Synthesis and in vitro antitumor and antimicrobial activity of some 2,3-diaryl-7-methyl-4,5,6,7-tetrahydroindazole and 3,3a,4,5,6,7- hexahydroindazole derivatives

Article abstract of DOI:10.3109/14756366.2011.653354

The synthesis of a series of 2,3-diaryl-7-methyl-4,5,6,7-tetrahydroindazole and 3,3a,4,5,6,7-hexahydroindazole derivatives substituted with various biologically-active function groups with anticipated chemotherapeutic activity is described. 4-(7-methyl-3-aryl-3,3a,4,5,6,7-hexahydro-indazol-2-yl) benzenesulfonamides 2a-c, which were employed as key intermediates in this study, were synthesized by cyclocondensation of 6-arylidene-2- methylcyclohexanones 1a-c with 4-hydrazinobenzenesulfonamide hydrochloride. A detailed discussion of the reactions utilized in the preparation of the intermediate and target compounds is reported, and the structures of the newly synthesized compounds were substantiated with IR, ¹H and ¹³C NMR spectral data and elementary microanalyses. Twenty of the newly synthesized compounds were selected by National Cancer Institute (NCI), Maryland, USA, to be evaluated for their antitumor activity and the results revealed that six compounds 3c, 4d,e, 5a,d and 8c exhibited broad spectrum of antitumor activity against most of the tested tumor cell lines. In addition, the in vitro antibacterial and antifungal activities of a number of the target compounds were also tested using the Agar-diffusion method. Some of these compounds have shown significant antibacterial and mild to moderate antifungal activities.

Full text of DOI:10.3109/14756366.2011.653354

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 1–14, Early Online
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.653354
RESEARCH ARTICLE
Synthesis and in vitro antitumor and antimicrobial activity
of some 2,3-diaryl-7-methyl-4,5,6,7-tetrahydroindazole and
3,3a,4,5,6,7-hexahydroindazole derivatives
Hassan M. Faidallah1, Khalid A. Khan1, Sherif A. F. Rostom2,3, and Abdullah M. Asiri1,4
1Department of Chemistry, Faculty of Science, 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
King Abdulaziz University, Jeddah, Saudi Arabia, 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria, Egypt, and 4Center of Excellence for Advanced Materials Research,
King Abdulaziz University, Jeddah, Saudi Arabia
Abstract
The synthesis of a series of 2,3-diaryl-7-methyl-4,5,6,7-tetrahydroindazole and 3,3a,4,5,6,7-hexahydroindazole
derivatives substituted with various biologically-active function groups with anticipated chemotherapeutic activity is
described. 4-(7-methyl-3-aryl-3,3a,4,5,6,7-hexahydro-indazol-2-yl)benzenesulfonamides 2a–c, which were employed
as key intermediates in this study, were synthesized by cyclocondensation of 6-arylidene-2-methylcyclohexanones
1a–c with 4-hydrazinobenzenesulfonamide hydrochloride. A detailed discussion of the reactions utilized in the
preparation of the intermediate and target compounds is reported, and the structures of the newly synthesized
compounds were substantiated with IR, 1H and 13C NMR spectral data and elementary microanalyses. Twenty of the
newly synthesized compounds were selected by National Cancer Institute (NCI), Maryland, USA, to be evaluated for
their antitumor activity and the results revealed that six compounds 3c, 4d,e, 5a,d and 8c exhibited broad spectrum
of antitumor activity against most of the tested tumor cell lines. In addition, the in vitro antibacterial and antifungal
activities of a number of the target compounds were also tested using the Agar-diffusion method. Some of these
compounds have shown significant antibacterial and mild to moderate antifungal activities.
Keywords: Tetrahydroindazoles, hexahydroindazoles, benzenesulfonylureas, thioureas, antitumor and
antimicrobial activity
Introduction
Over the past two decades, much evidence has been
accumulated on the importance of pyrazole deriva-
tives and their related fused heterocycles as chemo-
therapeutic agents. e pyrazole moiety is an important
pharmacophore showing a multitude of biological and
pharmacological properties. However, benzo-fused
derivatives of pyrazole (indazole) are probably one of
the least studied nuclei owing to its scarcity in nature.
e indazole ring system is regarded as a bioisostere of
indole. However, a large number of synthetically pre-
pared indazoles are known to show a variety of biologi-
cal activities, such as high binding affinity for estrogen
receptor1,2, inhibition of protein kinase C-β3, 5-HT2
receptor agonism4, human immunodeficiency virus
(HIV) protease inhibition5, anti-leukemic activity6, and
non-steroidal anti-inflammatory activity7. Other com-
pounds of interest are YC-1, a benzylindazole which has
been shown to activate soluble guanylate cyclase8, nitric
oxide synthase inhibition activity9,10, to induce cellular
apoptosis11 and a MCHr1 antagonism for the treatment
of obesity12. e most notable one is Lonidamine which
was found to induce apoptosis via a direct effect on the
mitochondrial permeability transition pore11.
Similarly, tetrahydroindazoles are known to show
activity against cancer13 and inflammation14 and hexa-
hydroindazoles, a novel heterocyclic nucleus has been
Address for correspondence: Prof. Hassan M. Faidallah, Department of Chemistry, P O Box 80203, Faculty of Science, King Abdulaziz
University, Jeddah-21589, Saudi Arabia. Tel: +966 567743180. E-mail: hfaidallahm@hotmail.com
(Received 17 November 2011; revised 20 December 2011; accepted 21 December 2011)
1

2
H.M. Faidallah et al.
reported to have antimicrobial15,16 and anti-inflammatory
activities17,18. In a molecular docking study of 4,5,6,7-
tetrahydro-2H-indazoles when position-2 is substituted
with 4-(trifluoromethyl)phenylhydrazine group it was
found to display more anti-inflammatory activity when
compared with 2-Hydrazino-4-(trifluoromethyl)pyrimi-
dine group. e difference in the anti-inflammatory
activity of these two compounds may be attributed to an
interplay between hydrogen bonding and hydrophobic
interactions at the catalytic site of COX-214. Minu et al.
reported in a QSAR study of hexahydroindazoles as
antimicrobial agents when substituted with an electron
withdrawing group in position-3 of the indazole ring, the
compounds were found more active than other deriva-
tives19. In another study by Gokhan-Kelekci et al. substi-
tution of a furan ring in position-3 of the indazole ring,
produces a MAO inhibitor20. Compounds carrying phenyl
substituents on nitrogen atom (position-2 in indazole)
were found to be highly potent MAO inhibitors. Further,
it was also noted that the presence of longer and volumi-
nous groups on thiocarbamoyl nitrogen was selectively
active towards MAO-A isoform while in case of MAO-B
inhibitors the length of the lateral chain of the aryl diazo
derivatives should not be longer than two atoms.
In view of the aforementioned findings, it was con-
ferred that the position-2 and position-3 are crucial
in the activity modulation of tetrahydro and hexahy-
droindazoles. erefore, in continuation of our ongoing
interest in the synthesis novel heterocycles with chemo-
therapeutic activities21–30, it was considered of interest to
prepare some new tetrahydro- and hexahydroindazole
derivatives, bearing functionalities that are reported to
contribute to potential chemotherapeutic activities such
as the sulfonamide, sulfonylurea and thiourea groups.
Besides incorporating an electron withdrawing group at
position-3 we have also introduced a methyl substituent
in position-7. Some target compounds were planned to
comprise other heterocyclic rings that are known to pos-
sess significant antibacterial and anticancer activities
such as the thiazole and the thiazine ring systems.
uncorrected (Table 1)36. e infrared (IR) spectra were
recorded on Perkin-Elmer 297 infrared spectrophotom-
eter, using the NaCl plate technique. e H-NMR and
1
13C-NMR spectra were recorded on Bruker DPX-400-FT
spectrometer using CDCl3 and DMSO-d6 as a solvent and
tetramethylsilane as the internal standard (Chemical
shifts in δ, ppm). Splitting patterns were designated as
follows: s: singlet; d: doublet; m: multiplet. Elemental
analyses were performed at the Microanalytical Unit,
Faculty of Science, King Abdul-Aziz University, Jeddah,
Saudi Arabia, and the found values were within 0.4%
of the theoretical values. Follow up of the reactions and
checking the homogeneity of the compounds were made
by TLC on silica gel-protected aluminium sheets (Type 60
F254, Merck) and the spots were detected by exposure to
UV-lamp at λ 254. e starting chalcone 1 was prepared
according to a literature procedure.
4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonamide (2a–c)
A solution of the appropriate chalcone 1a–c (20 mmol)
in ethanol (25mL) was refluxed with 4-hydrazinobenze-
nesulfonamide hydrochloride (4.9 g, 22 mmol) for 4 h.
On concentration, the separated product was filtered,
washed with cold ethanol and recrystallized from a mix-
1
ture of benzene-ethanol (1:1). H NMR data are listed
in Table 2. 13C NMR (DMSO/CDCl3) 2a δ= 14.0 (CH3),
25.9, 27.6, 29.9, 33.3 (cyclohexyl-C), 42.6 (C-3a), 56.0
(C-3), 112.6, 125.7, 126.3, 127.7, 128.3, 139.4, 146.7, 155.6
(Ar-C). 2b δ= 14.5 (CH3), 26.9, 29.7, 32.8 (cyclohexyl-C),
43.2 (C-3a), 56.6 (C-3), 113.0, 126.8, 127.4, 128.2, 129.7,
131.0, 137.8, 147.3, 154.6 (Ar-C). 2c δ= 13.9 (CH3), 20.9
(CH3), 25.2, 27.2, 29.4, 33.4 (cyclohexyl-C), 42.8 (C-3a),
56.8 (C-3), 112.2, 126.1, 127.3, 128.4, 129.0, 134.7, 135.9,
146.5, 152.8 (Ar-C).
4-(7-Methyl-3-aryl-4,5,6,7-tetrahydroindazol-2-yl)
benzenesulfonamide (3a–c)
To a stirred suspension of the indazoline derivative 2 (10
mmol) in water (10 mL), bromine water (5%, 15 mL) was
gradually added over a period of 30 min. at 25°C. After
stirring for 3 h at room temperature, the pyrazole deriva-
tives thus formed, were collected by filtration, thoroughly
washed with water and dried. ey were recrystallized
from ethanol. 1H NMR data are listed in Table 2. 13C NMR
(DMSO/CDCl3) 3a δ= 21.9 (CH3), 18.1, 31.5, 32.2, 41.3
(cyclohexyl-C), 117.7, 119.1, 126.1, 127.0, 128.5, 129.0,
136.5, 136.8, 142.9, 145.4, 152.5 (Ar-C). 3c δ= 20.2 (CH3),
21.6 (CH3), 18.4, 31.2, 32.6, 41.4 (cyclohexyl-C), 117.5,
118.8, 126.0, 126.9, 129.7, 133.5, 136.0, 137.7, 141.8, 144.6,
151.9.
e structures of the newly synthesized compounds
1
were confirmed by IR, H and 13C NMR data. e target
compounds have been subjected to the National Cancer
Institute NCI in vitro disease-oriented human cells screen-
ing panel assay, Maryland, USA, to screen their anticancer
activity31–33. In addition, in vitro antibacterial and antifun-
gal activities were also evaluated using the Agar-diffusion
method34. Finally, owing to the increase of reported cases
of tuberculosis even in developed countries, along with the
emergence of multidrug-resistant (MDR) Mycobacterium
tuberculosis strains, it was thought of interest to screen the
in vitro antimycobacterial activity of the new analogs35.
N1-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-
2-yl)benzenesulfonyl]- N3-substituted ureas (4a-f) and
N1-[4-(7-Methyl-3-aryl-4,5,6,7-tetrahydroindazoline-2-yl)
benzenesulfonyl]-N3-substituted ureas (5a–f)
Experimental
Chemistry
Melting points were determined in open glass capillar-
ies, on a Gallenkamp melting point apparatus, and were
A mixture of the appropriate pyrazoline 2 or pyrazole 3
(10 mmol) and anhydrous potassium carbonate (2.8 g, 20
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives
3
Table 1. Physicochemical and analytical data of compounds 2–10.
Analysis
Calculated/Found (%)
Compound
number
Mol. Formula
X
R
M.P. (°C)
199–200
Yield (%)
88
(Mol. Weight)
C20H23N3O2S
(369.48)
C
H
N
S
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
H
65.01
64.95
59.47
59.30
65.77
65.58
65.37
65.40
59.77
59.85
66.12
66.12
65.56
65.45
66.37
66.15
69.12
69.03
66.11
66.20
66.91
67.01
69.54
69.48
65.83
66.01
66.65
66.54
69.38
69.12
67.18
66.95
69.80
69.69
64.26
64.07
64.84
65.02
60.15
60.23
60.80
60.44
59.30
59.21
64.84
65.00
65.38
68.18
63.71
63.90
6.27
6.33
5.49
5.60
6.57
6.74
5.76
5.70
5.02
4.96
6.08
6.08
6.93
7.10
5.78
5.92
5.61
5.80
7.13
7.05
6.02
5.92
5.84
5.96
6.55
6.39
5.39
5.47
5.26
5.44
5.64
5.82
5.49
5.60
5.59
5.74
5.83
5.68
5.05
4.98
5.28
5.37
4.80
4.89
5.83
5.67
6.05
6.22
5.53
5.48
11.37
11.45
10.40
10.27
10.96
11.20
11.44
11.35
10.46
10.50
11.01
11.01
11.33
11.50
11.47
11.55
10.40
10.50
11.01
10.88
11.15
11.33
10.14
10.20
11.37
11.26
11.51
11.60
10.44
10.60
11.19
11.36
10.17
10.25
11.10
10.96
10.80
10.89
10.39
10.26
10.13
10.06
9.88
8.68
8.59
7.94
8.05
8.36
8.15
8.73
8.90
7.98
8.04
8.41
8.41
6.48
6.40
6.56
6.77
5.95
5.78
6.30
6.46
6.38
6.30
5.80
5.76
6.51
6.70
6.59
6.65
5.98
5.73
6.41
6.27
5.82
5.92
12.71
12.90
12.36
12.21
11.90
12.10
11.59
11.78
11.31
11.25
12.36
12.25
12.04
12.13
11.73
11.68
Cl
202–203
204–206
150–152
148–149
126–127
149–150
138–139
196–198
206–207
158–160
169–170
134–136
162–164
148–149
172–173
156–157
162–163
128–129
172–174
136–137
140–142
122–123
160–161
140–142
84
85
72
70
68
72
75
77
74
75
78
70
72
75
78
75
89
72
75
72
70
88
66
72
C20H22ClN3O2S
(403.93)
CH3
H
C21H25N3O2S
(383.51)
C20H21N3O2S
(367.46)
Cl
C20H20ClN3O2S
(401.91)
CH3
H
C21H23N3O2S
(381.49)
cyclohexyl
C6H5
C27H34N4O3S
(494.65)
H
C27H28N4O3S)
(488.60)
H
1-naphthyl
cyclohexyl
C6H5
C31H30N4O3S
(538.66)
CH3
CH3
CH3
H
C28H36N4O3S
9508.68)
C28H30N4O3S
(502.63)
1-naphthyl
cyclohexyl
C6H5
C32H32N4O3S
(552.69)
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
C27H32N4O3S
(492.63)
H
C27H26N4O3S
(486.59)
H
1-naphthyl
C6H5
C31H28N4O3S
(536.64)
CH3
CH3
H
C28H28N4O3S
(500.61)
1-naphthyl
C6H5
C32H30N4O3S
(550.67)
C27H28N4O2S2
(504.67)
H
CH2C6H5
C6H5
C28H30N4O2S2
(518.69)
Cl
C27H27ClN4O2S2
(539.11)
Cl
CH2C6H5
COC6H5
C6H5
C28H29ClN4O2S2
(553.14)
Cl
C28H27ClN4O3S2
(567.12)
10.01
10.80
10.90
10.52
10.44
10.25
10.47
CH3
CH3
CH3
C28H30N4O2S2
(518.69)
6g
6h
CH2C6H5
COC6H5
C29H32N4O2S2
(532.72)
C29H30N4O3S2
(546.70)
(Continued)
© 2012 Informa UK, Ltd.

4
H.M. Faidallah et al.
Table 1. Continued.
Compound
Analysis
Calculated/Found (%)
Mol. Formula
(Mol. Weight)
number
X
R
M.P. (°C)
212–214
Yield (%)
78
C
H
N
S
7a
H
C6H5
C29H28N4O3S2
(544.69)
63.95
64.04
64.49
64.22
59.35
59.52
64.49
64.77
65.01
64.88
63.46
63.21
69.51
69.86
69.87
70.06
69.87
69.99
64.49
64.30
65.01
64.86
65.50
65.77
64.49
64.27
65.01
64.85
65.0164.91
5.18
5.10
5.41
5.60
4.48
4.21
5.41
5.23
5.63
5.92
5.15
5.36
5.33
5.18
5.54
5.39
5.54
5.35
5.41
5.67
5.63
5.99
5.84
5.60
5.41
5.70
5.63
5.81
5.63
10.29
10.06
10.03
9.89
9.23
9.44
10.03
10.12
9.78
9.54
9.55
9.40
9.26
9.14
9.05
9.17
9.05
9.21
10.03
9.95
9.78
9.90
9.55
9.37
10.03
9.89
9.78
10.01
9.78
11.77
12.01
11.48
11.63
10.56
10.10
11.48
11.39
11.20
11.47
10.93
11.09
10.60
10.82
10.36
10.24
10.36
10.18
11.48
11.64
11.20
11.03
10.93
10.74
11.48
11.66
11.20
11.14
11.20
7b
7c
H
CH2C6H5
COC6H5
C6H5
126–128
162–164
187–188
137–138
150–152
168–169
112–113
122–123
134–135
130–132
122–124
174–176
136–138
162–164
65
72
74
70
72
75
62
64
67
65
62
70
69
74
C29H28N4O3S2
(558.71)
Cl
C30H27ClN4O4S2
(607.14)
7d
7e
CH3
CH3
CH3
H
C30H30N4O3S2
(558.71)
CH2C6H5
COC6H5
C6H5
C31H32N4O3S2
(572.74)
7f
C31H30N4O4S2
(586.72)
8a
8b
8c
C35H32N4O2S2
(604.78)
H
CH2C6H5
C6H5
C36H34N4O2S2
(618.81)
CH3
H
C36H34N4O2S2
(618.81)
9a
9b
9c
C6H5
C30H30N4O3S2
(558.71)
CH3
CH3
H
C6H5
C31H32N4O3S2
(572.74)
CH2C6H5
C6H5
C32H34N4O3S2
(586.77)
10a
10b
10c
C30H30N4O3S2
(558.71)
H
CH2C6H5
C6H5
C31H32N4O3S2
(572.74)
CH3
C31H32N4O3S2
(572.74)
64.91
5.77
9.92
11.08
mmol) in dry acetone (25 mL) was heated under reflux
with stirring with the appropriate isocyanate (11 mmol)
for 18 h. e solvent was removed under reduced pres-
sure and the remaining solid residue was dissolved in
water (30 mL). After acidification of the resulting solution
with 2N hydrochloric acid, the precipitated crude product
was filtered, washed with water, dried and recrystallized
from ethanol. 1H NMR data are listed in Table 2. 13C NMR
(DMSO/CDCl3) 4a δ= 14.3 (CH3), 21.6, 25.7, 27.1, 27.6,
29.2, 32.7, 33.4, 46.9 (2 cyclohexyl-C), 41.8 (C-3a), 55.7
(C-3), 119.1, 121.2, 124.6, 126.3, 128.5, 136.7, 138.4, 142.4,
155.2 (Ar-C), 162.6 (CO). 4b δ= 14.2 (CH3), 25.6, 27.3,
29.4, 33.2 (cyclohexyl-C), 42.0 (C-3a), 56.8 (C-3), 119.3,
120.4, 124.1, 126.0, 127.2, 128.3, 128.7, 129.0, 129.3, 136.5,
136.7, 142.0, 155.1 (Ar-C), 161.3 (CO). 4d δ= 14.4 (CH3),
20.4 (CH3), 21.2, 25.6, 26.9, 27.2, 29.4, 32.3, 33.2, 46.4 (2
cyclohexyl-C), 42.6 (C-3a), 55.8 (C-3), 118.8, 124.8, 126.4,
126.9, 129.7, 133.2, 137.0, 142.9, 155.6 (Ar-C), 163.1 (CO).
5a δ= 15.2 (CH3), 18.1, 22.0, 27.3, 31.6, 32.2, 33.5, 41.6,
44.4 (2 cyclohexyl-C), 117.7 (C-3a), 119.7, 126.8, 127.1,
128.5, 129.3, 135.9, 136.3, 145.2 (C-3), 145.4 (Ar-C), 150.6
(C-7a), 163.0 (CO). 5b δ= 21.6 (CH3), 18.4, 31.6, 32.8, 41.2
(cyclohexyl-C), 117.2 (C-3a), 119.3, 120.2, 124.6, 126.1,
127.2, 128.3, 128.7, 129.1, 136.4, 136.6, 139.0, 142.4, 145.0
(C-3), 152.5 (C-7a), 159.3 (CO). 5d δ= 14.6 (CH3), 20.7
(CH3), 18.6, 31.2, 32.1, 40.9 (cyclohexyl-C), 118.3 (C-3a),
119.0, 126.1, 126.8, 127.0, 128.2, 129.0, 130.1, 133.1, 135.8,
136.7, 137.9, 142.6, 144.2 (C-3), 152.0 (C-7a), 160.3 (CO).
N1-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonyl]- N3-substituted thioureas (6a–h)
A solution of the appropriate isothiocyanate (11 mmol)
in dry acetone (5 mL) was added to a stirred mixture
of the suitable indazoline 2 (10 mmol) and anhydrous
potassium carbonate (2.8 g, 20 mmol) in dry acetone
(25 mL), and the mixture was heated under reflux with
stirring for 10 h. e solvent was removed under reduced
pressure and the remaining solid residue was dissolved
in water (30 mL) and acidified with 2N hydrochloric acid.
e precipitated product was filtered, washed with water,
1
dried and recrystallized from ethanol. HNMR data are
listed in Table 2. 13C NMR (DMSO/CDCl3) 6a δ= 14.8
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives
5
Table 2. 1H-NMR spectral data of compoundsa (2–10).
H-4,5,6
Compound
number
cyclohexyl
(m, 6H)
CH3
H-7
H-3a
H-3
X
R
(d, 3H) (m, 1H) (m, 1H)
(d, 1H)
5.38
4.95
5.31
–
Ar-H (m)
Others
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
H
1.46–1.74
1.42–1.75
1.38–1.69
1.52–2.35
1.45–1.62
1.62–2.28
1.14–2.74c
1.36–1.72
1.38–1.80
1.22–2.68c
1.28
1.28
1.27
1.26
1.27
1.30
1.24
1.27
1.25
1.23
3.04
2.94
3.03
3.08
3.10
3.12
3.10
2.98
3.02
3.14
3.67
3.70
3.64
–
6.85–7.58 (11H)b
6.82–7.88 (10H)b
6.76–7.92 (10H)b 2.31 (s, 3H, Tol-CH3)
6.82–7.68 (11H)b
6.77–7.82 (10H)b
6.90–7.82 (10H)b 2.35 (s, 3H, Tol-CH3)
Cl
CH3
H
Cl
CH3
H
–
–
–
–
cyclohexyl
C6H5
3.78
3.62
3.71
3.65
5.44
5.32
5.38
5.52
7.14–8.11 (9H)
9.1 (s, 1H, NH), 9.6 (s, 1H, NH)
H
6.83–7.55 (14H) 8.93 (s, 1H, NH), 9.25 (s, 1H, NH)
6.98–8.02 (16H) 9.11 (s, 1H, NH), 9.72 (s, 1H, NH)
H
1-naphthyl
CH3 cyclohexyl
7.23–7.92 (8H)
2.32 (s, 3H, Tol-CH3), 9.23 (s, 1H, NH),
9.58 (s, 1H, NH)
4e
CH3 C6H5
1.39–1.79
1.27
3.06
3.63
5.42
7.02–7.98 (13H) 2.35 (s, 3H, Tol-CH3), 9.32 (s, 1H, NH),
9.63 (s, 1H, NH)
5a
5b
5c
5d
H
H
H
cyclohexyl
C6H5
1.18–2.60c
1.35–2.28
1.38–2.17
1.42–2.30
1.27
1.28
1.28
1.27
3.11
3.05
3.07
3.10
–
–
–
–
–
–
–
–
7.04–7.99 (8H)
9.25 (s, 1H, NH), 9.48 (s, 1H, NH)
7.02–8.05 (13H) 9.32 (s, 1H, NH), 9.43 (s, 1H, NH)
7.18–8.06 (15H) 9.04 (s, 1H, NH), 9.65 (s, 1H, NH)
1-naphthyl
CH3 C6H5
7.12–8.15 (13H) 2.38 (s, 3H, Tol-CH3), 9.26 (s, 1H, NH),
9.42 (s, 1H, NH)
5e
CH3 1-naphthyl
1.29–2.26
1.26
3.18
–
–
7.24–8.12 (15H) 2.34 (s, 3H, Tol-CH3), 9.15 (s, 1H, NH),
9.61 (s, 1H, NH)
6a
6b
H
H
C6H5
1.48–1.75
1.39–1.72
1.24
1.26
3.68
3.58
3.65
3.92
5.24
5.25
6.92–7.80 (14H) 9.02 (s, 1H, NH), 9.78 (s, 1H, NH)
CH2C6H5
7.02–7.82 (14H) 4.75 (d, 2H, CH2- C6H5), 9.13 (s, 1H, NH),
9.92 (s, 1H, NH)
6c
Cl
Cl
C6H5
1.41–1.78
1.39–1.67
1.24
1.23
3.42
3.38
3.70
3.62
5.21
5.14
6.87–7.84 (13H) 9.24 (s, 1H, NH), 9.63 (s, 1H, NH)
6d
CH2C6H5
7.11–7.89 (13H) 4.72 (d, 2H, CH2), 9.08 (s, 1H, NH), 9.52
(s, 1H, NH)
6e
6f
Cl
COC6H5
1.40–1.82
1.37–1.67
1.25
1.25
3.02
3.00
3.74
3.72
5.16
5.32
7.16–7.79 (13H) 9.18 (s, 1H, NH), 9.63 (s, 1H, NH)
CH3 C6H5
6.78–7.90 (13H) 2.32 (s, 3H, Tol-CH3), 9.21 (s, 1H, NH),
9.78 (s, 1H, NH)
6g
6h
CH3 CH2C6H5
CH3 COC6H5
1.34–1.69
1.44–1.78
1.28
1.31
3.14
3.06
3.66
3.72
5.04
5.12
7.13–8.11 (13H) 2.29 (s, 3H, Tol-CH3), 4.29 (d, 2H, CH2),
9.03 (s, 1H, NH), 9.49 (s, 1H, NH)
7.16–8.03 (13H) 2.34 (s, 3H, Tol-CH3), 9.14 (s, 1H, NH),
9.55 (s, 1H, NH)
7a
7b
7c
7d
7e
H
H
Cl
C6H5
1.42–1.72
1.45–1.75
1.40–1.74
1.43–1.69
1.39–1.72
1.28
1.29
1.28
1.26
1.24
3.10
3.03
3.06
3.12
3.34
3.64
3.78
3.77
3.69
3.84
5.34
5.22
5.25
5.43
5.68
6.92–7.85 (14H) 4.30 (s, 2H, CH2)
CH2C6H5
COC6H5
7.01–8.10 (14H) 4.16 (s, 2H, CH2), 4.82 (s, 2H, CH2- C6H5)
6.88–7.82 (13H) 4.33 (s, 2H, CH2)
CH3 C6H5
7.14–7.92 (13H) 2.31 (s, 3H, Tol-CH3), 4.13 (s, 2H, CH2)
CH3 CH2C6H5
7.12–7.85 (13H) 2.34 (s, 3H, Tol-CH3), 4.24 (s, 2H, CH2),
4.80 (s, 2H, CH2)
7f
CH3 COC6H5
1.40–1.75
1.38–1.85
1.37–1.75
1.36–1.78
1.25
1.29
1.26
1.28
3.24
2.98
3.01
3.05
3.75
3.65
3.68
3.72
5.52
5.28
5.10
5.15
7.09–7.88 (13H) 2.30 (s, 3H, Tol-CH3), 4.19 (s, 2H, CH2)
6.80–7.75 (19H) 5.78 (s, 1H, H-5 thiazole)
8a
8b
8c
H
H
C6H5
CH2C6H5
6.70–7.88 (19H) 4.75(s, 2H,CH2), 5.89 (s, 1H, H-5 thiazole)
CH3 C6H5
6.80–7.95 (18H) 2.35 (s, 3H, Tol-CH3), 5.74 (s, 1H, H-5
thiazole)
9a
9b
9c
H
C6H5
1.38–1.85
1.35–1.90
1.41–1.72
1.26
1.32
1.23
3.25d
3.19d
3.09d
3.68
3.72
3.74
5.10
5.30
5.48
6.70–7.88 (14H)
CH3 C6H5
6.92–7.82 (13H) 2.32 (s, 3H, Tol-CH3)
CH3 CH2C6H5
6.88–7.90 (13H) 2.31 (s, 3H, Tol-CH3), 4.75 (d, 2H,
CH2- C6H5)
10a
10b
10c
H
H
C6H5
1.34–1.78
1.37–1.79
1.40–1.68
1.29
1.20
1.29
2.99
3.12
3.20
3.70
3.78
3.69
5.29
5.34
5.21
6.88–7.82 (14H) 4.40 (s, 2H, H-4 thiazine), 5.42 (s, 2H, H-6
thiazine)
CH2C6H5
6.94–7.82 (14H) 4.42 (s, 2H, H-4 thiazine), 5.62 (s, 2H, H-6
thiazine)
CH3 C6H5
6.86–7.78 (13H) 2.34 (s, 3H, Tol-CH3), 4.38 (s, 2H, H-4
thiazine), 5.54 (s, 2H, H-6 thiazine)
aSolutions in a mixture of CDCl3 and DMSO-d6; b(Ar-H + NH2); c(m, 16H, 2cyclohexyl); d(m, 5H, H-7 and H-5 & H-6 of thiazine).
© 2012 Informa UK, Ltd.

6
H.M. Faidallah et al.
(CH3), 25.6, 27.4, 29.7, 33.1 (cyclohexyl-C), 42.6 (C-3a),
55.8 (C-3), 119.1, 124.5, 125.3, 126.0, 127.2, 128.4, 128.8,
129.0, 136.5, 137.2, 140.0, 143.1, 155.2 (Ar-C), 186.3 (CS).
6b δ= 14.2 (CH3), 25.9, 27.9, 29.7, 33.2 (cyclohexyl-C),
42.6 (C-3a), 56.0 (C-3), 55.5 (CH2), 119.3, 126.1, 126.5,
127.2, 127.5, 128.3, 128.6, 129.0, 136.8, 137.1, 141.3, 142.9,
154.8 (Ar-C), 188.2 (CS).
was cooled to room temperature, it was poured into ice-
cold water (30 mL). e solid product thus formed was
filtered, washed with water, dried and recrystallized from
ethanol-benzene mixture (1:1). 1H NMR data are listed in
Table 2. 13C NMR (DMSO/CDCl3) 9a δ= 14.2 (CH3), 24.8
(C-6, thiazine), 35.7 (C-5, thiazine), 25.7, 27.6, 29.5, 33.2
(cyclohexyl-C), 42.6 (C-3a), 56.3 (C-3), 114.3, 120.4, 124.1,
125.7, 126.6, 127.2, 128.2, 128.3, 128.7, 139.2, 140.8, 148.5,
155.6 (Ar-C), 163.0 (C=N), 173.4 (CO). 9b δ= 14.8 (CH3),
21.2 (CH3), 25.6 (C-6, thiazine), 36.2 (C-5, thiazine), 43.0
(C-3a), 55.2 (C-3), 114.0, 120.3, 124.6, 125.2, 126.7, 127.0,
128.2, 128.3, 128.8, 138.9, 140.6, 149.4, 154.9 (Ar-C), 164.8
(C=N), 174.3 (CO).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonylimino]-3-substituted thiazolidin-4-ones
(7a–f)
To a solution of the appropriate thiourea derivative 6
(10 mmol) in absolute ethanol (20 mL) was added ethyl
bromoacetate (1.84 g, 11 mmol) and anhydrous sodium
acetate (1.64 g, 20 mmol), and the reaction mixture
was heated under reflux for 2 h. e mixture was left
to attain room temperature then poured into ice-cold
water (30 mL), and the solid product thus formed was
filtered, washed with water, dried and recrystallized from
ethanol-benzene mixture (1:1). 1H NMR data are listed in
Table 2. 13C NMR (DMSO/CDCl3) 7a δ= 14.0 (CH3), 32.8
(CH2), 26.0, 27.7, 30.1, 33.5 (cyclohexyl-C), 42.6 (C-3a),
56.0 (C-3), 113.3, 120.4, 124.2, 125.7, 126.9, 127.1, 128.3,
128.9, 139.4, 140.8, 148.5, 155.8 (Ar-C), 163.3 (C=N), 168.8
(CO). 7b δ= 14.1 (CH3), 33.0 (CH2), 25.8, 27.6, 29.9, 33.2
(cyclohexyl-C), 41.8 (C-3a), 55.9 (C-3), 45.5 (Ph-CH2),
112.9, 125.5, 126.4, 126.9, 127.1, 127.4, 128.3, 128.5, 128.7,
138.8, 142.4, 146.9, 154.2 (Ar-C), 164.0 (C=N), 169.9 (CO).
7c δ= 14.6 (CH3), 32.8 (CH2), 25.7, 27.8, 29.6, 33.1 (cyclo-
hexyl-C), 42.0 (C-3a), 56.2 (C-3), 113.4, 125.6, 126.8, 127.3,
128.2, 128.3, 128.6, 129.7, 131.6, 133.5, 137.5, 139.2, 148.4,
155.6 (Ar-C), 163.0 (C=N), 167.7 (CO-Ph), 172.3 (CO).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonylimino]-3-substituted-[1,3]thiazinan-5-ones
(10a–c)
To a solution of the appropriate thiourea derivative 6 (10
mmol) in absolute ethanol (20 mL) was added 1,3-dichlo-
roacetone (1.4 g, 11 mmol) and anhydrous sodium ace-
tate (2 g, 25 mmol) and the reaction mixture was heated
under reflux for 5 h. After the mixture was cooled to room
temperature, it was poured into ice-cold water (30mL).
e solid product thus formed was filtered, washed with
water, dried and recrystallized from ethanol-benzene
mixture (1:1). 1H NMR data are listed in Table 2. 13C NMR
(DMSO/CDCl3) 10a δ= 14.1 (CH3), 25.8, 27.6, 29.7, 33.1
(cyclohexyl-C), 37.4 (C-6, thiazine), 59.4 (C-4, thiazine),
112.3, 115.8, 116.8, 126.0, 126.8, 127.0, 127.4, 128.3, 129.2,
137.4, 143.5, 148.1, 155.2 (Ar-C), 164.6 (C=N). 10c δ= 14.3
(CH3), 20.8 (CH3), 25.7, 27.8, 30.0, 33.2 (cyclohexyl-C),
36.9 (C-6, thiazine), 58.1 (C-4, thiazine), 113.4, 114.2,
117.1, 125.4, 126.7, 127.1, 128.1, 128.3, 129.4, 137.1, 138.2,
148.4, 155.9 (Ar-C), 163.3 (C=N).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonylimino]- 4-phenyl-3-substituted thiazolines
(8a–c)
A solution of the appropriate thiourea derivative 6 (10
mmol) in absolute ethanol (20 mL) was refluxed with
ω-bromoacetophenone (2.2 g, 11 mmol) and anhydrous
sodium acetate (1.64 g, 20 mmol) for 3 hr during which the
solid product was partially crystallized out. e mixture
was left to attain room temperature then filtered, washed
with cold ethanol, dried and recrystallized from ethanol.
1H NMR data are listed in Table 2. 13C NMR (DMSO/
CDCl3) 8c δ= 14.3 (CH3), 20.7 (CH3), 25.8, 27.7, 29.4, 33.3
(cyclohexyl-C), 42.3 (C-3a), 56.0 (C-3), 88.4 (C-2, thiazo-
line), 115.9, 120.4, 124.8, 125.7, 126.2, 126.8, 127.3, 127.8,
128.3, 128.5, 128.6, 134.9, 136.2, 140.2, 141.3, 148.5, 152.8
(C-3, thiazoline), 155.4 (Ar-C), 163.8 (C=N).
Biological activity
In vitro antitumor screening
Twenty of the newly synthesized compounds have been
selected by the NCI in vitro disease-oriented human cells
screening panel assay to be evaluated for their in vitro
antitumor activity. Primary in vitro one dose anticancer
assay was performed using the 3-cell line panel consist-
ing of NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)
according to the protocol of the Drug Evaluation Branch,
NCI, Bethesda31–33. All the compounds that reduced the
growth of any one of the cell lines to 32% or less namely
3c, 4d,e, 5a,d and 8c were passed on for evaluation in
the full panel of 60 human tumor cell lines of 9 tumor
subpanels including leukemia, non-small cell lung can-
cer, colon, central nervous system, melanoma, ovarian,
renal, prostate, and breast cancer cell lines. ese cell
lines were incubated with five concentrations (0.01–100
μM) for each compound. A 48h continuous drug expo-
sure protocol was used, and a SRB protein assay was
employed to estimate cell viability or growth. Subpanel
and full panel mean-graph midpoint values (MG-MID)
for certain agents are the average of individual real and
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)
benzenesulfonylimino]-3-substituted-[1,3]thiazinan-4-ones
(9a–c)
To a solution of the appropriate thiourea derivative 6
(10 mmol) in absolute ethanol (20 mL) was added ethyl
3-bromopropionate (2 g, 11 mmol) and anhydrous
sodium acetate (1.64 g, 20 mmol) and the reaction mix-
ture was heated under reflux for 4 h. After the mixture
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives
7
default GI50, TGI, or LC50 values of all cell lines in the sub-
panel or the full panel, respectively.
mL. A 100 µL sample of the mycobacterial suspension
was inoculated onto each compound-containing quad-
rant. Control quadrant consisted of agar alone, culture
medium with DMSO and culture medium with reference
antimycobacterial drugs; rifampicin and 1HN was per-
formed. All plates were then incubated at 37°C in a CO2
(5% CO2 / 95% humidified air) incubator, for 3–4 weeks.
e MIC was defined as the lowest chemical dilution
associated with at least a 99% reduction in the number of
visible colonies.
Antimicrobial screening
Inhibition zone (IZ) measurement Standard sterilized
filter paper discs (5 mm diameter) impregnated with a
solution of the test compound in DMSO (1 μg/mL) was
placed on an agar plate seeded with the appropriate
test organism in triplicates. e utilized test organisms
were Staphylococcus aureus (ATCC 25923) and Bacillus
subtilis (ATCC 6051) as examples of Gram positive bac-
teria, Escherichia coli (ATCC 25922) and Pseudomonas
aeruginosa (ATCC 27853) as examples of Gram negative
bacteria, Candida albicans (ATCC 10231) and Aspergillus
niger (recultured) as representatives of fungi. Ampicillin
trihydrate and Clotrimazole were used as standard anti-
bacterial and antifungal agents, respectively. DMSO
alone was used as control at the same above-mentioned
concentration. e plates were incubated at 37°C for 24 h
for bacteria and 72 h for fungi. e results were recorded
for each tested compound as the average diameter of
inhibition zones of bacterial growth around the discs in
mm (Table 5).
Results and discussion
Chemistry
e starting compounds in this study are 6-arylidene-2
-methylcyclohexanones 1a–c could be synthesized via
Claisen-Schmidt condensation of aromatic aldehydes
with 2-methylcyclohexanone. ese chalcones were
reacted with 4-hydrazinobenzenesulfonamide hydro-
chloride to afford the desired hexahydroindazole deriva-
tives2a–c(Scheme1). eirIRspectrarevealedtwobands
at 3365–3388 cm−1 and 3220–3258 cm−1 corresponding to
the amino group and two bands at 1350–1378cm−1 and
1180–1195 cm−1 attributed to the SO2N function. e
structures of these compounds were further confirmed
by the 1H-NMR which exhibited a characteristic dou-
blet at δ 4.95–5.38 ppm attributed to the H-3 proton and
a multiplet at δ 3.64–3.70 ppm due to the H-3a proton
which substantiate the closure of the indazoline ring
(Table 2). Mild oxidation of the indazoline 2a–c with
bromine water led to the formation of the correspond-
ing indazoles 3a–c. Such oxidation was confirmed by the
1H-NMR spectra, which lacked the characteristic doublet
and multiplet of H-3 and H-3a protons of the parent inda-
zolines (Table 2). e ureido derivatives 4a–f and 5a–e
were prepared in good yields by reacting the indazoline
2a–c or the indazole 3a–c derivatives with the appropri-
ate isocyanate in the presence of anhydrous potassium
carbonate as a mild basic catalyst. e IR spectra of these
compounds showed beside the absorption bands corre-
sponding to the NH group at 3216–3288cm−1, two bands
at 1345–1372 cm−1 and 1177–1190 cm−1 for the SO2N func-
tion, in addition to a characteristic urea carbonyl band at
1645–1665 cm−1. In a similar fashion, the synthesis of the
thioureido derivatives 6a–h was achieved by refluxing
2a–c with the appropriate isothiocyanate in dry acetone
in the presence of anhydrous potassium carbonate as a
mild basic catalyst (Scheme 2). eir IR spectra revealed
beside the characteristic absorption bands correspond-
ing to the amino group and the SO2N function lying at
the same range of their structurally-related derivatives, a
characteristic C=S band at 1155–1165 cm−1. Cyclization of
the thioureido derivatives 6a–h with ethyl bromoacetate
afforded the corresponding 4-oxothiazolidines 7a–f in
good yields. e IR of these compounds was character-
ized by the appearance of a new band at 1720–1735cm−1
due to the carbonyl group at the thiazol-C4 and two bands
at 1338–1365 cm−1 and 1169–1188 cm−1 for the SO2N
Minimalinhibitoryconcentration(MIC)measurement MICs
were measured for compounds that showed significant
growth inhibition zones (≥ 12 mm) using the two-fold
serial dilution technique34. e micro-dilution suscepti-
bility test in Muller-Hinton Broth (Oxoid) and Sabouraud
Liquid Medium (Oxoid) was used for the determination
of antibacterial and antifungal activity, respectively. Stock
solutions of the tested compounds, ampicillin trihydrate
and Clotrimazole were prepared in DMSO at concentra-
tion of 1600 µg/mL followed by two-fold dilution at con-
centrations of 800, 400, 6.25 µg/mL). e microorganism
suspensions at 106 CFU/mL (Colony Forming Unit/mL)
concentration were inoculated to the corresponding
wells. Plates were incubated at 36°C for 24 h to 48 h and
the minimal inhibitory concentrations (MIC) were deter-
mined. Control experiments were also done.
Antimycobacterial screening
e available Mycobacterium tuberculosis strain was
cultured in tubes with 10 µL of a suspension of the
strain in physiological saline. e strain was collected
from the slant after four weeks incubation33. e inocu-
lum was prepared with 3–5 weeks old M. tuberculosis
colonies from Loewenstein-Jensen slants, emulsified in
dilution fluid containing 2% fatty acid free albumin and
0.02% Tween 80, pH 6.9. Suspensions were then diluted
in saline to a turbidity of McFarland no.1 standard and
then diluted to obtain inocula of 3 × 105 cells per well. e
MICs measurement were determined by agar dilution
technique Agar supplemented with 10% OADC (oleic
acid-albumin-dextrose-catalase) enrichment, was used
to prepare quadrant plates with serial DMSO two-fold
dilutions of the test compounds. e following concen-
trations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4 µg/
© 2012 Informa UK, Ltd.

8
H.M. Faidallah et al.
moiety. Refluxing 6 with phenacyl bromide afforded
the targeted thiazolines 8a–c. Whereas, reacting the
same compounds 6a–c with ethyl 3-bromopropionate
or 1,2-dichloroacetone resulted in the formation of the
corresponding 4-oxo-5,6-dihydrothiazines 9a–c and the
5-oxo-5,6-dihydrothiazines 10a–c, respectively. eir
IR spectra were characterized by the presence of a car-
bonyl band at 1720–1730 cm−1 as well as two bands at
1340–1373 cm−1 and 1174–1185 cm−1 for the SO2N group.
active against leukemia RPMI-8226 and non-small cell
lung NCI-H322 cell lines with GI50 values of 5.56 and 4.98
μM, respectively and compound 4e exhibited distinct
activity against the colon HCT-116 cancer cells (GI50
3.40 μM). Furthermore, 5d displayed potential activity
against all the leukemia cell lines with a GI50 values range
of 2.46–24.62 μM. A special activity pattern was shown
by compound 5a which was remarkably active against
most of the tested 60 cell lines with GI50 values range of
0.02–28.4 μM. It revealed super potent activity towards
leukemia CCRF-CEM, SR, melanoma SK-MEL-5 and
renal A498, TK-10, UO-31 with GI50 values of 0.55, 0.80,
1.36, 0.02, 1.72 and 1.35 μM, respectively (Figures 1–3).
Concerning the broad spectrum of antitumor activ-
ity, the active compounds displayed effective growth
inhibition GI50 (MG-MID), total growth inhibitory TGI
(MG-MID) and cytotoxic LC50 (MG-MID) activities
(<100 μM) (Table 4). Compound 5a having GI50, TGI,
and LC50 MG-MID values of 9.22, 26.61 and 69.80 μM,
respectively, proved to be the most active member in this
study. It revealed potential activity against all the tested
subpanel tumor cell lines with special high potency
on the leukemia subpanel at the GI50 level (2.45 μM)
(Table 4). Furthermore, the compound showed almost
the same level of antitumor activity against the non-small
cell lung, colon, melanoma, renal and prostate cancer
subpanels (GI50 range 10.41–14.20 μM). Moderate activ-
ity has been shown by this compound against the CNS,
ovarian and breast subpanel tumor cell lines (Table 4).
On the other hand, compounds 3c and 4d were almost
equipotent at the GI50 level (15.60 and 15.91 μM, respec-
tively), while they showed some difference in the TGI and
LC50 MG-MID values (Table 4). Moreover, compounds
4e, 5d and 8c displayed appreciable antitumor activity
towards most of the tested tumor cell lines at the GI50,
TGI, and LC50 MG-MID levels (Table 4).
A close examination of the structures of the active
compounds revealed that, while the hexahydroinda-
zole derivative 2c was totally inactive, its correspond-
ing tetrahydroindazole analog 3c showed significant
antitumor activity against most of the tested subpanel
tumor cell lines with GI50, TGI and LC50 MG-MID values
of 15.6, 39.6 and 78.3 μM, respectively. Condensation
of 3c with different isocyanates to produce the dihy-
drosulfonylureas 4d (X = CH3, R1 = cyclohexyl) and 4e
(X = CH3, R1 = phenyl), did not significantly improve
the anticancer activity at the GI50 MG-MID (15.91 and
14.40 μM, respectively), TGI MG-MID (38.4 and 49.62
μM, respectively) and the LC50 MG-MID levels (77.80
and 88.23 μM, respectively). On the contrary, oxida-
tion of compound 4d resulted in a highly active inda-
zolesulfonylurea 5a (X = H, R1 = cyclohexyl), which is
proved to be the most active member in this study with
potential activity against all the tested subpanel tumor
cell lines (GI50, TGI and LC50 MG-MID values 9.22, 26.61
and 69.80 μM, respectively). In addition, 5a was able to
exert potential inhibitory activity against the leukemia
subpanel (GI50 2.45 μM, Table 4), when compared with
1
In addition, their H-NMR spectra showed the appear-
ance of new methylene groups of the thiazine rings at a
range of δ 3.19–5.42 ppm (Table 2).
In vitro antitumor evaluation
Primary in vitro 3-cell line assay
Twenty of the newly synthesized compounds were cho-
sen by the NCI to be evaluated for their in vitro antitumor
activity. Primary in vitro anticancer assay was performed
using the 3-cell line panel consisting of NCI-H460 (lung),
MCF7 (breast), and SF-268 (CNS) in accordance with the
protocol of the Drug Evaluation Branch, National Cancer
Institute, Bethesda31–33. e compounds were added at a
single concentration (10−4 M) and the culture was incu-
bated for 48 h. Compounds which reduced the growth of
any one of the cell lines to 32% or less, were passed on for
the full panel 60-cell lines assay. Six compounds 3c, 4d,
4e, 5a, 5d and 8c out of the 16 tested compounds have
been selected for a 60-cell panel assay.
In vitro full panel 60-cell line assay
About 60 cell lines of nine tumor subpanels including leu-
kemia, non-small cell lung cancer, colon, central nervous
system, melanoma, ovarian, renal, prostate, and breast
cancer cell lines were incubated with five concentrations
(0.01–100 μM) for each compound and were used to cre-
ate log concentration-% growth inhibition curves. ree
response parameters (GI50, TGI, and LC50) were calculated
for each cell line. e GI50 value corresponds to the con-
centration of the compounds causing 50% decrease in net
cell growth, the TGI value is the concentration of the com-
pounds resulting in total growth inhibition and the LC50
value is the concentration of the compounds causing net
50% loss of initial cells at the end of the incubation period
(48h). Subpanel and full panel mean-graph midpoint val-
ues (MG-MID) for certain agents are the average of indi-
vidual real and default GI50, TGI, or LC50 values of all cell
lines in the subpanel or the full panel, respectively31–33
.
From the results obtained it could be noticed that,
only six compounds, namely; 3c, 4d,e, 5a,d and 8c
exhibited remarkable antitumor activity against most of
the tested subpanel tumor cell lines (GI50 and TGI values
< 100 μM), whereas the rest of the compounds proved
to be totally inactive. ose six compounds showed an
obvious sensitivity against some individual cell lines
(Table 3). Compound 3c proved to be significantly active
towards leukemia CCRF-CEM, colon SW-620 and renal
A498 cell lines with GI50 values of 3.65, 4.82 and 6.28 μM
respectively. However, compound 4d was found to be
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives
9
Table 3. Growth inhibitory concentration (GI50, μM) of the active compounds.
Cell Lines
Leukaemia
CCRF-CEM
HL-60 (TB)
K-562
3c
4d
4e
5a
5d
8c
3.65
18.43
23.02
13.36
7.70
18.67
18.51
12.70
8.23
0.55
6.20
3.23
1.90
3.20
0.80
2.46
24.62
12.24
6.11
16.71
15.50
16.01
13.72
13.63
12.06
a
NT
17.4
MOLT-4
10.2
18.2
14.5
RPMI-8226
SR
5.56
15.41
3.01
7.43
6.53
2.35
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
24.74
20.21
21.41
14.60
17.12
23.12
19.80
21.32
11.75
13.91
21.10
46.75
13.33
20.82
14.85
4.98
13.21
13.92
18.95
15.30
16.01
14.72
20.62
22.03
12.66
22.27
11.62
17.55
2.79
27.90
18.52
33.15
18.83
16.46
16.90
15.84
19.91
11.22
19.81
22.00
42.22
19.21
22.22
NT
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
21.81
11.90
4.95
29.01
16.20
24.61
21.81
6.80
8.93
12.84
22.14
8.62
13.51
11.82
16.52
20.83
23.86
11.92
16.60
19.01
NT
11.81
NT
20.00
13.81
20.92
16.45
36.78
17.51
12.40
24.32
NT
11.53
20.62
22.15
16.40
4.82
3.40
4.02
17.90
24.32
NT
15.52
25.35
18.03
12.92
17.42
24.94
13.52
2.82
HT29
KM12
23.20
15.9
SW-620
CNS Cancer
SF-268
21.31
21.14
19.01
23.24
18.54
21.21
22.32
44.73
12.85
18.54
13.07
13.92
25.78
18.36
13.94
12.07
17.62
10.06
20.92
21.64
11.57
19.77
33.22
18.87
31.32
23.48
18.50
15.47
18.92
18.35
66.77
NT
SF-295
SF-539
SNB-19
384
SNB-75
17.94
20.14
U251
19.64
Melanoma
LOX IMVI
M14
19.35
20.20
12.67
18.67
12.78
21.22
13.74
18.43
35.70
17.56
18.32
14.97
22.44
11.94
12.54
17.41
15.32
23.5
1.95
19.47
15.67
17.30
1.36
11.37
23.74
17.15
20.44
13.23
17.70
11.82
18.40
32.52
22.01
NT
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
14.26
18.49
12.28
14.1
24.72
18.08
21.43
15.05
3.3
14.6
16.4
26.2
35.4
48.2
5.8
14.9
18.6
28.3
19.7
21.2
11.7
13.7
16.5
13.1
22.4
17.9
11.3
19.7
16.8
28.4
27.0
19.5
3.8
33.3
20.4
31.9
18.9
17.7
26.9
16.2
NT
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal Cancer
786-0
NT
22.3
35.1
16.12
6.28
29.40
22.51
14.77
17.40
18.78
17.06
12.34
12.61
16.0
2.33
0.02
22.84
19.70
14.8
32.57
14.76
21.82
17.16
23.80
16.76
19.19
A498
ACHN
19.89
18.17
17.80
18.11
20.12
13.81
13.66
16.70
14.10
16.60
15.6
CAKI-1
19.50
22.73
18.94
1.72
16.04
16.59
17.00
11.32
RXF 393
SN12C
TK-10
(Continued)
© 2012 Informa UK, Ltd.

10 H.M. Faidallah et al.
Table 3. Continued.
Cell Lines
3c
4d
4e
5a
5d
8c
UO-31
6.64
11.16
11.72
1.35
8.75
17.74
Prostate Cancer
PC-3
17.12
15.26
10.66
24.61
14.95
14.22
12.61
15.53
17.10
15.93
22.53
23.79
DU-145
Breast Cancer
MCF7
32.43
22.44
13.85
14.57
17.92
NT
11.35
12.74
28.52
24.76
15.06
17.78
31.45
18.11
16.17
11.58
18.74
17.17
15.47
35.98
5.94
17.00
21.89
21.15
19.54
12.14
26.47
21.54
33.81
17.55
23.81
28.22
18.09
31.05
5.67
NT
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
15.19
19.02
18.16
19.28
36.85
MDA-MB-435
BT-549
T-47D
72.23
aNT: Not Tested.
Figure 1. Leukaemia Growth inhibitory concentration (GI50, μM)
of the active compounds.
Figure 3. Renal Cancer Growth inhibitory concentration (GI50,
μM) of the active compounds.
Figure 2. Melanoma Growth inhibitory concentration (GI50, μM)
of the active compounds.
Figure 4. Structures of sulofenur A, its congeners B–D and the
general structures of the new compounds E and F. (See colour
version of this figure online at www.informahealthcare.com/enz)
sulofenur; (NSC 656667; GI50 1.27 μM, Table 4); a known
sulfonylurea derivative with potential antineoplastic
activity (Figure 4).Replacing the cyclohexyl moiety in
5a with a phenyl moiety and introduction of a methyl
group as in 5d (X = CH3, R1 = phenyl), resulted in about
2-fold decrease in activity at the GI50 and TGI levels
(16.64 and 54.22, respectively). However, to substanti-
ate the postulate a detailed protein assay is required
in order to outline the mechanism of their anticancer
activity. Moreover, in a related study 3-substituted-phe-
nyl-1H-indole-5-sulfonamides37 have shown inhibitions
of some of the isoforms of carbonic anhydrase enzyme.
is involves interfering with pH regulations of various
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives 11
Table 4. Median growth inhibitory concentrations (GI50, μM), total growth inhibitory concentrations (TGI, μM) and lethal concentrations
(LC50, μM) of in vitro subpanel tumor cell lines.
Subpanel Tumor Cell Linesa
MG-MIDb
Compound
number
I
II
III
IV
V
VI
VII
VIII
16.84
16.40
13.84
12.21
15.65
23.00
NTd
IX
GI50
15.60
15.91
14.40
9.22
TGI
LC50
78.30
77.80
88.23
69.80
91.23
90.11
1.99
3c
4d
4e
5a
5d
8c
Sc
13.08
10.43
12.55
2.45
18.13
16.54
14.37
11.21
18.29
23.61
1.05
15.38
16.40
15.25
12.00
19.41
20.67
1.21
22.2
15.5
24.02
18.14
16.05
20.51
20.65
25.52
1.163
14.81
17.42
15.03
10.41
16.51
20.82
1.16
30.0
20.5
18.6
17.1
21.8
18.2
NT
39.61
38.40
49.62
26.61
54.22
51.63
1.63
23.6
19.6
17.01
15.82
24.71
18.58
1.21
15.72
14.20
16.67
20.62
1.30
8.70
16.64
20.04
1.19
14.19
1.27
aGI50 values against: I, Leukaemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII,
renal cancer; VIII, prostate cancer; IX, breast cancer.
bGI50, TGI and LC50 (μM) full panel mean-graph midpoint (MG-MID)=the average sensitivity of all cell lines towards the test agent.
cSulofenur; (NSC 656667): NCI cancer screen; August 2004.
dNT=Not tested.
Table 5. Antimicrobial activity of the target compounds 2–10.
S. aureus
(ATCC 25923)
B. subtilis
(ATCC 6051)
E.coli
(ATCC 25922)
P. aeruginosa
(ATCC 27853)
C. albicans
(ATCC 10231)
Compound
number
IZa
MICb
>200 (540)
100 (247.6)
100 (260.7)
100 (272.1)
100 (248.8)
50 (131)
IZ
9
MIC
...c
IZ
7
MIC
…
IZ
NAd
MIC
IZ
MIC
2a
2b
2c
3a
3b
3c
4a
4b
4d
4e
5a
5b
5d
6a
6b
6c
10
13
15
19
22
24
19
16
18
17
28
20
24
8
…
6
12
14
9
…
14
16
18
17
20
18
15
16
15
22
16
18
6
100 (247.6)
14
8
200 (495.1)
10
…
...
200 (521.5)
100 (272.1)
100 (248.8)
50 (131)
100 (202.2)
200 (409.3)
200 (393.2)
200 (397.9)
50 (101.5)
100 (205.4)
100 (200)
…
…
…
NA
NA
10
…
100
10
14
15
6
…
…
200 (497.6)
200 (524)
…
…
16
23
8
100 (248.8)
11
…
100 (262)
50 (101.1)
100 (204.7)
100 (196.6)
100 (198.9)
12.5 (25.4)
50 (102.7)
50 (100)
NA
NA
12
…
…
9
...
...
10
15
9
...
14
12
17
12
16
NA
6
200 (393.2)
200 (397.9)
50 (101.5)
200 (410.8)
100 (200)
…
100 (196.6)
100 (196.6)
10
…
...
14
200 (406)
29
18
20
NA
NA
NA
9
50 (101.5)
10
…
50 (102.7)
11
…
50 (100)
...
10
…
…
7
…
8
...
…
5
…
…
9
…
6
…
NA
10
8
...
NA
6
…
…
6f
10
12
14
16
18
16
18
12
18
14
16
36
…
...
8
...
...
…
…
7a
7b
7c
7d
8a
8c
9a
9b
10a
10b
A*
C**
>200 (367.2)
100 (179)
100 (164.7)
100 (179)
100 (165.3)
100 (161.6)
>200 (358)
100 (174.6)
200 (358)
100 (174.6)
12.5 (31)
…
10
12
14
15
18
17
9
...
...
NA
10
…
10
12
9
...
200 (358)
200 (329.4)
200 (358)
100 (165.3)
100 (161.6)
...
15
12
14
14
16
8
200 (358)
200 (329.4)
200 (358)
200 (330.6)
200 (323.2)
...
…
100 (179)
10
…
…
10
…
12
8
100 (179)
16
200 (330.6)
…
NA
NA
NA
13
…
6
…
…
NA
NA
NA
6
...
17
12
14
30
…
50 (87.3)
200 (358)
200 (349.2)
25 (62)
14
12
10
32
…
200 349.2)
...
…
…
...
...
…
NA
27
…
…
…
25 (62)
…
50 (124)
…
…
…
…
42
12.5 (36.2)
aInhibition zone (mm).
bMinimal Inhibitory Concentration: μg/mL(µM).
cNot tested.
dNot active.
A*: Ampicillin trihydrate; C**: Clotrimazole.
forms of tumours and represents a new anticancer
drug discovery strategy. Finally, although the thiourea
derivatives 6f and 6g were inactive in this test, one of
the cyclized products namely 8c showed some moder-
ate antitumor activity (GI50, TGI and LC50 MG-MID val-
ues 20.04, 51.63 and 90.11 μM, respectively).
© 2012 Informa UK, Ltd.

12 H.M. Faidallah et al.
Scheme 1. Scheme 1 Reagents and reaction conditions: (i) ethanol, reflux, 4 h; (ii, iv) Br2 water, r.t., 3h; (iii, v) RNCO, anhyd.K2CO3, acetone,
reflux, 18h; (vi) RNCS, anhyd. K2CO3, acetone, reflux, 10h.
Scheme 2. Reagents and reaction conditions: (i) ethyl bromoacetate, anhyd. Na acetate, ethanol, reflux, e h; (ii) phenacyl bromide, anhyd.
Na acetate, ethanol, reflux, 3 h; (iii) ethyl 3-bromopropionate, anhyd. Na acetate, ethanol, reflux, 4 h; (iv) 1,3-dichloroacetone, anhyd. Na
acetate, ethanol, areflux, 5h.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antitumor and antimicrobial activity of indazole derivatives 13
Egypt) was utilized in this assay. Rifampicin and isoni-
cotinic acid hydrazide (INH) were used as reference anti-
mycobacterial drugs. e MIC was defined as the lowest
concentration of the tested compound that yielded no vis-
ible growth on the plate. Among the compounds tested,
only compound 7a was able to exert weak growth inhibi-
tory effect (MIC 250 µg/mL) against the Mycobacterium
tuberculosis used in this screening, while the rest of the
synthesized compounds were totally inactive.
Antimicrobial screening
Compounds 2a-c, 3a-c, 4a,b,d,e, 5a,b,d, 6a-c,f, 7a-d, 8a,c,
9a,band10a,cwereevaluatedfortheirinvitroantimicrobial
activity34 against Staphylococcus aureus (ATCC 25923) and
Bacillus subtilis (ATCC 6051) as examples of Gram positive
bacteria, Escherichia coli (ATCC 25922) and Pseudomonas
aeruginosa (ATCC 27853) as examples of Gram negative
bacteria, Candida albicans (ATCC 10231) and Aspergillus
niger (recultured) as representatives of fungi.
e results revealed that most of the tested com-
pounds displayed greater inhibitory effect on the growth
of the tested Gram positive strain compared to Gram
negative ones. Most of the compounds showed weak or
no antibacterial activity against the Gram negative, P.
aeruginosa. Moreover, few compounds were able to exert
mild to moderate antifungal activity against C. albicans,
while, all the tested compounds lacked antifungal activ-
ity against Aspergillus niger. A close examination of the
structures of the active compounds revealed that the anti-
microbial profile of the tetrahydroindazole compounds 3
seemed to be more interesting than their corresponding
hexahydroindazole derivatives 2, as evidenced by their
IZ diameters and MIC values recorded in Table 5.
Among the hexahydroindazole series, compound
2a showed weak antimicrobial activity against all the
tested microbial strains (IZ ≤ 12 mm). Introduction of
chlorine atom or methyl group in the phenyl ring in
position 3 of the hexahydroindazole derivative as in 2b
and 2c resulted in a slight improvement in the activity
against the Gram positive S. aureus and B. subtilis (MIC
values 100 and 200 µg/mL, respectively). Whereas,
replacement of the hexahydroindazole moiety in 2a–c
with tetrahydroindazole resulted in more potent com-
pounds 3a–c, with an appreciable broad spectrum of
antibacterial activity against the tested Gram positive,
Gram negative bacteria (MIC values 100–200 µg/mL),
and moderate antifungal activity towards C. albicans
(MIC 100 µg/mL). e replacement of amino group in
3a with a urea moiety as in 5a produced the most potent
antimicrobial activity in the current series of com-
pounds which was as potent as ampicillin (MIC 12.5
µg/mL) against S. aureus, whereas its activity against B.
subtilis and E.Coli was 50% lower than that of ampicil-
lin (MIC 50 νs 25 µg/mL, respectively). It displayed a
moderate antifungal activity towards C. albicans, which
was about 50% of that of Clotrimazole (MIC 25 νs 12.5
µg/mL, respectively). However, it is worthy to mention
that, structure modification of the urea derivatives to
thiourea ones led to almost complete abolishment of
the antimicrobial activity (IZ ≤ 10 mm).
Conclusion
In conclusion, the objective of the present study was to
synthesize and investigate the antitumor, antimicrobial
and antimycobacterial activities of new compounds
incorporating the sulfonamido, N1,N3-disubstituted
sulfonylurea and thiourea pharmacophores structurally
related to a well-documented sulfonylurea anticancer
agentSulofenurAanditsstructurecongenersB-D(Figure
1). is aim has been verified by the synthesis of hybrid
compounds comprising the above mentioned pharma-
cophores substituted essentially with a 3-(4-toly)-[1,2-c]
pyrazol(in)e counterpart at the N3 aryl moiety having the
general structure E and F (Figure 1), for synergistic pur-
pose. e results revealed that six compounds namely,
3c, 4d,e, 5a,d and 8c have exhibited broad spectrum
of antitumor activity against most of the tested tumor
cell lines. Compound 5a proved to be the most active
antitumor agent in the present study with GI50, TGI
and LC50 MG-MID values of 9.22, 26.61 and 69.80 μM,
respectively, with high sensitivity towards some leuke-
mia, melanoma and renal cell lines. e other five active
compounds showed variable degrees of appreciable
antitumor activity (GI50 and TGI MG-MID values range
14.40–20.04 and 38.40–54.22 μM, respectively). In addi-
tion, the in vitro antibacterial and antifungal activities of
a number of the target compounds revealed that some
of them have significant antibacterial and mild to mod-
erate antifungal activities. Compound 5a produced the
most potent antimicrobial activity in the current series
of compounds with equipotency to ampicillin (MIC
12.5 µg/mL) against S. aureus, and 50% of ampicillin’s
activity against B. subtilis and E.coli (MIC 50 νs 25 µg/
mL, respectively), beside a moderate antifungal activ-
ity against C. albicans, which was about 50% of that of
Clotrimazole (MIC 25 νs 12.5 µg/mL, respectively).
e broad spectrum antitumor activity displayed by
these compounds will be of interest for future derivati-
zation in the hope of finding more active and selective
antitumor and/or antimicrobial agents.
Antimycobacterial screening
Determination of in νitro antimycobacterial activity of
the target compounds 2a–c, 3a–c, 4a,b,d,e, 5a,b,d, 6a–c,f,
7a–d, 8a,c, 9a,b and 10a,c was performed by employ-
ing the two-fold agar dilution method slightly modified
from that described by Mamolo and Vio34. A strain of
Mycobacterium tuberculosis (locally isolated, Alexandria,
Declaration of interest
e authors report no conflicts of interest.
© 2012 Informa UK, Ltd.

14 H.M. Faidallah et al.
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Journal of Enzyme Inhibition and Medicinal Chemistry