Synthesis and QSAR study of novel 6-chloro-3-(2-Arylmethylene-1-methylhydrazino)-1,4,2-benzodithiazine 1,1-dioxide derivatives with anticancer activity.

A series of new 6-chloro-3-(2-arylmethylene-1-methylhydrazino)-1,4,2-benzodithiazine 1,1-dioxide derivatives were effectively synthesized from N-methyl-N-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazines. The intermediate compounds as well as the products, were evaluated for their cytotoxic effects toward three human cancer cell lines. All compounds shown moderate or weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compound 16 exhibited the most potent cytotoxic activity against the HeLa cell line, with an IC50 value of 10 µM, while 14 was the most active against the MCF-7 and HCT-116 cell lines, affording IC50 values of 15 µM and 16 µM, respectively. The structure-activity relationship was evaluated based on QSAR methodology. The QSAR MCF-7 model indicated that natural charge on carbon atom C13 and energy of highest occupied molecular orbital (HOMO) are highly involved in cytotoxic activity against MCF-7 cell line. The cytotoxic activity of compounds against HCT-116 cell line is dependent on natural charge on carbon atom C13 and electrostatic charge on nitrogen atom N10. The obtained QSAR models could provide guidelines for further development of novel anticancer agents.

1. Introduction

The 1,1-dioxo-1,4,2-benzodithiazines constitute a fundamental class of compounds in our laboratories since 1984, they have a wide range of biological activity and are useful substrates for many syntheses. It has been demonstrated that many 6-chloro-1,1-dioxo-1,4,2-benzodithiazine derivatives (I, Figure 1) possess low acute toxicity in mice and rats and depending on their structure, they act as potential diuretic [1,2,3,4,5,6] radioprotective [4], cholagogue [6,7], antiarrhythmic [4,6], or hypotensive [4,5,6,7] agents. It has also been shown that some 6-chloro-1,1-dioxo-1,4,2-benzodithiazines exhibit remarkable antitumor activities (I [8,9,10], II and III [10,11,12], Figure 1) or anti-HIV-1 activity (I and IV [13,14,15] Figure 1). This prompted us to investigate new potential antiproliferative agents with the general structure of type V (Figure 1) in which moieties attached to positions 3 and 7 of 6-chloro-1,1-dioxo-1,4,2-benzodithiazine ring were varied in molecular shape and electronic properties.

Figure 1

Chemical structures of biological active 1,1-dioxo-1,4,2-benzodithiazines I–IV [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15], and V.

Thus, we synthesized new series of 6-chloro-7-R1-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazines V and evaluated their in vitro anticancer activity against human breast (MCF-7), colon (HCT-116) and cervical (HeLa) cancer cell lines. To explain how structural features influence the biological activities the quantitative structure-activity relationship (QSAR) method was applied [16].

2. Results and Discussion

2.1. Chemistry

A series of the desired 6-chloro-7-R1-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazine derivatives 5‒18 was prepared in a two-step process as shown in Scheme 1 and Scheme 2. Thus, the starting N-methyl-N-(6-chloro-7-R1-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazines 2‒4 were synthesized by the reaction of the appropriate 3-methylthiobenzodithiazines 1a‒c with N-methylhydrazine in boiling dry acetonitrile (Scheme 1). It is well known that alkylations of alkyl-substituted hydrazines take place at the more substituted nitrogen atom [17,18,19], therefore in our investigations competition between N1 and N2 alkylation was not observed, and the regioselective substitution product, i.e., N-methyl-N-substituted hydrazines, were obtanined in high yields (90%‒93%). Subsequent condensation of the hydrazine derivatives with an aldehyde in the presence of a catalytic amount of glacial acetic acid furnished the expected final products 5‒18 in good yields (87%‒98%) (Scheme 2).

Scheme 1

Synthesis of N-methyl-N-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazines 2‒4.

Scheme 2

Synthesis of 6-chloro-7-R1-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazines 5‒18.

Reagents and conditions: (a) methylhydrazine (1.04 M equiv.), dry acetonitrile, r.t. 8 h, reflux 10–16 h.

The structures of the compounds 2‒4 and the final substances 5‒18 were confirmed by IR, 1H-NMR and 13C-NMR spectroscopy. For example, in the 1H-NMR spectra, the presence of N-CH3 and N-NH2 groups in 2–4 was identified from the singlet signals at 3.36–3.35 and 5.72–5.85 ppm, respectively. Meanwhile, the appearance of N=CH signals at 8.21‒8.66 ppm in the spectra of 5‒18 proved the presence of this group and conformed the proposed structure of the final compounds.

2.2. Cytotoxic Activity

Compounds 6‒18 and the intermediates 2 and 4 were evaluated in vitro for their effects on the viability of three human cancer cell lines: MCF-7 (breast cancer), HCT-116 (colon cancer) and HeLa (cervical cancer). The concentration required for 50% inhibition of cell viability IC50 was calculated and compared with the reference drug cisplatin, and the results are given in Table 1.

Table 1

Cytotoxicity of compounds 2, 4 and 6‒18 toward human cancer cell lines.

Compounds	IC50 [µM]
	MCF-7	HCT-116	HeLa
2	250 ± 7	280 ± 1	125 ± 7
4	155 ± 2	240 ± 5	99 ± 4
6	45 ± 0.5	31 ± 1	66 ± 2
7	270 ± 13	150 ± 4	>>100 a
8	84 ± 3	38 ± 1	80 ± 1
9	95 ± 1	24 ± 1	32 ± 2
10	73 ± 2	39 ± 1	64 ± 3
11	70 ± 3	38 ± 1	75 ± 3
12	63 ± 1	84 ± 1	100 ± 3
13	140 ± 1	115 ± 2	100 ± 1
14	15 ± 0.5	16 ± 0.5	135 ± 9
15	105 ± 2	70 ± 2	170 ± 5
16	150 ± 3	23 ± 0.5	10 ± 0.5
17	83 ± 1	74 ± 1	62 ± 1
18	165 ± 6	260 ± 3	140 ± 7
Cisplatin	3.0 ± 0.1	3.8 ± 0.2	2.2 ± 0.1

a viability of HeLa cell line at 100 µM of compd 7 was 100%.

The most active compounds possess at N2 of the 3-hydrazinobenzodithiazine scaffold a 2,4-dihydroxyphenyl (compound 6) or 2-hydroxy-5-nitrophenyl (compound 9) moiety and showed outstanding anticancer activity against all tested cell lines, whereas compounds 2, 4, 7, 13 and 18 demonstrated relatively weak activity, with IC50 values over 100 µM.

Reagents and conditions: (a) appropriate aryl aldehyde (1.25 M equiv.), EtOH, AcOH (catalytic amount), r.t. 3 h, reflux 25 h; (b) appropriate aryl aldehyde (1.33 M equiv.), MeOH, AcOH (catalytic amount), r.t. 4 h, reflux 28 h; (c) appropriate aryl aldehyde (1.33 M equiv.), 2-methoxyethanol, AcOH (catalytic amount), r.t. 2 h, reflux 9 h.

Interestingly, compound 16 (Ar = 2,5-dihydroxyphenyl) exhibited the strongest cytotoxic activity against the HeLa cell line with an IC50 value of 10 µM, however shifting of the hydroxyl functionality from position 5 (compound 16) to position 4 (compound 15) on the phenyl ring caused the loss of cytotoxic activity (IC50 = 170 µM) against this cell line. On the other hand, the presence of Ar = 5-nitrothiophene ring (compound 14) determined the highest cytotoxic activities against MCF-7 and HCT-116 cell lines, and affording the IC50 values of 15 µM and 16 µM, respectively, which changed significantly after its replacement by a 5-nitrofuran ring (compound 13) resulting in poor activity (IC50 = 140 µM, MCF-7, and IC50 = 115 µM, HCT-116).

Nevertheless, considering the compounds’ activities against each single cell line, the nature of substituents R1 and Ar have varying influences on the biological activity of these compounds. For instance, cytotoxic activity in the series of N2-methylenesubstituted 3-hydrazino-7-methyl-1,1-dioxo-1,4,2-benzodithiazines against the MCF-7 cell line decreased in the following order: 5-nitrothiophenyl (14) > 2,4-dihydroxyphenyl (6) > pyrrolyl (12) > 3-bromo-5-chloro-2-hydroxyphenyl (11) ≈ 5-bromo-2-hydroxy-3-methoxyphenyl (10) > 5-bromo-2-hydroxyphenyl (8) > 2-hydroxy-5-nitrophenyl (9), suggesting the necessity to choose other and more objective methods of establishing the structure-activity relationships, such as QSAR methodology.

2.3. Quantitative Structure-Activity Relationships (QSARs) of Cytotoxic Activity

QSAR analysis was performed in order to correlate the cytotoxic activity of the 1,1-dioxo-1,4,2-benzodithiazine derivatives with their chemical structure and to determine the most important parameters controlling their pharmacological effects. The optimized structures of tested compounds were sketched using the geometry optimization function of Spartan software (Spartan ’08, Wavefunction, Inc., Irvine, CA, USA) [20]. The electronic, steric, hydrophilic and hydrophobic features of molecules obtained from molecular modelling descriptors (Table 2 and Table 3) have been applied to the statistical calculations. The QSAR models for each cell line were developed separately. Compound 7 was excluded from the data set of QSAR analysis because of very low cytotoxic activity.

Table 2

Theoretical molecular parameters of compounds 6, 8‒18: CPK Area, CPK Volume, PSA, MW, CFD (HBD), CFD (HBA), Angle (i, j, k), Dihedral (i, j, k, l), Distance (i, j), LogP, Hardness.

Compd	CPK Area (Å2)	CPK Volume (Å3)	PSA (Å2)	MW (amu)	CFD (HBD)	CFD (HBA)	Angle (C3,N9,N10)	Angle (N9,N10,C11)	Dihedral (C3,N9,N10,C11)	Dihedral (N9,N10,C11,C12)	Distance (S4,C12)	Distance (S1,C12)	LogP	Hardness
6	378.32	351.11	89.6827	411.890	2	8	119.59	115.50	69.29	−179.26	6.053	5.332	4.8599	0.199531
8	389.58	362.09	69.9921	474.787	1	7	119.89	116.30	65.86	−179.35	6.053	5.325	6.0783	0.197595
9	395.72	366.01	109.1826	440.888	1	10	120.39	117.25	61.89	−179.55	6.052	5.320	5.2833	0.201345
10	417.97	388.93	74.8555	504.813	1	8	119.77	116.17	66.77	−179.57	6.055	5.335	5.9519	0.195736
11	402.80	375.17	68.4649	509.232	1	7	119.73	116.36	65.77	−179.05	6.050	5.317	6.6365	0.197948
12	346.63	318.50	63.7233	368.869	1	7	121.27	117.32	59.23	179.13	6.044	5.315	2.9324	0.190920
13	370.32	337.44	99.2438	414.850	0	10	120.74	118.17	58.08	−179.47	6.027	5.308	3.6225	0.193076
14	377.41	345.96	91.1344	430.917	0	10	121.28	118.67	55.91	179.99	6.045	5.329	4.3443	0.186695
15	411.87	380.93	110.6991	455.899	2	9	119.50	115.35	69.58	−179.19	6.053	5.334	4.1929	0.188761
16	411.80	380.88	110.6926	455.899	2	9	119.90	116.04	66.55	−179.41	6.058	5.337	4.1929	0.184929
17	370.21	345.35	85.0143	406.874	1	8	119.29	115.11	63.61	−177.70	6.017	5.199	4.7956	0.185247
18	371.53	338.82	114.5075	425.833	0	11	120.30	117.52	60.87	−179.34	6.027	5.303	3.1687	0.194908

CPK Area—surface area of a space-filling model; CPK Volume—volume of a space-filling model; PSA—polar surface area (N, O + attached hydrogens); MW—molecular weight; CFD (HBD)—number of hydrogen bond donors; CFD (HBA)—number of hydrogen bond acceptors; Angle (i, j, k)—angle involving atoms i, j, k (degrees); Dihedral (i, j, k, l)—dihedral angle involving atoms i, j, k, l (degrees); Distance (i, j)—distance involving atoms i, j (Å); LogP—lipophilicity estimated from Crippen model [22]; Hardness—-(HOMO—LUMO)/2 (eV).

Table 3

Theoretical molecular parameters of compounds 6, 8‒18: E, E HOMO, E LUMO, Elect (i), Mull (i), Nat (i), Electronegativity.

Compd	E	E HOMO	E LUMO	LUMO-HOMO	Elect (C13)	Mull (C13)	Nat (C13)	Elect (C11)	Mull (C11)	Nat (C11)	Elect (N10)	Mull (N10)	Nat (N10)	Electronegativity
	(kcal/mol)
6	−1462850	−194.26	56.16	250.42	−0.579	−0.303	−0.410	0.330	0.224	0.226	−0.384	−0.352	−0.376	0.110038
8	−3029531	−201.90	46.09	247.99	−0.608	−0.306	−0.411	0.299	0.217	0.213	−0.365	−0.338	−0.355	0.124147
9	−1543557	−214.66	38.03	252.69	−0.588	−0.308	−0.411	0.253	0.215	0.203	−0.338	−0.338	−0.350	0.140737
10	−3100993	−196.45	49.20	245.65	−0.587	−0.306	−0.411	0.297	0.217	0.213	−0.355	−0.337	−0.353	0.117330
11	−3317493	−208.48	39.95	248.43	−0.582	−0.307	−0.411	0.301	0.218	0.209	−0.350	−0.334	−0.349	0.134281
12	−1355166	−186.14	53.47	239.61	−0.662	−0.306	−0.410	0.175	0.157	0.183	−0.384	−0.327	−0.351	0.105707
13	−1495275	−218.12	24.20	242.32	−0.609	−0.311	−0.412	0.143	0.131	0.141	−0.376	−0.297	−0.313	0.154513
14	−1697748	−218.76	15.55	234.31	−0.602	−0.311	−0.411	0.189	0.175	0.163	−0.340	−0.301	−0.317	0.161921
15	−1580568	−195.25	41.65	236.90	−0.595	−0.304	−0.411	0.350	0.225	0.228	−0.386	−0.351	−0.377	0.122384
16	−1580564	−191.14	40.95	232.09	−0.574	−0.307	−0.411	0.347	0.212	0.215	−0.375	−0.334	−0.355	0.119665
17	−1448937	−206.78	25.71	232.49	−0.575	−0.307	−0.411	0.326	0.158	0.208	−0.399	−0.310	−0.350	0.144282
18	−1528335	−224.84	19.78	244.62	−0.595	−0.313	−0.413	0.150	0.139	0.150	−0.383	−0.296	−0.316	0.163393

E—total energy; E HOMO—energy of highest-occupied molecular orbital; E LUMO—energy of lowest-occupied molecular orbital; Elect (i)—electrostatic charge on atom i; Mull (i)—Mulliken charge on atom i; Nat (i)—natural charge on atom i; Electronegativity—-(HOMO + LUMO)/2 (eV).

The QSAR model generation was done using the Statistica package (v.10, Statsoft, Tulsa, OK, USA) [21], and to search for optimal QSAR models multiple linear regression (MLR) along with stepwise algorithm were employed. In order to improve the statistical performance of the HCT-116 model, compounds 12 and 18 were removed from dataset. Both compounds were identified as outliers based on the initial experimental versus predicted activity plot. A much higher difference between experimental and predicted activity in comparison to the rest of compounds, suggests that these compounds possess some additional features explaining their biological activity, that was not covered in the applied descriptor dataset.

QSAR models were validated using the leave-one-out cross validation technique. In the case of the quantitative structure-activity relationships for the HeLa cell line a suitable statistical model was not found.

Details of the predictive performance of constructed QSAR models are shown in Table 4. Predicted IC50 values were described by equations as the function of significant descriptor values. The observed as well as the predicted cytotoxic activities are given in Table 5. The statistical significance of equations, as well as high Rcv value and value of RMSECV comparable to s, suggests that the obtained QSAR model can be used to explain relationships between chemical structure and activity.

Table 4

The QSAR equations and their predictive performance in predicting cytotoxic activity of the 1,1-dioxo-1,4,2-benzodithiazine derivatives against MCF-7 and HCT-116 cell lines.

Cell Line	Equation	N	R	s	Rcv	RMSECV	F	p
MCF-7	IC50 = −72361.3 (Nat C13) + 2.8 (E HOMO) − 29088.3	12	0.874	23.52	0.750	28.66	14.592	0.001499
HCT-116	IC50 = −45749.1 (Nat C13) − 937.3 (Elect N10) − 19099.8	10	0.902	14.97	0.715	21.54	15.303	0.002782

N—number of compounds in data set; R—a correlation coefficient; s—a standard error of estimate; Rcv—a correlation coefficient of leave-one-out cross validation (LOO-CV); RMSECV—a root mean square error LOO-CV; F—Fisher test value; p—significance level of F test.

Table 5

The cytotoxic activity against MCF-7 and HCT-116 obtained from experiments (observed) and from statistical calculations (predicted).

Compounds	IC50 [µM]
	MCF-7		HCT-116
	Observed	Predicted	Observed	Predicted
6	45 ± 0.5	41	31 ± 1	17
8	84 ± 3	92	38 ± 1	45
9	95 ± 1	57	24 ± 1	20
10	73 ± 2	108	39 ± 1	36
11	70 ± 3	74	38 ± 1	31
12	63 ± 1	64	84 ± 1	- a
13	140 ± 1	120	115 ± 2	101
14	15 ± 0.5	46	16 ± 0.5	22
15	105 ± 2	111	70 ± 2	65
16	150 ± 3	122	23 ± 0.5	54
17	83 ± 1	79	74 ± 1	77
18	165 ± 6	174	260 ± 3	- a

a compound was identified as a statistical outlier.

The statistical MCF-7 model indicated that natural charge on carbon atom C13 and energy of highest occupied molecular orbital (HOMO) are highly involved in cytotoxic activity against MCF-7 cell line. It was found that natural charge on carbon atom C13 has a negative weight in the correlation, whereas energy of HOMO positively correlates with cytotoxic activity of compounds. The significant influence of electronic properties of atom C13 on biological activity has been illustrated by optimized structures including marked natural charges and isosurface plots of HOMO, that show the energy level of HOMO (Figure 2). The anticancer properties of compounds 6 and 12 have been determined by low natural charge of C13 in spite of low HOMO energy. Apparently differences in charges of C13 had the influence on totally different biological activities of compounds 13 and 14, despite their similar HOMO energy.

Figure 2

Isosurface plots of HOMO of compounds 6, 12, 13 and 14, and natural charges on atom C13.

As shown the QSAR model for HCT-116 cell line, natural charge on carbon atom C13 as well as electrostatic charge on nitrogen atom N10 correlates with cytotoxic activity of compound. According to the equation, high anticancer activity is affected by low natural charge of C13 as the most important descriptor and low electrostatic charge of N10. As indicated in Table 3 inactive compounds exhibited relatively larger natural charges on C13 atom.

The QSAR results indicate that the cytotoxic activities of 1,1-dioxo-1,4,2-benzodithiazines against MCF-7 and HCT-116 are related to their molecular structure and especially the nature of the Ar group. Taken together the SAR and QSAR results on the cytotoxic activities of these analogs may provide valuable information for the further design of novel anticancer agents.

3. Experimental Section

3.1. General Information

The melting points were determined on a Boethius PHMK apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Thermo Mattson Satellite FTIR spectrophotometer. The NMR spectra were recorded on a Varian Gemini 200 spectrometer at 200 MHz (1H-NMR) or on a Varian Unity 500 Plus apparatus 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 dimethyl sulfoxide (DMSO-d). Elemental analyses were performed on PerkinElmer 2400 Series II CHN Elemental Analyzer and were in agreement with the theoretical values within ±0.4% range. The commercially unavailable new substrates were obtained according to the following methods described previously: 1a [23], 1b [24] and 1c [25].

3.2. Chemistry

3.2.1. Procedure for the Preparation of N-Methyl-N-(6-chloro-7-R1-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazines 2–4

A mixture of methylhydrazine (2.39 g, 0.052 mol) and the corresponding 3-methylthio-1,4,2-benzodithiazine 1a–c (0.05 mol) in dry acetonitrile (60 mL) was stirred at room temperature for 8 h, followed by reflux until the evolution of MeSH had ceased (10–16 h) (Caution: because of its high toxicity, MeSH should be trapped in aqueous NaOH solution). After cooling to room temperature the reaction mixture was stirred for 4 h. The precipitate of the benzodithiazinyl hydrazines obtained was filtered off, washed with acetonitrile (2 × 5 mL) and dried. In this manner the following hydrazines were obtained.

N-Methyl-N-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazine (2). Starting from 6-chloro-7-methyl-3-methylthio-1,1-dioxo-1,4,2-benzoditiazine 1a (14.7 g), the title compound 2 was obtained (13.5 g, 93%): mp 271–272 °C dec.; IR (KBr) νmax 3235 (N-NH2), 1645 (C=N), 1345, 1155 (SO2) cm−1; 1H-NMR (500 MHz, DMSO-d) δ 2.40 (s, 3H, CH3-7), 3.31 (s, 3H, N-CH3), 5.70 (s, 2H, N-NH2), 7.86 (s, 1H, H-5), 7.92 (s, 1H, H-8) ppm; 13C-NMR (125 MHz, DMSO-d) δ 19.95, 41.37, 126.66, 128.48, 130.56, 130.59, 137.40, 137.43, 166.82 ppm; anal. C 37.12, H 3.58, N 14.44% calcd for C9H10ClN3O2S2, C 37.04, H 3.45, N 14.40%.

Methyl 6-chloro-3-(1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylate (3). Starting from 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylate 1b (16 g), the title compound 3 was obtained (15.1 g, 90%): mp 252–253 °C dec.; IR (KBr) νmax 3360 (N-NH2), 1740 (C=O), 1340, 1155 (SO2) cm−1; 1H-NMR (500 MHz, DMSO-d) δ 3.30 (s, 3H, N-CH3), 3.88 (s, 3H, CH3O), 5.78 (s, 2H, N-NH2), 8.09 (s, 1H, H-5), 8.29 (s, 1H, H-8) ppm; 13C-NMR (125 MHz, DMSO-d) δ 41.48, 53.68, 127.17, 129.90, 130.41, 130.88, 135.73, 137.28, 164.25, 166.17 ppm; anal. C 35.72, H 3.10, N 12.57% calcd for C10H10Cl N3O4S2, C 35.77, H 3.00, N 12.51%.

N-Methyl-N-(6-chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazine (4). Starting from 6-chloro-7-cyano-3-methylthio-1,1-dioxo-1,4,2-benzodithiazine 1c (15.2 g), the title compound 4 was obtained (13.7 g, 91%): mp 285–286 °C dec.; IR (KBr) νmax 3320, 2245 (N-NH2), 2235 (C≡N), 1340, 1155 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 3.35 (s, 3H, N-CH3), 5.82 (s, 2H, N-NH2), 8.27 (s, 1H, H-5), 8.50 (s, 1H, H-8) ppm; 13C-NMR (125 MHz, DMSO-d) δ 41.52, 112.64, 115.41, 129.95, 130.53, 130.98, 138.70, 139.43, 165.96 ppm; anal. C 35.70, H 2.40, N 18.52% calcd for C9H7Cl N4O2S2, C 35.70, H 2.33, N 18.50%.

3.2.2. General Procedure for the Preparation of 6-Chloro-7-methyl-3-(2-arylmethylene-1-methyl-hydrazino)-1,1-dioxo-1,4,2-benzodithiazines 5–14

A mixture of the benzodithiazinyl hydrazine 2 (1.17 g, 4 mmol), the appropriate aryl carbaldehyde (5 mmol) and glacial acetic acid (0.5 mL, catalytic amount) in ethanol (25 mL) was stirred at room temperature for 3 h, followed at reflux for 25 h. After cooling to room temperature and standing overnight, the precipitate was filtered off, washed with ethanol (4 × 2 mL) and dried. In this manner the following benzodithiazines were obtained.

6-Chloro-7-methyl-3-[2-(hydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzo-dithiazine) (5). Starting from 2-hydroxybenzaldehyde (0.61 g), the title compound 5 was obtained (1.45 g, 92%): mp 313–315 °C dec.; IR (KBr) νmax 3225 (OH), 1340, 1160 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.44 (s, 3H, CH3-7), 3.66 (s, 3H, N-CH3), 6.91–7.00 (m, 2H, Ph), 7.31–7.40 (m, 1H, Ph), 7.85 (dd, Jortho = 7.7 Hz, Jmeta = 1.6 Hz, 1H, H-3, Ph), 8.03 (s, 2H, H-5 and H-8, benzodithiazine), 8.45 (s, 1H, N=CH), 10.35 (br.s, 1H, OH) ppm; anal. C 48.60, H 3.61, N 10.60% calcd for C16H14ClN3O3S2, C 48.54, H 3.56, N 10.61%.

6-Chloro-7-methyl-3-[2-(2,4-dihydroxybenzylidene-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (6). Starting from 2,4-dihydroxybenzaldehyde (0.69 g), the title compound 6 was obtained (1.62 g, 98%): mp 318–319 °C dec.; IR (KBr) νmax 3395, 3310 (OH), 1630 (C=N), 1340, 1310, 1165, 1150 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.45 (s, 3H, CH3-7), 3.65 (s, 3H, N-CH3), 6.38–6.45 (m, 2H, H-3 and H-5, Ph), 7.70 (d, J = 9.1 Hz, 1H, H-6, Ph), 8.01 (s, 1H, H-5, benzodithiazine), 8.03 (s, 1H, H-8, benzodithiazine), 8.37 (s, 1H, N=CH), 10.10 (s, 1H, OH-4, Ph), 10.24 (s, 1H, OH-2, Ph) ppm.; 13C-NMR (125 MHz, DMSO-d) δ 19.98, 32.73, 103.16, 109.05, 111.67, 126.93, 128.70, 128.90, 129.82, 130.50, 137.87, 138.11, 143.83, 159.78, 162.62, 164.53 ppm; anal. C 46.60, H 3.50, N 10.19% calcd for C16H14ClN3O4S2, C 46.65 H, 3.42 N, 10.20%.

6-Chloro-7-methyl-3[2-(2,5-dihydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (7). Starting from 2,5-dihydroxybenzaldehyde (0.69 g) the title compound 7 was obtained (1.60 g, 97%): mp 314–315 °C dec.; IR (KBr) νmax 3485 (OH), 3385 (OH), 1615 (C=N), 1345, 1305, 1160 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d6) δ 2.45 (s, 3H, CH3-7), 3.66 (s, 3H, N-CH3), 6.80 (s 2H, H-3 and H-4, Ph), 7.23 (s, 1H, H-6, Ph), 7.98 (s, 1H, H-5, benzodithiazine), 8.04 (s, 1H, H-8, benzodithiazine), 8.39 (s, 1H, N=CH), 9.06 (s, 1H, OH-5, Ph), 9.63 (s, 1H, OH-2, Ph) ppm; 13C-NMR (125 MHz, DMSO-d) δ 20.01, 32.71, 111.95, 118.09, 120.12, 121.27, 126.95, 128.58, 129.59, 130.06, 138.10, 138.38, 143.12, 150.64, 151.04, 165.13 ppm; anal. C 45.66, H 3.51, N 10.28% calcd for C16H14Cl N3O4S2, C 46.65, H 3.42, N 10.20%.

6-Chloro-7-methyl-3-[2-(5-bromo-2-hydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (8). Starting from 5-bromo-2-hydroxybenzaldehyde (1.0 g), the title compound 8 was obtained (1.75 g, 92%): mp 330–331 °C dec.; IR (KBr) νmax 3235 (OH), 1610 (C=N), 1335, 1315, 1160 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.44 (s, 3H, CH3-7), 3.66 (s, 3H, N-CH3), 6.94 (d, J = 8.7 Hz, 1H, H-3, Ph), 7.50 (dd, Jortho = 8.7 Hz, Jmeta = 2.5 Hz, 1H, H-4, Ph), 7.88 (d, Jmeta = 2.5 Hz, 1H, H-8, Ph), 8.03 (s, 1H, H-5, benzodithiazine), 8.10 (s, 1H, H-8, benzodithiazine), 8.35 (s, 1H, N=CH), 10.70 (s, 1H, OH) ppm; anal. C 40.45, H 2.80, N 8.90% calcd for C16H13BrClN3O3S2, C 40.47, H 2.76, N 8.85%.

6-Chloro-7-methyl-3-[2-(2-hydroxy-5-nitrobenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzo-dithiazine (9). Starting from 2-hydroxy-5-nitrobenzaldehyde (0.84 g), the title compound 9 was obtained (1.68 g, 95%): mp 327–328 °C dec.; IR (KBr) νmax 3420 (OH), 1610 (C=N), 1340, 1320, 1165 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.44 (s, 3H, CH3-7), 3.69 (s, 3H, N-CH3), 7.15 (d, J = 9.1 Hz, 1H, H-3, Ph), 8.04 (s, 2H, H-5 and H-8, benzodithiazine), 8.24 (dd, Jortho = 9.1 Hz, Jmeta = 2.9 Hz, 1H, H-4, Ph), 8.40 (s, 1H, N=CH), 8.60 (d, Jmeta = 2.9 Hz, 1H, H-6, Ph), 11.95 (s, 1H, OH) ppm; anal. C 43.57, H 3.02, N 12.76% calcd for C16H13ClN4O5S2, C 43.58, H 2.97, N 12.70%.

6-Chloro-7-methyl-3-[2-(5-bromo-2-hydroxy-3-methoxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (10). Starting from 5-bromo-2-hydroxy-5-methoxybenzaldehyde (1.16 g), the title compound 10 was obtained (1.95 g, 97%): mp 311–312 °C dec.; IR (KBr) νmax 3500 (OH), 1610 (C=N), 1350, 1310, 1165 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.44 (s, 3H, CH3-7), 3.66 (s, 3H, N-CH3), 3.88 (s, 3H, N-CH3O), 7.24 (d, Jmeta = 1.8 Hz, 1H, Ph), 7.50 (d, Jmeta = 1.8 Hz, 1H, Ph), 8.03 (s, 1H, H-5, benzodithiazine), 8.09 (s, 1H, H-8, benzodithiazine), 8.38 (s, 1H, N=CH), 10.01 (s, 1H, OH) ppm; anal. C 40.51, H 3.00, N 8.36% calcd for C17H15BrClN3O4S2, C 40.45, H 2.99, N 8.32%.

6-Chloro-7-methyl-3-[2-(3-bromo-5-chloro-2-hydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (11). Starting from 3-bromo-5-chloro-2-hydroxybezaldehyde (1.18 g), the title compound 11 was obtained (1.95 g, 93%): mp 337–338 °C dec.; IR (KBr) νmax 3425 (OH), 1605 (C=N), 1335, 1315, 1165 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.45 (s, 3H, CH3-7), 3.67 (s, 3H, N-CH3), 7.72 (d, Jmeta = 2.5 Hz, 1H, Ph), 7.83 (d, Jmeta = 2.5 Hz, 1H, Ph), 8.05 (s, 1H, H-5, benzodithiazine), 8.11 (s, 1H, H-8, benzodithiazine), 8.47 (s, 1H, N=CH), 10.40 (s, 1H, OH) ppm; anal. C 37.78, H 2.40, N 8.26% calcd for C16H12BrCl2N3O3S2, C 37.73, H 2.37, N 8.25%.

6-Chloro-7-methyl-3-[2-(1H-pyrrol-2-yl)methylene-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (12). Starting from 1H-pyrrole-2-carbaldehyde (0.48 g), the title compound 12 was obtained (1.30 g, 88%): mp 245–246 °C dec.; IR (KBr) νmax 3420 (NH), 1615 (C=N), 1345, 1310, 1155 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.44 (s, 3H, CH3-7), 3.62 (s, 3H, NCH3), 6.20–6.25 (m,1H, H-5, pyrrole), 6.69 (t, J = 10 Hz, 1H, H-4, pyrrole), 7.10 (s, 1H, H-3, pyrrole), 7.78 (s, 1H, H-5, benzodithiazine), 8.02 (s, 1H, H-8, benzodithiazine), 8.21 (s, 1H, N=CH), 11.46 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d) δ 19.98, 32.95, 110.77, 116.53, 124.83, 126.81, 126.90, 128.35, 130.12, 130.45, 137.87, 138.12, 139.89, 164.40 ppm; anal. C 45.61, H 3.60, N 15.18% calcd for C14H13ClN4O2S2, C 45.58, H 3.55, N 15.19%.

6-Chloro-7-methyl-3-[2-(5-nitrofurfurylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzo-dithiazine (13). Starting from 5-nitrofuran-2-carbaldehyde (0.7 g), the title compound 13 was obtained (1.44 g, 87%): mp 281–282 °C dec.; IR (KBr) νmax 1615 (C=N), 1350, 1315, 1165 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.45 (s, 3H, CH3-7), 3.65 (s, 3H, NCH3), 7.31 (d, J = 3.9 Hz, 1H, furan), 7.86 (d, J = 3.9 Hz, 1H, furan), 8.04 (s, 1H, H-5, benzodithiazine), 8.06 (s, 1H, H-8, benzodithiazine), 8.38 (s, 1H, N=CH) ppm; 13C-NMR (125 MHz, DMSO-d) δ 20.01, 33.26, 115.22, 117.70, 127.10, 128.80, 129.12, 129.44, 135.18, 138.41, 138.78, 151.16, 152.93, 165.56 ppm; anal. C 40.55, H 2.68, N 13.55% calcd for C14H11ClN4O5S2, C 40.53, H 2.67, N 13.50%.

6-Chloro-7-methyl-3-[2-(5-nitrothiophen-2-ylmethylene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzo-dithiazine (14). Starting from 5-nitrothiophene-2-carbaldehyde (0.79 g), the title compound 14 was obtained (1.50 g, 87%): mp 335–336 °C dec.; IR (KBr) νmax 1600 (C=N), 1335, 1305, 1160 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 2.45 (s, 3H, CH3-7), 3.65 (s, 3H, NCH3), 7.62 (d, J = 4.3 Hz, 1H, thiophene), 8.05 (s, 1H, H-5, benzodithiazine), 8.14 (s, 1H, H-8, benzodithiazine), 8.18 (d, J = 4.3 Hz, 1H, thiophene), 8.62 (s, 1H, N=CH) ppm; anal. C 39.11, H 2.59, N 13.06% calcd for C14H11ClN4O4S3, C 39.02, H 2.57, N 13.00%.

3.2.3. Procedure for the Preparation of Methyl 6-Chloro-3-[2-(2,4-dihydroxybenzylidene)-1-methyl-hydrazino]-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylates 15, 16

A mixture of methyl 6-chloro-3-(1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylate 3 (1.01 g, 3 mmol), and the corresponding 2,4- or 2,5-dihydroxbenzaldehyde (0.55 g, 4 mmol) glacial acetic acid (0.4 mL, catalytic amount) and methanol (30 mL) was stirred at room temperature for 4 h, followed at reflux for 28 h. After cooling to room temperature and standing overnight, the precipitate was filtered off, washed with methanol (4 × 3 mL), and dried. In this manner the following benzodithiazines were obtained.

Methyl 6-chloro-3-[2-(2,4-dihydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylate (15). Starting from 2,4-dihydroxybenzaldehyde, the title compound 15 was obtained (1.30 g, 95%): mp 310–311 °C dec.; IR (KBr) νmax 3320 wide (OH), 1715, (C=O), 1615 (C=N), 1330, 1310, 1150, 1130 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 3.60 (s, 3H, N-CH3), 3.92 (s, 3H, CH3O-C=O), 6.37–6.42 (m, 2H, H-5 and H-6, Ph), 7.71(d, Jortho = 9.1 Hz, 1H, H-3, Ph), 8.23 (s, 1H, H-5, benzodithiazine), 8.38 (s, 1H, H-8, benzodithiazine), 8.40 (s, 1H, N=CH), 10.15 (s, 1H), 10.25 (s, 1H, OH) ppm; anal. C 44.78, H 3.12, N 9.26% calcd for C17H14ClN3O6S2, C 44.78, H 3.09, N 9.21%.

Methyl 6-chloro-3-[2-(2,5-dihydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylate (16). Starting from 2,5-dihydroxybenzaldehyde, the title compound 16 was obtained (1.28 g, 94%): mp 303–304 °C dec.; IR (KBr) νmax 3420, 3310 (OH), 1610 (C=N), 1340, 1305, 1170, 1165 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 3.68 (s, 3H, N-CH3), 3.91 (s, 3H, CH3O-C=O), 6.65–6.85 (m, 2H, H-3 and H-4, Ph), 7.23 (d, Jmeta = 1.6 Hz, 1H, H-6, Ph), 8.21 (s, 1H, H-5, benzodithiazine), 8.38 (s, 1H, H-8, benzodithiazine), 8.41 (s, 1H, N=CH), 9.09 (s, 1H, OH), 9.68 (s, 1H, OH) ppm; 13C-NMR (125 MHz, DMSO-d) δ 32.99, 53.72, 112.00, 118.12, 120.04, 121.50, 127.55, 130.26, 130.61, 131.06, 136.22, 136.25, 143.89, 150.74, 151.24, 164.14, 164.35 ppm; anal. C 44.77, H 3.14, N 9.24% calcd for C17H14ClN3O6S2, C 44.78, H 3.09, N 9.21%.

3.2.4. Procedure for the Preparation of 6-Chloro-7-cyano-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazines 17, 18

A mixture of N-methyl-N-(6-chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazine 4, (0.9 g, 3 mmol) and the corresponding aryl or heteroaryl carbaldehyde (4 mmol), glacial acetic acid (0.5 mL) and 2-methoxyethanol (10 mL) was stirred at room temperature for 2 h, followed at reflux for 9 h. After cooling to room temperature and standing overnight, the precipitate was filtered off, washed with methanol (5 × 2 mL), and dried. In this manner the following benzodithiazines were obtained.

6-Chloro-7-cyano-3-[2-hydroxybenzylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (17). Starting from 2-hydroxybenzaldehyde (0.49 g), the title compound 17 was obtained (1.10 g, 90%): mp 314–315 °C dec.; IR (KBr) νmax 2235 (C≡N), 1605 (C=N), 1330, 1160 (SO2) cm−1; 1H-NMR (200 MHz, DMSO-d) δ 3.69 (s, 3H, N-CH3), 6.90–7.00 (m, 2H, Ph), 7.33–7.41 (m, 1H, Ph), 7.84 (d, J = 7.9 Hz, 1H, Ph), 8.41 (s, 1H, H-5, benzodithiazine), 8.61 (s, 1H, H-8, benzodithiazine), 8.61 (s, 1H, N=CH), 10.39 (s, 1H, OH) ppm; anal. C 47.20, H 2.79, N 13.85% calcd for C16H11ClN4O3S2, C 47.23, H 2.72, N 13.77%.

6-Chloro-7-cyano-3-[2-(5-nitrofurfurylidene)-1-methylhydrazino]-1,1-dioxo-1,4,2-benzodithiazine (18). Starting from 5-nitrofuran-2-carbaldehyde (0.56 g), the title compound 18 was obtained (1.15 g, 90%): mp 302–303 °C dec.; IR (KBr) νmax 2235 (C≡N), 1615 (C=N), 1360, 1325, 1165 (SO2) cm−1; 1H-NMR (DMSO-d) δ 3.67 (s, 3H, N-CH3), 7.34 ( d, J = 3.9 Hz, 1H, furan), 7.86 (d, J = 3.9 Hz, 1H, furan), 8.43 (s, 1H, H-5, benzodithiazine), 8.44 (s, 1H, H-8, benzodithiazine), 8.66 (s, 1H, N=CH) ppm; 13C NMR (125 MHz, DMSO-d) δ 33.60, 113.64, 115.18, 118.30, 130.20, 130.41, 131.35, 136.17, 137.85, 139.37, 150.84, 153.09, 164.56 ppm; anal. C 39.53, H 1.91, N 16.55% calcd for C14H8ClN5O5S2, C 39.48, H 1.89, N 16.44%.

3.3. Cell Culture and Cell Viability Assay

All chemicals, if not stated otherwise, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MCF-7 cell line was purchased from Cell Lines Services (Eppelheim, Germany), the HeLa and HCT-116 cell lines were obtained from the Department of Microbiology, Tumor and Cell Biology, Karolinska Institute (Stockholm, Sweden). Cells were cultured in in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C in an incubator (Heraceus, HeraCell).

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay. Cells were seeded in 96-well plates at a density of 5 × 103 cells/well and treated for 72 h with the examined compounds in the concentration range 1–100 μM. Cisplatin was used as a control compound and was examined in the concentration range 0.01–10 μM. Following treatment, MTT (0.5 mg/mL) was added to the medium and cells were further incubated for 2 h at 37 °C. Cells were lysed with DMSO and the absorbance of the formazan solution was measured at 550 nm with a plate reader (Victor, 1420 multilabel counter). The optical density of the formazan solution was measured at 550 nm with a plate reader (Victor, 1420 multilabel counter). The experiment was performed in triplicate. Results are expressed as IC50 values. Values are expressed as the mean ± SD of at least three independent experiments.

3.4. Molecular Modeling Methodology/Calculations

Quantum chemical calculations were carried out to study the molecular geometry and electronic structure of 6-chloro-7-R1-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazines using the Spartan '08 Software [20]. The full optimized geometries of compounds 6‒18 in vacuum were calculated using ab-initio method at the Hartree-Fock (RHF) with 6-31G* polarization basis set.

Multiple linear regression (MLR, along with stepwise algorithm) analysis were performed using Statistica software [21]. The molecular descriptors were used as independent variables. The dependent variable was cytotoxic activity expressed as IC50 values.

In the leave-one-out cross-validation procedure, a data point (compound) was removed from the analyzed set, the regression was recalculated, and then the predicted value for that point was compared to its observed value. This process was repeated until each datum had been omitted once, and then the sum of squares of these errors for the omitted data were used to calculate the cross-validated root-mean-square error (RMSECV). The RMSECV was calculated using following equation: where yi is observed activity, ŷi is predicted activity, N is the sample size of the data set.

4. Conclusions

We have developed facile methods for the synthesis of new 6-chloro-7-R1-3-(2-arylmethylene-1-methylhydrazino)-1,1-dioxo-1,4,2-benzodithiazines from N-methyl-N-(6-chloro-7-R1-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazines and the appropriate aldehydes. The newly synthesized compounds were tested for their in vitro cytotoxic activity against the MCF-7, HCT-116 and HeLa cell lines. The most active compounds 6 and 9 shown the best average cytotoxic activities against all cancer cell lines. Compound 16 exhibited the strongest cytotoxic activity against HeLa cell line with IC50 value of 10 µM, while 14 was the most cytotoxic compound against MCF-7 and HCT-116 cell lines, giving the IC50 values of 15 µM and 16 µM, respectively. Performed QSAR analysis demonstrate the importance of some electronic properties of molecules in the cytotoxic activities against MCF-7 and HCT-116 cell lines. The QSAR equation showed that a low charge on the C13 carbon atom and high energy of HOMO are likely required for potent cytotoxic activity against MCF-7 cell line. On the other hand, QSAR HCT-116 model indicated that low natural charge on the C13 atom and low electrostatic charge on the N10 atom have an impact on the cytotoxic activity of compounds against the HCT-116 cell line. The QSAR results on the cytotoxic activities of the 1,1-dioxo-1,4,2-benzodithiazine analogs will surely provide useful information for the design of new anticancer agents.