http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.922554
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Synthesis and antitumor activity of tetrahydrocarbazole hybridized with dithioate derivatives Hala Bakr El-Nassan Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Abstract
Keywords
The present study reported the synthesis of tetrahydrocarbazoles hybridized with dithioate derivatives. Three series were synthesized namely alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). The synthesized compounds were tested in vitro on human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116). Most of the synthesized compounds exploited potent antitumor activity, especially compound 6f [4-chlorophenylpiperazine derivative], which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 value of 7.24 nM/mL.
Antitumor activity, dithiocarbamates, HCT116, MCF7, tetrahydrocarbazole
Introduction During the past decade, dithiocarbamates have received considerable attention owing to their diverse and excellent biological activities. Thus, this class of compounds has been reported to possess antibacterial1–3, antiviral4,5, anti-inflammatory6, antihistaminic7, antioxidant8 as well as antitumor activity2,8–17. The story of dithiocarbamates started with the discovery of the natural product brassinin (I, Figure 1). Brassinin is a phytoalexin first isolated from cabbage and was reported as antifungal and antiviral agent that displayed potential cancer preventive activity4,5,18. Structural modification of brassinin led to the identification of a series of dithiocarbamate derivatives which proved to exhibit various biological activities. For example, the heterocyclic dithiocarbamate derivative [pyrrolidinedithiocarbamate, PDTC (II)] is a well known antioxidant. PDTC was reported to strongly inhibit the replication of human rhinoviruses and coxsackie virus myocarditis. PDTC also showed inhibitory activity against murine colon adenocarcinoma bearing mice through the inhibition of nuclear factor kB in the tumor tissue19,20. Dithiocarbamates were previously thought to exert their antibacterial and antitumor activities through their reaction with HS-groups of physiologically important enzymes in which the alkyl group of the dithioester was transferred to the HS-function of the enzyme21. Later on, it was found that dialkyl dithiocarbamates and heterocyclic dithiocarbamates are unstable at low pH and they decompose rapidly to produce CS2 and a dialkylamine or cyclic secondary amine. Whilst, heterocyclic dithiocarbamates, e.g. pyrrolidinedithiocarbamate (PDTC), are more stable at serum
Address for correspondence: Hala Bakr El-Nassan, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini street, Cairo 11562, Egypt. Tel: 00202-23632245. Fax: 00202-23635140. E-mail: [email protected]
History Received 31 March 2014 Revised 30 April 2014 Accepted 5 May 2014 Published online 3 June 2014
pH22. The resulting CS2 mediates protein cross-linking23. Besides, the free thiol groups of dithiocarbamates can oxidize glutathione through a glutathione peroxidase-like activity24,25. Recently, the inhibitory activity of dithiocarbamates against EGFR, ErbB-2 kinases9 and carbonic anhydrase10,26,27 was reported and the combination of dithiocarbamates with EGFR9 or antifolates11) was reported to exert synergistic antitumor effect. These after-mentioned trials utilized molecular hybridization approach, in which two or more drug pharmacophores are covalently combined into a single molecule. Molecular hybridization approach represents an effective tool to design novel and highly active drugs since these hybridized molecules can act on multiple targets and thus can overcome drug resistance28,29. Many literature reported the molecular hybridization and anticancer activity of dithiocarbamates with thalidomide8, butenolide12, 1,2,3-triazoles14,15, quinazolines9,11,16,17, chromones13, sulphonamides10 and saccharin derivatives2. However, to the best of our knowledge, their molecular hybridization with tetrahydrocarbazoles to afford anticancer agents have not been reported before. Recently, considerable interest has been focused on tetrahydrocarbazoles which were reported to display broad spectrum of biological activities especially antitumor30–32 activity. Tetrahydrocarbazole ring can be considered as an isostere to b-carboline ring which is present in many naturally occurring anticancer agents, such as the vinca alkaloids, vincamine (III) and (–)-eburnamonine (IV) (Figure 1) isolated from the Madagascar periwinkle (Catharanthus roseus)33–35. Although (–)-eburnamonine (IV) was devoid of anticancer activity even at the high dose of 100 M, its synthetic analogue 15-methylene-eburnamonine (V) displayed in vitro micromolar activity against prostate and myeloma cell lines36. Furthermore, the marine alkaloid Eudistomin K (VI), isolated from the Caribbean ascidian Eudistoma olivaceum, demonstrated potent antitumor activity against L-1210, A-549, HCT-8 and P-388 cell lines37,38. In view of the above data, the present study reported the first synthesis of tetrahydrocarbazoles hybridized with dithioate
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Figure 1. Structures of biologically active dithiocarbamates and carbazoles.
derivatives. Upon designing the dithioate derivatives, three series were chosen, namely alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). By doing this, the effect of the alkyl group substituted on dithioates on the antitumor activity could be determined. The synthesized compounds were evaluated for their possible anticancer activity against MCF7 and HCT116 cell lines.
Results and discussion Chemistry The synthetic route towards the target tetrahydrocarbazoledithioate derivatives is shown in Scheme 1. The starting compound, 2-chloro-1-(1,2,3,4-tetrahydro-9Hcarbazol-9-yl)ethanone (2) was prepared via acylation of 1,2,3,4-tetrahydrocarbazole (1) with chloroacetyl chloride in refluxing toluene according to the procedure reported by Ettel and Myska39. In the present work, two methods were adopted for the synthesis of the target compounds. The first method (method A)
was a two-step method which involved the reaction of the appropriate primary alcohol or secondary amine with carbon disulfide in the presence of potassium hydroxide to give O-alkyl dithiocarbonates (3a–d) and N,N-disubstituted dithiocarbamates (5a–h) following the previously reported procedures1,2,7,40–42. The latter compounds 3 and 5 were reacted with equimolar amount of the chloromethyl derivative 2 at room temperature to afford carbodithioate derivatives (4a–d) and carbamodithioates (6a–g) and (7) in poor to good yields (39–89%). The second method (method B) was a one-step procedure comprised the reaction of the appropriate secondary amine, carbon disulfide and chloromethyl derivative 2 in acetone in the presence of catalytic amount of sodium phosphate. This procedure afforded the same compounds 6a–g and 7 in higher yields and reduced time (4 h) and comprised more efficient use of the chemical reagents. The formation of compounds 4a–d, 6a–g and 7 was confirmed by IR and NMR spectroscopy. The IR spectra revealed the presence of C¼O band at 1678–1701 cm1. In addition, C¼S band appeared at 1215–1267 cm1.
Tetrahydrocarbazole hybridized with dithioates
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DOI: 10.3109/14756366.2014.922554
3
Scheme 1. Synthesis of tetrahydrocarbazoles hybridized with dithioate derivatives.
The 1H-NMR spectra of all the compounds revealed the presence of the carbazole CH2 protons at position 2 and 3 as multiplet signals at d 1.79–1.99 ppm. While the carbazole CH2 protons at position 1 and 4 appeared as triplet signals at d 2.63–2.71 ppm and d 3.03–3.15 ppm, respectively (see Figure 2). Besides, a singlet signal appeared at d 4.65–4.95 ppm corresponding to CH2CO protons. Compounds 6a–g [containing cyclic secondary amine moieties (piperidine, morpholine and piperazine)] and compound 7 [containing diethylamine] revealed the appearance of N-(CH2)2 protons as two signals at d 3.85–4.22 ppm and d 4.05–4.46 ppm. This magnetic non-equivalence might be attributed to the conjugation between the C¼S group and the nitrogen atom, which increased the double bond character of C–N bond sufficiently to restrict the rotation at room temperature (Figure 3). Consequently, one of the CH2 groups is cis to the S atom and the other is trans to the S atom. The anisotropy of C¼S is sufficient to influence the chemical shift of the cis protons which appeared at lower chemical shift than the trans protons. This phenomenon resembles that reported for the protons of the two CH3 groups of DMF43.
On the other hand, 13C-NMR spectra of the synthesized compounds revealed the presence of C¼O carbon at d 166– 167 ppm and C¼S carbon at d 195–213 ppm. Figure 2 shows the 1 H-NMR and 13C-NMR chemical shifts for compound 6b. In vitro anticancer screening All the synthesized derivatives were evaluated for their in vitro cytotoxic activity against two cell lines; human breast cancer cell line, MCF7; and human colon tumor cell line, HCT116, using Sulforhodamine-B stain (SRB) assay44. Doxorubicin, which is one of the most effective anticancer agents was used as the reference drug. The relationship between surviving fraction of MCF7 or HCT116 and drug concentration was plotted and the response parameter IC50 was calculated. IC50 value corresponds to the concentration required for 50% inhibition of cell viability. The IC50 values of the test compounds and the reference drug on MCF7 and HCT116 are shown in Table 1 and the results are represented graphically in Figure 4.
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124.3
1.83-1.96 (m)
7.23-7.30 (m)
1.83-1.96 (m) 7.40 (m)
O
130.4 117.8 135.9
N
21.8 135.0 26.8
166.6
S
S
21.2
115.8
119.1
3.10 (t)
N
23.8
123.4
2.67 (t)
8.15 (m) 7.23-7.30 (m)
O
4.92 (s)
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3.78 (t)
N 4.06 (br s)
51.2
3.78 (t)
66.2
S
CH2R
51.2 66.2
O
O Figure 2. The 1H-NMR and triplet; m: multiplet.
195.6
45.0
N 4.30 (br s)
S
S
13
C-NMR chemical shifts for compound 6b. The data are expressed as d in ppm unit, br s: broad singlet; s: singlet; t:
S
CH2R
cis protons
CH2R
trans protons
7.24 nM/mL. Its cytotoxic activity against HCT116 was comparable to doxorubicin with IC50 value of 8.23 nM/mL.
+
N
N CH2R
Experimental
Figure 3. Effect of conjugation between C¼S and nitrogen atom.
The data presented in Table 1 revealed that most of the synthesized compounds showed potent to moderate antitumor activity. In general, MCF7 cell line was more sensitive to the cytotoxic action of the test compounds than HCT116 cell line. Structurally, the test compounds belonged to three series, namely; alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). Examining the data on MCF7 cell line pointed out that all the test compounds showed moderate to potent cytotoxic activity with IC50 values of 7.24–37.70 nM/mL. The alkyl dithiocarbonates 4a–d displayed more potent antitumor activity than the heterocyclic dithiocarbamates containing piperidine (6a) or morpholine moieties (6b) but were less potent than the heterocyclic dithiocarbamates containing piperazine (6c–g) or the dialkyl dithiocarbamate (7). Regarding the substituted piperazine derivatives 6c–g, it was found that substitution on the piperazine ring enhanced the antitumor activity in the following order: 4-chlorophenyl4C2H5 44-methoxyphenyl4CH3 4phenyl. The dialkyl dithiocarbamate (7) displayed cytotoxic activity comparable to the piperazine analogues. On the other hand, the data obtained on HCT116 cell line revealed that the alkyl dithiocarbonates (4a–d) exhibited lower cytotoxic activity (higher IC50 values) than the heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). Furthermore, the heterocyclic dithiocarbamates (6a–g) were more potent than the dialkyl dithiocarbamate (7). Here also, the heterocyclic dithiocarbamates containing piperazine (6c–g) displayed more potent antitumor activity than the heterocyclic dithiocarbamates containing piperidine (6a) or morpholine moieties (6b). Besides, substitution on the piperazine ring enhanced the antitumor activity against HCT116 in the following order: 4-chlorophenyl 4 CH3 4 C2H5 4 4-methoxyphenyl 4 phenyl. The most potent compound in this study was the 4-chlorophenylpiperazine derivative (6f) which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 value of
General Melting points were determined using a Griffin apparatus and were uncorrected. IR spectra were recorded on Shimadzu IR 435 spectrophotometer and values were represented in cm1. 1HNMR and 13C-NMR spectra were carried out on Bruker 400 MHz and 100 MHz spectrophotometer, respectively, Faculty of Pharmacy, Cairo University, Cairo, Egypt, using TMS as an internal standard and chemical shifts were recorded in ppm on d scale. Elemental analyses were carried out at the regional center for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques. The starting compounds 1,2,3,4-tetrahydrocarbazole (1) and 2-chloro-1-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethanone (2) were synthesized according to the reported procedures39,45. Potassium salts of O-alkyl dithiocarbonates (3a–d)2,41 A mixture of potassium hydroxide (2.25 g, 40 mmol) and the appropriate alcohol (methanol, ethanol, n-butanol and cyclohexanol) (7.5 mL) was refluxed for 1h. The reaction was cooled in an ice bath and carbon disulphide (3 mL, 40 mmol) was added to the mixture drop-wise with stirring. The reaction mixture was stirred at room temperature for 1 h. The solid formed was collected, washed with diethyl ether (2 25 mL) and used without further purification. Potassium salts of N,N-disubstitued dithiocarbamic acids (5a–h)1,2,7,40,42 Potassium hydroxide (0.28 g, 5 mmol) was dissolved in ethanol (20 mL) then the appropriate secondary amine (5 mmol) was added and the mixture was cooled in an ice bath. Carbon disulphide (4 mL, 50 mmol) was added to the mixture drop-wise with stirring. The reaction mixture was stirred for 1 h at room temperature and then left overnight. The solvent was removed under reduced pressure, and the solid formed was collected, washed with diethyl ether (2 25 mL) and used without further purification.
Tetrahydrocarbazole hybridized with dithioates
DOI: 10.3109/14756366.2014.922554
Table 1. Results of in vitro cytotoxic activity of the synthesized compounds on human breast adenocarcinoma cell line (MCF7) and human colon tumor cell line (HCT116).
N S
S
O
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R
Compound No.
R
MCF7
HCT116
Doxorubicin 4a 4b 4c
– –OCH3 –OCH2CH3 –OCH2CH2CH2CH3
8.43 14.10 10.51 26.87 12.14
6.86 4150 4150 66.20 87.08
O
N
29.9
11.63
19.20
9.22
19.28
16.70
45.88
O-Butyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4c)
N CH2CH3
N
6f
7
37.70
Yield: (method A: 75%); m.p.: 85–86 C; IR (cm1): 2926, 2839 (CH aliphatic), 1680 (C¼O), 1244 (C¼S); 1H-NMR (CDCl3) d ppm 1.40 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.83–1.96 (m, 4H, carbazole CH2), 2.66 (t, 2H, carbazole CH2), 3.03 (t, 2H, carbazole CH2), 4.62 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 4.67 (s, 2H, CH2CO), 7.24–8.11 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 13.7 (CH3CH2), 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 43.8 (CH2CO), 70.8 (CH3CH2), 115.6, 117.9, 119.2, 123.5, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.0 (C¼O), 213.2 (C¼S); Anal. Calcd. for C17H19NO2S2: C, 61.23; H, 5.74; N, 4.20. Found: C, 61.41; H, 5.78; N, 4.28.
N CH3
N
6e
6g
38.17
O
N
6d
N
N
N
Yield: (method A: 74%); m.p.: 117–118 C; IR (cm1): 2939, 2839 (CH aliphatic), 1685 (C¼O), 1228 (C¼S); 1H-NMR (CDCl3) d ppm 1.83–1.95 (m, 4H, carbazole CH2), 2.65 (m, 2H, carbazole CH2), 3.03 (m, 2H, carbazole CH2), 4.19 (s, 3H, OCH3), 4.68 (s, 2H, CH2CO), 7.25–8.10 (m, 4H, Ar-H); Anal. Calcd for C16H17NO2S2: C, 60.16; H, 5.36; N, 4.38. Found: C, 60.22; H, 5.34; N, 4.47.
21.77 N
6c
A mixture of the appropriate amine (1 mmol) and Na3PO4 12H2O (0.23 g, 0.6 mmol) in acetone (10 mL) was stirred at room temperature for 30 min. Carbon disulfide (0.3 mL, 5 mmol) was added drop-wise and the reaction mixture was stirred at room temperature for 30 min. Then, compound 2 (0.25 g, 1 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was poured onto water (100 mL) and the solid product was filtered, dried and crystallized from ethyl acetate.
O-Ethyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4b)
6a
6b
Method B
O-Methyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4a) IC50 in nM/mL*
4d
5
N
N
7.24
8.23
9.39
26.10
10.28
44.72
Cl
OCH3
N
*The given values are means of three experiments.
General procedures for the synthesis of dithioate derivatives 4a–d, 6a–g and 7 Method A A mixture of 2-chloro-1-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethanone (2) (0.25 g, 1 mmol) and the appropriate potassium salts of O-alkyl dithiocarbonates (3a–d) or N,N-disubstituted dithiocarbamic acids (5a–h) (1 mmol) in absolute ethanol (10 mL) was boiled till all the solid dissolved and then the reaction mixture was left overnight at room temperature. The resulting solid was filtered, washed with water (50 mL) and crystallized from ethyl acetate.
Yield: (method A: 39%); m.p.: 86–87 C; IR (cm1): 2937, 2862 (CH aliphatic), 1685 (C¼O), 1215 (C¼S); 1H-NMR (CDCl3) d ppm 0.95 (t, 3H, CH3CH2, J ¼ 7.4 Hz), 1.39 (sextet, 2H, CH3CH2, J ¼ 7.4 Hz), 1.74 (quintet, 2H, CH3CH2CH2, J ¼ 7.4 Hz), 1.88– 1.99 (m, 4H, carbazole CH2), 2.70 (s, 2H, carbazole CH2), 3.06 (s, 2H, carbazole CH2), 4.61 (t, 2H, CH2CH2O, J ¼ 7.4 Hz), 4.67 (s, 2H, CH2CO), 7.26–8.13 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 13.6 (CH3CH2), 19.1 (CH3CH2), 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 30.3 (CH3CH2CH2), 43.7 (CH2CO), 74.7 (CH2O), 115.6, 117.9, 119.2, 123.5, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.0 (C¼O), 213.2 (C¼S); Anal. Calcd. for C19H23NO2S2: C, 63.12; H, 6.41; N, 3.87. Found: C, 63.21; H, 6.47; N, 3.98. O-Cyclohexyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol9-yl)ethyl] carbonodithioate (4d) Yield: (method A: 46%); m.p.: 106–107 C; IR (cm1): 2935, 2856 (CH aliphatic), 1701 (C¼O), 1224 (C¼S); 1H-NMR (CDCl3) d ppm 1.27–2.01 (m, 14H, carbazole CH2 and cyclohexyl CH2), 2.71 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.06 (s, 2H, carbazole CH2, J ¼ 6 Hz), 4.65 (s, 2H, CH2CO), 5.52 (quintet, 1H, cyclohexyl CH), 7.26–8.13 (m, 4H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) d ppm 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 23.5, 25.2, 30.8 (CH2CH2CH2 of cyclohexyl), 43.4 (CH2CO), 83.4 (CHO), 115.6, 117.9, 119.2, 123.4, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.1 (C¼O),
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100 90 80 70 60 MCF7
50
HCT116
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10 0 Dox
4a
4b
4c
4d
6a
6b
6c
6d
6e
6f
6g
7
Figure 4. IC50 in nM/mL of compounds 4–7 and doxorubicin against MCF7 and HCT116 cell lines.
212.1 (C¼S); Anal. Calcd. for C21H25NO2S2: C, 65.08; H, 6.50; N, 3.61. Found: C, 65.19; H, 6.48; N, 3.78. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl piperidine-1-carbodithioate (6a) Yield: (method A: 76%; method B: 81%); m.p.: 166–167 C; IR (cm1): 2931, 2848 (CH aliphatic), 1695 (C¼O), 1230 (C¼S); 1 H-NMR (CDCl3) d ppm 1.59–1.65 (m, 6H, piperidine CH2), 1.79–1.87 (m, 4H, carbazole CH2), 2.63 (s, 2H, carbazole CH2), 3.06 (s, 2H, carbazole CH2), 3.97 (s, 2H, piperidine CH2), 4.14 (s, 2H, piperidine CH2), 4.93 (s, 2H, CH2CO), 7.22–8.14 (m, 4H, ArH); Anal. Calcd. for C20H24N2OS2: C, 64.48; H, 6.49; N, 7.52. Found: C, 64.56; H, 6.57; N, 7.68. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl morpholine-4-carbodithioate (6b) Yield: (method A: 82%; method B: 86%); m.p.: 200–201 C; IR (cm1): 2929, 2858 (CH aliphatic), 1681 (C¼O), 1224 (C¼S); 1 H-NMR (CDCl3) d ppm 1.83–1.96 (m, 4H, carbazole CH2), 2.67 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.10 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.78 (t, 4H, morpholine CH2O, J ¼ 4.8 Hz), 4.06 (br s, 2H, morpholine CH2N), 4.30 (br s, 2H, morpholine CH2N), 4.92 (s, 2H, CH2CO), 7.23-8.15 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.0 (CH2CO), 51.2 (morpholine CH2N), 66.2 (morpholine CH2O), 115.8, 117.8, 119.1, 123.4, 124.3, 130.4, 135.0, 135.9 (aromatic carbons), 166.6 (C¼O), 195.6 (C¼S); Anal. Calcd. for C19H22N2O2S2: C, 60.93; H, 5.92; N, 7.48. Found: C, 61.08; H, 5.97; N, 7.62. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-methylpiperazine-1-carbodithioate (6c) Yield: (method A: 64%; method B: 80%); m.p.: 162–163 C; IR (cm1): 2933, 2846 (CH aliphatic), 1681 (C¼O), 1232 (C¼S); 1 H-NMR (CDCl3) d ppm 1.81–1.95 (m, 4H, carbazole CH2), 2.3 (s, 3H, CH3), 2.51 (t, 4H, piperazine CH2), 2.65 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.08 (t, 2H, carbazole CH2, J ¼ 6 Hz), 4.02 (br s, 2H, piperazine CH2), 4.33 (br s, 2H, piperazine CH2), 4.89 (s, 2H, CH2CO), 7.22–8.16 (m, 4H, Ar-H); Anal. Calcd. for C20H25N3OS2: C, 61.98; H, 6.50; N, 10.84. Found: C, 62.12; H, 6.48; N, 10.92. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-ethylpiperazine-1-carbodithioate (6d) Yield: (method A: 64%; method B: 82%); m.p.: 167–168 C; IR (cm1): 2935, 2843 (CH aliphatic), 1681 (C¼O), 1234 (C¼S);
1
H-NMR (CDCl3) d ppm 1.14 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.87– 1.97 (m, 4H, carbazole CH2), 2.48 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 2.59 (t, 4H, piperazine CH2), 2.70 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.14 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 4.07 (br s, 2H, piperazine CH2), 4.37 (br s, 2H, piperazine CH2), 4.95 (s, 2H, CH2CO), 7.26–8.20 (m, 4H, Ar-H); Anal. Calcd. for C21H27N3OS2: C, 62.81; H, 6.78; N, 10.46. Found: C, 62.89; H, 6.82; N, 10.61. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-phenylpiperazine-1-carbodithioate (6e) Yield: (method A: 88%; method B: 88%); m.p.: 132–133 C; IR (cm1): 2927, 2852 (CH aliphatic), 1678 (C¼O), 1226 (C¼S); 1 H-NMR (CDCl3) d ppm 1.85–1.95 (m, 4H, carbazole CH2), 2.66 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.10 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.32 (t, 4H, piperazine CH2), 4.18 (br s, 2H, piperazine CH2), 4.46 (br s, 2H, piperazine CH2), 4.94 (s, 2H, CH2CO), 6.908.16 (m, 9H, Ar-H); Anal. Calcd. for C25H27N3OS2: C, 66.78; H, 6.05; N, 9.35. Found: C, 66.93; H, 6.13; N, 9.52. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-(4-chlorophenyl)piperazine-1-carbodithioate (6f) Yield: (method A: 74%; method B: 86%); m.p.: 164–165 C; IR (cm1): 2924, 2835 (CH aliphatic), 1685 (C¼O), 1228 (C¼S); 1 H-NMR (CDCl3) d ppm 1.83–1.97 (m, 4H, carbazole CH2), 2.68 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.11 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.30 (t, 4H, piperazine), 4.22 (br s, 2H, piperazine), 4.45 (br s, 2H, piperazine), 4.94 (s, 2H, CH2CO), 6.82–8.18 (m, 8H, Ar-H); Anal. Calcd. for C25H26ClN3OS2: C, 62.03; H, 5.41; N, 8.68. Found: C, 62.17; H, 5.46; N, 8.73. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-(4-methoxyphenyl)piperazine-1-carbodithioate (6g) Yield: (method A: 85%; method B: 86%); m.p.: 156–157 C; IR (cm1): 2935, 2833 (CH aliphatic), 1697 (C¼O), 1224 (C¼S); 1 H-NMR (CDCl3) d ppm 1.86–1.96 (m, 4H, carbazole CH2), 2.65 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.09 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.16 (t, 4H, piperazine, J ¼ 5.2 Hz), 3.77 (s, 3H, CH3O), 4.18 (br s, 2H, piperazine), 4.46 (br s, 2H, piperazine), 4.92 (s, 2H, CH2CO), 6.84–8.18 (m, 8H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.2 (CH2CO), 50.4, 51.6 (piperazine carbons), 55.5 (CH3O), 114.6, 115.8, 117.8, 118.9, 119.1, 123.4, 124.3, 130.4, 135.0, 135.9, 144.5, 154.5 (aromatic carbons), 166.7
Tetrahydrocarbazole hybridized with dithioates
DOI: 10.3109/14756366.2014.922554
(C¼O), 195.1 (C¼S); Anal. Calcd. for C26H29N3O2S2: C, 65.10; H, 6.09; N, 8.76. Found: C, 65.23; H, 6.13; N, 8.92.
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2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl diethyl carbamodithioate (7) Yield: (method A: 54%; method B: 90%); m.p.: 97–98 C; IR (cm1): 2929, 2850 (CH aliphatic), 1683 (C¼O), 1267 (C¼S); 1 H-NMR (CDCl3) d ppm 1.25 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.38 (t, 3H, CH3CH2, J ¼ 6.8 Hz), 1.85–1.99 (m, 4H, carbazole CH2), 2.70 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.15 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.85 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 4.05 (q, 2H, CH3CH2, J ¼ 6.8 Hz), 4.95 (s, 2H, CH2CO), 7.26–8.22 (m, 4H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) d ppm 11.5, 12.6 (CH3CH2N), 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.5 (CH2CO), 47.2, 50.0 (CH3CH2N), 115.9, 117.8, 119.0, 123.3, 124.2, 130.4, 135.1, 135.9, (aromatic carbons), 167.0 (C¼O), 193.7 (C¼S); Anal. Calcd. for C19H24N2OS2: C, 63.30; H, 6.71; N, 7.77. Found: C, 63.39; H, 6.77; N, 7.94.
7
dithiocarbamates (6c–g) [piperazine analogues] was comparable to dialkyl dithiocarbamate (7). Besides, the alkyl dithiocarbonates (4a–d) displayed more potent antitumor activity than the heterocyclic dithiocarbamates (6a,b) [piperidine and morpholine analouges]. Whilst, SAR study of the test compounds on HCT116 cell line indicated that heterocyclic dithiocarbamates (6a–g) afforded more potent cytotoxic agents than alkyl dithiocarbonate (4a–d) or dialkyl dithiocarbamate (7). On both cell lines, heterocyclic dithiocarbamates bearing piperazine moiety afforded more potent cytotoxic agents than piperidine or morpholine analogues. Within the piperazine derivatives, the antitumor activity was affected by the N-substituent in the following order: 4-chlorophenyl 4 alkyl 4 4-methoxyphenyl 4 phenyl. Further work need to be done to determine the effect of the linker between tetrahydrocarbazole and dithioate on the anticancer activity and also to determine the mechanism inherited behind the antitumor effect of these compounds.
Biological testing
Acknowledgements
Materials and methods
The author is grateful to all members of the department of Cancer Biology, National Cancer Institute, Cairo, Egypt, for carrying out the cytotoxicity testing.
The human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116) were obtained as a gift from NCI, Bethesda, MD. All chemicals and solvents were purchased from SigmaAldrich (Munich, Germany). Measurement of potential cytotoxicity The cytotoxic activity of the newly synthesized compounds was measured in vitro on human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116) using Sulforhodamine-B stain (SRB) assay applying the method described by Skehan et al.44. Cells were plated in a 96-multiwell plate (104 cells/well) for 24 h before treatment with the test compounds to allow attachment of the cells to the wall of the plate. Test compounds were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the test compounds (5, 12.5, 25 and 50 mg/mL) were added to the cell monolayer. Triplicate wells prepared for each individual dose. Monolayer cells were incubated with the test compound for 48 h at 37 C in atmosphere of 5% CO2. After 48 h, cells were fixed with trichloroacetic acid, washed with water and stained for 30 min with 0.4% (wt/vol) SRB stain dissolved with 1% acetic acid. Excess stain was removed by four washes with 1% acetic acid and attached stain was recovered with Tris EDTA buffer. The color intensity was measured in a ELISA reader. The relation between surviving fraction and compound concentration was plotted and IC50 [the concentration required for 50% inhibition of cell viability] was calculated for each compound and results are given in Table 1 and represented graphically in Figure 4.
Conclusion The objective of the present study was to synthesize new tetrahydrocarbazoles hybridized with dithioate derivatives and to study the effect of the alkyl group of the dithioate moiety on the antitumor activity. Most of the synthesized compounds exploited potent antitumor activity, especially compound 6f [4-chlorophenylpiperazine derivative] which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 7.24 nM/mL. MCF7 cell line was more susceptible to the action of the test compounds than HCT116 cell line. SAR study of the test compounds on MCF7 cell line pointed out that the antitumor activity of the heterocyclic
Declaration of interest The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.
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Synthesis and antitumor activity of tetrahydrocarbazole hybridized with dithioate derivatives Hala Bakr El-Nassan Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Abstract
Keywords
The present study reported the synthesis of tetrahydrocarbazoles hybridized with dithioate derivatives. Three series were synthesized namely alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). The synthesized compounds were tested in vitro on human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116). Most of the synthesized compounds exploited potent antitumor activity, especially compound 6f [4-chlorophenylpiperazine derivative], which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 value of 7.24 nM/mL.
Antitumor activity, dithiocarbamates, HCT116, MCF7, tetrahydrocarbazole
Introduction During the past decade, dithiocarbamates have received considerable attention owing to their diverse and excellent biological activities. Thus, this class of compounds has been reported to possess antibacterial1–3, antiviral4,5, anti-inflammatory6, antihistaminic7, antioxidant8 as well as antitumor activity2,8–17. The story of dithiocarbamates started with the discovery of the natural product brassinin (I, Figure 1). Brassinin is a phytoalexin first isolated from cabbage and was reported as antifungal and antiviral agent that displayed potential cancer preventive activity4,5,18. Structural modification of brassinin led to the identification of a series of dithiocarbamate derivatives which proved to exhibit various biological activities. For example, the heterocyclic dithiocarbamate derivative [pyrrolidinedithiocarbamate, PDTC (II)] is a well known antioxidant. PDTC was reported to strongly inhibit the replication of human rhinoviruses and coxsackie virus myocarditis. PDTC also showed inhibitory activity against murine colon adenocarcinoma bearing mice through the inhibition of nuclear factor kB in the tumor tissue19,20. Dithiocarbamates were previously thought to exert their antibacterial and antitumor activities through their reaction with HS-groups of physiologically important enzymes in which the alkyl group of the dithioester was transferred to the HS-function of the enzyme21. Later on, it was found that dialkyl dithiocarbamates and heterocyclic dithiocarbamates are unstable at low pH and they decompose rapidly to produce CS2 and a dialkylamine or cyclic secondary amine. Whilst, heterocyclic dithiocarbamates, e.g. pyrrolidinedithiocarbamate (PDTC), are more stable at serum
Address for correspondence: Hala Bakr El-Nassan, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini street, Cairo 11562, Egypt. Tel: 00202-23632245. Fax: 00202-23635140. E-mail: [email protected]
History Received 31 March 2014 Revised 30 April 2014 Accepted 5 May 2014 Published online 3 June 2014
pH22. The resulting CS2 mediates protein cross-linking23. Besides, the free thiol groups of dithiocarbamates can oxidize glutathione through a glutathione peroxidase-like activity24,25. Recently, the inhibitory activity of dithiocarbamates against EGFR, ErbB-2 kinases9 and carbonic anhydrase10,26,27 was reported and the combination of dithiocarbamates with EGFR9 or antifolates11) was reported to exert synergistic antitumor effect. These after-mentioned trials utilized molecular hybridization approach, in which two or more drug pharmacophores are covalently combined into a single molecule. Molecular hybridization approach represents an effective tool to design novel and highly active drugs since these hybridized molecules can act on multiple targets and thus can overcome drug resistance28,29. Many literature reported the molecular hybridization and anticancer activity of dithiocarbamates with thalidomide8, butenolide12, 1,2,3-triazoles14,15, quinazolines9,11,16,17, chromones13, sulphonamides10 and saccharin derivatives2. However, to the best of our knowledge, their molecular hybridization with tetrahydrocarbazoles to afford anticancer agents have not been reported before. Recently, considerable interest has been focused on tetrahydrocarbazoles which were reported to display broad spectrum of biological activities especially antitumor30–32 activity. Tetrahydrocarbazole ring can be considered as an isostere to b-carboline ring which is present in many naturally occurring anticancer agents, such as the vinca alkaloids, vincamine (III) and (–)-eburnamonine (IV) (Figure 1) isolated from the Madagascar periwinkle (Catharanthus roseus)33–35. Although (–)-eburnamonine (IV) was devoid of anticancer activity even at the high dose of 100 M, its synthetic analogue 15-methylene-eburnamonine (V) displayed in vitro micromolar activity against prostate and myeloma cell lines36. Furthermore, the marine alkaloid Eudistomin K (VI), isolated from the Caribbean ascidian Eudistoma olivaceum, demonstrated potent antitumor activity against L-1210, A-549, HCT-8 and P-388 cell lines37,38. In view of the above data, the present study reported the first synthesis of tetrahydrocarbazoles hybridized with dithioate
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Figure 1. Structures of biologically active dithiocarbamates and carbazoles.
derivatives. Upon designing the dithioate derivatives, three series were chosen, namely alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). By doing this, the effect of the alkyl group substituted on dithioates on the antitumor activity could be determined. The synthesized compounds were evaluated for their possible anticancer activity against MCF7 and HCT116 cell lines.
Results and discussion Chemistry The synthetic route towards the target tetrahydrocarbazoledithioate derivatives is shown in Scheme 1. The starting compound, 2-chloro-1-(1,2,3,4-tetrahydro-9Hcarbazol-9-yl)ethanone (2) was prepared via acylation of 1,2,3,4-tetrahydrocarbazole (1) with chloroacetyl chloride in refluxing toluene according to the procedure reported by Ettel and Myska39. In the present work, two methods were adopted for the synthesis of the target compounds. The first method (method A)
was a two-step method which involved the reaction of the appropriate primary alcohol or secondary amine with carbon disulfide in the presence of potassium hydroxide to give O-alkyl dithiocarbonates (3a–d) and N,N-disubstituted dithiocarbamates (5a–h) following the previously reported procedures1,2,7,40–42. The latter compounds 3 and 5 were reacted with equimolar amount of the chloromethyl derivative 2 at room temperature to afford carbodithioate derivatives (4a–d) and carbamodithioates (6a–g) and (7) in poor to good yields (39–89%). The second method (method B) was a one-step procedure comprised the reaction of the appropriate secondary amine, carbon disulfide and chloromethyl derivative 2 in acetone in the presence of catalytic amount of sodium phosphate. This procedure afforded the same compounds 6a–g and 7 in higher yields and reduced time (4 h) and comprised more efficient use of the chemical reagents. The formation of compounds 4a–d, 6a–g and 7 was confirmed by IR and NMR spectroscopy. The IR spectra revealed the presence of C¼O band at 1678–1701 cm1. In addition, C¼S band appeared at 1215–1267 cm1.
Tetrahydrocarbazole hybridized with dithioates
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DOI: 10.3109/14756366.2014.922554
3
Scheme 1. Synthesis of tetrahydrocarbazoles hybridized with dithioate derivatives.
The 1H-NMR spectra of all the compounds revealed the presence of the carbazole CH2 protons at position 2 and 3 as multiplet signals at d 1.79–1.99 ppm. While the carbazole CH2 protons at position 1 and 4 appeared as triplet signals at d 2.63–2.71 ppm and d 3.03–3.15 ppm, respectively (see Figure 2). Besides, a singlet signal appeared at d 4.65–4.95 ppm corresponding to CH2CO protons. Compounds 6a–g [containing cyclic secondary amine moieties (piperidine, morpholine and piperazine)] and compound 7 [containing diethylamine] revealed the appearance of N-(CH2)2 protons as two signals at d 3.85–4.22 ppm and d 4.05–4.46 ppm. This magnetic non-equivalence might be attributed to the conjugation between the C¼S group and the nitrogen atom, which increased the double bond character of C–N bond sufficiently to restrict the rotation at room temperature (Figure 3). Consequently, one of the CH2 groups is cis to the S atom and the other is trans to the S atom. The anisotropy of C¼S is sufficient to influence the chemical shift of the cis protons which appeared at lower chemical shift than the trans protons. This phenomenon resembles that reported for the protons of the two CH3 groups of DMF43.
On the other hand, 13C-NMR spectra of the synthesized compounds revealed the presence of C¼O carbon at d 166– 167 ppm and C¼S carbon at d 195–213 ppm. Figure 2 shows the 1 H-NMR and 13C-NMR chemical shifts for compound 6b. In vitro anticancer screening All the synthesized derivatives were evaluated for their in vitro cytotoxic activity against two cell lines; human breast cancer cell line, MCF7; and human colon tumor cell line, HCT116, using Sulforhodamine-B stain (SRB) assay44. Doxorubicin, which is one of the most effective anticancer agents was used as the reference drug. The relationship between surviving fraction of MCF7 or HCT116 and drug concentration was plotted and the response parameter IC50 was calculated. IC50 value corresponds to the concentration required for 50% inhibition of cell viability. The IC50 values of the test compounds and the reference drug on MCF7 and HCT116 are shown in Table 1 and the results are represented graphically in Figure 4.
4
H. B. El-Nassan
J Enzyme Inhib Med Chem, Early Online: 1–8
124.3
1.83-1.96 (m)
7.23-7.30 (m)
1.83-1.96 (m) 7.40 (m)
O
130.4 117.8 135.9
N
21.8 135.0 26.8
166.6
S
S
21.2
115.8
119.1
3.10 (t)
N
23.8
123.4
2.67 (t)
8.15 (m) 7.23-7.30 (m)
O
4.92 (s)
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3.78 (t)
N 4.06 (br s)
51.2
3.78 (t)
66.2
S
CH2R
51.2 66.2
O
O Figure 2. The 1H-NMR and triplet; m: multiplet.
195.6
45.0
N 4.30 (br s)
S
S
13
C-NMR chemical shifts for compound 6b. The data are expressed as d in ppm unit, br s: broad singlet; s: singlet; t:
S
CH2R
cis protons
CH2R
trans protons
7.24 nM/mL. Its cytotoxic activity against HCT116 was comparable to doxorubicin with IC50 value of 8.23 nM/mL.
+
N
N CH2R
Experimental
Figure 3. Effect of conjugation between C¼S and nitrogen atom.
The data presented in Table 1 revealed that most of the synthesized compounds showed potent to moderate antitumor activity. In general, MCF7 cell line was more sensitive to the cytotoxic action of the test compounds than HCT116 cell line. Structurally, the test compounds belonged to three series, namely; alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). Examining the data on MCF7 cell line pointed out that all the test compounds showed moderate to potent cytotoxic activity with IC50 values of 7.24–37.70 nM/mL. The alkyl dithiocarbonates 4a–d displayed more potent antitumor activity than the heterocyclic dithiocarbamates containing piperidine (6a) or morpholine moieties (6b) but were less potent than the heterocyclic dithiocarbamates containing piperazine (6c–g) or the dialkyl dithiocarbamate (7). Regarding the substituted piperazine derivatives 6c–g, it was found that substitution on the piperazine ring enhanced the antitumor activity in the following order: 4-chlorophenyl4C2H5 44-methoxyphenyl4CH3 4phenyl. The dialkyl dithiocarbamate (7) displayed cytotoxic activity comparable to the piperazine analogues. On the other hand, the data obtained on HCT116 cell line revealed that the alkyl dithiocarbonates (4a–d) exhibited lower cytotoxic activity (higher IC50 values) than the heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). Furthermore, the heterocyclic dithiocarbamates (6a–g) were more potent than the dialkyl dithiocarbamate (7). Here also, the heterocyclic dithiocarbamates containing piperazine (6c–g) displayed more potent antitumor activity than the heterocyclic dithiocarbamates containing piperidine (6a) or morpholine moieties (6b). Besides, substitution on the piperazine ring enhanced the antitumor activity against HCT116 in the following order: 4-chlorophenyl 4 CH3 4 C2H5 4 4-methoxyphenyl 4 phenyl. The most potent compound in this study was the 4-chlorophenylpiperazine derivative (6f) which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 value of
General Melting points were determined using a Griffin apparatus and were uncorrected. IR spectra were recorded on Shimadzu IR 435 spectrophotometer and values were represented in cm1. 1HNMR and 13C-NMR spectra were carried out on Bruker 400 MHz and 100 MHz spectrophotometer, respectively, Faculty of Pharmacy, Cairo University, Cairo, Egypt, using TMS as an internal standard and chemical shifts were recorded in ppm on d scale. Elemental analyses were carried out at the regional center for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques. The starting compounds 1,2,3,4-tetrahydrocarbazole (1) and 2-chloro-1-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethanone (2) were synthesized according to the reported procedures39,45. Potassium salts of O-alkyl dithiocarbonates (3a–d)2,41 A mixture of potassium hydroxide (2.25 g, 40 mmol) and the appropriate alcohol (methanol, ethanol, n-butanol and cyclohexanol) (7.5 mL) was refluxed for 1h. The reaction was cooled in an ice bath and carbon disulphide (3 mL, 40 mmol) was added to the mixture drop-wise with stirring. The reaction mixture was stirred at room temperature for 1 h. The solid formed was collected, washed with diethyl ether (2 25 mL) and used without further purification. Potassium salts of N,N-disubstitued dithiocarbamic acids (5a–h)1,2,7,40,42 Potassium hydroxide (0.28 g, 5 mmol) was dissolved in ethanol (20 mL) then the appropriate secondary amine (5 mmol) was added and the mixture was cooled in an ice bath. Carbon disulphide (4 mL, 50 mmol) was added to the mixture drop-wise with stirring. The reaction mixture was stirred for 1 h at room temperature and then left overnight. The solvent was removed under reduced pressure, and the solid formed was collected, washed with diethyl ether (2 25 mL) and used without further purification.
Tetrahydrocarbazole hybridized with dithioates
DOI: 10.3109/14756366.2014.922554
Table 1. Results of in vitro cytotoxic activity of the synthesized compounds on human breast adenocarcinoma cell line (MCF7) and human colon tumor cell line (HCT116).
N S
S
O
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R
Compound No.
R
MCF7
HCT116
Doxorubicin 4a 4b 4c
– –OCH3 –OCH2CH3 –OCH2CH2CH2CH3
8.43 14.10 10.51 26.87 12.14
6.86 4150 4150 66.20 87.08
O
N
29.9
11.63
19.20
9.22
19.28
16.70
45.88
O-Butyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4c)
N CH2CH3
N
6f
7
37.70
Yield: (method A: 75%); m.p.: 85–86 C; IR (cm1): 2926, 2839 (CH aliphatic), 1680 (C¼O), 1244 (C¼S); 1H-NMR (CDCl3) d ppm 1.40 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.83–1.96 (m, 4H, carbazole CH2), 2.66 (t, 2H, carbazole CH2), 3.03 (t, 2H, carbazole CH2), 4.62 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 4.67 (s, 2H, CH2CO), 7.24–8.11 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 13.7 (CH3CH2), 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 43.8 (CH2CO), 70.8 (CH3CH2), 115.6, 117.9, 119.2, 123.5, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.0 (C¼O), 213.2 (C¼S); Anal. Calcd. for C17H19NO2S2: C, 61.23; H, 5.74; N, 4.20. Found: C, 61.41; H, 5.78; N, 4.28.
N CH3
N
6e
6g
38.17
O
N
6d
N
N
N
Yield: (method A: 74%); m.p.: 117–118 C; IR (cm1): 2939, 2839 (CH aliphatic), 1685 (C¼O), 1228 (C¼S); 1H-NMR (CDCl3) d ppm 1.83–1.95 (m, 4H, carbazole CH2), 2.65 (m, 2H, carbazole CH2), 3.03 (m, 2H, carbazole CH2), 4.19 (s, 3H, OCH3), 4.68 (s, 2H, CH2CO), 7.25–8.10 (m, 4H, Ar-H); Anal. Calcd for C16H17NO2S2: C, 60.16; H, 5.36; N, 4.38. Found: C, 60.22; H, 5.34; N, 4.47.
21.77 N
6c
A mixture of the appropriate amine (1 mmol) and Na3PO4 12H2O (0.23 g, 0.6 mmol) in acetone (10 mL) was stirred at room temperature for 30 min. Carbon disulfide (0.3 mL, 5 mmol) was added drop-wise and the reaction mixture was stirred at room temperature for 30 min. Then, compound 2 (0.25 g, 1 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was poured onto water (100 mL) and the solid product was filtered, dried and crystallized from ethyl acetate.
O-Ethyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4b)
6a
6b
Method B
O-Methyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethyl] carbonodithioate (4a) IC50 in nM/mL*
4d
5
N
N
7.24
8.23
9.39
26.10
10.28
44.72
Cl
OCH3
N
*The given values are means of three experiments.
General procedures for the synthesis of dithioate derivatives 4a–d, 6a–g and 7 Method A A mixture of 2-chloro-1-(1,2,3,4-tetrahydro-9H-carbazol-9yl)ethanone (2) (0.25 g, 1 mmol) and the appropriate potassium salts of O-alkyl dithiocarbonates (3a–d) or N,N-disubstituted dithiocarbamic acids (5a–h) (1 mmol) in absolute ethanol (10 mL) was boiled till all the solid dissolved and then the reaction mixture was left overnight at room temperature. The resulting solid was filtered, washed with water (50 mL) and crystallized from ethyl acetate.
Yield: (method A: 39%); m.p.: 86–87 C; IR (cm1): 2937, 2862 (CH aliphatic), 1685 (C¼O), 1215 (C¼S); 1H-NMR (CDCl3) d ppm 0.95 (t, 3H, CH3CH2, J ¼ 7.4 Hz), 1.39 (sextet, 2H, CH3CH2, J ¼ 7.4 Hz), 1.74 (quintet, 2H, CH3CH2CH2, J ¼ 7.4 Hz), 1.88– 1.99 (m, 4H, carbazole CH2), 2.70 (s, 2H, carbazole CH2), 3.06 (s, 2H, carbazole CH2), 4.61 (t, 2H, CH2CH2O, J ¼ 7.4 Hz), 4.67 (s, 2H, CH2CO), 7.26–8.13 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 13.6 (CH3CH2), 19.1 (CH3CH2), 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 30.3 (CH3CH2CH2), 43.7 (CH2CO), 74.7 (CH2O), 115.6, 117.9, 119.2, 123.5, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.0 (C¼O), 213.2 (C¼S); Anal. Calcd. for C19H23NO2S2: C, 63.12; H, 6.41; N, 3.87. Found: C, 63.21; H, 6.47; N, 3.98. O-Cyclohexyl S-[2-oxo-2-(1,2,3,4-tetrahydro-9H-carbazol9-yl)ethyl] carbonodithioate (4d) Yield: (method A: 46%); m.p.: 106–107 C; IR (cm1): 2935, 2856 (CH aliphatic), 1701 (C¼O), 1224 (C¼S); 1H-NMR (CDCl3) d ppm 1.27–2.01 (m, 14H, carbazole CH2 and cyclohexyl CH2), 2.71 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.06 (s, 2H, carbazole CH2, J ¼ 6 Hz), 4.65 (s, 2H, CH2CO), 5.52 (quintet, 1H, cyclohexyl CH), 7.26–8.13 (m, 4H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) d ppm 21.1, 21.8, 23.8, 26.7 (carbazole C-3, C-2, C-4, C-1), 23.5, 25.2, 30.8 (CH2CH2CH2 of cyclohexyl), 43.4 (CH2CO), 83.4 (CHO), 115.6, 117.9, 119.2, 123.4, 124.3, 130.4, 134.8, 135.8 (aromatic carbons), 166.1 (C¼O),
6
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J Enzyme Inhib Med Chem, Early Online: 1–8
100 90 80 70 60 MCF7
50
HCT116
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10 0 Dox
4a
4b
4c
4d
6a
6b
6c
6d
6e
6f
6g
7
Figure 4. IC50 in nM/mL of compounds 4–7 and doxorubicin against MCF7 and HCT116 cell lines.
212.1 (C¼S); Anal. Calcd. for C21H25NO2S2: C, 65.08; H, 6.50; N, 3.61. Found: C, 65.19; H, 6.48; N, 3.78. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl piperidine-1-carbodithioate (6a) Yield: (method A: 76%; method B: 81%); m.p.: 166–167 C; IR (cm1): 2931, 2848 (CH aliphatic), 1695 (C¼O), 1230 (C¼S); 1 H-NMR (CDCl3) d ppm 1.59–1.65 (m, 6H, piperidine CH2), 1.79–1.87 (m, 4H, carbazole CH2), 2.63 (s, 2H, carbazole CH2), 3.06 (s, 2H, carbazole CH2), 3.97 (s, 2H, piperidine CH2), 4.14 (s, 2H, piperidine CH2), 4.93 (s, 2H, CH2CO), 7.22–8.14 (m, 4H, ArH); Anal. Calcd. for C20H24N2OS2: C, 64.48; H, 6.49; N, 7.52. Found: C, 64.56; H, 6.57; N, 7.68. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl morpholine-4-carbodithioate (6b) Yield: (method A: 82%; method B: 86%); m.p.: 200–201 C; IR (cm1): 2929, 2858 (CH aliphatic), 1681 (C¼O), 1224 (C¼S); 1 H-NMR (CDCl3) d ppm 1.83–1.96 (m, 4H, carbazole CH2), 2.67 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.10 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.78 (t, 4H, morpholine CH2O, J ¼ 4.8 Hz), 4.06 (br s, 2H, morpholine CH2N), 4.30 (br s, 2H, morpholine CH2N), 4.92 (s, 2H, CH2CO), 7.23-8.15 (m, 4H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.0 (CH2CO), 51.2 (morpholine CH2N), 66.2 (morpholine CH2O), 115.8, 117.8, 119.1, 123.4, 124.3, 130.4, 135.0, 135.9 (aromatic carbons), 166.6 (C¼O), 195.6 (C¼S); Anal. Calcd. for C19H22N2O2S2: C, 60.93; H, 5.92; N, 7.48. Found: C, 61.08; H, 5.97; N, 7.62. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-methylpiperazine-1-carbodithioate (6c) Yield: (method A: 64%; method B: 80%); m.p.: 162–163 C; IR (cm1): 2933, 2846 (CH aliphatic), 1681 (C¼O), 1232 (C¼S); 1 H-NMR (CDCl3) d ppm 1.81–1.95 (m, 4H, carbazole CH2), 2.3 (s, 3H, CH3), 2.51 (t, 4H, piperazine CH2), 2.65 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.08 (t, 2H, carbazole CH2, J ¼ 6 Hz), 4.02 (br s, 2H, piperazine CH2), 4.33 (br s, 2H, piperazine CH2), 4.89 (s, 2H, CH2CO), 7.22–8.16 (m, 4H, Ar-H); Anal. Calcd. for C20H25N3OS2: C, 61.98; H, 6.50; N, 10.84. Found: C, 62.12; H, 6.48; N, 10.92. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-ethylpiperazine-1-carbodithioate (6d) Yield: (method A: 64%; method B: 82%); m.p.: 167–168 C; IR (cm1): 2935, 2843 (CH aliphatic), 1681 (C¼O), 1234 (C¼S);
1
H-NMR (CDCl3) d ppm 1.14 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.87– 1.97 (m, 4H, carbazole CH2), 2.48 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 2.59 (t, 4H, piperazine CH2), 2.70 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.14 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 4.07 (br s, 2H, piperazine CH2), 4.37 (br s, 2H, piperazine CH2), 4.95 (s, 2H, CH2CO), 7.26–8.20 (m, 4H, Ar-H); Anal. Calcd. for C21H27N3OS2: C, 62.81; H, 6.78; N, 10.46. Found: C, 62.89; H, 6.82; N, 10.61. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-phenylpiperazine-1-carbodithioate (6e) Yield: (method A: 88%; method B: 88%); m.p.: 132–133 C; IR (cm1): 2927, 2852 (CH aliphatic), 1678 (C¼O), 1226 (C¼S); 1 H-NMR (CDCl3) d ppm 1.85–1.95 (m, 4H, carbazole CH2), 2.66 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.10 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.32 (t, 4H, piperazine CH2), 4.18 (br s, 2H, piperazine CH2), 4.46 (br s, 2H, piperazine CH2), 4.94 (s, 2H, CH2CO), 6.908.16 (m, 9H, Ar-H); Anal. Calcd. for C25H27N3OS2: C, 66.78; H, 6.05; N, 9.35. Found: C, 66.93; H, 6.13; N, 9.52. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-(4-chlorophenyl)piperazine-1-carbodithioate (6f) Yield: (method A: 74%; method B: 86%); m.p.: 164–165 C; IR (cm1): 2924, 2835 (CH aliphatic), 1685 (C¼O), 1228 (C¼S); 1 H-NMR (CDCl3) d ppm 1.83–1.97 (m, 4H, carbazole CH2), 2.68 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.11 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.30 (t, 4H, piperazine), 4.22 (br s, 2H, piperazine), 4.45 (br s, 2H, piperazine), 4.94 (s, 2H, CH2CO), 6.82–8.18 (m, 8H, Ar-H); Anal. Calcd. for C25H26ClN3OS2: C, 62.03; H, 5.41; N, 8.68. Found: C, 62.17; H, 5.46; N, 8.73. 2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl 4-(4-methoxyphenyl)piperazine-1-carbodithioate (6g) Yield: (method A: 85%; method B: 86%); m.p.: 156–157 C; IR (cm1): 2935, 2833 (CH aliphatic), 1697 (C¼O), 1224 (C¼S); 1 H-NMR (CDCl3) d ppm 1.86–1.96 (m, 4H, carbazole CH2), 2.65 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.09 (t, 2H, carbazole CH2, J ¼ 6 Hz), 3.16 (t, 4H, piperazine, J ¼ 5.2 Hz), 3.77 (s, 3H, CH3O), 4.18 (br s, 2H, piperazine), 4.46 (br s, 2H, piperazine), 4.92 (s, 2H, CH2CO), 6.84–8.18 (m, 8H, Ar-H); 13C-NMR (100 MHz, CDCl3) d ppm 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.2 (CH2CO), 50.4, 51.6 (piperazine carbons), 55.5 (CH3O), 114.6, 115.8, 117.8, 118.9, 119.1, 123.4, 124.3, 130.4, 135.0, 135.9, 144.5, 154.5 (aromatic carbons), 166.7
Tetrahydrocarbazole hybridized with dithioates
DOI: 10.3109/14756366.2014.922554
(C¼O), 195.1 (C¼S); Anal. Calcd. for C26H29N3O2S2: C, 65.10; H, 6.09; N, 8.76. Found: C, 65.23; H, 6.13; N, 8.92.
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2-Oxo-2-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)ethyl diethyl carbamodithioate (7) Yield: (method A: 54%; method B: 90%); m.p.: 97–98 C; IR (cm1): 2929, 2850 (CH aliphatic), 1683 (C¼O), 1267 (C¼S); 1 H-NMR (CDCl3) d ppm 1.25 (t, 3H, CH3CH2, J ¼ 7.2 Hz), 1.38 (t, 3H, CH3CH2, J ¼ 6.8 Hz), 1.85–1.99 (m, 4H, carbazole CH2), 2.70 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.15 (t, 2H, carbazole CH2, J ¼ 6.0 Hz), 3.85 (q, 2H, CH3CH2, J ¼ 7.2 Hz), 4.05 (q, 2H, CH3CH2, J ¼ 6.8 Hz), 4.95 (s, 2H, CH2CO), 7.26–8.22 (m, 4H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) d ppm 11.5, 12.6 (CH3CH2N), 21.2, 21.8, 23.8, 26.8 (carbazole C-3, C-2, C-4, C-1), 45.5 (CH2CO), 47.2, 50.0 (CH3CH2N), 115.9, 117.8, 119.0, 123.3, 124.2, 130.4, 135.1, 135.9, (aromatic carbons), 167.0 (C¼O), 193.7 (C¼S); Anal. Calcd. for C19H24N2OS2: C, 63.30; H, 6.71; N, 7.77. Found: C, 63.39; H, 6.77; N, 7.94.
7
dithiocarbamates (6c–g) [piperazine analogues] was comparable to dialkyl dithiocarbamate (7). Besides, the alkyl dithiocarbonates (4a–d) displayed more potent antitumor activity than the heterocyclic dithiocarbamates (6a,b) [piperidine and morpholine analouges]. Whilst, SAR study of the test compounds on HCT116 cell line indicated that heterocyclic dithiocarbamates (6a–g) afforded more potent cytotoxic agents than alkyl dithiocarbonate (4a–d) or dialkyl dithiocarbamate (7). On both cell lines, heterocyclic dithiocarbamates bearing piperazine moiety afforded more potent cytotoxic agents than piperidine or morpholine analogues. Within the piperazine derivatives, the antitumor activity was affected by the N-substituent in the following order: 4-chlorophenyl 4 alkyl 4 4-methoxyphenyl 4 phenyl. Further work need to be done to determine the effect of the linker between tetrahydrocarbazole and dithioate on the anticancer activity and also to determine the mechanism inherited behind the antitumor effect of these compounds.
Biological testing
Acknowledgements
Materials and methods
The author is grateful to all members of the department of Cancer Biology, National Cancer Institute, Cairo, Egypt, for carrying out the cytotoxicity testing.
The human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116) were obtained as a gift from NCI, Bethesda, MD. All chemicals and solvents were purchased from SigmaAldrich (Munich, Germany). Measurement of potential cytotoxicity The cytotoxic activity of the newly synthesized compounds was measured in vitro on human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116) using Sulforhodamine-B stain (SRB) assay applying the method described by Skehan et al.44. Cells were plated in a 96-multiwell plate (104 cells/well) for 24 h before treatment with the test compounds to allow attachment of the cells to the wall of the plate. Test compounds were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the test compounds (5, 12.5, 25 and 50 mg/mL) were added to the cell monolayer. Triplicate wells prepared for each individual dose. Monolayer cells were incubated with the test compound for 48 h at 37 C in atmosphere of 5% CO2. After 48 h, cells were fixed with trichloroacetic acid, washed with water and stained for 30 min with 0.4% (wt/vol) SRB stain dissolved with 1% acetic acid. Excess stain was removed by four washes with 1% acetic acid and attached stain was recovered with Tris EDTA buffer. The color intensity was measured in a ELISA reader. The relation between surviving fraction and compound concentration was plotted and IC50 [the concentration required for 50% inhibition of cell viability] was calculated for each compound and results are given in Table 1 and represented graphically in Figure 4.
Conclusion The objective of the present study was to synthesize new tetrahydrocarbazoles hybridized with dithioate derivatives and to study the effect of the alkyl group of the dithioate moiety on the antitumor activity. Most of the synthesized compounds exploited potent antitumor activity, especially compound 6f [4-chlorophenylpiperazine derivative] which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 7.24 nM/mL. MCF7 cell line was more susceptible to the action of the test compounds than HCT116 cell line. SAR study of the test compounds on MCF7 cell line pointed out that the antitumor activity of the heterocyclic
Declaration of interest The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.
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