, H-4⁰, H-5⁰), 3.81 (s, 3H, OCH₃); EI-MS: m/z (rel.

abund.%), 271 (M⁺, 98), 149 (74), 106 (100), 78 (84); HREI-MS calcd

for C₁₄H₁₃N₃O₃: m/z = 271.0957, found 271.0961; Anal. Calcd for

@CH), 8.25 (br.d, 1H, J_4,5= 8.0 Hz, H-4), 7.77 (d, 2H, J_{2 ,3 /6 ,5}= 8.0

Hz, H-2⁰, H-6⁰), 7.58 (m, 1H, H-5), 7.54 (d, 2H, J_{3 ,2 /5 ,6}= 8.0 Hz,

0

H-3⁰, H-5⁰); EI-MS: m/z (rel. abund.%), 259 (M⁺, 7), 261 (M + 2, 3),

122 (69), 106 (100), 78 (63); Anal. Calcd for

C₁₃H₁₀ClN₃O

C

₁₄H₁₃N₃O₃(271.27): C, 61.99; H, 4.83; N, 15.49; Found: C,

(259.69): C, 60.12; H, 3.88; N, 16.18; Found: C, 60.10; H, 3.89; N,

16.17.

61.97; H, 4.84; N, 15.50.

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18

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

4.13.23. (E)-N’-(2ʹ,4ʹ-Dichlorobenzylidene) nicotinohydrazide (23)^18c

Yield: 81%; m.p. 139–141 °C (lit. 138–140 °C); ¹H NMR

8.28 (dd, 1H, J_{4 ,5}= 8.5 Hz, J_{4 ,2}= 2.5 Hz, H-4⁰), 8.18 (d, 1H,

0

J_{6 ,5}= 8.5 Hz, H-6⁰), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.76 (t,

0

+

(400 MHz, DMSO-d₆):

d

12.28 (s, 1H, NH), 9.07 (d, 1H,

1H, J_{5 (4 ,6 )}= 8.5 Hz, H-5⁰); EI-MS: m/z (rel. abund.%), 270 (M , 04),

122 (95), 106 (100), 78 (80); Anal. Calcd for C₁₃H₁₀N₄O₃(270.24):

C, 57.78; H, 3.73; N, 20.73; Found: C, 57.75; H, 3.75; N, 20.74.

0

J_2,6= 1.2 Hz, H-2), 8.79 (s, 1H, @CH), 8.78 (d, 1H, J_4,5= 3.6 Hz, H-

4), 8.27 (d, 1H, J_6,5= 8.5 Hz, H-6), 8.03 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰),

0

7.75 (d, 1H, J_{5 ,6}= 8.0 Hz, H-3⁰), 7.59 (m, 2H, H-5, H-3⁰); EI-MS:

m/z (rel. abund.%), 293 (M⁺, 8), 295 (M + 2, 5), 258 (30), 122 (88),

106 (100); Anal. Calcd for C₁₃H₉Cl₂N₃O (294.14): C, 53.08; H,

3.08; N, 14.29; Found: C, 53.07; H, 3.07; N, 14.31.

0

4.13.30. (E)-N’-(2ʹ-Nitrobenzylidene)isonicotinohydrazide (30)¹⁸ⁿ

Yield: 92%; m.p. 234–236 °C (lit. 235–236 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 12.39 (s, 1H, NH), 8.88 (s, 1H, @CH), 8.79

(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.13 (m, 2H, H-3⁰, H-4⁰), 7.84

4.13.24. (E)-N’-(2ʹ-Methylbenzylidene) nicotinohydrazide (24)^18l

Yield: 83%; m.p. 152–154 °C; ¹H NMR (400 MHz, DMSO-d₆): d

11.97 (s, 1H, NH), 9.07 (d, 1H, J_2,6= 1.5 Hz, H-2), 8.76 (dd, 1H,

J_6,5= 4.8 Hz, J_6,2= 1.5 Hz, H-6), 8.73 (s, 1H, @CH), 8.26 (td, 1H,

J_4,5= 5.0 Hz, J_4(2,6)= 1.5 Hz, H-4), 7.58 (m, 1H, H-5), 7.29 (m, 4H,

H-3⁰, H-4⁰, H-5⁰, H-6⁰), 2.17 (s, 3H, CH₃); EI-MS: m/z (rel. abund.

%), 239 (M⁺, 23), 123 (90), 106 (100), 78 (87); Anal. Calcd for

(m, 3H, H-6⁰, H-2, H-6), 7.72 (t, 1H, J_{5 (4 ,6 )}= 8.5 Hz, H-5⁰); EI-MS:

m/z (rel. abund.%), 270 (M⁺, 2), 122 (60), 106 (100), 78 (75); Anal.

Calcd for C₁₃H₁₀N₄O₃(270.24): C, 57.78; H, 3.73; N, 20.73; Found:

C, 57.77; H, 3.71; N, 20.75.

0

4.13.31. (E)-N’-(4ʹ-Nitrobenzylidene)isonicotinohydrazide (31)¹⁸ⁿ

Yield: 77%; m.p. 280–282 °C (lit. 281–282 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

C₁₄H₁₃N₃O (239.27): C, 70.28; H, 5.48; N, 17.56; Found: C 70.27;

d 12.20 (s, 1H, NH), 8.80 (d, 2H,

H, 5.47; N, 17.58.

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.56 (s, 1H, @CH), 8.32 (d, 2H,

J_{3 ,2 /5 ,6}= 8.5 Hz, H-3⁰, H-5⁰), 8.01 (d, 2H, J_{2 ,3 /6 ,5}= 8.5 Hz, H-2⁰, H-

6⁰), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6); EI-MS: m/z (rel. abund.

%), 270 (M⁺, 8), 122 (98), 106 (100), 78 (88); Anal. Calcd for

0

4.13.25. (E)-N’-(2ʹ-Hydroxy-5ʹ-methylbenzylidene) nicotinohydrazide

(25)^18h

Yield: 78%; m.p. 200–202 °C; ¹H NMR (300 MHz, DMSO-d₆): d

12.20 (s, 1H, NH), 10.87 (s, 1H, OH), 9.07 (s, 1H, H-2), 8.76 (d, 1H,

J_6,5= 4.2 Hz, H-6), 8.59 (s, 1H, @CH), 8.26 (d, 1H, J_4,5= 8.0 Hz, H-

C

₁₃H₁₀N₄O₃(270.24): C, 57.78; H, 3.73; N, 20.73; Found: C,

57.77; H, 3.75; N, 20.72.

4), 7.38 (s, 1H, H-6⁰), 7.11 (d, 1H, J_{4 ,3}= 8.0 Hz, H-4⁰), 6.82 (d, 1H,

0

4.13.32. (E)-N’-(4ʹ-Hydroxybenzylidene)isonicotinohydrazide (32)¹⁸ⁿ

Yield: 74%; m.p. 294–296 °C (lit. 293–294 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 11.84 (s, 1H, NH), 9.96 (s, 1H, OH), 8.76

(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.34 (s, 1H, @CH), 7.80 (d, 2H,

J_{3 ,4}= 8.0 Hz, H-3⁰), 2.24 (s, 3H, CH₃); EI-MS: m/z (rel. abund.%),

0

255 (M⁺, 100), 122 (80), 106 (100), 78 (100); Anal. Calcd for

C₁₄H₁₃N₃O₂(255.27): C, 65.87; H, 5.13; N, 16.46; Found: C,

65.85; H, 5.14; N, 16.45.

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.58 (d, 2H, J_{2 ,3 /6 ,5}= 8.5 Hz, H-2⁰, H-6⁰),

0

6.84 (d, 2H, J_{3 ,2 /5 ,6}= 8.5 Hz, H-3⁰, H-5⁰); EI-MS: m/z (rel. abund.

0

4.13.26. (E)-N’-(4ʹ-(Methylthio)benzylidene) nicotinohydrazide

(26)^18m

%), 241 (M⁺, 23), 165 (17), 106 (100), 78 (71); Anal. Calcd for

C

₁₃H₁₁N₃O₂(241.25): C, 64.72; H, 4.60; N, 17.42; Found: C,

64.70; H, 4.59; N, 17.44.

Yield: 81%; m.p. 149–151 °C; ¹H NMR (400 MHz, DMSO-d₆): d

11.97 (s, 1H, NH), 9.04 (s, 1H, H-2), 8.76 (d, 1H, J_6,5= 4.2 Hz, H-6),

8.38 (s, 1H, @CH), 8.24 (d, 1H, J_4,5= 8.0 Hz, H-4), 7.67 (d, 2H,

4.13.33. (E)-N’-(3ʹ-Hydroxybenzylidene)isonicotinohydrazide (33)¹⁸ⁿ

Yield: 78%; m.p. 269–271 °C (lit. 268–269 °C); ¹H NMR:

(300 MHz, DMSO-d₆): d 11.99 (s, 1H, NH), 9.65 (s, 1H, OH), 8.78

(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.36 (s, 1H, @CH), 7.81 (d, 2H,

J_{3 ,2 /5 ,6}= 8.0 Hz, H-3⁰, H-5⁰) 7.57 (m, 1H, H-5), 7.33 (d, 2H,

0

J_{2 ,3 /6 ,5}= 8.0 Hz, H-2⁰, H-6⁰), 2.51 (s, 3H, SCH₃); EI-MS: m/z (rel.

abund.%), 271 (M⁺, 67), 149 (100), 106 (65), 78 (38); Anal. Calcd

for C₁₄H₁₃N₃OS (271.34); C, 61.97; H, 4.83; N, 15.49; Found: C,

61.98; H, 4.81; N, 15.51.

0

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.26 (t, 1H, J_{5 (4 ,6 )}= 8.5, H-5⁰), 7.22 (s,

0

1H, H-2⁰), 7.11 (d, 1H, J_{4 ,5}= 8.5, H-4⁰), 6.84 (d, 1H, J_{6 ,5}= 8.5, H-

6⁰); EI-MS: m/z (rel. abund.%), 239 (M⁺, 58), 122 (92), 91 (34), 44

(22); Anal. Calcd for C₁₃H₁₁N₃O₂(241.25): C, 64.72; H, 4.60; N,

17.42; Found: C, 64.71; H, 4.61; N, 17.40.

0

4.13.27. (E)-N’-(4ʹ-(Dimethylamino)benzylidene) nicotinohydrazide

(27)^18b

Yield: 78%; m.p. 144–146 °C (lit. 145–147 °C); ¹H NMR

(400 MHz, DMSO-d₆):

J_2,6= 1.2 Hz, H-2), 8.72 (dd, 1H, J_6,5= 4.8 Hz, J_6,2= 1.2 Hz, H-6),

d 11.67 (s, 1H, NH), 9.03 (d, 1H,

4.13.34. (E)-N’-(2ʹ,4ʹ-Dihydroxybenzylidene)isonicotinohydrazide

(34)¹⁸ⁿ

8.29 (s, 1H, @CH), 8.21 (d, 1H, J_4,6= 1.2 Hz, H-2), 7.54 (m, 3H, H-

Yield: 70%; m.p. 296–298 °C (lit. 294–295 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 12.09 (s, 1H, OH), 11.25 (s, 1H, NH), 9.99

(s, 1H, OH), 8.77 (s, 1H, H-3⁰), 8.52 (s, 1H, @CH), 7.81 (d, 2H,

5, H-2⁰, H-6⁰), 6.75 (d, 2H, J_{3 ,2 /5 ,6}= 8.0 Hz, H-3⁰, H-5⁰), 2.97 (s, N

(CH₃)₂); EI-MS: m/z (rel. abund.%), 268 (M⁺, 24), 146 (32), 83

(23), 43 (15); Anal. Calcd for C₁₅H₁₆N₄O (268.31): C, 67.15; H,

6.01; N, 20.88; Found: C, 67.14; H, 6.02; N, 20.87.

0

J_3,2/5,6= 6.0 Hz, H-3, H-5), 7.35 (d, 1H, J_{6 ,5}= 7.5 Hz, H-6⁰), 6.33 (m,

3H, H-2, H-6, H-5⁰); EI-MS: m/z (rel. abund.%), 257 (M⁺, 51), 135

(100), 106 (63), 78 (42); Anal. Calcd for C₁₃H₁₁N₃O₃(257.24): C,

60.70; H, 4.31; N, 16.33; Found: C, 60.71; H, 4.30; N, 16.32.

0

4.13.28. (E)-N’-Benzylideneisonicotinohydrazide (28)¹⁸ⁿ

Yield: 71%; m.p. 200–202 °C (lit. 201–202 °C); Yield: 0.20 (68%);

¹H NMR: (400 MHz, DMSO-d₆): d 12.04 (s, 1H, NH), 8.78 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.46 (s, 1H, @CH), 7.82 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.75 (m, 2H, H-2⁰, H-6⁰), 7.47 (m, 3H, H-

2⁰, H-3⁰, H-4⁰); EI-MS: m/z (rel. abund.%), 225 (M⁺, 15), 122 (94),

106 (100), 78 (64); Anal. Calcd for C₁₃H₁₁N₃O (225.25): C, 69.32;

H, 4.92; N, 18.66; Found: C, 69.31; H, 4.93; N, 18.65.

4.13.35. (E)-N’-(3ʹ,4ʹ-Dihydroxybenzylidene)isonicotinohydrazide

(35)^18o

Yield: 73%; m.p. 221–223 °C (lit. 224–225 °C); ¹H NMR:

(300 MHz, DMSO-d₆):

d 11.92 (s, 1H, NH), 8.73 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.38 (s, 1H, @CH), 7.80 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.35 (d, 1H, J_{2 ,6}= 2.5 Hz, H-2⁰), 7.22

0

4.13.29. (E)-N’-(3ʹ-Nitrobenzylidene)isonicotinohydrazide (29)¹⁸ⁿ

Yield: 92%; m.p. 231–233 °C (lit. 231–232 °C); ¹H NMR:

(dd, 1H, J_{6 ,5}= 8.5 Hz, J_{6 ,2}= 2.5 Hz, H-6⁰), 7.03 (d, 1H, J_{5 ,6}= 8.5 Hz,

H-5⁰); EI-MS: m/z (rel. abund.%), 257 (M⁺, 23), 163 (100), 106 (25),

78 (28); Anal. Calcd for C₁₅H₁₅N₃O₃(285.30): C, 63.15; H, 5.30; N,

14.73; Found: C, 63.17; H, 5.29; N, 14.71.

0

(400 MHz, DMSO-d₆):

d 12.31 (s, 1H, NH), 8.80 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.57 (s, 1H, @CH), 8.56 (s, 1H, H-2⁰),

Please cite this article in press as: Zafar H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.044

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

19

4.13.36. (E)-N’-(2ʹ,4ʹ,6ʹ-Trihydroxybenzylidene)isonicotinohydrazide

(36)^18p

H-5⁰), 3.81 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃); EI-MS: m/z (rel. abund.

%), 285 (M⁺, 77), 163 (100), 148 (44), 106 (39); Anal. Calcd For

Yield: 77%; m.p 239–241 °C (lit. 238–240 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 12.11 (s, 1H, OH), 11.05 (s, 1H, OH), 9.86

(s, 1H, OH), 8.81 (s, 1H, @CH), 8.77 (d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-

5), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 5.85 (s, 2H, H-3⁰, H-5⁰);

EI-MS: m/z (rel. abund.%), 273 (M⁺, 100), 151 (79), 106 (89), 78

(55); Anal. Calcd for C₁₃H₁₁N₃O₄(273.24): C, 57.14; H, 4.06; N,

15.38; Found: C, 57.12; H, 4.08; N, 15.37.

C₁₅H₁₅N₃O₃(285.11): C, 63.15; H, 5.30; N, 14.73; Found: C,

63.16; H, 5.33; N, 14.71.

4.13.42. (E)-N’-(4ʹ-Ethoxy-3ʹ-hydroxybenzylidene)

isonicotinohydrazide (42) CAS # 680578-78-3

Yield: 73%; m.p. 225–227 °C; ¹H NMR: (400 MHz, DMSO-d₆): d

11.85 (s, 1H, NH), 8.76 (d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.32 (s,

1H, @CH), 7.79 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.30 (d, 1H,

4.13.37. (E)-N’-(2ʹ-Ethoxybenzylidene)isonicotinohydrazide (37)¹⁸ⁿ

Yield: 81%; m.p. 201–203 °C (lit. 204–205 °C); ¹H NMR:

(300 MHz, DMSO-d₆): d 12.05 (s, 1H, NH), 8.81 (s, 1H, @CH), 8.77

J_{2 ,6}= 2.0 Hz, H-2⁰), 7.10 (dd, 1H, J_{6 ,5}= 8.5 Hz, J_{6 ,2}= 2.0 Hz, H-6⁰),

0

6.85 (d, 1H, J_{5 ,6}= 8.5 Hz, H-5⁰), 4.05 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-

0

00 00

1⁰⁰), 1.34 (t, 3H, J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰); EI-MS: m/z (rel. abund.%),

00 00

(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 7.88 (dd, 1H, J_{6 ,4}= 2.0 Hz,

285 (M⁺, 81), 163 (98), 135 (100) 106 (88), 78 (40); Anal. Calcd

for C₁₅H₁₅N₃O₃(285.30): C, 63.15; H, 5.30; N, 14.73; Found: C,

63.16; H, 5.31; N, 14.71.

0

J_{6 ,5}= 8.0 Hz, H-6⁰), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.40 (t,

0

1H, J_{5 (4 ,6 )}= 8.5 Hz, H-5⁰), 7.10 (d, 1H, J_{3 ,4}= 8.5 Hz, H-3⁰), 7.01 (t,

0

1H, J_{4 (3 ,5 )}= 8.5 Hz, H-4⁰), 4.15 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-1⁰⁰),

0

00 00

1.40 (t, 3H, J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰); EI-MS: m/z (rel. abund.%), 269

4.13.43. (E)-N’-(3ʹ-Hydroxy-4ʹ-methoxybenzylidene)

isonicotinohydrazide (43)^18t

00 00

(M⁺, 07), 147 (94), 119 (100), 106 (74), 78 (59); Anal. Calcd for

C₁₅H₁₅N₃O₂(269.30): C, 66.90; H, 5.61; N, 15.60; Found: C,

Yield: 73%; m.p. 204–206 °C; ¹H NMR: (400 MHz, DMSO-d₆): d

11.86 (s, 1H, NH), 8.74 (d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.30 (s,

1H, @CH), 7.80 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.27 (d, 1H,

66.91; H, 5.63; N, 15.58.

4.13.38. (E)-N’-(4ʹ-Ethoxybenzylidene)isonicotinohydrazide (38)^18q

Yield: 74%; m.p. 176–178 °C (lit. 177 °C); ¹H NMR: (400 MHz,

DMSO-d₆): d 11.90 (s, 1H, NH), 8.76 (d, 2H, J_3,2/5,6= 6.0 Hz, H-3,

H-5), 8.39 (s, 1H, @CH), 7.80 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6),

J_{2 ,6}= 1.5 Hz, H-2⁰), 7.08 (dd, 1H, J_{6 ,5}= 8.5 Hz, J_{6 ,2}= 1.5 Hz, H-6⁰),

0

6.98 (d, 1H, J_{5 ,6}= 8.5 Hz, H-5⁰), 3.80 (s, 3H, OCH₃); EI-MS: m/z

(rel. abund.%), 271 (M⁺, 40), 149 (100), 106 (75), 78 (60); Anal.

Calcd for C₁₄H₁₃N₃O₃(271.27): C, 61.99; H, 4.83; N, 15.49; Found:

C, 61.97; H, 4.84; N, 15.50.

0

7.67 (d, 2H, J_{2 ,3 /6 ,5}= 8.5 Hz, H-2⁰, H-6⁰), 7.00 (d, 2H, J_{3 ,2 /5 ,6}= 8.5

0

Hz, H-3⁰, H-5⁰), 4.10 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-1⁰⁰), 1.35 (t, 3H,

00 00

+

J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰); EI-MS: m/z (rel. abund.%), 269 (M , 38),

4.13.44. (E)-N’-(2ʹ-Hydroxy-5ʹ-methoxybenzylidene)

isonicotinohydrazide (44)^18h

00 00

147 (93), 119 (100), 78 (36); Anal. Calcd for

C₁₅H₁₅N₃O₂

(269.30): C, 66.90; H, 5.61; N, 15.60; Found: C, 66.88; H, 5.62; N,

15.61.

Yield: 85%; m.p. 251–253 °C; ¹H NMR: (400 MHz, DMSO-d₆): d

12.24 (s, 1H, NH), 10.48 (s, 1H, OH), 8.78 (d, 2H, J_3,2/5,6= 6.0 Hz,

H-3, H-5), 8.65 (s, 1H, @CH), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-

6), 7.16 (d, 1H, J_{6 ,4}= 2.0 Hz, H-6⁰), 6.93 (dd, 1H, J_{4 ,3}= 8.0 Hz,

0

4.13.39. (E)-N’-(3ʹ-Ethoxy-2ʹ-hydroxybenzylidene)

isonicotinohydrazide (39)^18r

J_{4 ,6}= 2.0 Hz, H-4⁰), 6.87 (d, 1H, J_{3 ,4}= 8.0 Hz, H-3⁰), 3.72 (s, 3H,

OCH₃); EI-MS: m/z (rel. abund.%), 271 (M⁺, 100), 149 (83), 106

(79), 78 (68); Anal. Calcd for C₁₄H₁₃N₃O₃(271.27): C, 61.99; H,

4.83; N, 15.49; Found: C, 61.97; H, 4.84; N, 15.48.

0

Yield: 87%; m.p. 228–230 °C; ¹H NMR: (400 MHz, DMSO-d₆): d

12.25 (s, 1H, NH), 10.66 (s, 1H, -OH), 8.78 (d, 2H, J_3,2/5,6= 6.0 Hz,

H-3, H-5), 8.67 (s, 1H, @CH), 7.84 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-

6), 7.16 (d, 1H, J_{6 ,5}= 8.0 Hz, H-6⁰), 7.04 (d, 1H, J_{4 ,5}= 8.0 Hz, H-

0

4⁰), 6.84 (t, 1H, J_{5 (4 ,6 )}= 8.0 Hz, H-5⁰), 4.09 (q, 2H, J_{1 ,2}= 7.2 Hz,

4.13.45. (E)-N’-(2ʹ-Hydroxy-3ʹ-methoxybenzylidene)

isonicotinohydrazide (45)¹⁸ⁿ

0

00 00

OCH₂-1⁰⁰), 1.36 (t, 3H, J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰); EI-MS: m/z (rel.

00 00

abund.%), 285 (M⁺, 45), 163 (63), 135 (97), 106 (100); Anal. Calcd

for C₁₅H₁₅N₃O₃(285.30): C, 63.15; H, 5.30; N, 14.73; Found: C,

63.14; H, 5.32; N, 14.72.

Yield: 81%; m.p. 235–237 °C (lit. 236–237 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

d 11.80 (s, 1H, NH), 8.78 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.69 (s, 1H, @CH), 7.84 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.17 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰), 7.04 (d,

0

4.13.40. (E)-N’-(3ʹ,4ʹ-Diethoxybenzylidene)isonicotinohydrazide

(40)¹⁸ⁿ

1H, J_{4 ,5}= 8.5 Hz, H-4⁰), 6.85 (t, 1H, J_{5 (4 ,6 )}= 8.5 Hz, H-4⁰), 3.81 (s,

3H, OCH₃); EI-MS: m/z (rel. abund.%), 271 (M⁺, 71), 149 (95), 106

(100), 78 (93); Anal. Calcd for C₁₄H₁₃N₃O₃(271.27): C, 61.99; H,

4.83; N, 15.49; Found: C, 61.98; H, 4.85; N, 15.48.

0

Yield: 84%; m.p. 193–195 °C (lit. 193–194 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

d 11.92 (s, 1H, NH), 8.77 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.38 (s, 1H, @CH), 7.80 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.34 (d, 1H, J_{2 ,6}= 1.5 Hz, H-2⁰), 7.35

4.13.46. (E)-N’-(2ʹ-Chlorobenzylidene)isonicotinohydrazide (46)¹⁸ⁿ

Yield: 77%; m.p. 222–224 °C (lit. 221–222 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 12.26 (s, 1H, NH), 8.87 (s, 1H, @CH), 8.79

0

(dd, 1H, J_{6 ,5}= 8.5 Hz, J_{6 ,2}= 1.5 Hz, H-6⁰), 7.03 (d, 1H, J_{5 ,6}= 8.5 Hz,

0

H-5⁰), 4.12 (q, 2H, J_{1 ,2}= 7.2 Hz, OCH₂-1⁰⁰), 4.10 (q, 2H, J₁

= 7.2

00 00

⁰⁰⁰,2⁰⁰⁰

Hz, OCH₂-1⁰⁰⁰), 1.37 (t, 3H, J_{2 ,1}= 6.8 Hz, CH₃-2⁰⁰), 1.35 (t, 3H,

(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.04 (dd, 1H, J_{3 ,4}= 8.5 Hz,

00 00

0

= 6.8 Hz, CH₃-2⁰⁰⁰); EI-MS: m/z (rel. abund.%), 313 (M⁺, 08),

J_{3 ,5}= 1.5 Hz, H-3⁰), 7.84 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.55 (d,

⁰⁰⁰,1⁰⁰⁰

J₂

0

1H, J_{6 ,5}= 8.5 Hz, H-6⁰), 7.49 (m, 2H,H-4⁰, H-5⁰); EI-MS: m/z (rel.

abund.%), 257 (M⁺, 4), 259 (M + 2, 3.5), 122 (80), 106 (100), 78

(80); Anal. Calcd for C₁₃H₁₀ClN₃O (259.69): C, 60.12; H, 3.88; N,

16.18; Found: C, 60.10; H, 3.87; N, 16.19.

0

202 (69), 140 (100),83 (100); Anal. Calcd for

C₁₇H₁₉N₃O₃

(313.35): C, 65.16; H, 6.11; N, 13.41; Found: C, 65.15; H, 6.13; N,

13.40.

4.13.41. (E)-N’-(3ʹ,4ʹ-Dimethoxybenzylidene)isonicotinohydrazide

(41)^18s

4.13.47. (E)-N’-(2ʹ-Fluorobenzylidene)isonicotinohydrazide (47)^18p

Yield: 86%; m.p. 190–191 °C (lit. 191–193 °C); ¹H NMR:

Yield: 70%; m.p. 190–192 °C (lit. 189–190 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

d

11.92 (s, 1H, NH), 8.77 (d, 2H,

(400 MHz, DMSO-d₆):

d

12.16 (s, 1H, NH), 8.79 (d, 2H,

0

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.38 (s, 1H, @CH), 7.80 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.70 (s, 1H, @CH), 7.95 (t, 1H, J₅

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.35 (d, 1H, J_{2 ,6}= 2.0 Hz, H-2⁰), 7.24

_{(4 ,6 )}= 8.0, H-5⁰), 7.83 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.52 (m,

1H, H-4⁰), 7.31 (m, 2H, H-3⁰, H-6⁰); EI-MS: m/z (rel. abund.%), 243

0

(dd, 1H, J_{6 ,5}= 8.0 Hz, J_{6 ,2}= 2.0 Hz, H-6⁰), 7.04 (d, 1H, J_{5 ,6}= 8.5 Hz,

0

Please cite this article in press as: Zafar H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.044

20

H. Zafar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

(M⁺, 19), 122 (96), 106 (98), 78 (100); Anal. Calcd for C₁₃H₁₀FN₃O

(243.24): C, 64.19; H, 4.14; N, 17.28; Found: C, 64.17; H, 4.15; N,

17.29.

4.13.54. (E)-N’-(4ʹ-(Dimethylamino)benzylidene) isonicotinohydrazide

(54)¹⁸ⁿ

Yield: 70%; m.p. 206–208 °C (lit. 206–207 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

d 11.73 (s, 1H, NH), 8.75 (d, 2H,

4.13.48. (E)-N’-(3ʹ-Chlorobenzylidene)isonicotinohydrazide (48)^18u

Yield: 85%; m.p. 222–224 °C (lit. 224–225 °C); ¹H NMR:

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.31 (s, 1H, @CH), 7.78 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.54 (d, 2H, J_{2 ,3 /6 ,5}= 8.5 Hz, H-2⁰, 6⁰),

0

6.75 (d, 2H, J_{3 ,2 /5 ,6}= 8.5 Hz, H-3⁰, H-5⁰), 2.92 (s, 6H, 2CH₃); EI-

MS: m/z (rel. abund.%), 268 (M⁺, 83), 146 (100), 78 (17), 44 (45);

Anal. Calcd for C₁₅H₁₆N₄O (268.31): C, 67.15; H, 6.01; N, 20.88;

Found: C, 67.14; H, 6.00; N, 20.89.

0

(400 MHz, DMSO-d₆):

d 12.24 (s, 1H, NH), 8.78 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.46 (s, 1H, @CH), 7.83 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.79 (s, 1H, H-2⁰), 7.71 (d, 1H, J_{6 ,5}= 8.5

Hz, H-6⁰), 7.50 (m, 2H, H-4⁰, H-5⁰); EI-MS: m/z (rel. abund.%), 259

(M⁺, 12), 122 (91), 106 (100), 78 (52); Anal. Calcd for C₁₃H₁₀ClN₃O

(259.69): C, 60.12; H, 3.88; N, 16.18; Found: C, 60.13; H, 3.86; N,

16.19.

0

Acknowledgments

The authors acknowledge to the Higher Education Commission

(HEC), Pakistan, for the ﬁnancial support for this research study

under the National Research Program for Universities (Project No.

20-1910).

4.13.49. (E)-N’-(4ʹ-Chlorobenzylidene) isonicotinohydrazide (49)¹⁸ⁿ

Yield: 92%; m.p. 216–218 °C (lit. 217–218 °C); ¹H NMR:

(400 MHz, DMSO-d₆):

d 12.10 (s, 1H, NH), 8.78 (d, 2H,

J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.45 (s, 1H, @CH), 7.81 (d, 2H,

J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.77 (d, 2H, J_{2 ,3 /6 ,5}= 8.5 Hz, H-2⁰, H-6⁰),

A. Supplementary data

0

7.54 (d, 2H, J_{3 ,2 /5 ,6}= 8.5 Hz, H-3⁰, H-5⁰); EI-MS: m/z (rel. abund.

%), 259 (M⁺, 13), 122 (83), 106 (100), 78 (55); Anal. Calcd for C₁₃

₁₀ClN₃O (259.69): C, 60.12; H, 3.88; N, 16.18; Found: C, 60.11;

H, 3.86; N, 16.19.

0

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.044.

-

H

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Yield: 78%; m.p. 230–231 °C (lit. 228–229 °C); ¹H NMR:

(400 MHz, DMSO-d₆): d 12.32 (s, 1H, NH), 8.76 (s, 1H, @CH), 8.74

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(d, 2H, J_3,2/5,6= 6.0 Hz, H-3, H-5), 8.04 (d, 1H, J_{6 ,5}= 8.5 Hz, H-6⁰),

0

7.84 (d, 2H, J_2,3/6,5= 6.0 Hz, H-2, H-6), 7.70 (d, 1H, J_{3 ,5}= 2.5 Hz,

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(51)^18v

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12.51 (s, 1H, NH), 8.90 (s, 1H, @CH), 8.81 (d, 2H, J_3,2/5,6= 6.0 Hz,

H-3, H-5), 8.73 (d, 1H, J_{6 ,4}= 2.0 Hz, H-6⁰), 8.26 (dd, 1H, J_{4 ,3}= 8.0

0

Hz, J_{4 ,6}= 2.0 Hz, H-4⁰), 7.86 (m, 3H, H-2, H-6, H-3⁰); EI-MS: m/z

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18.39; Found: C, 51.24; H, 2.96; N, 18.41

0

4.13.52. (E)-N’-(2ʹ-Hydroxybenzylidene)isonicotinohydrazide (52)¹⁸ⁿ

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Xanthine oxidase inhibitory activity of nicotino/isonicotinohydrazides: A systematic approach from in vitro, in silico to in vivo studies

DOI: 10.1016/j.bmc.2017.02.044

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