Novel 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives with anticancer activity: Synthesis and QSAR study

Article abstract of DOI:10.3390/molecules201219821

A series of new 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5a-j have been synthesized and evaluated in vitro for their antiproliferative activity at the U.S. National Cancer Institute. The most active compound 5h showe

Full text of DOI:10.3390/molecules201219821

Article
Novel 3-Amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-
1,4,2-benzodithiazine Derivatives with Anticancer
Activity: Synthesis and QSAR Study
1
1
Aneta Pogorzelska 1,*, Jarosław Sławin´ski , Kamil Brozewicz , Szymon Ulenberg 2 and
˙
Tomasz Ba˛czek 2
Received: 30 October 2015 ; Accepted: 1 December 2015 ; Published: 9 December 2015
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
1
Department of Organic Chemistry, Medical University of Gdan´sk, Al. Gen. J. Hallera 107,
80-416 Gdan´sk, Poland; jaroslaw@gumed.edu.pl (J.S.); kamil@gumed.edu.pl (K.B.)
Department of Pharmaceutical Chemistry, Medical University of Gdan´sk, Al. Gen. J. Hallera 107,
2
80-416 Gdan´sk, Poland; szymon.ulenberg@gumed.edu.pl (S.U.); tbaczek@gumed.edu.pl (T.B.)
*
Correspondence: anetapogorzelska@gumed.edu.pl; Tel.: +48-58-349-1237; Fax: +48-58-349-1277
Abstract: A series of new 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine
derivatives 5a have been synthesized and evaluated in vitro for their antiproliferative activity
–
j
at the U.S. National Cancer Institute. The most active compound 5h showed significant cytotoxic
effects against ovarian (OVCAR-3) and breast (MDA-MB-468) cancer (10% and 47% cancer cell death,
respectively) as well as a good selectivity toward prostate (DU-145), colon (SW-620) and renal (TK-10)
cancer cell lines. To obtain a deeper insight into the structure-activity relationships of the new
compounds 5a–j QSAR studies have been applied. Theoretical calculations allowed the identification
of molecular descriptors belonging to the RDF (RDF055p and RDF145m in the MOLT-4 and UO-31
QSAR models, respectively) and 3D-MorSE (Mor32m and Mor16e for MOLT-4 and UO-31 QSAR
models) descriptor classes. Based on these data, QSAR models with good robustness and predictive
ability have been obtained.
Keywords: 1,4,2-benzodithiazines; sulfonamide; QSAR; anticancer activity
1. Introduction
1,4,2-Benzodithiazines are very attractive lead structures for designing new compounds as
potential pharmaceutical agents. This can be attributed to their wide range of biological activity
as well as to their facility for chemical transformation into 2-mercaptobenzenesulfonamides that would
not otherwise be easily obtainable.
Considering their biological properties, compounds containing a 1,4,2-benzodithiazine scaffold
are widely recognized as having a great number of activities, such as diuretic [ ], cholagogue [
1
–
6
6,7],
radioprotective [ ], antiarrhythmic [ ], hypotensive [ ] and anti-HIV [8–10]. Of particular
4
4,
6
4–
7
interest is that much research on the use of 6-chloro-1,1-dioxo-1,4,2-benzodithiazines as potential
therapeutic agents has demonstrated that some of them exhibit remarkable anticancer activity (Figure 1,
I
[
11
–
14], II
,
III
[
13
,
15
,
16] and IV
[
17]). With regard to these reports we have designed novel
(Figure 1) that vary according to
benzodithiazine derivatives of the general structure of type
V
both the nitrogen-containing 5-membered heterocycle scaffold at position 7 and also the substituent
bearing either a condensed indazole or indole ring attached to the amine group at position 3 of the
6-chloro-1,1-dioxo-1,4,2-benzodithiazine ring. These modifications were selected not only based on
the biological properties of benzodithiazines but also the significant pharmacological importance of
heterocycles with a high nitrogen content.
Molecules 2015, 20, 21960–21970; doi:10.3390/molecules201219821
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Molecules 2015, 20, 21960–21970
Figure 1. General structures of 1,1-dioxo-1,4,2-benzodithiazines I–IV [11–17] and V with anticancer activity.
Thus, herein we report the synthesis and anticancer activity of the new series of
3-(R2-amino)-7-(azol-2 or 5-yl)-6-chloro-1,1-dioxo-1,4,2-benzodithiazines (
, Figure 1). The new
V
compounds have been investigated for in vitro activity against 60 human cancer cell lines from different
organs of origin. To correlate the chemical structure of compounds with their potency to inhibit the
growth of cancer cells quantitative structure-activity relationship (QSAR) analysis was applied. As
a result, the most important parameters controlling the biological properties have been determined
using statistical approaches.
2. Results
2.1. Chemistry
The synthetic routes for the preparation of the desired 3-(R2-amino)-7-(azol-2 or 5-yl)-6-chloro-
1,1-dioxo-1,4,2-benzodithiazine derivatives 5a–j are shown in Schemes 1 and 2.
The essential substrates for the synthesis of novel compounds 5a
6-chloro-7-heteroaryl-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4a , were obtained using
previously described methods which are briefly summarized in Scheme 1 [18 22]. Thus, the
starting 3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4a could be converted to the desired
3-(R2-amino-7-azolyl-6-chloro-1,1-dioxo-1,4,2-benzodithiazine derivatives 5a
by nucleophilic
substitution reactions with one molar equivalent of a primary amine, as outlined in Scheme 2.
–j, the already known
–
e
–
–
e
–
j
Scheme 1. Synthesis of 7-(azol-2 or 5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4a
Reagents and conditions: ( ) SOCl2 excess, benzene, reflux; (
) 12% NH3(aq), benzene 5–10 ˝C;
) arylhydrazide, benzene, 5 ) N-hydroxybenzamidine, toluene, 0 ) Lawesson
20 ˝C; ( 20 ˝C; (
) SOCl2, rt reflux; ( ) LR, toluene, 20
100 ˝C; ( ) 110 ˝C;
–e.
a
b
(
c
Ñ
d
Ñ
e
reagent (LR, 0.5 equiv.), toluene; (
(i) ω-halogenoacetophenone, MeOH, 20Ñ65 ˝C.
f
Ñ
g
Ñ
h
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Molecules 2015, 20, 21960–21970
Scheme 2. Synthesis of 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives
5a
–j. Reagents and Conditions: (A) reflux, 30–50 h; (B) 24 h at room temperature, then reflux, 7.5–48 h;
(C) room temperature, 52 h.
The structures of the final compounds 5a
–
j
were confirmed by elemental analyses and
1
spectroscopic (IR, H-NMR and 13C-NMR) data, in particular the presence of the IR absorption
´1
bands corresponding to the stretching vibration of the NH group in the 3228–3408 cm range as well
as two singlet signals in the 1H-NMR spectra related to the NH protons, one in the 10.04–11.79 ppm
range for the proton of the NH group attached directly at the position 3 of the benzodithiazine scaffold
and another at 10.89–13.24 ppm for the NH linked to the indazole or indole ring (see Supplementary
Material, Figures S1–S16).
2.2. Anticancer Activity
Compounds 5a
at a single dose of 10
–
j
µ
were tested in vitro at the U.S. National Cancer Institute (Bethesda, MD, USA)
M against the NCI panel of 60 cell lines derived from nine different types of
cancer, including leukemia, non-small-cell lung cancer (NSCLC), colon, central nervous system (CNS),
melanoma, ovarian, renal, prostate and breast. The results, obtained as an inhibition growth percent
(IGP), are shown in Table 1. The control was performed via the comparison with no-drug cell growth.
The best anticancer activity was noticed for compounds 5a and 5h. The derivative 5a has inhibited
the growth of 21 cancer cell lines with mean IGP value at 19.33%. In the case of 5h the activity against
28 cancer cell lines has been observed with mean IGP at 43.93%.
2.3. QSAR Studies
To further elucidate the structure-activity relationships among the series of compounds 5a–j
QSAR methodology was applied. Statistical analysis is essential for QSAR work and therefore,
in order to avoid statistical outliers, we selected cancer cell lines which were sensitive toward
all studied derivatives 5a–j (0% < IGP < 100%)—leukemia MOLT-4 and renal cancer UO-31. In
presented study, the QSAR models for each cell line were developed separately. Descriptors have been
calculated using DRAGON, SPARTAN and Gaussian software. After excluding descriptors with no
variation or value and adding descriptors from Spartan software and dihedral angle values, a final
set comprised of 2765 molecular descriptors. Using Data Mining feature selection, only 490 most
statistically important descriptors were chosen for further analysis. After importing the previously
mentioned set of descriptors into STATISTICA 10.0 software (Statsoft, Tulsa, OK, USA) Stepwise
Multiple Linear Regression (MLR) was performed. The IGP was taken as a dependent value for
analysis, while molecular descriptors were independent values. Achieved models were validated
using Leave-One-Out Cross-validation and the results are presented in Table 2. Molecular descriptors
that entered the model, along with their meaning and values have been presented in Table 3.
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Molecules 2015, 20, 21960–21970
Table 1. The inhibition growth percent of selected NCI-60 cancer cells (IGP) at a single concentration of 10´5 M of novel 1,4,2-benzodithiazine derivatives 5a–j.
IGP (%) of Compound
Panel
Cell Line
5a
5c
5e
5g
5i
5j
5b
5d
5f
5h
-
4.20
4.05
*
-
*
-
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
31.62
31.12
16.23
24.20
-
11.56
36.36
17.07
2.85
-
3.41
25.24
*
23.59
18.21
26.54
13.73
69.38
9.06
81.00
1.76
6.77
*
0.44
1.18
6.36
10.91
*
*
3.64
*
*
Leukemia
12.96
-
10.64
0.99
53.71
24.25
*
-
21.80
*
22.08
HOP-62
NCI-H226
NCI-H522
15.32
13.09
-
13.98
26.50
-
*
*
*
*
*
*
6.75
17.09
22.52
*
34.63
14.78
27.30
*
1.10
*
*
*
*
NSCLC
6.62
15.60
3.34
5.34
5.67
-
9.47
*
-
HCC-2998
HCT-116
SW-620
*
*
*
*
0.68
*
*
*
*
5.10
3.30
*
*
7.37
*
60.93
43.60
50.98
*
*
*
*
8.42
*
Colon cancer
CNS cancer
2.28
9.09
SNB-19
SNB-75
U251
6.57
7.09
12.89
9.45
-
16.48
*
-
*
4.26
11.44
*
*
4.47
*
17.47
-
17.55
9.70
32.19
5.97
3.40
76.93
21.68
2.53
12.31
*
*
8.52
*
LOX IMVI
UACC-257
UACC-62
3.56
11.40
16.61
10.33
1.35
*
*
*
*
2.14
*
*
*
*
1.30
*
8.79
*
53.68
33.69
23.80
3.02
*
*
*
*
Melanoma
Ovarian cancer
Renal cancer
4.08
5.83
0.29
2.39
10.27 a
OVCAR-3
OVCAR-8
*
7.74
15.64
*
*
*
*
*
*
*
*
*
*
15.93
2.07
0.66
8.81
24.08
1.69
SN12C
TK-10
UO-31
2.62
*
4.78
14.33
*
36.34
1.69
5.41
16.35
4.46
*
23.53
*
*
17.11
*
34.00
11.87
*
29.54
15.54
52.33
6.57
1.69
*
24.73
*
*
8.75
9.74
-
*
PC-3
DU-145
19.12
*
11.45
*
1.37
*
6.74
*
*
*
1.87
*
51.71
98.79
*
*
0.36
*
Prostate cancer
MCF-7
MDA-MB-231/ATCC
T-47D
35.97
25.84
49.10
53.37
6.77
15.13
12.27
18.73
8.25
1.75
*
4.44
*
*
*
*
10.50
15.78
*
35.19
4.93
23.52
42.05
5.32
10.35
*
*
*
*
*
*
*
*
*
*
*
Breast cancer
37.64
47.18 a
MDA-MB-468
5.58
9.62
* IGP ď 0%; - not tested; a—cytotoxic effect.
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Molecules 2015, 20, 21960–21970
Table 2. Developed QSAR models and their performance in predicting anticancer activity of
3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5a–j against MOLT-4
and UO-31 cell lines.
Cell Line
Equation
N
R
Rcv
0.927 2.34
0.861 11.44 1.371126
s
RMSECV
p
F
IGP = 0.815(RDF055p) ´
MOLT-4
10
0.967
0.859
0.00006
51.34
0.55(Mor32m) ´ 41.34
IGP = 0.770(Mor16e) ´
UO-31
10
0.943
0.0006
28.33
0.497(RDF145m) + 67.048
N—number of compounds; R—correlation coefficient; Rcv—correlation coefficient of leave-one-out
cross-validation (LOO-CV); s—a standard error of estimate; RMSECV—a root mean square error LOO-CV,
p—significance level of F-test; F—Fisher test value
Table 3. Molecular descriptors along with their values and interpretation used for developing
QSAR equations. RDF055p—Radial Distribution Function—055/weighted by polarizability;
Mor32m—signal 32/weighted by mass; Mor16e—signal 16/weighted by Sanderson electronegativity;
RDF145m—Radial Distribution Function—145/weighted by mass.
MOLT-4
UO-31
Compd.
RDF055p
Mor32m
Mor16e RDF145m
5a
5b
5c
5d
5e
5f
5.903
6.251
4.882
5.670
6.278
6.038
8.458
5.958
5.521
7.891
´0.701
´0.775
´0.627
´0.616
´0.202
´0.486
´0.549
´0.448
´0.400
´0.551
1.632
1.243
1.529
1.744
1.765
1.252
1.260
1.662
1.289
1.565
3.349
5.546
3.010
5.816
3.392
3.104
3.370
1.985
3.139
1.986
5g
5h
5i
5j
Good correlation between the data obtained by in vitro studies and the one predicted by QSAR
model application has been obtained as presented in Table 4.
Table 4. Comparison of observed IGP values (anticancer activity) and those predicted by QSAR models
for MOLT-4 and UO-31 cell lines.
IGP [%]
MOLT-4
UO-31
Compd.
Observed Predicted Observed Predicted
5a
5b
5c
5d
5e
5f
5g
5h
5i
16.23
17.07
4.05
12.96
3.64
10.64
23.59
9.06
13.82
19.33
6.96
10.15
2.13
9.13
28.91
7.55
4.78
36.34
16.35
23.53
8.75
34
29.54
6.57
14.44
43.11
15.78
11.71
7.11
25.97
28.72
5.05
0.44
24.25
4.77
21.63
24.73
9.74
27.15
9.61
5j
3. Discussion
3.1. Anticancer Activity
Considering the anticancer activity results (see Table 1) it has been observed that the nature and
structure of substituents located at positions 3 and 7 of benzodithiazine have varying influences on the
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Molecules 2015, 20, 21960–21970
compounds’ anticancer activity. However, the most important features seem to be both the electronic
character of the substituent at the position 7 and the substitution pattern of the heterocyclic ring
attached to the amine group in position 3.
Derivative 5h, possessing a 3-phenyl-1,2,4-oxadiazol-5-yl moiety in position 7 as well as a
1H-indazol-5-yl scaffold attached directly to the amine group in position 3, shows the best anticancer
properties. Particularly noteworthy is the cytotoxic effect observed against ovarian cancer OVCAR-3
and breast cancer MDA-MB-468. Moreover, only compound 5h displayed the ability to inhibit
growth of the DU-145 prostate cancer cell line at a level of 98.79%. A similar, although slightly
lower, selectivity has been observed in the case of colon cancer SW-620 (IGP = 50.98%) and renal cancer
TK-10 (IGP = 52.33%).
Good anticancer properties have been observed for compounds bearing a 5-phenyl-
1,3,4-oxadiazol-2-yl scaffold at position 7 of the benzodithiazine and either 1H-indazol-5-yl (5a
)
or 1H-indazol-6-yl (5b) fragments in position 3. On the other hand, a significant decrease in
activity was observed after incorporation of substituents such as (1H-indazol-7-yl)amino (5c) or
2-(1H-indol-3-yl)ethylamino (5d) at position 3. These findings prove the importance of the structure in
this position. However, in the case of compounds 5e and 5f bearing 5-phenyl-1,3,4-thiadiazol-2-yl at
position 7 (instead of an oxadiazole ring), modification of position 3 seems to have much lower
impact. The influence of the electronic character of the substituent located at position 7 of the
benzodithiazine scaffold could be especially observed in the case of derivative 5e. The replacement
of 5-phenyl-1,3,4-oxadiazol-2-yl (5a) by 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl (5e) led to an almost
completely lack of anticancer activity.
3.2. QSAR Studies
The application of QSAR methodology led to the obtaining of descriptors for building QSAR
models which provide not only information useful for further chemical synthesis, but also enable the
prediction of pharmacological activity of novel derivatives. It should be emphasized that developed
models showed good correlation of chosen descriptors with activity of compounds. Moreover QSAR
models for both MOLT-4 and UO-31 cell lines showed good predictability, as presented in Table 2.
Difficulty in practical interpretation of model descriptors for both cell lines shows the complex
nature of mode of action of the studied derivatives. Further study on a larger set of compounds might
reveal structure-activity relationships that are easier to interpret, providing valuable guidelines for
further synthesis. However, when comparing observed anticancer activity with that predicted by
statistical analysis (Table 4), the developed models already show good performance and prove to be
useful in this kind of study
4. Experimental Section
4.1. General Information
The melting points were determined on a Boethius PHMK apparatus (Veb Analytic, Dresden,
Germany) and are uncorrected. Infrared (IR) spectra were taken on a Thermo Mattson Satellite FTIR
spectrophotometer (Thermo Mattson, Madison, WI, USA). The NMR spectra were recorded on a Varian
Gemini 200 apparatus (Varian, Palo Alto, CA, USA) at 200 MHz (1H-NMR) and 50 MHz (13C-NMR)
or on a Varian Unity 500 Plus apparatus (Varian, Palo Alto, CA, USA) at 500 MHz (1H-NMR) and
125 MHz (13C-NMR). Chemical shifts are expressed as
δ values in parts per million (ppm) relative to
TMS as an internal standard. Spectra were acquired in deuterated dimethylsulfoxide (DMSO-d6). The
results of elemental analyses for C, H and N were in agreement with the theoretical values within
˘
0.4% range. The commercially unavailable substrates were obtained according to the following
previously described methods : 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylic
acid 18], 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carbonyl chloride (2a) 19],
(
1
)
[
[
1
6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carboamide (2b) [20], N -(6-chloro-3-methylthio-
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Molecules 2015, 20, 21960–21970
1,1-dioxo-1,4,2-benzodithiazine-7-carbonyl)benzhydrazides (3a–b) [21], 6-chloro-7-(5-aryl-1,3,4-oxa
or 1,3,4-thiadiazol-2-yl)-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines (4a–c) [21], 7-(3-phenyl-1,2,4-
oxadiazol-5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine (4d) [22] and 7-(4-phenylthiazol-
2-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine (4e) [22]. The NMR spectra of newly
synthesized compounds 5a–j have been given as Figures S1–S16 in Supplementary Material.
4.2. Synthesis
General Procedure for the Preparation of 3-(R2-Amino)-7-(azol-2 or 5-yl)-6-chloro-1,1-dioxo-1,4,2-
benzodithiazines 5a–j
To a suspension of the 7-(azol-2 or 5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine
4a
reaction mixture was refluxed for 30–50 h (method A—5a
and then refluxed for 7.5–48 h (method B—5g 5j) or stirred at room temperature for 52 h (method
–
e
(1.0 mmol) in methanol (10 mL) the appropriate primary amine (1.0 mmol) was added. The
–f,
5h), stirred for 24 h at room temperature
,
C—5i) until the methanethiol was released. The precipitated solid was filtered off and washed several
times with methanol. The crude product was purified by crystallization from the appropriate solvent.
6-Chloro-7-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-[(1H-indazol-5-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine (5a).
Starting from 4a (0.424 g) and 5-amino-1H-indazole (0.133 g) after refluxing for 48 h the title compound
5a was obtained (0.443 g, 87%), mp 325–328 ˝C (DMF–MeOH, 1:3); IR (KBr)
ν
3387, 3296 (NH),
max
2925, 2854 (CH), 1589, 1548, 1531, 1505, 1450 (C=N, C=C), 1312, 1159 (SO2) cm´1; 1H-NMR (200 MHz,
DMSO-d6) 7.50–7.60 (m, 1H, Ar H), 7.62–7.69 (m, 4H, Ar H), 8.11–8.18 (m, 4H, Ar H), 8.37 (s, 1H,
δ
H-5), 8.63 (s, 1H, H-8), 11.67 (s, 1H, NH), 13.22 (s, 1H, NH) ppm; anal. C 51.92, H 2.57, N 16.51% calcd
for C22H13ClN6O3S2, C 52.03 H 2.85 N 16.80%.
6-Chloro-7-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-[(1H-indazol-6-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine (5b).
Starting from 4a (0.424 g) and 6-amino-1H-indazole (0.133 g) after refluxing for 30 h the title compound
˝
5b was obtained (0.244 g, 48%) mp 336–338 C (DMF–MeOH, 1:3); IR (KBr)
νmax 3288 (NH), 2923 (CH),
1632, 1609, 1588, 1539, 1476, 1450 (C=N, C=C), 1305, 1160 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
7.23–7.24 (m, 1H, Ar H), 7.61–7.63 (m, 3H, Ar H), 7.79–7.80 (m, 1H, Ar H), 8.07–8.14 (m, 4H, Ar H), 8.32
(s, 1H, H-5), 8.62 (s, 1H, H-8), 11.77 (s, 1H, NH), 13.17 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ
102.62, 115.50, 121.23, 122.91, 122.97, 126.37, 126.90, 129.50, 130.92, 130.98, 132.39, 133.60, 133.84,
135.09, 135.54, 139.76, 160.88, 164.57 ppm; anal. C 51.92, H 2.57, N 16.51% calcd for C22H13ClN6O3S2,
C 52.12, H 2.63, N 16.75%.
6-Chloro-7-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-[(1H-indazol-7-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine (5c).
Starting from 4a (0.424 g) and 7-amino-1H-indazole (0.133 g) after refluxing for 50 h the title compound
˝
5c was obtained (0.224 g, 44%) mp 329–332 (dec.) C (DMF–MeOH, 1:3); IR (KBr)
(CH), 1649, 1581, 1551, 1488, 1459 (C=N, C=C), 1316, 1162 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
7.18–7.19 (m, 1H, Ar H), 7.40–7.41 (m, 1H, Ar H), 7.62–7.67 (m, 3H, Ar H), 7.78–7.79 (m, 1H, Ar H),
8.11–8.17 (m, 3H, Ar H), 8.40 (s, 1H, H-5), 8.61 (s, 1H, H-8), 11.69 (s, 1H, NH), 13.12 (s, 1H, NH) ppm;
13C-NMR (125 MHz, DMSO-d6)
120.30, 120.44, 122.45, 122.80, 122.95, 126.44, 126.93, 126.98, 129.54,
νmax 3289 (NH), 2927
δ
δ
130.80, 131.33, 132.42, 134.07, 134.22, 135.00, 135.39, 160.90, 164.61 ppm; anal. C 51.92, H 2.57, N 16.51%
calcd for C22H13ClN6O3S2, C 52.03, H 2.91 N 16.60%.
6-Chloro-7-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-[2-(1H-indol-3-yl)ethylamino]-1,1-dioxo-1,4,2-benzodithiazine
(
5d). Starting from 4a (0.424 g) and 2-(1H-indol-3-yl)ethanamine (0.160 g) after refluxing for 30 h the
˝
title compound 5d was obtained (0.278 g, 53%) mp 262–264 C (DMF–MeOH, 1:3); IR (KBr)
3408 (NH), 3080 (CH Ar), 2925 (CH), 1568, 1488, 1450 (C=N, C=C), 1303, 1159 (SO2) cm´1; 1H-NMR
(500 MHz, DMSO-d6) 3.00–3.02 (m, 2H, CH2), 3.65–3.68 (m, 2H, CH2), 6.95–6.98 (m, 1H, Ar H), 7.06(t,
ν
max
δ
J = 7.3 Hz, 1H, Ar H), 7.20 (s, 1H, Ar H), 7.33 (d, J = 8.3 Hz, 1H, Ar H), 7.55 (d, J = 8.3 Hz, 1H, Ar H),
7.63–7.70 (m, 3H, Ar H), 8.13–8.14 (m, 2H, Ar H), 8.26 (s, 1H, H-5), 8.60 (s, 1H, H-8), 10.04 (s, 1H, NH),
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Molecules 2015, 20, 21960–21970
10.89 (s, 1H, NH) ppm; anal. C 56.02, H 3.38, N 13.07% calcd for C25H18ClN5O3S2, C 56.31, H 3.52,
N 13.24%.
6-Chloro-7-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-3-[(1H-indazol-5-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine
(
5e). Starting from 4b (0.458 g) and 5-amino-1H-indazole (˝0.133 g) after refluxing for 44 h the title
compound 5e was obtained (0.212 g, 39%) mp 352–354 (dec.) C (DMF–MeCN, 3:4); IR (KBr) νmax 3383
(NH), 3079 (CH Ar), 2925, 2854 (CH), 1604, 1530, 1506, 1482, 1459 (C=N, C=C), 1320, 1159 (SO2) cm
1H-NMR (200 MHz, DMSO-d6)
´1
;
δ
7.66–7.74 (m, 4H, Ar H), 8.09–8.18 (m, 4H, Ar H), 8.37 (s, 1H, H-5),
8.64 (s, 1H, H-8), 11.66 (s, 1H, NH), 13.22 (s, 1H, NH) ppm; anal. C 48.63, H 2.23, N 15.47% calcd for
C22H12Cl2N6O3S2, C 48.95, H 2.39 N 15.62%.
6-Chloro-7-(5-phenyl-1,3,4-thiadiazol-2-yl)-3-[(1H-indazol-5-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine (5f).
Starting from 4c (0.440 g) and 5-amino-1H-indazole (0.133 g) after refluxing for 45 h the title compound
5f was obtained (0.415 g, 79%) mp >360 ˝C (DMF–MeOH, 1:3); IR (KBr)
ν
max 3299 (NH), 2925, 2853
(CH), 1611, 1575, 1531, 1504, 1457 (C=N, C=C), 1305, 1157 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
: 7.50–7.52 (m, 1H, Ar H), 7.57–7.62 (m, 5H, Ar H), 8.03–8.10 (m, 2H, Ar H), 8.16 (s, 1H, Ar H), 8.29 (s,
1H, H-5), 8.76 (s, 1H, H-8), 11.67 (s, 1H, NH), 13.21 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ
δ
111.42, 114.58, 122.61, 123.21, 126.41, 128.48, 129.46. 129.69, 130.26, 131.08, 132.14, 132.45, 133.64, 134.76,
135.37, 138.55, 160.63, 161.96, 170.41 ppm; anal. C 50.33, H 2.50 N 16.01% calcd for C22H13ClN6O2S3,
C 50.52, H 2.81 N 15.95%.
6-Chloro-7-(5-phenyl-1,3,4-thiadiazol-2-yl)-3-[2-(1H-indol-3-yl)ethylamino]-1,1-dioxo-1,4,2-benzodithiazine
(5g). Starting from 4c (0.440 g) and 2-(1H-indol-3-yl)ethanamine (0.160 g) after stirring for 24 h in room
tempera˝ture followed by refluxing for 7.5 h the title compound 5g was obtained (0.488 g, 93%) mp
3228 (NH), 2962, 2929, 2873 (CH), 1649, 1602 1575, 1486,
max
227–229 C (DMF–MeOH, 1:3); IR (KBr)
ν
1452 (C=N, C=C), 1316, 1164 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ 3.01 (m, 2H, CH2), 3.67 (m,
2H, CH2), 6.97–6.98(m, 1H, Ar H), 7.04–7.07 (m, 1H, Ar H), 7.20 ( s, 1H, Ar H), 7.33–7.34 ( m, 1H, Ar H),
7.55–7.57 (m, 4H, Ar H), 8.04–8.05 (m, 2H, Ar H), 8.19 (s, 1H, H-5), 8.74 (s, 1H, H-8), 10.02 (s, 1H, NH),
10.89 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ 23.80, 44.28, 110.73, 111.45, 118.17, 118.40,
121.06, 122.99, 125.59, 127.04, 127.78, 128.50, 129.02, 129.57, 130.13, 131.75, 132.01, 132.93, 134.44, 136.24,
161.33, 161.64, 169.67 ppm; anal. C 54.39, H 3.29, N 12.69% calcd for C25H18ClN5O2S3, C 54.58, H 3.35,
N 12.73%.
6-Chloro-7-(3-phenyl-1,2,4-oxadiazol-5-yl)-3-[(1H-indazol-5-yl)amino]-1,1-dioxo-1,4,2-benzodithiazine (5h).
Starting from 4d (0.424 g) and 5-amino-1H-indazole (0.133 g) after refluxing for 50 h the title compound
˝
5h was obtained (0.341 g, 67%) mp 347–349 C (70% DMFaq); IR (KBr)
ν
max 3385 (NH), 1595, 1565, 1531
7.55 (m, 1H, Ar H), 7.62–7.66
(C=N, C=C), 1321, 1158 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
(m, 4H, Ar H), 8.11 (s, 1H, Ar H), 8.14–8.15 (m, 2H, Ar H), 8.19 (s, 1H, Ar H), 8.41 (s, 1H, H-5), 8.71
(s, 1H, H-8), 11.75 (s, 1H, NH), 13.24 (s, 1H, NH) ppm; anal. C 51.92, H 2.57, N 16.51% calcd for
C22H13ClN6O3S2, C 52.08, H 2.69, N 16.62%.
6-Chloro-7-(3-phenyl-1,2,4-oxadiazol-2-yl)-3-[2-(1H-indol-3-yl)ethylamino]-1,1-dioxo-1,4,2-benzodithiazine
(
5i). Starting from 4d (0.424 g) and 2-(1H-indol-3-yl)ethanamine (0.160 g) after stirring for 52 h in room
˝
temperature the title compound 5i was obtained (0.306 g, 57%) mp 205–207 C (MeOH); IR (KBr)
ν
max
3403, 3283 (NH), 3049 (CH Ar), 2921, 2854 (CH), 1599, 1567, 1475, 1450 (C=N, C=C), 1314, 1141 (SO2)
cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
3.00–3.03 (m, 2H, CH2), 3.66–3.69 (m, 2H, CH2), 6.95–6.98 (m,
1H, Ar H), 7.05–7.08 (m, 1H, Ar H), 7.20 ( s, 1H, Ar H), 7.34 (d, J = 8.3 Hz, 1H, Ar H), 7.54–7.58 (m, 1H,
Ar H), 7.60–7.62 (m, 3H, Ar H), 8.09–8.10 (m, 2H, Ar H), 8.24 (s, 1H, H-5), 8.64 (s, 1H, H-8), 10.07 (s,
1H,NH), 10.89 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ 24.44, 45.02, 111.40, 112.14, 118.85,
119.09, 121.74, 123.44, 123.69, 126.37, 127.73, 127.89, 127.91, 130.04, 131.50, 132.48, 132.57, 135.91, 136.07,
136.92, 162.13, 168.71, 173.28 ppm; anal. C 56.02, H 3.38, N 13.07% calcd for C25H18ClN5O3S2, C 56.38,
H 3.50,N 13.31%.
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Molecules 2015, 20, 21960–21970
6-Chloro-7-(4-phenylthiazol-2-yl)-3-[2-(1H-indol-3-yl)ethylamino]-1,1-dioxo-1,4,2-benzodithiazine (5j). Starting
from 4e (0.439 g) and 2-(1H-indol-3-yl)ethanamine (0.160 g) after stirring for 24 h in room t˝emperature
followed by refluxing for 48 h the title compound 5j was obtained (0.518 g, 94%) mp 145–147 C (MeOH);
1
IR (KBr)
ν
max
3408 (NH), 2925, 2853 (CH), 1563, 1458 (C=N, C=C), 1306, 1156 (SO2) cm´1; H-NMR
(500 MHz, DMSO-d6)
δ
3.01–3.03 (m, 2H, CH2), 3.67 (m, 2H, CH2), 6.96–6.99 (m, 1H, Ar H), 7.05–7.08 (m,
1H, Ar H), 7.20 (s, 1H, Ar H), 7.33–7.40 (m, 2H, Ar H), 7.47–7.50 (m, 2H, Ar H), 7.55–7.57 (m, 1H, Ar H),
8.03–8.04 (m, 2H, Ar H), 8.12 (s, 1H, H-5), 8.38 (s, 1H, Ar H), 8.88 (s, 1H, Ar H), 9.98 (s, 1H, NH), 10.89 (s,
1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ 23.80, 44.24, 110.76,.11.45, 117.64, 118.18, 118.40, 121.06,
122.98, 125.24, 126.19, 127.06, 128.52, 128.95, 130.19, 131.15, 131.99, 133.40, 133.57, 136.24, 154.26, 160.05,
161.76 ppm; anal. C 56.66, H 3.48, N 10.17% calcd for C26H19ClN4O2S3, C 56.80, H 3.61, N 10.42%.
4.3. In Vitro Anticancer Screening
Antitumor evaluation of compounds 5a–j was performed at the National Cancer Institute
according to NCI-60 DTP Human Tumor Cell Line Screen procedure [23–26].
4.4. Methodology of Molecular Modeling and QSAR Models Development
Studied compounds were manually drawn using ACD ChemSketch (Advanced Chemistry
Development, Inc., Toronto, ON, Canada), and geometrically optimized afterwards. AM1
pre-optimization was conducted using HyperChem (v 8.0.8, HyperCube, Gainesville, FL, USA). DFT
calculations were conducted using Gaussian software [27], at the B3LYP/6–311 G(d) level of theory.
Molecular descriptors were calculated using DRAGON 6.0 Software (Talete, Milano, Italy),
SPARTAN software (Spartan ’08, Wavefunction, Inc., Irvine, CA, USA) and Gaussian software [27].
Statistical analysis, feature selection and chemometric calculations (Stepwise Multiple Linear
Regression) were all conducted using STATISTICA 10.0 software. IGP was taken as a dependent value
for analysis, while molecular descriptors were independent values.
Created models were validated using Leave-one-out cross-validation (LOO-CV). This procedure
assumed removing single data point (cytotoxic value) from analyzed set, recalculating regression
on the rest of the dataset, and comparing predicted cytotoxic value of the omitted compound with
experimental value. The procedure was repeated until each compound’s cytotoxic value has been
omitted once. To evaluate developed model’s performance, sum of squares of each omitted data errors
were used to calculate the cross-validated root-mean-square error (RMSECV).
5. Conclusions
We have developed a facile method for the synthesis of new 3-amino-6-chloro-7-(azol-2 or
5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives. The compounds were evaluated in vitro for their
antiproliferative activity at the U.S. National Cancer Institute. We have found that the novel
compounds displayed moderate anticancer activity related to their structure. The best antiproliferative
properties have been observed for compound 5h, especially against the ovarian (OVCAR-3) and
breast (MDA-MB-468) cancer cell lines. Moreover, good selectivity against prostate (DU-145),
colon (SW-620) and renal (TK-10) cancer cell lines have also been observed for derivative 5h. To
summarize the structure-activity relationship very briefly, it could be stated that derivatives possessing
a 3-phenyl-1,2,4-oxadiazol-5-yl or 5-phenyl-1,3,4-oxadiazol-2-yl moiety attached directly to position
7 as well as a 1H-indazol-5-yl scaffold incorporated in the position 3 display the best anticancer
properties. The QSAR studies have revealed that the atomic masses and atomic polarizability weighted
descriptors played a significant role in addressing compounds activity against the leukemia (MOLT-4)
cell line. On the other hand, atomic masses and atomic Sanderson electronegativity have a greater
impact on the anticancer activity toward renal cancer (UO-31) cell line. The comparison of the cytotoxic
activity with the one predicted by statistical analysis has shown that the obtained QSAR models display
a good correlation with Rcv values of 0.927 and 0.861 for MOLT-4 and UO-31 respectively, suggesting
these models can be used in order to design new structures with interesting anticancer activity.
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Molecules 2015, 20, 21960–21970
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/
12/19821/s1.
Acknowledgments: The authors are very grateful to Joel Morris, Chief of Drug Synthesis and Chemistry Branch
(DSCB), National Cancer Institute (Bethesda, MD, USA) for the in vitro screening. The publishing fee covering the
cost to publish in open access was supported by the Ministry of Science and Higher Education of the Republic of
Poland, from the quality promoting subsidy under the Leading National Research Centre (KNOW) program for
the years 2012–2017.
Author Contributions: A.P. and J.S. created the concept, and designed the study. A.P. and K.B. performed
chemical research. A.P. and K.B. under J.S. supervision analyzed both chemical and biological data. A.P. and J.S.
wrote the manuscript together. S.U. and T.B. preformed statistical analysis and interpreted the statistical models.
All Authors read and approved the final version of the article.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5a–j are available from the authors.
©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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