Synthesis, antibacterial and antifungal activity of some new pyrazoline and pyrazole derivatives

Molecules 2013, 18, 2683-2711; doi:10.3390/molecules18032683

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molecules

ISSN 1420-3049

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Article

Synthesis, Antibacterial and Antifungal Activity of Some New

Pyrazoline and Pyrazole Derivatives

Seham Y. Hassan

Department of Chemistry, Faculty of Science, University of Alexandria, PO Box 426,

Ibrahimia 21321, Alexandria, Egypt; E-Mail: sehamyassen@yahoo.com; Tel.: +20-01-286-525-997;

Fax: +20-3-5932-488

Received: 31 October 2012; in revised form: 7 January 2013 / Accepted: 9 January 2013 /

Published: 28 February 2013

Abstract: A series of 2-pyrazolines 5–9 have been synthesized from α,β-unsaturated

ketones 2–4. New 2-pyrazoline derivatives 13–15 bearing benzenesulfonamide moieties

were then synthesized by condensing the appropriate chalcones 2–4 with 4-hydrazinyl

benzenesulfonamide hydrochloride. Ethyl [1,2,4] triazolo[3,4-c][1,2,4]triazino[5,6-b]-5H-

indole-5-ethanoate (26) and 1-(5H-[1,2,4]triazino[5,6-b] indol-3-yl)-3-methyl-1H-pyrazol-

5(4H)-one (32) were synthesized from 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24).

On the other hand ethyl[1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5,10-dihydroquinoxaline-

5-ethanoate (27) and 1-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H-

pyrazol-5(4H)-one (33) were synthesized from 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-

b]quinoxaline (25) by reaction with diethyl malonate or ethyl acetoacetate, respectively.

Condensation of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1') with

compound 24 or 25 afforded the corresponding Schiff's bases 36 and 37, respectively.

Reaction of the Schiff's base 37 with benzoyl hydrazine or acetic anhydride afforded

benzohydrazide derivative 39 and the cyclized compound 40, respectively. Furthermore,

the pyrazole derivatives 42–44 were synthesized by cyclization of hydrazine derivative 25

with the prepared chalcones 2–4. All the newly synthesized compounds have been

1

characterized on the basis of IR and H-NMR spectral data as well as physical data.

Antimicrobial activity against the organisms E. coli ATCC8739 and P. aeruginosa ATCC

9027 as examples of Gram-negative bacteria, S. aureus ATCC 6583P as an example of

Gram-positive bacteria and C. albicans ATCC 2091 as an example of a yeast-like fungus

have been studied using the Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA)

diffusion methods. The best performance was found for the compounds 16, 17, 19 and 20.

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Keywords: chalcones; pyrazoline; pyrazole; carbothioamide; benzenesulfonamide;

thiazolidine; indole; quinoxaline

1. Introduction

Chalcones have been recently the subject of great interest due to their interesting pharmacological

activities, including antioxidant [1,2], antibacterial [3], antileishmanial [4], anticancer [5],

antiangiogenic [6], anti-infective, anti-inflammatory [7], antifungal [8], anti-malarial [9], anti-tumor [10],

anti-protozoal [11] and cytotoxic properties [12]. Many pyrazole derivatives are reported to have a

broad spectrum of biological activities, such as anti-inflammatory [13], antifungal [14], antiviral [15],

cytotoxic [12], A3 adenosine receptor antagonists [16], antioxidant [13], antihypertensive [17],

tranquilizing, muscle relaxant, psychoanaleptic, hypnotic, ulcerogenic, antidepressant, antibacterial

and analgesic effects [18]. Pharmacologically-interesting heterocyclic systems like pyrazolines have

been widely studied owing to their pharmacological activities, which include anti-tumor [19,20],

anti-inflammatory [21–32], anti-parasitary [33], anticonvulsant [34], antimicrobial [35–39],

antinociceptives [40], antimalarial [41], nitric oxide synthase inhibitory, associated with diseases such

as Alzheimer, Huntington, and inflammatory arthritis [42], antidepressant [43,44], anticancer [45–47],

antibacterial [48], antitubercular, analgesic [49], antiviral [46], antioxidant [50], antiamoebic [51–53],

cytotoxic [53], antidiabetic [20], antifungal [54,55], antinociceptive [56], antimycobacterial [57],

antihepatotoxic [58] and pesticidal properties [59].

Substituted 2-pyrazolines have been synthesized from α,β-unsaturated ketones and hydrazine

hydrate with acetic/formic acid in ethanol/dimethyl sulfoxide (DMSO) [60], hydrazine in dimethyl

formamide (DMF) or acetic acid [46], nicotinic acid hydrazide in n-butanol [41], phenyl hydrazine

hydrochloride in the presence of sodium acetate [39], hydrazine hydrate in ethanol and DMF [25], and

phenyl hydrazine in the presence of hot pyridine [27]. Some new substituted 2-pyrazoline derivatives

bearing benzenesulfonamide moieties [21–23,26] were synthesized by condensing appropriate chalcones

with 4-hydrazinobenzenesulfonamide hydrochloride. In view of these observations and in continuation

of our research programme on the synthesis of five-membered heterocyclic compounds [61–66], we

report herein the synthesis of some new pyrazoline and pyrazole derivatives bearing an indoline and

quinoxaline moiety, which have been found to possess an interesting profile of antimicrobial activity.

2. Results and Discussion

2.1. Chemistry

2.1.1. Preparation of the Chalcones 2–4

The chalcones 2–4 were prepared as starting material to obtain the desired pyrazoline and pyrazole

derivatives. The sequence leading to the title compounds is outlined in Scheme 1. The desired

compounds were prepared by the reaction of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-

carbaldehyde (1') [67] with different acetophenones (p-bromo-, p-chloro-, or p-methoxyacetophenones)

1

in aqueous ethanolic KOH in good yield (Scheme 1). Their H-NMR spectra showed the -CH=CH-

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protons as a multiplet in the 7.52–7.63 ppm range for compound 3, and two doublet peaks at 7.54, 7.60

and 7.48, 7.60 ppm with coupling constants of 15.3 Hz for compounds 2 and 4, respectively. The ¹³C-NMR

spectrum of prototypical compound 2 showed the two carbonyl carbons at 187.8 and 192.7 ppm.

2.1.2. Synthesis of Pyrazoline Derivatives 5–9 and Isoxazoline Derivatives 10–12

The compounds 2–4 were converted into the corresponding 3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-

tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamides 5–7 by treatment with

thiosemicarbazide (Scheme 1).

Scheme 1. Synthesis of chalcones 2–4, pyrazoline derivatives 5–9 and isoxazoline derivatives 10–12.

1

Their H-NMR spectra showed multiplets within the 3.33–4.82 range corresponding to H₄, H_4'of

the pyrazoline ring, where a multiplet at 6.92–6.98 ppm is observed for compound 7 corresponding to

H₅. A doublet of doublets at 6.73–6.88 corresponding to H₅of the pyrazoline ring was observed for

compounds 5 and 6, respectively. In addition to a broad signal corresponding to the exchangeable NH₂

protons was observed in the 7.25–8.01 ppm range. The ¹³C-NMR spectrum of compound 6 chosen as a

prototype showed C=S and C=O peaks at 180.0 and 204.6 ppm, respectively. Reaction of compounds

2 and 3 with hydrazinum chloride gave rise to 2-(3-(aryl)-4,5-dihydro-1H-pyrazol-5-yl)-6,6-dimethyl-

1

6,7-dihydro-1H-indol-4(5H)-ones 8 and 9 (Scheme 1). In their H-NMR spectra, the appearance of

signals in the ranges 3.33–3.75 and 5.49–6.70 ppm corresponding to (H₄, H_4') and H₅of the pyrazoline

ring respectively was observed. The product of compound 4 with hydrazinium chloride could not be

separated in a pure form. The 2-(3-(aryl)-4,5-dihydroisoxazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-

indol-4(5H)-ones 10–12 were synthesized by cyclization of 2–4 in presence of hydroxylamine

hydrochloride. Their ¹H-NMR spectra showed three signals within the ranges 3.34–4.22 and 5.55–6.52

ppm corresponding to the (H₄, H_4') and H₅of the pyrazoline ring, respectively. The ¹³C-NMR spectrum

of compound 11 selected as a prototype showed the carbonyl carbon at 193.7 ppm.

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2.1.3. Synthesis of Benzenesulfonamide Derivatives 13–21

Reaction of the prepared chalcones 2–4 with 4-hydrazinyl benzenesulfonamide hydrochloride

afforded 4-(3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-

1-yl)benzenesulfonamides 13–15 (Scheme 2).

Scheme 2. Synthesis of benzenesulfonamide derivatives 13–21.

1

Their H-NMR spectra showed three signals at 3.15–3.40, 3.36–4.00, and 4.80–5.56 ppm

corresponding to the H₄, H_4', and H₅of the pyrazoline ring. In addition a broad singlet was observed in

13

the 6.63–7.35 ppm range corresponding to the NH₂protons. The C-NMR spectrum of compound 15

as a prototype showed C=O at 193.8 ppm. On the other hand, the reaction of pyrazolines 13–15 with

bromine in acetic acid [68] at room temperature in order to obtain the pyrazoles, afforded the

corresponding substituted 4-(4-bromo-3-(4-bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-

1H-indol-2-yl)-1H-pyrazol-1-yl)benzenesulfonamide derivatives 16–18, respectively in 79–84% yield.

Furthermore, the prepared compound 17 was treated with phenyl isothiocyanate to furnish 4-(4-

bromo-3-aryl-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-1H-pyrazol-1-yl)-N-(phenyl-

carbamothioyl)benzene sulfonamide 19 in 76.8% yield (Scheme 2). The proton NMR spectrum

showed three broad singlets at 8.62, 9.76, and 11.11 ppm corresponding to three NH protons.

The prepared substituted benzenesulfonamides 13 and 14 were allowed to react with phenyl

isothiocyanate to correspondingly furnish 4-(3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-

2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-(phenylcarbamothioyl)benzenesulfonamides 20 and 21 (Scheme 2).

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1

Their H-NMR spectra showed D₂O exchangeable signals at the ranges 9.00–9.15, 10.54–10.60, and

11.03–11.08 ppm, corresponding to three NH protons.

2.1.4. Synthesis of [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5-N-(phenylcarbamothioyl) Ethanoic

Acid Hydrazide Derivatives 30, 31 and 3-Methyl-4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one

Derivatives 34, 35

Reaction of indoline-2,3-dione (1'') [69,70] with thiosemicarbazide gave rise to 5H-[1,2,4]

1

triazino[5,6-b]indole-3-thiol (22) [70] (Scheme 3). The H-NMR spectrum showed two broad singlets

exchangeable with D₂O at 12.43 and 14.54 ppm, corresponding to the two NH protons, which confirm

the structure of 22. The ¹³C-NMR also confirmed the structure of 22 with a peak at 179.5

corresponding to the C=S group. Treatment of the thiol derivative 22 with hydrazine hydrate afforded

3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24) [70] in 92.6% yield (Scheme 3). The proton NMR

spectrum showed two broad singlets at 4.31 and 8.54 ppm corresponding to NH₂and NH protons of

hydrazine chain, in addition to a broad singlet at 11.82 ppm corresponding to the indole ring NH.

The reaction of 24 with diethyl malonate gave rise to the corresponding ester 26. The proton NMR

spectrum showed a triplet signal at 1.16 ppm corresponding to the CH₃protons, and a quartet signal at

4.31 ppm corresponding to CH₂of the ester moiety, and a singlet signal at 4.12 corresponding to CH₂

protons. Reaction of the ester 26 with hydrazine hydrate afforded the corresponding 5-ethanoic

hydrazide 28 (Scheme 3). From the proton NMR spectrum the disappearance of CH₃and CH₂protons

of the ester chain can be observed. Treatment of the prepared hydrazide 28 with phenyl isothiocyanate

afforded the corresponding 5-N-(phenylcarbamothioyl)ethanoic acid hydrazide 30 in 80.1% yield

1

(Scheme 3). Its structure was confirmed by H-NMR, ¹³C-NMR spectra, and elemental analysis.

The ¹³C-NMR spectrum showed C=S and C=O carbons at 168.7 and 189.4 ppm respectively

(see Experimental part).

On the other hand, treatment of the prepared hydrazine derivative 24 with ethyl acetoacetate in

acetic acid afforded 1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (32)

(Scheme 3). The proton NMR spectrum of this compound showed the CH₃protons as a singlet at 1.80

ppm, and the CH₂protons as a singlet at 2.43 ppm. 1-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-3-methyl-

4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one (34) was prepared from the previous pyrazoline-5-one

derivative 32 by its reaction with acetone (Scheme 3). The proton NMR spectrum showed two methyl

protons as a singlet at 1.87 ppm.

Cyclization of quinoxaline-2,3(1H,4H)-dione (1''') with thiosemicarbazide afforded 5,10-dihydro-

[1,2,4]triazino[5,6-b]quinoxaline-3-thiol (23) in good yield (Scheme 3). Its proton NMR spectrum

showed two broad singlets, exchangeable with D₂O, at 11.88 and 14.50 ppm, corresponding to the

three NH protons, which confirm the structure of 23. Treatment of this thiol derivative 23 with

hydrazine hydrate gave 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline (25, Scheme 3).

Its proton NMR spectrum showed NH₂protons as a broad singlet signal at 4.55 ppm, in addition to

three NH protons, see Experimental part. Treatment of the prepared hydrazine derivative 25 with

diethyl malonate gave rise to the corresponding ester 27 (Scheme 3). The proton NMR spectrum

showed the ester protons (CH₃, CH₂) as a triplet and a quartet signals at 1.09 and 4.09 ppm,

respectively, in addition to CH₂protons at 4.66 ppm as a singlet signal.

Molecules 2013, 18

Scheme 3. Synthesis of [1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5-N-(phenylcarbamothioyl)

2688

ethanoic acid hydrazide derivatives 30, 31 and 3-methyl-4-(propan-2-ylidene)-1H-pyrazol-

5(4H)-one derivatives 34, 35.

Reaction of the ester 27 with hydrazine hydrate leads to the corresponding 5-ethanoic hydrazide 29.

The proton NMR spectrum showed the disappearance of CH₃and CH₂protons of the ester chain, and

NH and NH₂protons at 8.90 and 9.61 ppm were observed as a two broad singlets. Its ¹³C-NMR

spectrum showed the carbonyl carbon at 168.4 ppm. Treatment of the prepared hydrazide 29 with

phenyl isothiocyanate leads to corresponding 5-N-(phenylcarbamothioyl) ethanoic acid hydrazide 31

(Scheme 3). Its structure was also confirmed by ¹H-NMR, and elemental analysis.

Reaction of the hydrazine derivative 25 with ethyl acetoacetate in acetic acid afforded 1-(5,10-

dihyro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (33, Scheme 3). The

proton NMR spectrum showed the CH₃protons at position 3 of the pyrazoline ring as a singlet signal

at 2.29 ppm, and the CH₂protons (H₄) of the pyrazoline ring as a singlet signal at 2.92 ppm, with the

disappearance of the peak corresponding to NH₂protons. ¹³C-NMR spectrum showed the carbonyl

carbon at 170.0 ppm. Reaction of the prepared pyrazoline-5-one 33 with acetone gave rise to 1-(5,10-

dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one

(35, Scheme 3). Its proton NMR spectrum showed three methyl groups at 2.30, 2.42, and 2.93 ppm.

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2.1.5. Synthesis of Schiff’s Bases 36, 37, 4-Oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)thiazolidin-4-one (38),

Benzohydrazide Derivative 39, 1,2,4]Triazolo[3,4-c]-5,10-dihydro [1,2,4]triazino[5,6-b] quinoxaline

(40), and Pyrazole Derivatives 41–44

Condensation of hydrazine derivative 24 with 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-

carbaldehyde (1') afforded the corresponding Schiff's base 36 (Scheme 4). Its proton NMR spectrum

showed the disappearance of the NH₂signal, and a singlet signal corresponding to a CH=N proton at

8.03 ppm was observed. The ¹³C-NMR spectrum showed the carbonyl carbon at 192.6 ppm. Treatment

of the prepared compound 36 with thioglycolic acid in dry benzene gave rise to corresponding 3-(5H-

[1,2,4]triazino[5,6-b]indol-3-yl)-2-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl) thiazolidin-

4-one (38, Scheme 4). The proton NMR spectrum showed the CH₂(H₅, H_5') protons of the thiazolidine

ring at 3.35–3.49 ppm as a multiplet and the CH proton of thiazolidine ring (H₂) as a singlet signal at

7.93 ppm.

Scheme 4. Synthesis of Schiff’s bases 36, 37, 4-oxo-4,5,6,7-tetrahydro-1H-indol-2-

yl)thiazolidin-4-one (38), benzohydrazide derivative 39, 1,2,4]triazolo[3,4-c]-5,10-dihydro

[1,2,4]triazino[5,6-b] quinoxaline (40), and pyrazole derivatives 41–44.

Condensation of the hydrazine derivative 25 with 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-

2-carbaldehyde (1') furnished to the corresponding Schiff's base 37 in 86.6% yield (Scheme 4). Its

proton NMR spectrum showed the CH=N proton as a singlet at 9.45 ppm. Condensation of 37 with

benzoyl hydrazine afforded the corresponding N'-2-((2-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-

3-yl)hydrazono)methyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-ylidene)benzohydrazide (39). Its

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¹³C-NMR spectrum showed the C=O group at 162.7 ppm. On the other hand, oxidative cyclization of

Schiff's base 37 with acetic anhydride afforded the cyclized compound 40 (Scheme 4). The proton

NMR spectrum showed the disappearance of CH=N proton. We expected to obtain the acetylated

1

product, but the H-NMR spectrum confirmed the structure of the cyclized compound 40 as shown,

with an exchangeable peak corresponding to three NH protons being observed at 12.23 ppm, and no

peak observed corresponding to the acetyl methyl group. Oxidative cyclization of 3-hydrazinyl-5H-

[1,2,4]triazino[5,6-b]indole (24) with 2-(3-(4-bromophenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-

1H-indol-4(5H)-one (2) afforded 2-(1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)-3-(4-bromo-phenyl)-1H-

pyrazol-5-yl)-(6,6-dimethyl-6,7-dihydro-1H-indol-4 (5H)-one (41) in 80.8% yield. The proton NMR

spectrum showed the CH proton of pyrazole ring as a singlet signal at 6.12 ppm. The ¹³C-NMR

spectrum showed C=O carbon 190.8 at ppm.

Scheme 5. Charge distribution on nitrogen atoms N₁, N₃of compound 37.

On the other hand, the hydrazine derivative 25 was allowed to react with the prepared chalcones

2–4 to correspondingly furnish 2-(3-(aryl)-1-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-1H-

pyrazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-ones 42–44, respectively (Scheme 4). Their

proton NMR spectra showed the CH protons of the pyrazole ring (H₄) and indole ring (H₃) at 6.80 and

13

6.91, 6.90 and 7.27 and 6.78 and 6.85, ppm respectively. The C-NMR spectra of compounds 42–44

showed the C=O of the indole ring at 192.8–193.3 ppm. According to the charge distribution

determined using ChemDraw Ultra, the N₁nitrogen atom has a better nucleophile character compared

to the N₃nitrogen atom, which is in accordance with the proposed structure of compound 40 (Scheme 5).

2.2. Pharmacological Screening

Four test organisms representing different groups of microorganisms were used to evaluate the

bioactivity of the designed products. The utilized test organisms were: Escherichia coli ATCC8739,

Pseudomonas aeruginosa ATCC 9027 as Gram-negative bacteria, Staphylococcus aureus ATCC

6538P as an example of Gram-positive bacteria, and Candida albicans ATCC 2091 as yeast-like fungi.

The inhibition zone (IZ) and minimal inhibitory zone (MIC) results are given in Table 1.

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Table 1. In vitro antimicrobial activity of the test compounds and evaluation of the

inhibition zone (IZ) and the minim. inhibitory concentration (MIC).

Escherichia

coli

Staphylococcus

aureus

Candida

albicans

Pseudomonas

aeruginosa

IZ MIC

Microorganism

IZ

MIC

25

IZ

22

30

-----

30

17

15

16

13

17

16

18

15

19

15

16

26

>50

20

16

8

MIC

12.5

25

IZ

MIC

------

12.5

----

ampicillin 10.0 µg/disc

18

28

----

26

18

19

18

19

18

19

18

19

18

19

18

19

18

19

17

19

-----

40

-----

21

24

23

25

21

22

24

23

22

27

>40

23

25

26

28

25

24

23

22

23

25

23

21

26

34

23

24

23

24

22

21

-----

38

-----

30

16

18

19

20

16

18

20

19

18

19

20

21

19

18

20

18

20

18

19

17

18

16

20

18

17

19

18

-----

25

ciprofloxacin 5.0 µg/disc

12.5

-----

200

clotrimazole 100.0 µg/disc

------

200

100

200

100

25

------

200

100

200

imipenam 10.0 µg/disc

2

200

50

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

100

200

100

200

50

200

12.5

50

200

25

16

15

13

17

15

17

16

17

16

17

15

17

200

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Table 1. Cont.

Escherichia

coli

Staphylococcus

aureus

Candida

albicans

Pseudomonas

aeruginosa

Microorganism

IZ

MIC

200

IZ

17

13

16

17

13

MIC

200

100

IZ

MIC

200

IZ

16

18

16

18

19

16

MIC

200

39

40

19

18

20

18

22

21

23

24

23

21

41

42

43

44

DMF

The compounds under investigation 2–44 did not show any activity against the test organisms

Escherichia coli and Pseudomonas aeruginosa. The inhibition Zone (IZ) listed in Table 1 showed that

compound 16 has good antimicrobial activity against Staphylococcus aureus, comparable to that of

ampicillin, while compound 17 has remarkable antimicrobial activity against Staphylococcus aureus

exceeding that of ampicillin, ciprofloxacin and imipenam.. The minimal inhibitory concentration

(MIC) value showed that compound 16 has good antimicrobial activity against Staphylococcus aureus,

comparable to that of ciprofloxacin, while its activity is about 50% of that of ampicillin. In addition,

compound 17 has an IZ against Candida albicans comparable to that of clotrimazole. The minimal

inhibitory concentration (MIC) of compound 17 against Candida albicans is about 25% of that

clotrimazole. On the other hand, the minimal inhibitory concentration (MIC) of compounds 19 and 20

against Candida albicans was good, and comparable to that of clotrimazole, while compound 31 has

50 % activity compared to that of clotrimazole.

3. Experimental

3.1. General Methods

Fresh solvents were used without purification. Melting points were obtained in open capillary tubes

by using a MEL-Temp II melting point apparatus and are uncorrected. Infrared spectra (IR) were

recorded on a Perkin-Elmer 1600 series Fourier Transform instrument with the samples as KBr pellets.

¹H-NMR and ¹³C-NMR spectra were recorded on a JEOL 500 MHz spectrometer at ambient

temperature using tetramethylsilane as an internal reference. Elemental analyses were carried out by

the University of Cairo Microanalytical Laboratories. The antimicrobial tests were carried out at the

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University. ChemDraw-

Ultra-11.0 has been used for the nomenclature of the prepared compounds.

3.2. General Procedure for the Preparation of Compounds 2–4

An equimolar mixture of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1', 1.91 g,

0.01 mol) [67] and the substituted acetophenone (0.01 mol) in 2% ethanolic KOH (20 mL) was stirred

at room temperature (R.T.) for 5 h. The solid product was cooled, collected by filtration, washed with

water, dried and recrystallized from chloroform/ethanol.

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2-(3-(4-Bromophenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (2). Yellow

1

crystals; yield 3.36 g, 90.5%; m.p. 269–270 °C; IR (KBr): 1651 (C=O), 3252 cm⁻¹(NH); H-NMR

(DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.23 (s, 2H, CH₂), 2.72 (s, 2H, CH₂), 6.91 (s, 1H, CH-pyrrole), 7.54

(d, 1H, COCH=; J = 15.3 Hz), 7.60 (d, 1H, CH=; J = 15.3 Hz), 7.76 (d, 2H, ArH; J = 8.4 Hz), 7.93 (d,

2H, ArH; J = 8.4 Hz), 12.14 (bs, 1H, NH; exchangeable with D₂O); ¹³C-NMR (DMSO-d₆) δ:28.6,

35.7, 36.5, 52.2, 113.5, 116.7, 121.0, 127.5, 130.6, 132.4, 134.6, 137.5, 147.8, 187.8, 192.7. Anal.

Calcd for C₁₉H₁₈BrNO₂(372.26): C, 61.30; H, 4.87; N, 3.76 Found: C, 61.42; H, 4.97; N, 3.53.

2-(3-(4-Chlorophenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (3). Yellow

1

crystals; yield 2.99 g, 91.5%; m.p. 261–262 °C ; IR (KBr): 1651 (C=O), 3250 cm⁻¹(NH); H-NMR

(DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.23 (s, 2H, CH₂), 2.72 (s, 2H, CH₂), 6.91 (s, 1H, CH-pyrrole),

7.52–7.63 (m, 4H, CH=CH, 2 ArH), 8.01 (d, 2H, ArH; J = 8.4 Hz), 12.12 (bs, 1H, NH; exchangeable

with D₂O). Anal. Calcd for C₁₉H₁₈ClNO₂(327.80): C, 69.62; H, 5.53; N, 4.27 Found: C, 69.82; H,

5.76; N, 3.99.

2-(3-(4-Methoxyphenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (4). Yellow

1

crystals; yield 3.00 g, 93.0%; m.p. 240–241 °C ; IR (KBr): 1651 (C=O), 3232 cm⁻¹(NH); H-NMR

(DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.23 (s, 2H, CH₂), 2.71 (s, 2H, CH₂), 3.82 (s, 3H, OCH₃), 6.85 (s,

1H, CH-pyrrole), 7.07 (d, 2H, ArH; J = 8.4 Hz), 7.48 (d, 1H, -COCH=; J = 15.3 Hz), 7.60 (d, 1H,

CH=; J = 15.3 Hz), 8.01 (d, 2H, ArH; J = 8.4 Hz), 12.03 (bs, 1H, NH; exchangeable with D₂O). Anal.

Calcd for C₂₀H₂₁NO₃(323.39): C, 74.28; H, 6.55; N, 4.33 Found: C, 74.40; H, 6.71; N, 4.11.

3.3. General Procedure for the Preparation of Compounds 5–7

A mixture of the appropriate 2-(3-aryl-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-

4(5H)-one 2–4 (0.002 mol) and thiosemicarbazide (0.003 mol) was dissolved in a mixture of acetone

and ethanol (30 mL), then potassium carbonate (0.004 mol) was added with vigorous stirring. Heating

under reflux was performed for 14 h. The solvent was removed under vacuum, and ice-water was

added to the reaction mixture. The solid product obtained was filtered, washed with ethanol, dried and

recrystallized from chloroform/ethanol.

3-(4-Bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazole-

1-carbothioamide 5. Buff crystals; yield 0.71 g, 80.0%; m.p. 192–193 °C ; IR (KBr): 1167 (C=S), 1651

1

(C=O), 3134, 3232, 3436 cm⁻¹(NH, NH₂); H-NMR (DMSO-d₆) δ 1.13 (s, 6H, 2 CH₃), 2.39 (s, 2H,

CH₂), 2.80 (s, 2H, CH₂), 3.86–3.89 (m, 2H, H_4,H_4'-pyrazoline), 6.73–6.88 (dd, 1H, H₅-pyrazoline;

J = 17.4, 12.0 Hz), 6.92 (s, 1H, CH-pyrrole), 6.97 (d, 2H, ArH; J = 8.6 Hz), 7.25 (bs, 2H, NH₂;

exchangeable with D₂O), 8.01 (d, 2H, ArH; J = 8.6 Hz), 12.05 (bs, 1H, NH; exchangeable with D₂O).

Anal. Calcd for C₂₀H₂₁BrN₄OS (445.38): C, 53.94; H, 4.75; N, 12.58 Found: C, 53.70; H, 4.60; N, 12.76.

3-(4-Chlorophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazole-1-

carbothioamide 6. Buff crystals; yield 0.63 g, 79.8%; m.p. 210–211 °C ; IR (KBr): 1092 (C=S), 1646

(C=O), 3237, 3428 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 0.95 (s, 3H, CH₃), 1.02 (s, 3H, CH₃), 2.24

(s, 2H, CH₂), 2.72 (s, 2H, CH₂), 3.33–3.41 (m, 2H, H_4,H_4’-pyrazoline), 6.80–6.88 (dd, 1H,

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H₅-pyrazoline; J = 17.4, 12.0 Hz), 7.58 (s, 1H, CH-pyrrole), 7.47 (bs, 2H, NH₂; exchangeable with

D₂O), 7.77 (d, 2H, ArH; J = 7.5 Hz), 7.93 (d, 2H, ArH; J = 7.5 Hz), 12.00 (bs, 1H, NH; exchangeable

with D₂O); ¹³C-NMR (DMSO-d₆) δ: 29.0, 31.4, 40.4, 41.0, 52.5, 63.5, 114.4, 120.2, 125.3, 129.3,

129.5, 130.6, 136.0, 140.0, 150.49, 180.0, 204.6. Anal. Calcd for C₂₀H₂₁ClN₄OS (400.92): C, 59.91; H,

5.28; N, 13.97 Found: C, 59.70; H, 5.11; N, 14.12.

5-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-

1-carbothioamide 7. Yellow crystals; yield 0.60 g, 76.7%; m.p. 259–260 °C ; IR (KBr): 1167 (C=S), 1651

1

(C=O), 3134, 3232, 3436 cm⁻¹(NH, NH₂); H-NMR (DMSO-d₆) δ 1.13 (s, 6H, 2 CH₃), 2.39 (s, 2H,

CH₂), 2.80 (s, 2H, CH₂), 3.86–3.89 (m, 4H, H₄-pyrazoline, OCH₃), 4.73–4.82 (m, 1H, H_4'-pyrazoline),

6.92–6.98 (m, 1H, H₅-pyrazoline), 7.15–7.25 (m, 5H, 4 ArH, CH-pyrrole), 8.01 (bs, 2H, NH₂;

exchangeable with D₂O), 11.59 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₂₁H₂₄N₄O₂S

(396.51): C, 63.61; H, 6.10; N, 14.13 Found: C, 63.80; H, 6.24; N, 13.90.

3.4. General Procedure for the Preparation of Compounds 8 and 9

A mixture of 2-(3-aryl-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one 2 or 3

(0.001 mol), hydrazinum chloride (0.003 mol) and anhydrous sodium acetate (0.003 mol), in ethanol

(15 mL) and glacial acetic acid (5 mL) was refluxed for 8 h. The reaction mixture was poured over

crushed ice. The solid obtained was filtered, washed with water, dried and recrystallized from ethanol.

2-(3-(4-Bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one

(8). Brown crystals; yield 0.31 g, 81.3%; m.p. 210–211 °C ; IR (KBr): 1641 (C=O), 3260, 3436 cm⁻¹

(NH); ¹H-NMR (DMSO-d₆) δ 0.98 (s, 6H, 2 CH₃), 2.24 (s, 2H, CH₂), 2.72 (s, 2H, CH₂), 3.33–3.75 (m,

2H, H₄, H_4'-pyrazoline), 6.50–6.70 (m, 1H, H₅-pyrazoline), 6.91 (s, 1H, CH-pyrrole), 7.77 (d, 2H,

ArH; J = 7.7 Hz), 7.93 (d, 2H, ArH; J = 7.7 Hz), 11.25 (bs, 1H, NH; exchangeable with D₂O), 12.08

(bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₁₉H₂₀BrN₃O (386.29): C, 59.08; H, 5.22; N,

10.88 Found: C, 59.26; H, 5.45; N, 10.65.

2-(3-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (9).

Buff crystals; yield 0.26 g, 78.8%; m.p. 195–196 °C ; IR (KBr): 1642 (C=O), 3209, 3423 cm⁻¹(NH);

¹H-NMR (DMSO-d₆) δ 0.98 (s, 3H, CH₃), 1.01 (s, 3H, CH₃), 2.20 (s, 2H, CH₂), 2.42 (s, 2H, CH₂),

3.53–3.75 (m, 1H, H₄-pyrazoline), 3.69–3.75 (m, 1H, H_4'-pyrazoline), 5.50 (m, 1H, H₅-pyrazoline),

6.69 (s, 1H, CH-pyrrole), 7.41 (d, 2H, ArH; J = 7.7 Hz), 7.78 (d, 2H, ArH; J = 7.7 Hz), 11.19 (bs, 1H,

NH; exchangeable with D₂O), 12.07 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for

C₁₉H₂₀ClN₃O (341.83): C, 66.76; H, 5.90; N, 12.29 Found: C, 66.53; H, 5.69; N, 12.50.

3.5. General Procedure for the Preparation of Compounds 10–12

A mixture of 2-(3-aryl-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-ones 2–4

(0.001 mol), hydroxylamine hydrochloride (0.003 mol) and anhydrous sodium acetate (0.003 mol), in

ethanol (15 mL) and glacial acetic acid (5 mL) was refluxed for 8 h. The reaction mixture was poured

over crushed ice. The solid obtained was filtered, washed with water, dried and recrystallized

from ethanol.

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2-(3-(4-Bromophenyl)-4,5-dihydroisoxazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (10).

Yellow crystals; yield 0.34 g, 90.1%; m.p. 115–116 °C ; IR (KBr): 1646 (C=O), 3423 cm⁻¹(NH);

¹H-NMR (DMSO-d₆) δ 0.95 (s, 6H, 2 CH₃), 2.42 (s, 2H, CH₂), 2.51 (s, 2H, CH₂), 3.34–3.46 (m, 2H,

H₄, H_4'-isoxazoline), 6.50–6.52 (m, 1H, H₅isoxazoline), 6.84 (s, 1H, CH-pyrrole), 7.49–7.56 (m, 4H,

ArH), 11.32 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₁₉H₁₉BrN₂O₂(387.27): C, 58.93;

H, 4.95; N, 7.23 Found: C, 58.75; H, 4.86; N, 7.39.

2-(3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (11).

1

Buff crystals; yield 0.26 g, 78.3%; m.p. 160–161 °C ; IR (KBr): 1643 (C=O), 3433 cm⁻¹(NH); H-

NMR (DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.35 (s, 2H, CH₂), 2.59 (s, 2H, CH₂), 3.50–3.55 (m, 1H, H₄-

isoxazoline), 3.66–3.72 (m, 1H, H_4'-isoxazoline), 5.66–5.70 (m, 1H, H₅isoxazoline), 6.81 (s, 1H, CH-

pyrrole), 7.51 (d, 2H, ArH; J = 6.7 Hz), 7.70 (d, 2H, ArH; J = 6.7 Hz), 11.97 (bs, 1H, NH;

exchangeable with D₂O). ¹³C-NMR (DMSO-d₆) δ: 27.9, 34.4, 36.6, 43.3, 55.0, 76.8, 108.1, 109.8,

128.1, 128.9, 129.4, 131.9, 135.2, 143.6, 156.5, 193.7. Anal. Calcd for C₁₉H₁₉ClN₂O₂(342.82): C,

66.57; H, 5.59; N, 8.17 Found: C, 66.40; H, 5.35; N, 8.34.

2-(3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one (12).

Buff crystals; yield 0.27 g, 81.0%; m.p. 170–171 °C ; IR (KBr): 1645 (C=O), 3427 cm⁻¹(NH);

¹H-NMR (DMSO-d₆) δ 0.98 (s, 6H, 2 CH₃), 2.46 (s, 4H, 2 CH₂), 3.34 (s, 3H, OCH₃), 3.46–3.63 (m,

1H, H₄-isoxazoline), 4.08–4.22 (m, 1H, H_4'-isoxazoline), 5.55–5.66 (m, 1H, H₅isoxazoline), 6.72 (s,

1H, CH-pyrrole), 6.98 (d, 2H, ArH; J = 6.9 Hz), 7.62 (d, 2H, ArH; J = 6.9 Hz), 11.16 (bs, 1H, NH;

exchangeable with D₂O). Anal. Calcd for C₂₀H₂₂N₂O₃(338.40): C, 70.99; H, 6.55; N, 8.28 Found: C,

70.79; H, 6.31; N, 8.47.

3.6. General Procedure for the Preparation of Compounds 13–15

A mixture of 2-(3-aryl-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one 2–4

(0.001 mol) and 4-hydrazinylbenzenesulfonamide hydrochloride (0.001 mol) in methanol (30 mL) was

heated under reflux for 6 h, partially concentrated and cooled. The separated solid product was filtered,

washed with ethanol, dried and recrystallized from ethanol.

4-(3-(4-Bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-

1-yl)benzenesulfonamide (13). Brown crystals; yield 0.48 g, 90.2%; mp 180–181 °C ; IR (KBr): 1219,

1374 (SO₂), 1651 (C=O), 3250, 3434 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 0.93 (s, 3H, CH₃), 1.01

(s, 3H, CH₃), 2.50 (s, 2H, CH₂), 2.66 (s, 2H, CH₂), 3.15–3.22 (m, 1H, H₄-pyrazoline), 3.36–3.40 (m,

1H, H_4'-pyrazoline), 5.53–5.56 (m, 1H, H₅- pyrazoline), 6.43 (s, 1H, CH-pyrrole), 6.63 (bs, 2H, NH₂;

exchangeable with D₂O), 7.00–7.26 (m, 2H, ArH), 7.40–7.70 (m, 2H, ArH), 7.73–7.93 (m, 2H, ArH),

8.05–8.18 (m, 2H, ArH), 11.61 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₂₅H₂₅BrN₄O₃S

(541.46): C, 55.46; H, 4.65; N, 10.35 Found: C, 55.60; H, 4.87; N, 10.15.

4-(3-(4-Chlorophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-

1-yl)benzenesulfonamide (14). Brown crystals; yield 0.43 g, 87.5%; m.p. 150–151 °C ; IR (KBr): 1219,

1374 (SO₂), 1651 (C=O), 3256, 3438 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.24

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(s, 2H, CH₂), 2.72 (s, 2H, CH₂), 3.20–3.40 (m, 1H, H₄-pyrazoline), 3.78–3.84 (dd, 1H, H_4'-pyrazoline;

J = 17.4, 12.0 Hz), 5.53–5.55 (m, 1H, H₅-pyrazoline), 6.85 (s, 1H, CH-pyrrole), 7.13 (d, 2H, ArH;

J = 9.1 Hz), 7.34 (bs, 2H, NH₂; exchangeable with D₂O), 7.43 (d, 2H, ArH; J = 9.1 Hz), 7.53 (d, 2H,

ArH; J = 8.4 Hz), 7.69 (d, 2H, ArH; J = 8.4 Hz), 12.18 (bs, 1H, NH; exchangeable with D₂O). Anal.

Calcd for C₂₅H₂₅ClN₄O₃S (497.01): C, 60.41; H, 5.07; N, 11.27 Found: C, 60.66; H, 5.30; N, 11.03.

4-(5-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-

1-yl)benzenesulfonamide (15). Buff crystals; yield 0.44 g, 91.1%; m.p. 224–225 °C ; IR (KBr): 1158,

1

1307 (SO₂), 1650 (C=O), 3263, 3345 cm⁻¹(NH, NH₂); H-NMR (DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃),

2.20 (s, 2H, CH₂), 2.68 (s, 2H, CH₂), 3.24–3.28 (m, 1H, H₄-pyrazoline), 3.34 (s, 3H, OCH₃), 3.76–4.00

(m, 1H, H_4'-pyrazoline), 4.80–5.53 (m, 1H, H₅-pyrazoline), 6.46 (s, 1H, CH-pyrrole), 7.00–7.04 (m, 4H,

2 ArH, NH₂), 7.13 (d, 2H, ArH; J = 8.4 Hz), 7.60 (d, 2H, ArH; J = 8.4 Hz), 7.70–7.73 (m, 2H, ArH),

11.65 (bs, 1H, NH; exchangeable with D₂O). ¹³C-NMR (DMSO-d₆) δ: 28.7, 35.1, 40.9, 43.7, 53.6,

55.7, 60.3, 106.4, 114.7, 118.4, 119.9, 125.4, 127.6, 127.9, 130.3, 141.6, 142.3, 153.2, 160.0, 193.8.

Anal. Calcd for C₂₆H₂₈N₄O₄S (492.59): C, 63.40; H, 5.73; N, 11.37 Found: C, 63.69; H, 5.90; N, 11.22.

3.7. General Procedure for the Preparation of Compounds 16–18

Solid 4-(3-aryl-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-1-

yl)benzenesulfonamide 13–15 (0.001 mol) was dissolved in hot acetic acid, and after cooling bromine

(0.3 mL) in acetic acid (10 mL) was added dropwise with shaking, then the reaction mixture was

allowed to stand at R.T. overnight. The solid mass obtained was separated by filtration, washed with

water, dried and recrystallized from ethanol.

4-(4-Bromo-3-(4-bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-1H-pyrazol-

1-yl)benzenesulfonamide (16). Buff crystals; yield 0.49 g, 79.3%; m.p. 200–201 °C ; IR (KBr): 1162,

1335 (SO₂), 1653 (C=O), 3253, 3433 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 1.09 (s, 6H, 2 CH₃), 2.26

(s, 2H, CH₂), 2.86 (s, 2H, CH₂), 3.58–3.88 (m, 1H, H₄-pyrazoline), 4.80–5.00 (m, 1H, H₅-pyrazoline),

7.44–7.46 (m, 3H, CH-pyrrole, NH₂), 7.71 (d, 2H, ArH; J = 7.7 Hz), 7.86 (d, 2H, ArH; J = 8.4 Hz), 7.92

(d, 2H, ArH; J = 8.4 Hz), 8.07 (d, 2H, ArH; J = 7.7 Hz), 9.02 (bs, 1H, NH; exchangeable with D₂O).

Anal. Calcd for C₂₅H₂₄Br₂N₄O₃S (620.36): C, 48.40; H, 3.90; N, 9.03 Found: C, 48.80; H, 3.76; N, 8.87.

4-(4-Bromo-3-(4-chlorophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-1H-pyrazol-

1-yl)benzenesulfonamide (17). Brown crystals; yield 0.47 g, 82.4%; m.p. 129–130 °C ; IR (KBr): 1163,

1315 (SO₂), 1670 (C=O), 3068, 3385 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 1.16 (s, 6H, 2 CH₃), 2.27

(s, 2H, CH₂), 2.47 (s, 2H, CH₂), 3.13–3.73 (m, 1H, H₄-pyrazoline), 3.88–4.16 (m, 1H, H₅-pyrazoline),

7.56 (s, 1H, H-pyrrole), 7.63 (bs, 2H, NH₂; exchangeable with D₂O), 7.80–7.85 (m, 4H, ArH), 8.00–

8.10 (m, 4H, ArH), 11.06 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₂₅H₂₄BrClN₄O₃S

(575.91): C, 52.14; H, 4.20; N, 9.73 Found: C, 52.25; H, 4.00; N, 9.58.

4-(4-Bromo-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-3-(4-methoxy-phenyl)-1H-pyrazol-

1-yl)benzenesulfonamide (18). Brown crystals; yield 0.48 g, 84.3%; m.p. 160–161 °C ; IR (KBr): 1164,

1340 (SO₂), 1650 (C=O), 3220, 3435 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 1.01 (s, 3H, CH₃), 1.02

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(s, 3H, CH₃), 2.26 (s, 2H, CH₂), 2.70 (s, 2H, CH₂), 3.34 (s, 3H, OCH3), 3.80–3.89 (m, 2H, H₄, H₅-

pyrazoline), 6.93 (d, 1H, ArH; J = 8.4 Hz), 7.03–7.07 (m, 2H, ArH, CH-pyrrole), 7.42 (d, 2H, ArH; J =

9.1 Hz), 7.82 (d, 2H, ArH; J = 9.1 Hz), 7.87–7.93 (m, 4H, 2ArH, NH₂), 12.25 (bs, 1H, NH;

exchangeable with D₂O). Anal. Calcd for C₂₆H₂₇BrN₄O₄S (571.49): C, 54.64; H, 4.76; N, 9.80 Found:

C, 55.01; H, 4.65; N, 9.68.

3.8. 4-(4-Bromo-3-aryl-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-1H-pyrazol-1-yl)-

N-(phenylcarbamothioyl)benzenesulfonamide (19)

A mixture of 4-(4-bromo-3-(4-chlorophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-

yl)-1H-pyrazol-1-yl) benzenesulfonamide (17, 0.005 mol) and anhydrous potassium carbonate (0.01

mol) in dry acetone (100 mL) was stirred under reflux for 15 h. A solution of phenyl isothiocyanate

(0.007 mol) in dry acetone was added drop by drop at this temperature, and refluxing was continued

for 12 h more. The acetone was distilled under reduced pressure and the solid residue was dissolved in

water, the product was isolated after acidification with 2 N HCl. The solid mass obtained was

separated by filtration, washed with water, dried and recrystallized from ethanol. The product was

obtained as buff crystals; yield 2.73 g, 76.8%; m.p. 92–93 °C; IR (KBr): 1092 (C=S), 1160, 1345

1

(SO₂), 1648 (C=O), 3435 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.19 (s, 6H, 2 CH₃), 1.99 (s, 2H, CH₂),

2.61 (s, 2H, CH₂), 3.87–4.03 (m, 1H, H₄-pyrazoline), 6.90–6.92 (m, 1H, H₅-pyrazoline), 7.09 (s, 1H,

CH-pyrrole), 7.22–7.27 (m, 2H, ArH), 7.29–7.32 (m, 3H, ArH), 7.40–7.45 (m, 4H, ArH), 7.85 (d, 2H,

ArH; J = 8.4 Hz), 8.06 (d, 2H, ArH; J = 8.4 Hz), 8.62 (bs, 1H, NH; exchangeable with D₂O), 9.76 (bs,

1H, NH; exchangeable with D₂O), 11.11 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for

C₃₂H₂₉BrClN₅O₃S₂(711.09): C, 54.05; H, 4.11; N, 9.85 Found: C, 54.00; H, 3.96; N, 10.04.

3.9. General Procedure for the Preparation of Compounds 20 and 21

A mixture of 4-(3-aryl-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-

pyrazol-1-yl)benzenesulfonamide 13 and 14 (0.005 mol) and anhydrous potassium carbonate

(0.01 mol) in dry acetone (100 mL) was stirred with refluxing for 15 h. A solution of phenyl

isothiocyanate (0.007 mol) in dry acetone was added drop by drop at this temperature, and refluxing

was continued for 12 h. The acetone was distilled under reduced pressure and the solid residue was

dissolved in water, the product was isolated after acidification with 2 N HCl. The solid mass obtained

was separated by filtration, washed with water, dried and crystallized from ethanol.

4-(3-(4-Bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-

1-yl)-N-(phenylcarbamothioyl)benzenesulfonamide (20). the product was obtained as brown crystals;

Yield 2.86 g, 84.6%; m.p. 210–211 °C ; IR (KBr): 1089 (C=S), 1160, 1355 (SO₂), 1646 (C=O),

1

3436 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.19 (s, 6H, 2 CH₃), 2.69 (s, 2H, CH₂), 2.85 (s, 2H, CH₂),

3.50–3.60 (m, 1H, H₄-pyrazoline), 4.08–4.15 (m, 1H, H_4'-pyrazoline), 4.40–4.55 (m, 1H, H₅-pyrazoline),

6.57 (s, 1H, CH-pyrrole), 7.10–7.18 (m, 2H, ArH), 7.20–7.33 (m, 2H, ArH), 7.40–7.55 (m, 1H, ArH),

7.60–7.72 (m, 2H, ArH), 7.80–7.93(m, 2H, ArH), 7.95–8.18 (m, 2H, ArH), 8.61–8.68 (m, 2H, ArH),

9.15 (bs, 1H, NH; exchangeable with D₂O), 10.54 (bs, 1H, NH; exchangeable with D₂O), 11.03 (bs,

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1H, NH; exchangeable with D₂O). Anal. Calcd for C₃₂H₃₀BrN₅O₃S₂(676.65): C, 56.80; H, 4.47; N,

10.35 Found: C, 56.62; H, 4.26; N, 10.54.

4-(3-(4-Chlorophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol-

1-yl)-N-(phenylcarbamothioyl)benzenesulfonamide (21). Brown crystals; yield 2.49 g, 78.9%; m.p.

1

168–169 °C ; IR (KBr): 1086 (C=S), 1160, 1355 (SO₂), 1653 (C=O), 3434 cm⁻¹(NH); H-NMR

(DMSO-d₆) δ 1.16 (s, 6H, 2 CH₃), 2.71 (s, 2H, CH₂), 2.87 (s, 2H, CH₂), 3.89–4.07 (m, 1H, H₄-

pyrazoline), 4.40–4.50 (m, 1H, H_4'-pyrazoline), 6.90–6.94 (m, 1H, H₅-pyrazoline), 7.02 (s, 1H, CH-

pyrrole), 7.11–7.23 (m, 3H, ArH), 7.40–7.43 (m, 4H, ArH), 7.90–7.94 (m, 4H, ArH), 8.66–8.73 (m, 2H,

ArH), 12.41 (bs, 2H, 2NH; exchangeable with D₂O), 12.73 (bs, 1H, NH; exchangeable with D₂O). Anal.

Calcd for C₃₂H₃₀ClN₅O₃S₂(632.20): C, 60.79; H, 4.78; N, 11.08 Found: C, 60.56; H, 4.62; N, 10.87.

3.10. 5H-[1,2,4]Triazino[5,6-b]indole-3-thiol (22)

A mixture of indoline-2,3-dione [69,70] (1'', 0.1 mol), thiosemicarbazide (0.11 mol) and anhydrous

potassium carbonate (0.15 mol) [71] were stirred in water (500 mL) for 2 h at R.T., and then refluxed

for 5 h. The mixture was cooled, filtered, and the filtrate was acidified with acetic acid. The solid mass

obtained was separated by filtration, washed with water and dried. The product was recrystallized from

ethanol. The product was obtained as yellow crystals; yield 19.29 g, 95.4%; m.p. 334–335 °C ; IR

1

(KBr): 3423 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 7.27 (t, 1H, ArH; J = 7.6 Hz), 7.37 (d, 1H, ArH; J =

7.6 Hz), 7.55 (t, 1H, ArH; J = 7.6 Hz), 7.92 (d, 1H, ArH; J = 7.6 Hz), 12.43 (bs, 1H, NH;

exchangeable with D₂O), 14.54 (bs, 1H, NH; exchangeable with D₂O); ¹³C-NMR (DMSO-d₆) δ:113.5,

118.1, 122.3, 123.3, 132.3, 136.1, 143.5, 149.6, 179.5. Anal. Calcd for C₉H₆N₄S (202.24): C, 53.45; H,

2.99; N, 27.70 Found: C, 53.60; H, 3.23; N, 27.58.

3.11. 5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxaline-3-thiol (23)

A mixture of quinoxaline-2,3(1H,4H)-dione (1''', 0.01 mol), thiosemicarbazide (0.011 mol) and

anhydrous potassium carbonate (0.015 mol) [71] was stirred in water (500 mL) for 2 h at R.T., and then

refluxed for 5 h. The mixture was cooled, filtered, and the filtrate was acidified with acetic acid. The

solid mass obtained was separated by filtration, washed with water and dried. The product was

recrystallized from ethanol. The product was obtained as off white crystals; yield 1.81 g, 83.6%; m.p.

1

>300 °C; IR (KBr): 3160, 3440 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 7.03 (d, 2H, ArH; J = 7.6 Hz),

7.08 (t, 2H, ArH; J = 7.6 Hz), 11.88 (bs, 2H, 2NH; exchangeable with D₂O), 14.50 (bs, 1H, NH;

exchangeable with D₂O). Anal. Calcd for C₉H₇N₅S (217.25): C, 49.76; H, 3.25; N, 32.24 Found: C,

49.90; H, 3.40; N, 32.00.

3.12. 3-Hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24)

A mixture of 5H-[1,2,4]triazino[5,6-b] indole-3-thiol (22, 0.01 mol) [71] and hydrazine hydrate

(10 mL, 98%) was heated on water bath for 5 h. The product was collected, washed with ethanol and

dried. It was recrystallized from ethanol. The product was obtained as yellow crystals; yield 1.85 g,

92.6%; m.p. 260–261 °C ; IR (KBr): 3176, 3242, 3300, 3404 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ

4.31 (bs, 2H, NH₂; exchangeable with D₂O), 7.24 (t, 1H, ArH; J = 7.9 Hz), 7.38 (d, 1H, ArH; J = 7.9 Hz),

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7.44 (t, 1H, ArH; J = 7.9 Hz), 8.07 (d, 1H, ArH; J = 7.9 Hz), 8.54 (bs, 1H, NH; exchangeable with

D₂O), 11.82 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₉H₈N₆(200.20): C, 53.99; H,

4.03 N, 41.98 Found: C, 54.10; H, 4.22; N, 41.80.

3.13. 3-Hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline (25)

A mixture of 5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline-3-thiol (23, 0.01 mol) and hydrazine

hydrate (10 mL, 98%) [71] was heated on water bath for 5 h. The product was collected, washed with

ethanol, dried and recrystallized from ethanol. The product was obtained as buff crystals; yield 1.83 g,

1

85.2%; m.p. 255–256 °C; IR (KBr): 3176, 3237, 3271, 3300, 3402 cm⁻¹(NH, NH₂); H-NMR

(DMSO-d₆) δ 4.55 (bs, 2H, NH₂; exchangeable with D₂O), 7.07 (t, 2H, ArH; J = 7.6 Hz), 7.29 (d, 2H,

ArH; J = 7.6 Hz), 8.90 (bs, 1H, NH; exchangeable with D₂O),11.89 (bs, 2H, 2 NH; exchangeable with

D₂O). Anal. Calcd for C₉H₉N₇(215.21): C, 50.23; H, 4.22; N, 45.56 Found: C, 50.40; H, 4.45; N, 45.39.

3.14. Ethyl [1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5H-indole-5-ethanoate (26)

A mixture of 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24, 0.01 mol) [70] and diethyl malonate

(20 mL) [72] was heated 10 h. The product was filtered off, washed with ethanol and dried. It was

recrystallized from ethanol. The product was obtained as buff crystals; yield 2.55g, 86.3%; m.p.

1

283–284 °C ; IR (KBr): 1737 (C=O), 3408 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.16 (t, 3H, CH₃-ester;

J = 6.7 Hz), 4.12 (s, 2H, CH₂), 4.31 (q, 2H, CH₂-ester; J = 6.7 Hz), 7.20–7.26 (m, 1H, ArH), 7.36–7.38

(m, 1H, ArH), 7.60–7.65 (m, 1H, ArH), 8.00–8.10 (m, 1H, ArH), 12.18 (bs, 1H, NH; exchangeable

with D₂O). Anal. Calcd for C₁₄H₁₂N₆O₂(296.28): C, 56.75; H, 4.08; N, 28.36 Found: C, 56.90; H,

4.24; N, 28.21.

3.15. Ethyl [1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5,10-dihydroquinoxaline-5-ethanoate (27)

A mixture of 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline (25, 0.01 mol) and

diethyl malonate (20 mL) [72] was heated 10 h. The product was filtered off, washed with ethanol,

dried and recrystallized from ethanol. The product was obtained as brown crystals; yield 2.77 g,

89.0%; m.p. 280–281 °C; IR (KBr): 1713 (C=O), 3150, 3430 cm⁻(NH); ¹H-NMR (DMSO-d₆) δ 1.09

(t, 3H, CH₃; J = 6.9 Hz), 4.09 (q, 2H, CH₂; J = 6.9 Hz), 4.66 (s, 2H, CH₂), 7.24 (d, 1H, ArH; J = 8.4

Hz), 7.37–7.41 (m, 2H, ArH), 7.74 (d, 1H, ArH; J = 8.4 Hz), 12.10 (bs, 2H, 2 NH; exchangeable with

D₂O). ¹³C-NMR (DMSO-d₆) δ: 14.4, 34.5, 62.0, 123.7, 128.5, 129.7, 145.2, 147.0, 152.1, 168.4. Anal.

Calcd for C₁₄H₁₃N₇O₂(311.30): C, 54.02; H, 4.21; N, 31.50 Found: C, 54.25; H, 4.41; N, 31.38.

3.16. [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5H-indole-5-ethanoic acid hydrazide (28)

A solution of ester 26 (0.01 mol) in ethanol (30 mL) and hydrazine hydrate 98% (10 mL) [72] was

refluxed for 6 h. The product was collected, washed with ethanol and dried. It was recrystallized from

ethanol. The product was obtained as buff crystals; yield 2.15 g, 76.5%; m.p. >300 °C , IR (KBr): 1657

(C=O), 3176, 3242, 3308, 3434 cm⁻¹(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 4.24 (s, 2H, CH₂), 7.24–7.30

(m, 1H, ArH), 7.36–7.41 (m, 1H, ArH), 7.62–7.68 (m, 1H, ArH), 8.03–8.10 (m, 1H, ArH), 9.20 (bs,

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1H, NH), 9.89 (bs, 2H, NH₂; exchangeable with D₂O), 12.18 (bs, 1H, NH; exchangeable with D₂O).

Anal. Calcd for C₁₂H₁₀N₈O (282.26): C, 51.06; H, 3.57; N, 39.70 Found: C, 51.20; H, 4.11; N, 39.48.

3.17. [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5,10-dihydroquinoxaline-5-ethanoic acid hydrazide (29)

A solution of ester 27 (0.01 mol) in ethanol (30 mL) and hydrazine hydrate 98% (10 mL) [72] was

refluxed for 6 h. The product was collected, washed with ethanol, dried and recrystallized from

ethanol. The product was obtained as orange crystals; yield 2.36 g, 79.6%; m.p. 250–251 °C; IR (KBr):

1675 (C=O), 3190, 3306, 3434 cm⁻⁽(NH, NH₂); ¹H-NMR (DMSO-d₆) δ 4.34 (s, 2H, CH₂), 7.24 (t, 1H,

ArH; J = 7.6 Hz), 7.38 (d, 1H, ArH; J = 7.6 Hz), 7.44 (t, 1H, ArH; J = 7.6 Hz), 7.87 (d, 1H, ArH;

J = 7.6 Hz), 8.90 (bs, 1H, NH; exchangeable with D₂O), 9.61 (bs, 2H, NH₂; exchangeable with D₂O),

12.08 (bs, 2H, 2 NH; exchangeable with D₂O). Anal. Calcd for C₁₂H₁₁N₉O (297.28): C, 48.48; H, 3.73;

N, 42.41 Found: C, 48.63; H, 3.90; N, 42.30.

3.18. [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5H-indole-5-N-(phenylcarbamothioyl) ethanoic acid

hydrazide (30)

A mixture of acid hydrazide 28 (0.01 mol) and anhydrous potassium carbonate (0.05 mol) [71] in

absolute ethanol (25 mL) was treated by drop wise addition of phenyl isothiocyanate (0.2 mol) in dry

acetone (10 mL) .The reaction mixture was heated under reflux for 12 h. The product was collected,

washed with ethanol and dried. The acetone was distilled under reduced pressure and the solid residue

was dissolved in water, the product was isolated after acidification with 2 N HCl. The solid mass

obtained was separated by filtration, washed with water, dried and recrystallized from ethanol The

product was obtained as orange crystals; yield 3.34 g, 80.1%; m.p. 270–271 °C ; IR (KBr): 1105 (C=S),

1

1651 (C=O), 3236, 3433 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 2.06 (s, 2H, CH₂), 6.89–6.92 (m, 2H,

ArH, NH), 7.24–7.28 (m, 4H, ArH), 7.50–7.53 (m, 4H, ArH), 9.88 (bs, 2H, NH; exchangeable with

D₂O), 11.82 (bs, 1H, NH; exchangeable with D₂O); ¹³C-NMR (DMSO-d₆) δ: 31.4, 117.5, 117.5, 117.6,

117.6, 121.7, 121.7, 122.1, 123.6, 127.2, 129.6, 129.7, 141.9, 155.0, 156.7, 178.7, 189.4. Anal. Calcd

for C₁₉H₁₅N₉OS (417.45): C, 54.67; H, 3.62; N, 30.20 Found: C, 54.40; H, 3.43; N, 30.42.

3.19. [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5,10-dihydroquinoxaline-5-N-(phenylcarbamothioyl)-

ethanoic acid hydrazide (31)

A mixture of acid hydrazide 35 (0.001 mol) and anhydrous potassium carbonate (0.005 mol) in

absolute ethanol (25 mL) was treated by dropwise addition of phenyl isothiocyanate (0.02 mol) [72] in

dry acetone (10 mL) .The reaction mixture was heated under reflux for 12 h. The product was

collected, washed with ethanol and dried. It was recrystallized from ethanol. The acetone was distilled

under reduced pressure and the solid residue was dissolved in water, the product was isolated after

acidification with 2 N HCl. The solid mass obtained was separated by filtration, washed with water,

dried and recrystallized from ethanol. The product was obtained as buff crystals; yield 0.34 g, 80.0%;

m.p. 275–276 °C; IR (KBr): 1109 (C=S), 1651 (C=O), 3236, 3433 cm⁻¹(NH); ¹H-NMR (DMSO-d₆) δ

4.25 (s, 2H, CH₂), 7.00–7.54 (m, 5H, 4ArH, NH), 7.56–8.00 (m, 7H, 5ArH, 2NH), 12.05 (m, 2H, 2NH;

Molecules 2013, 18

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exchangeable with D₂O). Anal. Calcd for C₁₉H₁₆N₁₀OS (432.46): C, 52.77; H, 3.73; N, 32.39 Found:

C, 52.49; H, 3.53; N, 32.52.

3.20. 1-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (32)

A mixture of 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24, 0.01 mol) and ethyl acetoacetate

(0.011 mol) [73] in acetic acid (20 mL) was stirred with refluxing for 4 h. The reaction mixture was

evaporated till dryness, the product was collected, washed with ethanol and dried. It was recrystallized

from ethanol. The product was obtained as buff crystals; yield 2.47 g, 93.0%; m.p. 330–331 °C ; IR

1

(KBr): 1712 (C=O), 3128, 3438 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.80 (s, 3H, CH₃), 2.43 (s, 2H,

CH₂), 7.45 (t, 1H, ArH; J = 6.7 Hz), 7.61 (d, 1H, ArH; J = 6.7 Hz), 7.72 (t, 1H, ArH; J = 6.7 Hz), 8.36

(d, 1H, ArH; J = 6.7 Hz), 12.00 (bs, 1H, NH; exchangeable with D₂O). Anal. Calcd for C₁₃H₁₀N₆O

(266.26): C, 58.64; H, 3.79; N, 31.56 Found: C, 58.80; H, 3.90; N, 31.43.

3.21. 1-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (33)

A mixture of 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline 25 (0.01 mol) and ethyl

acetoacetate (0.011 mol) [73] in acetic acid (20 mL) was stirred with refluxing for 4 h. Evaporation till

dryness, the product was collected, washed with ethanol and dried. It was recrystallized from ethanol.

The product was obtained as buff crystals; yield 2.45 g, 87.2%; m.p. 239–240 °C; IR (KBr): 1693

1

(C=O), 3161, 3438 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 2.29 (s, 3H, CH₃), 2.92 (s, 2H, CH₂), 7.23 (t,

1H, ArH; J = 7.6 Hz), 7.33 (d, 1H, ArH; J = 7.6 Hz), 7.39 (t, 1H, ArH; J = 7.6 Hz), 7.98 (d, 1H, ArH;

J = 7.6 Hz), 10.99 (bs, 1H, NH; exchangeable with D₂O), 11.68 (bs, 1H, NH; exchangeable with D₂O).

¹³C-NMR (DMSO-d₆) δ: 15.0, 43.8, 127.9, 130.7, 144.5, 152.9, 155.7, 161.8, 170.0. Anal. Calcd for

C₁₃H₁₁N₇O (281.27): C, 55.51; H, 3.94; N, 34.86 Found: C, 55.68; H, 4.03; N, 34.70.

3.22. 1-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-3-methyl-4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one (34)

A solution of 1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (32, 0.01 mol)

in acetone (25 mL) [73] was stirred with refluxing for 20 h. The reaction mixture was evaporated till

dryness, cooled. 20 mL of water was added in order to precipitate the product. The crude product was

filtered, washed with ethanol, dried, and recrystallized from ethanol. The product was obtained as off

white crystals; yield 2.47 g, 80.7%; m.p. 360–361 °C ; IR (KBr): 1713 (C=O), 3439 cm⁻¹(NH);

¹H-NMR (DMSO-d₆) δ 1.87 (s, 6H, 2 CH₃), 2.49 (s, 3H, CH₃), 7.44 (t, 1H, ArH; J = 7.6 Hz), 7.62 (d,

1H, ArH; J = 7.6 Hz), 7.71 (t, 1H, ArH; J = 7.6 Hz), 8.36 (d, 1H, ArH; J = 7.6 Hz), 13.08 (bs, 1H, NH;

exchangeable with D₂O). Anal. Calcd for C₁₆H₁₄N₆O (306.32): C, 62.74; H, 4.61; N, 27.44 Found: C,

62.97; H, 5.03; N, 27.70.

3.23. 1-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-4-(propan-2-ylidene)-1H-pyrazol-

5(4H)-one (35)

A solution of 1-(5,10-dihyro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H-pyrazol-5(4H)-one

(33, 0.01 mol) in acetone (25 mL) [73] was stirred with refluxing for 20 h. Evaporation till dryness,

cooled, water was added, filter the product was collected, washed with ethanol and dried. It was

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recrystallized from ethanol. The product was obtained as buff crystals; yield 2.66 g, 82.9%; crystals;

1

m.p. 263–264 °C ; IR (KBr): 1701 (C=O), 3202, 3433 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 2.30 (s, 3H,

CH₃), 2.42 (s, 3H, CH₃), 2.93 (s, 3H, CH₃), 7.00–7.05 (m, 2H, ArH), 7.35–7.39 (m, 2H, ArH), 11.88 (bs,

13

2H, 2 NH; exchangeable with D₂O); C-NMR (DMSO-d₆) δ:14.6, 19.2, 107.3, 115.6, 123.5, 126.1,

137.4, 153.0, 155.7, 159.5, 161.8. Anal. Calcd for C₁₆H₁₅N₇O (321.34): C, 59.80; H, 4.71; N, 30.51

Found: C, 60.03; H, 4.93; N, 30.37.

3.24. 2-((2-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)hydrazono)methyl)-6,6-dimethyl-6,7-dihydro-1H-indol-

4(5H)-one (36)

A mixture of 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24, 0.001 mol) and 6,6-dimethyl-4-oxo-

4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1', 0.0011 mol) was refluxed in ethanol (30 mL) for 6 h.

The product was collected, washed with ethanol and dried, it was recrystallized from ethanol. The

product was obtained as brown crystals; yield 0.32 g, 87.6%; m.p. 290–291 °C ; IR (KBr): IR (KBr):

1

1652 (C=O), 3234, 3439 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.00 (s, 6H, 2 CH₃), 2.21 (s, 2H, CH₂),

2.68 (s, 2H, CH₂), 6.53 (s, 1H, CH-pyrrole), 7.30 (t, 1H, ArH; J = 7.9 Hz), 7.46 (d, 1H, ArH;

J = 7.9 Hz ), 7.51 (t, 1H, ArH; J = 7.9 Hz), 8.03 (s, 1H, CH=N), 8.13 (d, 1H, ArH; J = 7.9 Hz), 8.34

(bs, 1H, NH; exchangeable with D₂O), 11.52 (bs, 1H, NH; exchangeable with D₂O), 11.89 (bs, 1H,

NH; exchangeable with D₂O). ¹³C-NMR (DMSO-d₆) δ: 28.7, 35.7, 36.6, 52.2, 108.2, 112.7, 119.4,

119.9, 120.6, 122.5, 129.4, 129.6, 136.1, 138.6, 140.3, 146.3, 148.8, 158.7, 192.6. Anal. Calcd for

C₂₀H₁₉N₇O (373.41): C, 64.33; H, 5.13; N, 26.26 Found: C, 64.63; H, 4.90; N, 26.30.

3.25. 2-((2-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)hydrazono)methyl)-6,6-dimethyl-6,7-

dihydro-1H-indol-4(5H)-one (37)

A mixture of 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b] quinoxaline (25, 0.001 mol) and 6,6-

dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1a, 0.0011 mol) was refluxed in ethanol

(30 mL) for 6 h. The product was collected, washed with ethanol, dried and recrystallized from

ethanol. The product was obtained as yellow crystals; yield 0.33 g, 86.6%; m.p. 155–156 °C; IR (KBr):

1651 (C=O), 3236, 3433 cm⁻¹(NH); ¹H-NMR (DMSO-d₆) δ 0.97 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 2.21

(s, 2H, CH₂), 2.69 (s, 2H, CH₂), 6.82 (s, 1H, CH-pyrrole), 7.03–7.06 (m, 2H, ArH), 7.07–7.10 (m, 2H,

ArH), 9.45 (s, 1H, CH=N), 10.34 (bs, 1H, NH; exchangeable with D₂O), 11.16 (bs, 1H, NH;

exchangeable with D₂O), 11.91 (bs, 1H, NH; exchangeable with D₂O), 12.11 (bs, 1H, NH;

exchangeable with D₂O). Anal. Calcd for C₂₀H₂₀N₈O (388.43): C, 61.84; H, 5.19; N, 28.85 Found: C,

62.00; H, 5.31; N, 28.70.

3.26. 3-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-2-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-

yl)thiazolidin-4-one (38)

A mixture of 2-((2-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)hydrazono)methyl)-6,6-dimethyl-6,7-dihydro-

1H-indol-4(5H)-one (36, 0.001 mol) and thioglycolic acid (0.0012 mol) was refluxed in dry benzene

(30 mL) on water bath for 10 h, cooled and poured onto water. The reaction mixture was extracted

with benzene three times, washed with sodium bicarbonate, water, dried over anhydrous sodium

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sulfate, then concentrated to half its volume. The separated solid product was filtered, washed ethanol,

dried and recrystallized from ethanol. The product was obtained as buff crystals; yield 0.33 g, 77.2%;

m.p. 215–216 °C ; IR (KBr): 1650, 1710 (C=O), 3427 cm⁻¹(NH); ¹H-NMR (DMSO-d₆) δ 1.00 (s, 6H,

2 CH₃), 2.20 (s, 2H, CH₂), 2.69 (s, 2H, CH₂), 3.35–3.49 (m, 2H, CH₂–thiazol.), 6.52 (s, 1H,

CH-pyrrole), 7.24 (t, 1H, ArH; J = 7.7 Hz), 7.43–7.49 (m, 2H, ArH), 7.93 (s, 1H, H-thiazol.), 8.08 (d,

1H, ArH; J = 7.7 Hz), 11.46 (bs, 1H, NH; exchangeable with D₂O), 12.03 (bs, 1H, NH; exchangeable

with D₂O). Anal. Calcd for C₂₂H₂₀N₆O₂S (432.50): C, 61.10; H, 4.66; N, 19.43 Found: C, 61.36; H,

4.86; N, 19.21.

3.27. N'-2-((2-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)hydrazono)methyl)-6,6-dimethyl-

6,7-dihydro-1H-indol-4(5H)-ylidene)benzohydrazide (39)

To a solution of 2-((2-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)hydrazono)methyl)-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (37, 0.001 mol) in ethanol (30 mL) was added benzoyl

hydrazine (0.163 g, 0.0012 mol) and two drops of acetic acid. The reaction mixture was heated under

reflux for 6 h, partially concentrated and cooled. The separated solid product was filtered, washed with

ethanol, dried and recrystallized from ethanol. The product was obtained as yellow crystals; yield 0.40 g,

79.7%; m.p. 175–176 °C; IR (KBr): 1644 (C=O), 3236, 3426 cm⁻¹(NH); ¹H-NMR (DMSO-d₆) δ 1.01

(s, 6H, 2 CH₃), 2.22 (s, 2H, CH₂), 2.69 (s, 2H, CH₂), 6.66 (s, 1H, CH-pyrrole), 7.17 (bs, 1H, NH;

exchangeable with D₂O), 7.49–7.51 (m, 5H, ArH), 7.87–7.90 (m, 4H, ArH), 8.25 (s, 1H, CH=N), 10.56

(s, 1H, NH; exchangeable with D₂O), 11.55 (s, 1H, NH; exchangeable with D₂O), 11.63 (s, 1H, NH;

13

exchangeable with D₂O), 12.00 (s, 1H, NH; exchangeable with D₂O); C-NMR (DMSO-d₆) δ: 28.8,

29.0, 35.8, 36.7, 110.6, 112.3, 120.2, 128.2, 128.3, 128.3, 128.3, 129.2, 129.2, 132.3, 132.6, 140.2,

146.6, 148. 3, 158.4, 172.7. Anal. Calcd for C₂₇H₂₆N₁₀O (506.56): C, 64.02; H, 5.17; N, 27.65 Found:

C, 63.89; H, 4.92; N, 27.80.

3.28. 3-(6,6-Dimethyl-6,7-dihydro-1H-indol-4(5H)-one)-1,5-dihydro-[1,2,4]triazolo[3,4-c]-5,10-

dihydro-[1,2,4]triazino[5,6-b]quinoxaline (40)

A mixture of 2-((2-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)hydrazono)methyl)-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (37, 0.001 mol) and acetic anhydride (15 mL) was heated

on a boiling water bath for 10 h. The reaction mixture was poured onto crushed ice, the precipitated

product was filtered, washed with water, dried and recrystallized from ethanol/chloroform. The

product was obtained as brown crystals; yield 0.28 g, 73.5%; m.p. 280–281 °C; IR (KBr): 1662 (C=O),

1

3234, 3429 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.08 (s, 6H, 2 CH₃), 2.32 (s, 2H, CH₂), 2.75 (s, 2H,

CH₂), 6.79 (s, 1H, CH-pyrrole), 7.09–7.12 (m, 1H, ArH), 7.31 (d, 1H, ArH; J = 7.6 Hz), 7.38–7.42 (m,

2H, ArH), 12.23 (bs, 3H, 3NH; exchangeable with D₂O). Anal. Calcd for C₂₀H₁₈N₈O(386.41): C,

62.17; H, 4.70; N, 29.00 Found: C, 61.92; H, 4.62; N, 29.19.

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3.29. 2-(1-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-3-(4-bromophenyl)-1H-pyrazol-5-yl)-(6,6-dimethyl-

6,7-dihydro-1H-indol-4(5H)-one (41)

To a solution of 2-(3-(4-bromophenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-

4(5H)-one (2, 0.001 mol) in ethanol (15 mL) was added 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole

(24, 0.0012 mol) and acetic acid (5 mL). The reaction mixture was heated under reflux for 7 h,

concentrated till dryness, poured onto H₂O (50 mL), 10% bromine water (5 mL) was added, the

mixture stirred overnight, and poured onto crushed ice. The separated solid product was filtered,

washed with ethanol, dried and recrystallized from ethanol. The product was obtained as brown

crystals; yield 0.44 g, 80.8%; m.p. 150–151 °C; IR (KBr): 1649 (C=O), 3439 cm⁻¹(NH);

¹H-NMR (DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.35 (s, 2H, CH₂), 2.67 (s, 2H, CH₂), 6.12 (s, 1H,

CH-pyrazole), 6.70 (s, 1H, CH-pyrrole), 7.30–7.37 (m, 2H, ArH), 7.50–7.54 (m, 4H, ArH), 7.73 (d,

2H, ArH; J = 7.7 Hz), 11.35 (s, 1H, NH; exchangeable with D₂O), 11.78 (s, 1H, NH; exchangeable

13

with D₂O); C-NMR (DMSO-d₆) δ: 28.9, 33.2, 45.4, 57.8, 108.2, 111.3, 113.6, 120.8, 121.8, 122.6,

124.7, 124.9, 125.2, 125.7, 126.9, 127.6, 128.9, 132.3, 142.2, 146.6, 148.5, 150.4, 157.6, 190.8. Anal.

Calcd for C₂₈H₂₂BrN₇O (552.42): C, 60.88; H, 4.01; N, 17.75 Found: C, 61.00; H, 4.20; N, 17.58.

3.30. General Procedure for the Preparation of Compounds 42–44

To a solution of 2-(3-aryl-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one 2–4

(0.001 mol) in ethanol (15 mL) was added 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b] quinoxaline

(25, 0.0012 mol) and acetic acid (5 mL). The reaction mixture was heated under reflux for 7 h,

concentrated till dryness, poured onto H₂O (50 mL), 10% bromine water (5 mL) was added, the

mixture stirred overnight, and poured onto crushed ice. The separated solid product was filtered,

washed with ethanol, dried and recrystallized from ethanol.

2-(3-(4-Bromophenyl)-1-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-1H-pyrazol-5-yl)-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (42). Orange crystals; yield 0.49 g, 88.0%; m.p. 289–290 °C ;

1

IR (KBr): 1680 (C=O), 3232, 3454 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.01 (s, 6H, 2 CH₃), 2.23 (s,

2H, CH₂), 2.71 (s, 2H, CH₂), 6.80 (s, 1H, CH-pyrazole), 6.91 (s, 1H, CH-pyrrole), 7.50–7.60 (m, 4H,

ArH), 7.76 (d, 2H, ArH; J = 8.4 Hz), 7.92 (d, 2H, ArH; J = 8.4 Hz), 12.06 (m, 3H, 3NH; exchangeable

with D₂O); ¹³C-NMR (DMSO-d₆) δ:.28.8, 34.5, 48.6, 49.7, 51.5, 99.1, 115.6, 122.4, 124, 125.8, 129.0,

129.8, 131.8, 138.0, 140.1, 1418, 143.9, 146.6, 151.7, 155.6, 193.3. Anal. Calcd for C₂₈H₂₃BrN₈O

(567.44): C, 59.27; H, 4.09; N, 19.75 Found: C, 59.15; H, 4.21; N, 19.50.

2-(3-(4-Chlorophenyl)-1-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-1H-pyrazol-5-yl)-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (43). Brown crystals; yield 0.48 g, 82.3%; m.p. 229–230 °C ;

1

IR (KBr): 1681 (C=O), 3250, 3405 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.00 (s, 6H, 2 CH₃), 2.23 (s,

2H, CH₂), 2.71 (s, 2H, CH₂), 6.90 (s, 1H, CH-pyrazole), 7.27 (s, 1H, CH-pyrrole), 7.50–7.55 (m, 2H,

ArH), 7.69–7.72 (m, 4H, ArH), 8.00–8.12 (m, 2H, ArH), 12.18 (bs, 3H, 3NH; exchangeable with

13

D₂O); C-NMR (DMSO-d₆) δ:.28.8, 39.8, 40.2, 40.6, 54.2, 64.1, 109.6, 115.8, 126.3, 129.5, 129.6,

129.6, 130.6 130.8, 137.4, 143.2, 143.7, 150.2, 155.9, 169.5, 192.8. Anal. Calcd for C₂₈H₂₃ClN₈O

(522.99): C, 64.30; H, 4.43; N, 21.43 Found: C, 64.26; H, 4.20; N, 21.66.

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2-(1-(5,10-Dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (44). Brown crystals; yield 0.45 g, 86.8%; m.p. 280–281 °C ;

1

IR (KBr): 1681 (C=O), 3151, 3436 cm⁻¹(NH); H-NMR (DMSO-d₆) δ 1.02 (s, 6H, 2 CH₃), 2.24 (s,

2H, CH₂), 2.72 (s, 2H, CH₂), 2.94 (s, 3H, OCH₃), 6.75–6.78 (m, 1H, CH-pyrazole), 6.85 (s, 1H,

CH-pyrrole), 7.06 (d, 2H, ArH; J = 8.4 Hz), 7.27–7.36 (m, 2H, ArH), 7.40 (d, 2H, ArH; J = 8.4 Hz),

8.01–8.09 (m, 2H, ArH), 11.95 (bs, 2H, 2NH; exchangeable with D₂O), 12.05 (bs, 1H, NH;

13

exchangeable with D₂O). C-NMR (DMSO-d₆) δ: 28.6, 35.0, 43.1, 53.8, 56.1, 100.1, 115.6, 117.1,

117.3, 121.5, 123.5, 123.8, 126.1, 129.5, 135.2, 140.1, 144.7, 149.7, 152.3, 155.7, 160.8, 193.0. Anal.

Calcd for C₂₉H₂₆N₈O₂(518.57): C, 67.17; H, 5.05; N, 21.61 Found: C, 67.30; H, 5.18; N, 21.41.

3.31. Biological Activity Assay

3.31.1. Inhibition Zone Measurement (IZ)

Compounds 2–44 were evaluated in vitro for antimicrobial activity against the following four

organisms: Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC 9027 as examples of Gram-

negative bacteria, Staphylococcus aureus ATCC 6583P as an example of Gram-positive bacteria, and

Candida albicans ATCC 2091 as an example of a yeast-like fungus have been studied by using the

Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA) diffusion methods [74], respectively, in

N,N-dmethylformamide as solvent. The bacteria were subcultured on Nutrient Agar medium (NA),

whereas, fungi were subcultured on Sabouraud Dextrose Agar (SDA). Petri plates

(150 mm × 15 mm) were prepared by pouring 60 mL of NA or SDA and allowing it to solidify. Plates

were dried and 1 mL of each standardized inoculums suspension was poured and uniformly spread.

The excess inoculums was drained and the inoculums was allowed to dry for 15 min. Eight equidistant

wells were made in the medium using a sterile cork borer (6 mm in diameter and 75 μL of the test

chemicals (1 mg/mL) diluted in DMF were placed into the wells. The plates containing bacterial and

fungi species were incubated at 37 °C for 24 h. The tests were carried in triplicate. Ampicillin

trihydrate (10.0 µg/disc), ciprofloxacin (5.0 µg/disc), impenam (10.0 µg/disc), and clotrimazole (100.0

µg/disc) were used as standard antibacterial and antifungal agents, respectively. (DMF) alone showed

no inhibition zone. The plates were incubated at 37 °C for 24 h. The results were recorded for each

tested compound as the average diameter of inhibition zones of bacterial growth around the disks in mm.

3.31.2. Minimal inhibitory concentration (MIC)

MIC measurements [75] were carried out for compounds that showed significant inhibition zones

using the twofold serial dilution technique. The compounds 2–44 were prepared in a concentration

range of 200, 100, 50, 25, and 12.5 µg/mL. The microdilution susceptibility test in Muller-Hinton

broth (oxoid) and Sabouraud Liquid Medium (oxoid) were used for the determination of antibacterial

and antifungal activity. The microorganism suspensions at 106 CFU/mL (colony forming unit/mL)

were used to inoculate the prepared test compounds in the above mentioned serial dilution broth. The

culture tubes were incubated at 37 °C for 24–48 h. At the end of the incubation period the growth of

bacteria was observed by turbidity measurements [75]. The MIC is defined as the lowest concentration

that showed no bacterial growth.

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4. Conclusions

The objective of the present study was to synthesize and investigate the antimicrobial and

antifungal activity of a new series of pyrazolines and pyrazoles in the hope of discovering new

structural leads serving as antimicrobial agents. Some new pyrazoline and pyrazole derivatives have

been prepared, and their physical properties were characterized. The biological activity of the

compounds 2–44 was evaluated by the agar diffusion method against Escherichia coli, Pseudomonas

aeruginosa, Staphylococcus aurous and Candida albicans. None of the investigated compounds not

showed any activity against the test organisms Escherichia coli and Pseudomonas aeruginosa.

Compound 16 has good antimicrobial activity against Staphylococcus aureus, comparable to that of

ampicillin and ciprofloxacin, while compound 17 has remarkable antimicrobial activity against

Staphylococcus aureus, exceeding that of ampicillin, ciprofloxacin and imipenam. In addition,

compound 17 has comparable IZ against Candida albicans comparable to that of clotrimazole. On the

other hand, the minimal inhibitory concentration (MIC) of compounds 19, 20 and 31 against Candida

albicans indicate good antifungal activity, comparable to that of clotrimazole. Based on the preliminary

results, it can be seen that all four compounds 16, 17, 19 and 20 showing good antimicrobial and

antifungal activity have benzenesulfonamide substituents as a common structural feature.

Supplementary Materials

Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/3/2683/s1.

Acknowledgments

Thanks are due to the Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria

University for the antimicrobial measurements.

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Synthesis, antibacterial and antifungal activity of some new pyrazoline and pyrazole derivatives

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