3-Methyl-2-(4-substituted phenyl)-4,5-dihydronaphtho[1,2-c]-pyrazoles: Synthesis and in-vitro biological evaluation as antitumour agents

Article abstract of DOI:10.1002/ardp.200700178

The synthetic strategies and characterization of some novel derivatives of 3-methyl-2-(4-substituted phenyl)-4,5-dihydronaphtho[1,2-c]pyrazoles carrying different pharmacophores and heterocyclic rings that are relevant to potential antitumour and cytotoxic activities are described. The antitumour activities of the newly synthesized compounds were evaluated according to the protocol of the National Cancer Institute (NCI) in-vitro disease-oriented human cells screening panel assay. The results revealed that six compounds, namely 6, 8, 11, 15, 17 and 18; displayed promising in-vitro antitumour activity in the 60-cell lines assay. The sulfonylthioureido group emerged as the most favourable pharmacophore. Incorporating such thioureido counterpart into the 6-membered 1,3-thiazinan-5-one resulted in better antitumour activities than those displayed by the 5-membered thiazoles and the 6-membered 1,3-thiazinan-4-one ring systems. Further ring expansion led to a total loss of the antitumour activity. The analog 18, 3-benzyl-2-[4-(3-methyl-4,5-dihydronaphtho-[1,2-c] pyrazol-2-yl)-benzenesulfonylimino]-1,3-thiazinan-5-one, proved to be the most active member identified in this series of compounds (GI₅₀, TGI, and LC₅₀ MG-MID values of 34.7, 85.1 and 97.7 μM, respectively). The differential cytotoxicity of the six active compounds to cancer and normal cells was studied utilizing the standard MTT cell viability assay. Compounds 17 and 18 were totally selective for the breast cancer cell line MCF7 (IC₅₀ 8.5 and 4.7 μM), without exerting any inhibitory effect on the normal breast cell line MCF-10A at the concentration level used (25 μM).

Full text of DOI:10.1002/ardp.200700178

Arch. Pharm. Chem. Life Sci. 2008, 341, 181–190
M. S. Al-Saadi et al.
181
Full Paper
3-Methyl-2-(4-substituted phenyl)-4,5-dihydronaphtho[1,2-c]-
pyrazoles: Synthesis and in-vitro Biological Evaluation as
Antitumour Agents
Mohamed S. Al-Saadi, Sherif. A. F. Rostom, and Hassan M. Faidallah
Department of Medicinal Chemistry, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
The synthetic strategies and characterization of some novel derivatives of 3-methyl-2-(4-substi-
tuted phenyl)-4,5-dihydronaphtho[1,2-c]pyrazoles carrying different pharmacophores and heter-
ocyclic rings that are relevant to potential antitumour and cytotoxic activities are described.
The antitumour activities of the newly synthesized compounds were evaluated according to the
protocol of the National Cancer Institute (NCI) in-vitro disease-oriented human cells screening
panel assay. The results revealed that six compounds, namely 6, 8, 11, 15, 17 and 18; displayed
promising in-vitro antitumour activity in the 60-cell lines assay. The sulfonylthioureido group
emerged as the most favourable pharmacophore. Incorporating such thioureido counterpart
into the 6-membered 1,3-thiazinan-5-one resulted in better antitumour activities than those dis-
played by the 5-membered thiazoles and the 6-membered 1,3-thiazinan-4-one ring systems. Fur-
ther ring expansion led to a total loss of the antitumour activity. The analog 18, 3-benzyl-2-[4-(3-
methyl-4,5-dihydronaphtho-[1,2-c]pyrazol-2-yl)-benzenesulfonylimino]-1,3-thiazinan-5-one,
proved to be the most active member identified in this series of compounds (GI50, TGI, and LC50
MG-MID values of 34.7, 85.1 and 97.7 lM, respectively). The differential cytotoxicity of the six
active compounds to cancer and normal cells was studied utilizing the standard MTT cell viabil-
ity assay. Compounds 17 and 18 were totally selective for the breast cancer cell line MCF7 (IC50
8.5 and 4.7 lM), without exerting any inhibitory effect on the normal breast cell line MCF-10A at
the concentration level used (25 lM).
Keywords: Antitumour activity / Cytotoxicity / Dihydronaphtho[1,2-c]pyrazoles / Thiazinanes / Thiazoles /
Received: August 22, 2007; accepted: September 14, 2007
DOI 10.1002/ardp.200700178
limited. This might be attributed to the difficulties faced
during the search of new anticancer agents; principally
the unknown and unexpected etiology of neoplasm and
the difficulty in designing selective agents against trans-
formed cells rather than normal cells [1]. Random screen-
ing continues to be one of the main routes to discover
new structure leads with antitumour activity and the
National Cancer Institute (NCI), Bethesda, MD, USA, still
playing a pivotal role in this field, through the selection
of novel compounds with unique chemical structures [2].
Therefore, new chemical classes of agents that have not
had extensive clinical evaluation are prime targets. A
common feature of several types of antitumour agents is
the presence of primary or secondary sulfonamide moi-
eties in their molecules [3–7]. In this regard, diarylsulfo-
Introduction
The design of new antitumour agents is one of the most
active areas in contemporary medicinal chemistry.
Nevertheless, the number of drugs available for the treat-
ment of some types of disseminated cancers is still very
Correspondence: Dr. S. A. F. Rostom, Associate Professor of Medicinal
Chemistry, Department of Medicinal Chemistry, Faculty of Medicine, King
Abdulaziz University, P. O. Box 80205, Jeddah 21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: +966 2-6400000-22327
Abbreviations: GI50 value (growth inhibitory activity); TGI value (cyto-
static activity); LC50 value (cytotoxic activity); mean-graph midpoint values
(MG-MID)
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182
M. S. Al-Saadi et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
nylureas and their congeners [8–10] have been focused and impairment of other functions [36]. Consequently, as
upon as new chemical entities with a distinct mode of a complementary part to the present research, it was
action, which has not been explored [11–13]. Meanwhile, thought worthwhile to investigate the differential cyto-
among the wide variety of heterocycles that have been toxic effect of the NCI-active antitumour analogs on both
explored for developing pharmaceutically important cancerous and normal cells. Their in-vitro cytotoxic effect
molecules, pyrazole-containing compounds have was determined via a modification of the MTT cell viabil-
attracted synthetic interest for being an essential struc- ity technique [37], against a panel of two cell types: breast
ture in many chemotherapeutic agents with potential cancer cell line MCF7 and normal breast cell line MCF-
antimicrobial [14–16], antiparasitic [17, 18], antimalarial 10A.
[19] and antiviral activities [20, 21]. As far as the anti-
cancer activity is concerned, literature citation revealed
that a wide range of pyrazole derivatives were reported
to contribute to a variety of antineoplastic potentials
Results and discussion
against a wide range of cancer cell lines [22–25].
Chemistry
During our ongoing studies aimed at the discovery of The suggested synthetic plans to obtain the target com-
new structures endowed with potential chemotherapeu- pounds are shown in Schemes 1–3. The key intermediate
tic activities [26–31], a series of 3-(4-chlorophenyl)- in this study is 3-methyl-2-(4-sulfamylphenyl)-4,5-dihydro-
indeno[1,2-c]pyrazol(in)es substituted with benzenesulfo- naphtho[1,2-c]pyrazole 2, which was synthesized by a
namide, sulfonylurea, sulfonylthiourea pharmacophores simple reproducible procedure involving the 1,3-cyclo-
and some derived thiazole-ring systems, was identified as condensation of 2-acetyl-1-tetralone 1 with 4-hydrazino-
promising antitumour agents [32]. Some compounds benzenesulfonamide hydrochloride. Condensation of 2
showed an interesting antitumour activity often associ- with different isocyanates and isothiocyanates yielded
ated with high or moderate specificity for certain human the corresponding substituted benzene-sulfonylureas 3–
tumour cell lines. In particular, it was found that the oxi- 5 and sulfonylthioureas 6–8, respectively (Scheme 1).
dized pyrazole derivatives showed better antitumour Cyclization of the sulfonylthioureido derivatives 6–8 to
activity than their parent pyrazoline analogs, whereas the corresponding 4-oxothiazolidines 9–11 and thiazo-
the benzenesulfonamides and sulfonylureas showed sig- lines 12–14 was effected by reacting the former with
nificant better antitumour spectrum than that of the sul- ethyl bromoacetate and phenacyl bromide in the pres-
fonylthioureido and derived thiazole analogs. These find- ence of sodium acetate, respectively. Analogously, it is
ings prompted the design and synthesis of a new series of considered of interest to cyclize the sulfonylthioureido
analogs in which the 4-chlorophenyl-indeno[1,2-c]pyrazo- derivatives 6, 7 to the respective 1,3-thiazinan-4-ones 15,
l(in)e moiety was replaced by a 3-methyl-dihydronaph- 16 and 1,3-thiazinan-5-ones 17, 18 target compounds
tho[1,2-c]pyrazole counterpart in order to evaluate the using ethyl 3-bromopropionate and 1,3-dichloroacetone,
impact of ring size and lipophilicity alteration on the respectively, as interesting structure variants (Scheme 2).
antitumour activity. All the newly synthesized com-
At this stage, it was attempted to prepare some con-
pounds comprise the pyrazole ring that proved to con- densed ring systems starting from the 4-oxothiazolidine
tribute to a better antitumour activity [32]. In an attempt analogs. In this view, when 11 (R = C6H5CO) was reacted
to add some synergism to the target compounds, it was with hydrazine hydrate in refluxing ethanol, the corre-
considered of interest to incorporate some of the sulfo- sponding thiazolo[4,3-c][1,2,4]triazole derivative 19 was
nylthioureido derivatives into 6-membered thiazinone, obtained. Moreover, condensing the 4-oxothiazolidine
thiazolo[4,3-c][1,2,4]triazole and pyrazolo[3,4-d]thiazole, analog 9 (R = C6H5) with benzaldehyde in the presence of
in addition to the 5-membered thiazole ring systems, in sodium hydroxide resulted in the formation of the aryli-
order to explore the influence of such structure rigidiza- dine compound 20. The latter chalcone 20 in its turn, was
tion on the anticipated biological activity. The antitu- condensed with methylhydrazine to afford the targeted
mour activities of the newly synthesized compounds pyrazolo[3,4-d]thiazole 21 (Scheme 3).
were evaluated according to the protocol of the National
Cancer Institute (NCI) in-vitro disease-oriented human In-vitro antitumour activity
cells screening panel assay [33–35]. It has been well estab- Primary in-vitro one-dose 3-cell line assay
lished that many adverse effects on normal cells and Out of the newly synthesized analogs, twelve com-
organs are common for most chemotherapeutic drugs, pounds, namely 2, 4, 6–9, 11, 15, 17–19 and 21 were
basically due to lack of specificity. These side effects selected by the National Cancer Institute (NCI) in-vitro dis-
include nephrotoxicity, hematotoxicity, neurotoxicity ease-oriented human cells screening panel assay to be
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Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Dihydronaphthopyrazoles as in-vitro Antitumour Agents
183
Scheme 1. Synthesis route of compounds 1–8.
Scheme 2. Synthesis route of compounds 9–18.
evaluated for their in-vitro antitumour activity. Primary cells. All the compounds which reduced the growth of
in-vitro one-dose anticancer assay was performed using any one of the cell lines to 32% or less were passed on for
the 3-cell line panel consisting of NCI-H460 (lung), MCF7 evaluation in the full panel of 60 human tumour cell
(breast) and SF-268 (CNS) in accordance with the protocol lines [33–35]. The results revealed that six compounds,
of the Drug Evaluation Branch, NCI, Bethesda, MD, USA namely 6, 8, 11, 15, 17 and 18 passed this primary anti-
[33–35]. The compounds were added at a single concen- cancer assay and consequently were carried over to be
tration (10– 4 M) and the culture was incubated for 48 h. tested against a panel of 60 different tumour cell lines.
End point determinations were made with a protein-
binding dye, sulphorhodamine B (SRB). Results for each In-vitro full-panel 60-cell line assay
compound were reported as the percent of growth of the About 60 cell lines of nine tumour subpanels, including
treated cells when compared to the untreated control leukemia, non-small cell lung, colon, CNS, melanoma,
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184
M. S. Al-Saadi et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Scheme 3. Synthesis of compounds 19–21.
ovarian, renal, prostate and breast cancer cell lines, were Moreover, compound 17 proved to be sensitive to the leu-
incubated with five concentrations (0.01–100 lM) for kemia subpanel with GI50 range of 1.88–28.10 lM. Fur-
each compound and were used to create: log concentra- thermore, compound 18 proved to be sensitive towards
tion - %growth inhibition curves. Three response param- some of the tested subpanel tumour cell lines including
eters (GI50, TGI, and LC50) were calculated for each cell the leukemia subpanel with GI50 range of 6.45–31.1 lM.
line. The GI50 value (growth inhibitory activity) corre- It showed noticeable sensitivity against melanoma LOX
sponds to the concentration of the compounds causing IMVI, SK-MEL-5 and breast MCF7 cell lines with GI50 values
50% decrease in net cell growth, the TGI value (cytostatic of 10.7, 9.09 and 3.74 lM, respectively (Table 1).
activity) is the concentration of the compounds resulting
With regard to the broad spectrum antitumour activ-
in total growth inhibition and the LC50 value (cytotoxic ity (Tables 2 and 3), the results revealed that all of the
activity) is the concentration of the compounds causing active compounds showed effective growth inhibition
net 50% loss of initial cells at the end of the incubation GI50 (MG-MID) beside a cytostatic activity TGI (MG-MID). In
period (48 h). Subpanel and full panel mean-graph mid- addition, only one compound, namely 18, exhibited
point values (MG-MID) for certain agents are the average weak cytotoxic activity LC50 (MG-MID). Further interpreta-
of individual real and default GI50, TGI, or LC50 values of tion of the results revealed that, compound 18 (GI50, TGI
all cell lines in the subpanel or the full panel, respec- and LC50 MG-MID values of 34.7, 85.1 and 97.7 lM, respec-
tively. The NCI antitumour drug discovery was designed tively) proved to be the most active member in this series
to distinguish between broad spectrum antitumour com- with a broad spectrum of activity against all the tested
pounds and tumours or subpanel-selective agents.
subpanel tumour cell lines and particular effectiveness
In the present study, the active compounds exhibited on the leukemia subpanel at the GI50 level (20.0 lM). On
remarkable antitumour activities against most of the the other hand, compounds 8, 11 and 17 displayed mod-
tested subpanel tumour cell lines (GI50, TGI and LC50 val- erate antitumour activity lying almost in the same range
ues a100 lM). These compounds showed a distinctive pat- (GI50 MG-MID values 42.6–44.8 lM), while their cytostatic
tern of sensitivity against some individual cell lines values were significantly lower (TGI MG-MID values
(Table 1), as well as a broad spectrum of antitumour activ- 79.4–91.4 lM). Finally, compound 15 proved to be the
ity (Tables 2 and 3).
least active member within this series as evidenced by its
With regard to the sensitivity against some individual GI50 MG-MID value (89.1 lM). Meanwhile, it was totally
cell lines, compound 6 showed high sensitivity against deprived of any cytostatic and cytotoxic activities.
melanoma MALME-3M and renal CAKI-1 cell lines with
The ratio obtained by dividing the full panel MG-MID
GI50 values of 1.15 and 1.85 lM, respectively. Whereas, (lM) of the compounds by their individual subpanel MG-
the analog 8 exhibited remarkable sensitivity against MID (lM) is considered as a measure of compound selec-
three leukemia cell lines, namely HL-60 (TB), K-562 and tivity. Ratios between 3 and 6 refer to moderate selectiv-
SR with GI50 values of 3.85, 6.09 and 0.76 lM, respectively. ity, ratios greater than 6 indicate high selectivity towards
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Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Dihydronaphthopyrazoles as in-vitro Antitumour Agents
185
Table 1. Growth inhibitory concentration (GI50, lM) values of the
in-vitro tumour cell lines.a)
Table 1. Continued
Cell Lines
UO-31
6
–
8
–
11
67.4
15
–
17
28.4
18
64.3
Cell Lines
6
8
11
15
17
18
Prostate Cancer
PC-3
DU-145
Breast Cancer
MCF7
NCI/ADR-RES
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
PRMI-8226
SR
39.9
–
42.0
23.9
42.1
38.6
–
–
20.4
43.9
27.7
50.5
c)
NTb)
–
–
–
15.5
–
–
21.0
65.5
79.2
–
NT
69.9
-
–
–
–
NT
65.0
28.1
–
17.6
19.6
7.85
1.88
6.45
34.1
30.1
9.62
31.1
8.35
3.85
6.09
–
36.3
0.76
11.5
–
35.9
–
42.5
NT
27.9
NT
44.9
NT
–
NT
–
35.1
NT
–
7.79
14.5
15.4
15.5
16.8
11.1
24.7
3.74
39.3
24.8
NT
20.6
22.5
82.9
MDA-MB-231/ATCC -
24.0
NT
26.1
38.6
NT
HS 578T
MDA-MB-435
BT-549
44.6
14.4
–
–
Non-Small Cell Lung Cancer
A 549 / ATCC
EKVX
HOP-62
HOP-92
NCI – H226
NCI – H23
NCI – H322M
NCI – H460
NCI – H522
–
–
–
–
NT
–
–
46.8
–
NT
21.5
54.1
NT
–
–
–
–
46.6
46.7
–
T-47D
40.5
33.5
30.9
24.0
27.7
–
94.0
46.4
81.3
84.4
–
23.5
–
91.4
a)
–
–
12.7
–
9.75
23.2
–
The data obtained from NCI's in-vitro disease-oriented human
–
tumour cell screen.
NT = not tested.
GI50 value A100 lM.
62.2
53.9
24.5
45.0
50.4
38.5
49.8
–
29.4
22.6
b)
c)
47.5
96.2
15.2
15.4
the corresponding cell line, while compounds not meet-
ing either of these criteria are rated non-selective [38]. In
this context, the active compounds in the present study
were found to be non-selective with broad spectrum anti-
tumour activity against the nine tumour subpanels
tested, with selectivity ratios ranging between 0.5–1.73.
A close examination of the structures of the active com-
pounds revealed that the benzene-sulfonamide parent-
compound 2 and the sulfonylureido derivative 4 were
totally inactive, whereas the sulfonylthioureido deriv-
atives 6 and 8 showed moderate antitumour activity
which varied greatly according to the nature of the sub-
stituent. Compound 6 (R1 = phenyl) showed GI50 MG-MID
value of 69.4 lM. Replacing the phenyl to a benzoyl moi-
ety as in compound 8 resulted in a remarkable improve-
ment in the growth inhibitory activity (GI50 MG-MID
value 69.4 lM). Meanwhile, at the cytostatic level, both
compounds 6 and 8 displayed almost the same level of
activity (TGI MG-MID values 98.0 and 93.3 lM, respec-
tively). Introducing a benzyl substituent as in compound
7 resulted in a complete abolishment in the antitumour
activity.
On the other hand, cyclization of the sulfonylthiour-
eido derivative 8 to the thiazolidinone 11 (R = phenyl) did
not result in a significant improvement in the antitu-
mour activity (GI50 MG-MID values 42.6 vs 44.7 lM, respec-
tively). However, the cytostatic potential of 11 has been
remarkably improved when compared with 8 (79.4 vs
93.3 lM, respectively). Nevertheless, a dramatic reduc-
tion in the antitumour activity was encountered with
ring enlargement to the 6-membered 1,3-thiazinan-4-one
15 (R1 = phenyl), which proved to be the least active mem-
ber within this series (GI50 MG-MID value 89.1 lM), and
without any cytostatic and cytotoxic effects. On the con-
Colon Cancer
COLO 205
HCC – 2998
HCT – 116
HCT – 15
HT29
–
44.9
NT
89.2
39.2
–
NT
–
–
–
–
–
–
NT
63.4
NT
NT
18.2
38.8
–
25.6
–
98.6
26.4
22.4
63.5
51.4
–
6.50
21.2
34.5
46.5
19.1
72.9
31.7
51.4
60.1
25.5
KM12
34.4
–
–
NT
SW – 620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
18.7
–
–
–
–
39.6
29.3
17.4
NT
96.4
59.5
43.3
44.6
21.6
NT
NT
NT
35.0
75.7
NT
–
NT
–
20.2
–
22.4
–
–
21.1
17.6
25.7
29.5
–
75.9
21.8
23.3
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
25.8
1.15
50.3
NT
–
–
NT
–
NT
–
–
–
95.5
–
–
5.59
–
10.7
41.4
46.5
58.2
24.6
9.09
38.2
28.3
–
–
–
–
–
–
22.9
–
52.5
25.1
92.5
18.7
13.4
18.2
28.5
8.11
45.3
16.0
–
41.1
30.7
10.7
29.0
Ovarian Cancer
IGROV1
20.6
36.8
–
–
–
–
–
13.7
–
–
72.4
–
–
–
–
–
–
89.6
–
8.31
–
37.6
–
–
43.3
24.0
25.2
97.6
–
41.0
88.9
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
15.8
77.5
49.9
68.8
NT
Renal Cancer
786-0
ACHN
CAKI-1
RXF-393
SN12C
26.4
–
1.85
–
–
10.0
–
28.9
24.6
26.6
28.8
19.2
98.0
81.7
–
–
70.9
87.5
–
13.3
24.1
18.1
31.5
17.8
–
89.2
32.9
26.6
22.6
26.7
50.4
41.0
16.1
NT
34.4
28.9
TK-10
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M. S. Al-Saadi et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Table 2. Median growth inhibitory concentrations (GI50, lM) of in-vitro subpanel tumour cell lines.a)
Cpd. No.
Subpanel tumour cell linesb)
MG-MIDc)
I
II
III
IV
V
VI
VII
VIII
IX
6
8
86.2
41.2
67.1
93.0
41.6
20.0
20.1
91.0
61.0
55.6
80.1
63.4
59.3
38.5
77.6
68.0
60.4
86.0
49.2
29.8
77.7
72.2
45.1
17.1
81.8
51.7
58.8
99.4
48.0
36.3
31.9
85.8
81.0
62.4
98.3
79.8
62.6
43.0
68.0
53.4
41.9
91.4
52.4
44.7
34.4
90.0
33.0
40.3
–
64.2
39.1
34.7
73.6
50.2
47.2
87.0
30.2
32.3
39.2
69.4
42.6
44.7
89.1
44.8
34.7
27.1
11
15
17
18
M*
d)
–
59.2
50.8
42.1
a)
Data obtained from NCI's in-vitro disease-oriented human tumour cell screen.
I, Leukemia; 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.
GI50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
Subpanel GI50 value >100 lM.
b)
c)
d)
* Melphalan (A reference standard antineoplastic agent).
Table 3. Median total growth inhibitory concentrations (TGI, lM) of in-vitro subpanel tumour cell lines.a)
Cpd. No.
Subpanel tumour cell linesb)
MG-MIDc)
I
II
III
IV
V
VI
VII
VIII
IX
d)
6
8
97.0
–
89.8
–
–
–
–
–
–
84.7
–
88.6 (96.4)
–
–
–
–
92.0
–
90.2
95.2
88.4
–
–
-
–
–
–
–
95.6
98.3
78.1
–
91.8
70.9
–
98.0
93.3
79.4
–
91.4
85.1 (97.7)
98.3
97.7
87.4
–
94.4
95.5
–
89.7
73.5
–
–
79.1
81.9
11
15
17
18
M*
76.2 (95.4)e) 87.7 (99.0) 76.7
–
86.8
–
86.0
–
–
91.7
–
75.0 (86.2) 94.7
73.9
66.3
–
a)
Data obtained from NCI's in-vitro disease-oriented human tumour cell screen.
b)
c)
d)
e)
For subpanel tumour cell lines, see footnote (&) of Table 2.
TGI (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent
Subpanel TGI value >100 lM.
LC50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
* Melphalan (A reference standard antineoplastic agent).
trary, the 1,3-thiazinan-5-one 17 (R1 = phenyl) showed an agent, melphalan. Compound 18 showed an overall
obvious higher activity than the parent sulfonyl-thiour- tumour growth inhibitory activity comparable with that
eido derivative 6 (GI50 MG-MID values 44.8 vs 69.4 lM, displayed by melphalan (Table 2). In addition, the same
respectively). At the cytostatic level, both compounds 6 compound proved to be nearly equipotent to melphalan
and 17 displayed almost the same level of activity (TGI against the leukaemia, melanoma, prostate and breast
MG-MID values 98.0 and 91.4 lM, respectively). Replacing subpanel tumour cell lines (Table 2). Meanwhile, except
the phenyl substituent with a benzyl one gave rise to the for compound 6, the other five active compounds were
analog 18, which proved to be the most active member more active than melphalan as cytostatic agents as indi-
identified in this series of compounds (GI50, TGI, and LC50 cated from their TGI MG-MID values (Table 3).
MG-MID values of 34.7, 85.1 and 97.7 lM, respectively).
Finally, further ring enlargement to the fused thia- In-vitro MTT cytotoxicity assay
zolo[4,3-c][1,2,4]triazole and pyrazolo[3,4-d]thiazole ring Chemotherapy very often induces severe side effects,
systems led to completely inactive derivatives 19 and 21.
which are in part a consequence of destruction of normal
The antitumour activities of the active compounds cells. As a complementary part for this study, it was
were compared with that of the known antineoplastic thought worthwhile to evaluate the differential cyto-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Dihydronaphthopyrazoles as in-vitro Antitumour Agents
187
toxic effect of the NCI-active antitumour analogs: 6, 8, 11, the ring size at the benzenesulfonylimino functionality
15, 17 and 18 on both cancerous and normal cells. Their together with their substitution pattern seemed be rele-
in-vitro cytotoxic effect was determined via a modifica- vant to their biological activity. Incorporating the thiour-
tion of the MTT method [37], against a panel of two cell eido counterpart into the 6-membered 1,3-thiazinan-5-
types: breast cancer cell line MCF7 and normal breast cell one ring system resulted in better antitumour activities
line MCF-10A.
than those displayed by the 5-membered thiazoles and
MTT assay is a standard colorimetric assay for measur- the 6-membered 1,3-thiazinan-4-ones. Further ring
ing cell growth. It is used to determine cytotoxicity of expansion led to a total loss of the antitumour activity. In
potential medicinal agents and other toxic materials. In general, the overall antitumour activity of such type of
brief, yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
dihydronaphtho[1,2-c]pyrazoles was less than their corre-
nyltetrazolium bromide) is reduced to purple formazane sponding indeno[1,2-c]pyrazol(in)es substituted with the
by mitochondrial dehydrogenases of living cells. A suit- same pharmacophores [32]. Interestingly, two com-
able solvent is added to dissolve the insoluble purple for- pounds, namely 17 and 18 were preferentially able to
mazane product into a coloured solution. The absorb- inhibit the growth of human breast cancer cell line
ance of this coloured solution can be quantified by meas- MCF7, as compared to normal epithelial cells MCF-10A in
uring at a certain wavelength. When the amount of pur- a modified MTT assay. However, the broad spectrum anti-
ple formazane produced by cells treated with an agent is tumour effects expressed by the active compounds
compared with that produced by untreated control cells, against most of the tested tumour cell lines increases the
the effectiveness of the agent in causing death of cells likelihood of their future derivatization in order to
can be deduced, through the production of a dose- explore the scope and limitations of their activities.
response curve. The results represent the mean of four
independent experiments and are expressed as IC50, i. e., The authors are deeply grateful to the authorities of the Insti-
the concentration that reduced by 50% the optical den- tute of Research and Consultation, King Abdulaziz University,
sity of treated cells with respect to untreated controls.
Jeddah, Kingdom Saudi Arabia, for the financial support of
Particular interest was focussed on the compounds this research project (425/006). Extendable thanks are due to
that were able to preferentially inhibit the growth of the the staff members of the Department of Health and Human
MCF7 human breast cancer cell line over the normal Services, National Cancer Institute (NCI), Bethesda, Maryland,
breast epithelial cell line MCF-10A. In this respect, com- USA for carrying out the anticancer screening of the newly syn-
pounds 17 and 18 were totally selective for the cancer cell thesized compounds. Sincere gratitude is conveyed to Dr. Y. R.
line at low micromolar concentrations (IC50 8.5 and Abdel-Fattah, Genetic Engineering and Biotechnology Research
4.7 lM), without any inhibitory effect on the normal Institute (GEBRI), Mubarak City for Scientific Research and
breast epithelial cell line MCF-10A (IC50 A 25 lM). On the Technology Applications, Bourg El-Arab, Alexandria, Egypt,
other hand, in the presence of compounds 5 and 8, for carrying out the MTT assay.
growth of the human breast cancer cell line MCF7 was
The authors have declared no conflict of interest.
inhibited with obviously higher IC50 values ranging from
16.3 to 19.7 lM. Cell growth in the normal breast epithe-
lial cell line MCF-10A showed almost the same level of
Experimental
growth inhibition at IC50 values of 22.4 and 24.1 lM,
respectively. Finally, cell growth in both cancerous and
normal breast cell lines appeared unaffected at the maxi-
mum concentration utilized (25 lM) in the presence of
the test compounds 11 and 15.
Chemistry
Melting points were determined in open glass capillaries on a
Gallenkamp melting point apparatus (Weiss-Gallenkamp, Lon-
don, UK) and were uncorrected. The infrared (IR) spectra were
recorded on Perkin-Elmer 297 infrared spectrophotometer (Per-
kin Elmer, Beaconsfield, UK) using the NaCl plate technique. The
1H-NMR spectra were recorded on a Varian EM 360 spectrometer
(Varian Inc., Palo Alto, CA, USA) using tetramethylsilane as the
internal standard and DMSO-d6 as the solvent (Chemical shifts in
(d, 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 l 0.4% of the theoretical values. Physicochemical and
analytical data are recorded in Table 4. Follow up of the reac-
tions and checking the homogeneity of the compounds were
Conclusion
In conclusion, according to the protocol of the NCI in-
vitro disease-oriented human cells screening panel assay,
the obtained data suggests that the sulfonylthioureido
group proved to be the most favourable pharmacophore
rather than the parent benzenesulfonamide and the cor-
responding sulfonylureido isosteres. On the other hand,
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188
M. S. Al-Saadi et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
made by TLC on silica gel-protected aluminium sheets (Type 60
F254, Merck, Darmstadt, Germany) and the spots were detected
by exposure to UV-lamp at k 254.
Table 4. Physicochemical and analytical data of compounds 2–
21.
Cpd.
R or R1
Mp.
Yield
(%)
Mol. formula
(Mol. weight)
(8C)
3-Methyl-2-(4-sulfanylphenyl)-4,5-dihydronaphtho[1,2-
c]pyrazole 2
2
3
-
270-2
A310
144-6
212-3
184-5
162-4
136-8
222-3
266-7
216-8
154-6
260-2
214-5
266-7
272-4
182-4
209-1
279-1
110-2
278-9
92
82
84
86
83
77
78
74
72
76
70
72
74
71
74
75
76
72
70
65
C18H17N3O2S
(339.41)
C25H28N4O3S
(464.58)
C25H22N4O3S
(458.53)
C29H24N4O3S
(508.59)
C25H22N4O2S2
(474.60)
C26H24N4O2S2
(488.63)
C26H22N4O3S2
(502.61)
C27H22N4O3S2
(514.62)
C28H24N4O3S2
(528.65)
C28H22N4O4S2
(542.63)
C33H26N4O2S2
(574.72)
C34H28N4O2S2
(588.74)
C34H26N4O3S2
(602.73)
C28H24N4O3S2
(528.65)
C29H26N4O3S2
(542.67)
C28H24N4O3S2
(528.65)
A solution of 2-acetyl-1-tetralone (3.8 g, 20 mmol) in ethanol
(30 mL) was heated under reflux with 4-hydrazinobenzenesulfo-
namide hydrochloride (4.9 g, 22 mmol) for 3 h. On concentra-
tion, the separated solid product was filtered, washed with cold
ethanol and recrystallized from ethanol. Physicochemical and
analytical data are recorded in Table 4. IR (cm– 1): 1310 and 1175
(SO2N), 3240 and 3345 (NH2). 1H-NMR (d, ppm): 2.29 (s, 3H, CH3),
2.67 (m, 2H, C4-2H), 3.02 (m, 2H, C5-2H), 6.84–8.02 (m, 10H, Ar-H
+ 2NH).
Cyclohexyl
Phenyl
1-Naphthyl
Phenyl
Benzyl
Benzoyl
Phenyl
Benzyl
Benzoyl
Phenyl
Benzyl
Benzoyl
Phenyl
Benzyl
Phenyl
Benzyl
–
4
5
6
7
N1-Substituted N3-[4-(3-methyl-4,5-dihydronaphtho[1,2-
c]pyrazol-2-yl)-benzenesulfonyl]-ureas 3–5
8
9
A mixture of the pyrazole 2 (1.7 g, 5 mmol) and anhydrous potas-
sium carbonate (1.38 g, 10 mmol) in dry acetone (20 mL) was
heated with the appropriate isocyanate (6 mmol) under reflux
with stirring for 18 h. The solvent was removed under reduced
pressure and the remaining solid residue was dissolved in water
(20 mL). After acidification of the resulting solution with 2 N
hydrochloric acid, the precipitated crude product was filtered,
washed with water, dried and recrystallized from ethanol. Phys-
10
11
12
13
14
15
16
17
18
19
20
21
icochemical and analytical data are recorded in Table 4. IR cm– 1
:
1
1300–1335 and 1165–1200 (SO2N), 1640–1655 (C=O). H-NMR
(d, ppm) or 3 (R = cyclohexyl): 1.58–2.13 (m, 11H, cyclohexyl-H),
2.28 (s, 3H, CH3), 2.64 (m, 2H, C4-2H), 3.19 (m, 2H, C5-2H), 6.39–
7.89 (m, 10H, 2 NH + 8 Ar-H); for 4 (R = phenyl): 2.25 (s, 3H, CH3),
2.63 (m, 2H, C4-2H), 3.05 (m, 2H, C5-2H), 6.70–8.04 (m, 15H, 2 NH
+ 13 Ar-H); for 5 (R = 1-naphthyl): 2.30 (s, 3H, CH3), 2.71 (m, 2H, C4-
2H), 3.08 (m, 2H, C5-2H), 6.88–8.22 (m, 17H, 2 NH + 15 Ar-H).
C29H26N4O3S2
(542.67)
C28H22N6O2S2
(538.65)
C34H26N4O3S2
(602.73)
C35H30N6O2S2
(630.78)
N1-Substituted N3-[4-(3-methyl-4,5-dihydronaphtho[1,2-
c]pyrazol-2-yl)-benzenesulfonyl]-thioureas 6–8
A solution of the appropriate isothiocyanate (6 mmol) in dry ace-
tone (5 mL) was added to a stirred mixture of the pyrazole 2
(1.7 g, 5 mmol) and anhydrous potassium carbonate (1.38 g,
10 mmol) in dry acetone (20 mL), and the reaction mixture was
heated under reflux with stirring for 10 h. The solvent was
removed under reduced pressure and the remaining residue was
dissolved in water (20 mL) and acidified with 2 N hydrochloric
acid. The precipitated crude product was filtered, washed with
water, dried and recrystallized from ethanol. Physicochemical
and analytical data are recorded in Table 4. IR cm– 1: 1300–1335
and 1165–1300 (SO2N), 1155–1165 (C=S), 1650 (C=O, 8: R1 = CO-
C6H5). 1H-NMR (d, ppm) for 6 (R1 = phenyl): 2.32 (s, 3H, CH3), 2.59
(m, 2H, C4-2H), 3.11 (m, 2H, C5-2H), 6.86–8.12 (m, 15H, 2 NH + 13
Ar-H); for 7 (R1 = benzyl): 2.28 (s, 3H, CH3), 2.61 (m, 2H, C4-2H), 3.08
(m, 2H, C5-2H), 4.75 (d, 2H, CH2-C6H5), 6.90-8.05 (m, 15H, 2 NH +
13 Ar-H); for 8 (R1 = benzoyl): 2.31 (s, 3H, CH3), 2.72 (m, 2H, C4-2H),
3.17 (m, 2H, C5-2H), 6.86–7.95 (m, 15H, 2 NH + 13 Ar-H).
–
–
tate (1 g, 6 mmol) and anhydrous sodium acetate (0.82 g,
10 mmol), and the reaction mixture was heated under reflux for
2 h. The reaction mixture was left to attain room temperature
then poured into ice-cold water (30 mL). The solid product thus
formed was filtered, washed with water, dried and recrystallized
from ethanol-benzene mixture (1 : 1). Physicochemical and ana-
lytical data are recorded in Table 4. IR cm– 1: 1720–1735 (thiazol-
1
C=O), 1660 (C=O, 11: R1 = CO-C6H5). H-NMR (d, ppm) for 9 (R1 =
phenyl): 2.29 (s, 3H, CH3), 2.66 (m, 2H, C4-2H), 3.01 (m, 2H, C5-2H),
4.24 (s, 2H, thiazol-CH2), 6.88–7.93 (m, 13H, Ar-H); for 10 (R1 =
benzyl): 2.27 (s, 3H, CH3), 2.68 (m, 2H, C4-2H), 3.05 (m, 2H, C5-2H),
4.22 (s, 2H, thiazol-CH2), 4.80 (s, 2H, CH2-C6H5), 6.87–7.98 (m,
13H, Ar-H); for 11 (R1 = benzoyl): 2.31 (s, 3H, CH3), 2.65 (m, 2H, C4-
2H), 3.02 (m, 2H, C5-2H), 4.28 (s, 2H, thiazol-CH2), 6.79–8.02 (m,
13H, Ar-H).
2-[4-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl)-
benzenesulfonylimino]-3-substituted-thiazolidin-4-one
9–11
To a solution of the appropriate thioureido derivative 6–8
(5 mmol) in absolute ethanol (20 mL) was added ethyl bromoace-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Dihydronaphthopyrazoles as in-vitro Antitumour Agents
189
2.99 (m, 2H, C5-2H), 4.22 (s, 2H, thiazol-CH2), 7.11–8.06 (m, 13H,
Ar-H).
2-[4-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl)-
benzenesulfonylimino]-4-phenyl-3-substituted-1,3-
thiazolines 12–14
A
solution of the appropriate thioureido derivative 6–8
5-Benzylidene-2-[4-(3-methyl-4,5-dihydronaphtho[1,2-
c]pyrazol-2-yl)-benzenesulfonylimino]-3-
phenylthiazolidin-4-one 20
(5 mmol) in absolute ethanol (20 mL) was refluxed with phe-
nacyl bromide (1.2 g, 6 mmol) and anhydrous sodium acetate
(0.82 g, 10 mmol), for 3 h during which a solid product was par-
tially crystallized out. The mixture was left to attain room tem-
perature then filtered, washed with cold ethanol, dried and
recrystallized from ethanol. Physicochemical and analytical
To a solution of the 3-phenylthiazolidin-4-one 9 (5 mmol) and
piperidine (3 drops) in absolute ethanol (15 mL) was added ben-
zaldehyde (0.53 g, 5 mmol). The mixture was heated under
reflux for 8 h, when a solid product partially crystallized out.
The reaction mixture was left to attain room temperature, and
the separated solid product was filtered, dried and recrystallized
from ethanol. Physicochemical and analytical data are recorded
in Table 4. 1H-NMR (d, ppm): 2.31 (s, 3H, CH3), 2.72 (m, 2H, C4-2H),
3.11 (m, 2H, C5-2H), 5.80 (s, 1H, thiazol-H), 7.01–8.09 (m, 19H,
benzylidin-CH + Ar-H).
1
data are recorded in Table 4. H-NMR (d, ppm) for 12 (R1 = phe-
nyl): 2.33 (s, 3H, CH3), 2.68 (m, 2H, C4-2H), 3.09 (m, 2H, C5-2H),
5.80 (s, 1H, thiazol-H), 6.91–8.18 (m, 18H, Ar-H); for 13 (R1 = ben-
zyl): 2.30 (s, 3H, CH3), 2.71 (m, 2H, C4-2H), 3.11 (m, 2H, C5-2H), 4.77
(s, 2H, CH2-C6H5), 5.83 (s, 1H, thiazol-CH), 6.85–8.10 (m, 18H, Ar-
H); for 14 (R1 = benzoyl): 2.33 (s, 3H, CH3), 2.68 (m, 2H, C4-2H), 3.05
(m, 2H, C5-2H), 5.75 (s, 1H, thiazol-CH), 6.80-8.05 (m, 18H, Ar-H).
2-[4-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl)-
benzenesulfonylimino]-3-substituted-1,3-thiazinan-4-
ones 15, 16
3,6-Diphenyl-5-[4-(3-methyl-4,5-dihydronaphtho[1,2-
c]pyrazol-2-yl)-benzenesulfonylimino]-2-methyl-3,3a,5,6-
tetrahydro-2H-pyrazolo[3,4-d]thiazole 21
To a solution of the appropriate thioureido derivative 6 or 7
(5 mmol) in absolute ethanol (20 mL) was added ethyl 3-bromo-
propionate (1.1 g, 6 mmol) and sodium acetate (0.82 g, 10 mmol)
and the reaction mixture was heated under reflux for 4 h. The
reaction mixture was worked up as described under compounds
9–11. Physicochemical and analytical data are recorded in
Table 4. IR cm– 1: 1720–1730 (C=O). 1H-NMR (d, ppm) for 15 (R1 =
phenyl): 2.29 (s, 3H, CH3), 2.64 (m, 2H, C4-H), 2.93 (m, 4H, C5-2H +
thiazinan-CH2), 3.20 (t, 2H, thiazinan-CH2), 6.82–8.00 (m, 13H,
Ar-H); for 16 (R1 = benzyl): 2.30 (s, 3H, CH3), 2.66 (m, 2H, C4-2H),
3.01 (m, 4H, C5-2H + thiazinan-CH2), 3.23 (t, 2H, thiazinan-CH2),
4.75 (s, 2H, CH2-C6H5), 6.85–7.99 (m, 13H, Ar-H).
A mixture of 20 (3 g, 5 mmol) and methylhydrazine (0.23 g,
5 mmol) in ethanol (15 mL) was heated under reflux for 30 min.
The solid product obtained on cooling was filtered, washed with
ethanol and recrystallized from ethanol. Physicochemical and
analytical data are recorded in Table 4. 1H-NMR (d, ppm): 2.28 (s,
3H, CH3), 2.51 (s, 3H, N-CH3), 2.75 (m, 2H, C4-2H), 3.06 (m, 2H, C5-
2H), 3.47 (d, J = 9 Hz, 1H, C3a-H), 4.65 (d, J = 9 Hz, 1H, C3-H), 6.65–
7.86 (m, 18H, Ar-H).
In-vitro antitumour activity
Out of the newly synthesized compounds, twelve compounds,
namely 2, 4, 6–9, 11, 15, 17–19 and 21 were selected by the
National Cancer Institute (NCI) in-vitro disease-oriented human
cells screening panel assay to be evaluated for their in-vitro anti-
tumour activity. Primary in-vitro one dose 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, NCI, Bethesda [33–35].
All the compounds that reduced the growth of any one of the
cell lines to 32% or less, namely 6, 8, 11, 15, 17 and 18 were
passed on for evaluation in the full panel of 60 human tumour
cell lines of nine tumour subpanels. These cell lines were incu-
bated with five concentrations (0.01–100 lM) for each com-
pound. A 48 h continuous drug exposure protocol was used, and
a SRB protein assay was employed to estimate cell viability or
growth [33–35].
2-[4-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl)-
benzenesulfonylimino]-3-substituted-1,3-thiazinan-5-
ones 17, 18
To a solution of the appropriate thioureido derivative 6 or 7
(5 mmol) in absolute ethanol (20 mL) was added 1,3-dichloroace-
tone (0.76 g, 6 mmol) and sodium acetate (0.82 g, 10 mmol) and
the reaction mixture was heated under reflux for 5 h. The reac-
tion mixture was worked up as described under compounds 9–
11. Physicochemical and analytical data are recorded in Table 4.
IR cm– 1: 1720–1730 (C=O). 1H-NMR (d, ppm) for 17 (R1 = phenyl):
2.33 (s, 3H, CH3), 2.68 (m, 2H, C4-2H), 3.03 (m, 2H, C5-2H), 4.39 (s,
2H, thiazinan-CH2), 5.38 (s, 2H, thiazinan-CH2), 6.79–8.06 (m,
13H, Ar-H); for 18 (R1 = benzyl): 2.35 (s, 3H, CH3), 2.69 (m, 2H, C4-
2H), 3.05 (m, 3H, C5-2H), 4.39 (s, 2H, thiazinan-CH2), 4.78 (s, 2H,
CH2-C6H5), 5.36 (s, 2H, thiazinan-CH2), 6.88–8.03 (m, 13H, Ar-H).
In-vitro MTT cytotoxicity assay
A modified MTT colorimetric assay [37] was employed to deter-
mine growth inhibition. Cell lines were obtained from the Amer-
ican Type Culture Collection (ATCC). The human breast cancer
cell line MCF7 was maintained in DMEM supplemented with 5%
fetal bovine serum (FBS), 2 mM glutamine and 100 units/mL pen-
icillin/streptomycin. The normal human breast epithelial cell
line MCF-10A was maintained in 5% and 10% horse serum,
respectively, supplemented with 2 mM glutamine, 100 units/mL
penicillin/streptomycin, 0.02 lg/mL EGF, 0.01 mg/mL insulin
and 0.1 lg/mL cholera toxin. Cells were incubated at 378C in a
5% CO2 atmosphere.
5-[4-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl)-
benzenesulfonylimino]-3-phenyl-7H-thiazolo[4,3-
c][1,2,4]triazole 19
A mixture of the 3-benzoylthiazolidin-4-one 11 (2.7 g, 5 mmol)
and hydrazine hydrate (0.35 g, 7 mmol) in ethanol (15 mL) was
heated under reflux for 30 min. The solid product obtained on
cooling was filtered, washed with ethanol and recrystallized
from ethanol. Physicochemical and analytical data are recorded
in Table 4. 1H-NMR (d, ppm): 2.35 (s, 3H, CH3), 2.70 (m, 2H, C4-2H),
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190
M. S. Al-Saadi et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 81–190
Cells were plated in 96-well plates and allowed to attach for
24 h. Test compounds were dissolved in DMSO at 10 mM. Cells in
quadruplicate wells were exposed to the individual test com-
pounds at 0–25 lM for 72 h. Thereafter, the medium was
replaced with 100 lL of 1 mg/mL MTT solution diluted in serum-
free DMEM in each well. After 4 h of incubation, the MTT solu-
tion was removed, and 200 lL of DMSO was added to each well
to dissolve the formed formazane crystals. The absorbance was
measured at 495 nm. Results were compared with those of
DMSO-treated cells as control. The results represent the mean of
four independent experiments and are expressed as IC50, i. e.,
the concentration that reduced by 50% the optical density of
treated cells with respect to untreated controls.
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