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Molecules 2015, 20(12), 21960-21970; 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
Department of Organic Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland
2
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland
*
Author to whom correspondence should be addressed.
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 30 October 2015 / Accepted: 1 December 2015 / Published: 9 December 2015

Abstract

:
A series of new 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5aj have been synthesized and evaluated in vitro for their antiproliferative activity 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 5aj 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 [1,2,3,4,5,6], cholagogue [6,7], radioprotective [4], antiarrhythmic [4,6], hypotensive [4,5,6,7] and anti-HIV [8,9,10]. Of particular 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 [11,12,13,14], II, III [13,15,16] and IV [17]). With regard to these reports we have designed novel benzodithiazine derivatives of the general structure of type V (Figure 1) that vary according to 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.
Figure 1. General structures of 1,1-dioxo-1,4,2-benzodithiazines IIV [11,12,13,14,15,16,17] and V with anticancer activity.
Figure 1. General structures of 1,1-dioxo-1,4,2-benzodithiazines IIV [11,12,13,14,15,16,17] and V with anticancer activity.
Molecules 20 19821 g001
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 (V, Figure 1). The new 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 5aj are shown in Scheme 1 and Scheme 2.
The essential substrates for the synthesis of novel compounds 5aj, the already known 6-chloro-7-heteroaryl-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4ae, were obtained using previously described methods which are briefly summarized in Scheme 1 [18,19,20,21,22]. Thus, the starting 3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4ae could be converted to the desired 3-(R2-amino-7-azolyl-6-chloro-1,1-dioxo-1,4,2-benzodithiazine derivatives 5aj by nucleophilic substitution reactions with one molar equivalent of a primary amine, as outlined in Scheme 2.
Scheme 1. Synthesis of 7-(azol-2 or 5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4ae. Reagents and conditions: (a) SOCl2 excess, benzene, reflux; (b) 12% NH3(aq), benzene 5–10 °C; (c) arylhydrazide, benzene, 5→20 °C; (d) N-hydroxybenzamidine, toluene, 0→20 °C; (e) Lawesson reagent (LR, 0.5 equiv.), toluene; (f) SOCl2, rt→reflux; (g) LR, toluene, 20→100 °C; (h) 110 °C; (i) ω-halogenoacetophenone, MeOH, 20→65 °C.
Scheme 1. Synthesis of 7-(azol-2 or 5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 4ae. Reagents and conditions: (a) SOCl2 excess, benzene, reflux; (b) 12% NH3(aq), benzene 5–10 °C; (c) arylhydrazide, benzene, 5→20 °C; (d) N-hydroxybenzamidine, toluene, 0→20 °C; (e) Lawesson reagent (LR, 0.5 equiv.), toluene; (f) SOCl2, rt→reflux; (g) LR, toluene, 20→100 °C; (h) 110 °C; (i) ω-halogenoacetophenone, MeOH, 20→65 °C.
Molecules 20 19821 g002
Scheme 2. Synthesis of 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5aj. 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.
Scheme 2. Synthesis of 3-amino-6-chloro-7-(azol-2 or 5-yl)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5aj. 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.
Molecules 20 19821 g003
The structures of the final compounds 5aj were confirmed by elemental analyses and spectroscopic (IR, 1H-NMR and 13C-NMR) data, in particular the presence of the IR absorption bands corresponding to the stretching vibration of the NH group in the 3228–3408 cm−1 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 5aj were tested in vitro at the U.S. National Cancer Institute (Bethesda, MD, USA) at a single dose of 10 μM 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 5aj 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 5aj (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.
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 5aj.
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 5aj.
PanelCell LineIGP (%) of Compound
5a5b5c5d5e5f5g5h5i5j
LeukemiaCCRF-CEM31.6211.56--3.41-25.2413.736.7710.91
HL-60(TB)31.1236.364.20****69.38**
MOLT-416.2317.074.0512.963.6410.6423.599.060.4424.25
RPMI-822624.202.85*-*0.9918.2181.001.18*
SR---21.80*53.7126.541.766.3622.08
NSCLCHOP-6215.3213.98***6.75*34.63**
NCI-H22613.0926.506.62**17.093.3414.781.10*
NCI-H522--15.60*5.6722.525.3427.30**
Colon cancerHCC-2998--***5.10*60.93**
HCT-1169.472.28*0.68*3.307.3743.60*8.42
SW-620*9.09*****50.98**
CNS cancerSNB-196.579.45*4.26*17.479.703.402.53*
SNB-757.09--11.444.47-32.1976.9312.318.52
U25112.8916.48***17.555.9721.68**
MelanomaLOX IMVI3.5610.33*2.14*1.308.7953.683.02*
UACC-25711.401.35*****33.69**
UACC-6216.61**4.08*5.830.2923.802.39*
Ovarian cancerOVCAR-3*7.74*****10.27 a**
OVCAR-815.9315.64*2.07*0.668.8124.081.69*
Renal cancerSN12C2.6214.331.694.46*17.1111.8715.541.69*
TK-10**5.41****52.33**
UO-314.7836.3416.3523.538.7534.0029.546.5724.739.74
Prostate cancerPC-319.1211.451.37-6.74*1.8751.71*0.36
DU-145*******98.79**
Breast cancerMCF-735.976.778.254.44*10.5015.7823.525.3210.35
MDA-MB-231/ATCC25.8415.131.75****42.05**
T-47D49.1012.27****35.1937.64**
MDA-MB-46853.3718.735.58**9.624.9347.18 a**
* IGP ≤ 0%; - not tested; a—cytotoxic effect.
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 5aj against MOLT-4 and UO-31 cell lines.
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 5aj against MOLT-4 and UO-31 cell lines.
Cell LineEquationNRRcvsRMSECVpF
MOLT-4IGP = 0.815(RDF055p) − 0.55(Mor32m) − 41.34100.9670.9272.340.8590.0000651.34
UO-31IGP = 0.770(Mor16e) − 0.497(RDF145m) + 67.048100.9430.86111.441.3711260.000628.33
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.
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.
Compd.MOLT-4UO-31
RDF055pMor32mMor16eRDF145m
5a5.903−0.7011.6323.349
5b6.251−0.7751.2435.546
5c4.882−0.6271.5293.010
5d5.670−0.6161.7445.816
5e6.278−0.2021.7653.392
5f6.038−0.4861.2523.104
5g8.458−0.5491.2603.370
5h5.958−0.4481.6621.985
5i5.521−0.4001.2893.139
5j7.891−0.5511.5651.986
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.
Table 4. Comparison of observed IGP values (anticancer activity) and those predicted by QSAR models for MOLT-4 and UO-31 cell lines.
Compd.IGP [%]
MOLT-4UO-31
ObservedPredictedObservedPredicted
5a16.2313.824.7814.44
5b17.0719.3336.3443.11
5c4.056.9616.3515.78
5d12.9610.1523.5311.71
5e3.642.138.757.11
5f10.649.133425.97
5g23.5928.9129.5428.72
5h9.067.556.575.05
5i0.444.7724.7327.15
5j24.2521.639.749.61

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 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 (1) [18], 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carbonyl chloride (2a) [19], 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carboamide (2b) [20], N′-(6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carbonyl)benzhydrazides (3ab) [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 (4ac) [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 5aj 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 5aj

To a suspension of the 7-(azol-2 or 5-yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine 4ae (1.0 mmol) in methanol (10 mL) the appropriate primary amine (1.0 mmol) was added. The reaction mixture was refluxed for 30–50 h (method A—5af, 5h), stirred for 24 h at room temperature and then refluxed for 7.5–48 h (method B—5g, 5j) or stirred at room temperature for 52 h (method 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) νmax 3387, 3296 (NH), 2925, 2854 (CH), 1589, 1548, 1531, 1505, 1450 (C=N, C=C), 1312, 1159 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d6) δ 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, 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) νmax 3289 (NH), 2927 (CH), 1649, 1581, 1551, 1488, 1459 (C=N, C=C), 1316, 1162 (SO2) cm−1; 1H-NMR (500 MHz, DMSO-d6) δ 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) δ 120.30, 120.44, 122.45, 122.80, 122.95, 126.44, 126.93, 126.98, 129.54, 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) νmax 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) δ 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, 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), 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−1; 1H-NMR (200 MHz, DMSO-d6) 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) νmax 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) δ: 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 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 temperature followed by refluxing for 7.5 h the title compound 5g was obtained (0.488 g, 93%) mp 227–229 °C (DMF–MeOH, 1:3); IR (KBr) νmax 3228 (NH), 2962, 2929, 2873 (CH), 1649, 1602 1575, 1486, 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) νmax 3385 (NH), 1595, 1565, 1531 (C=N, C=C), 1321, 1158 (SO2) cm−1; 1H-NMR (500 MHz, DMSO-d6) δ 7.55 (m, 1H, Ar H), 7.62–7.66 (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) νmax 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%.
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 temperature followed by refluxing for 48 h the title compound 5j was obtained (0.518 g, 94%) mp 145–147 °C (MeOH); IR (KBr) νmax 3408 (NH), 2925, 2853 (CH), 1563, 1458 (C=N, C=C), 1306, 1156 (SO2) cm−1; 1H-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 5aj was performed at the National Cancer Institute according to NCI-60 DTP Human Tumor Cell Line Screen procedure [23,24,25,26].

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.

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 5aj are available from the authors.
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