Design, Synthesis, Cytotoxicity, Physicochemical and In Vivo Studies of New Thalidomide Dithiocarbamate Analogs as Anticancer

Received: 22-Jan-2020 Accepted Date: Feb 17, 2020 ; Published: 26-Feb-2020

Introduction

Thalidomide was originally marketed as a hypnotic/sedative agent in the late of 1950s and the early 1960s; Thalidomide had caused severe congenital malformations in children whose mothers took this medicinal product during pregnancy. As a result, it was withdrawn from the market due to its teratogenic effect [1-3]. Subsequently, Thalidomide was discovered to have various biological effects against some diseases including Acquired Immunodeficiency Syndrome (AIDS) [4], Graft versus Host Disease (GvHD) [5], leprosy [6] and other related diseases [7]. The Effectiveness of thalidomide in these diseases has been attributed to its regulatory activity on Tumor Necrosis Factor-alpha (TNF-α) production [8] and has been identified as an effective agent for the treatment of Multiple Myloma (MM) [9,10] and cancer related pathologic angiogenesis [11]. Thalidomide teratogenicity was partially explained by its anti-angiogenic activity [12]. In addition, thalidomide may have suppression effects on the human lung fibroblasts [13]. On the other hand, thalidomide analogs have found recent application in the treatment of cancer [14-17]. Thalidomide is currently used in therapy as a racemate. It is expected to play an important and effective role in medicine so we should develop its physicochemical properties to enhance its bioavailability via using it in different dosage forms. In the current study, using a two-step synthesis, new thalidomide dithiocarbamate compounds were synthesized and tested for their cytotoxic activity against human cell line. These new compounds may be formulated as inhaler due to their better physicochemical properties (particle size).

Materials and Methods

1,2 dichloroethane and Sodium hydride (Merck-Schuchardt), 1,3 dichloropropane (ACROS organics), silica gel GF254 (type 60) for thin layer chromatography (Merck), dimethyl formamide, Chloroform and Methylene Chloride (Fischer Scientific UK Limited). The reagents RPMI-1640 medium, SRB (SulphoRhodamine-B), Sodium diethyl dithiocarbamate and DMSO (Dimethyl sulfoxide) were purchased from (Sigma-Aldrich). 1HNMR and 13C NMR spectra were recorded on a Varian Gemini 300 MHZ spectrometer, Cairo University. Elemental analyses were determined on a Yanaca CHN Corder MT-3 elemental analyzer in microanalysis laboratory at Cairo University. IR spectra were recorded (KBr) on a Perkin-Elmer FT-IR Spectrum One spectrophotometer, Arab Drug Company. MS spectra were run on GC MS-QP 1000 EX (SHIMADZU) Mass Spectrometer, Micro analytical Laboratory, Faculty of Science, Cairo University.

Melting Point Determination

The melting point was determined by using optimelt Automated Melting Point System SRS (Stanford Research Systems) model MPA100, at Arab Drug Company.

Geometric Particle Size Determination

Geometric particle size was determined for powder and Spray by Mastersizer X using lens 100 mm and beam length 14.3 or 2.4. Sample unit is MSX64 dry powder feeder in case of dry powder and is MS5 inhalers mounting unit in case of spray. Apparatus: Mastersizer x ver.2.15 serial no.33337-22, Malvern Instruments Ltd., Malvern, UK, at Arab Drug Company

General Synthesis Method for Chloroethyl Thalidomide and Chloropropyl Thalidomide

A solution of (4 mmol) sodium hydride (60 %) in 15 ml of absolute dimethyl formamide was reacted with (4 mmol) thalidomide while cooling, after the gas formation has ceased; (4 mmol) dichloro compounds (1, 2 dichloroethane or 1,3 dichloropropane) were added slowly and stirred for further 8 h, Insoluble materials were filtered off and the filtrate was evaporated under reduced pressure. The residue was purified by preparative TLC using methylene chloride and the yield is 52 % and 54 % for compounds 2 and 3 respectively.

General Synthesis Method for Compounds 4 and 5

Two mmol of compound 2 or 3 with a stoichiometric amount of sodium diethyl dithiocarbamate in 20 ml acetonitrile was heated under reflux with stirring at 120-130°C for 4 h, the solvent was evaporated under reduced pressure. The residue was purified by preparative TLC with chloroform and the yield is 79 % and 83 % for compounds 4 and 5 respectively.

2-(1-(2-Chloroethyl)-2,6-dioxo-piperidine-3-yl)-1,3-dihydro-2H-isoindole-1,3-dione (2): Yield (52 %), m.p. 148°C, white powder, IR (KBr) 2919, 1790, 1775, 1717, 1677, 1467, 1392, 1158, 719 cm^-1; 1H NMR (DMSO-d6) δ: 2.09-2.13 (m, 1H, H-4´), 2.51-2.63 (m, 1H, H-5´), 2.85-2.88 (m, 1H, H-4´), 3.00-3.08 (m, 1H, H-5´ CO), 3.88-3.91 (t, 2H, N-CH2CH2), 4.21-4.25 (t, 2H, N-CH2), 5.19-5.25 (m, 1H, H-3´), 7.8-7.84 (m, 4H, H aryl.); 13C NMR (DMSO-d6) δ: 21.97 (C-4´). 30.91 (C-5´), 40.68 (CH2CH2CL), 46.52, (N-CH2), 48.98 (C-3’), 123.37, 123.43 (C-4, C-7), 131.19 (C-3a, C-7a), 134.83, 134.88 (C-5, C-6), 167.04, 167.12 (C-1, C-3) 169.79 (C-2´) 172.71 (C-6´). EIMS m/z=322 [M+] Anal. Calc. for C15H13N2O4Cl: C, 56.17; H, 4.08; N, 8.73; Found: C, 55.95; H, 4.02; N, 8.65.

2-(1-(3-Chloropropyl)-2,6-dioxo-piperidine-3-yl)-1,3-dihydro-2H-isoindole-1, 3-dione (3): Yield (54 %), m.p. 133°C, off white powder, IR (KBr) 3098, 2912, 1785, 1772, 1711, 1692, 1610, 1386, 1341, 1113, 728 cm^-1; 1H NMR (DMSO-d6) δ: 1.88-1.93 (m, N-CH2CH2), 2.07-2.10 (m, 1H, H-4´), 2.50-2.63 (m, 1H, H-5´), 2.84-2.86 (m, 1H, H-4´), 2.88-2.96 (m, 1H, H-5´ CO), 3.75-3.77 (m, 2H, N-CH2CH2CH2), 3.95-4.03 (m, 2H, N-CH2), 5.23-5.29 (m, 1H, H-3´), 7.88-7.97 (m, 4H, H aryl.); 13C NMR (DMSO-d6): 21.81 (C-4´). 29.92 (C-5´), 31.78 (N-CH2CH2), 40.81 (CH2 CH2CH2Cl), 43.57 (N-CH2), 48.95 (C-3’), 123.31, 123.42 (C-4, C-7), 131.09 (C-3a, C-7a), 134.73, 134.77 (C-5, C-6), 167.1,167.19 (C-1, C-3),169.42 (C-2´), 172.81 (C-6´). EIMS m/z=335 [M+]. Anal. Calc. for C16H15N2O4Cl: C, 57.40; H, 4.51; N, 8.36; Found: C, 57.23; H, 4.36; N, 8.32.

[3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopiperidin-1-yl] ethyl diethyldithiocarbamate (4): Yield (79 %), m.p. 125°C,white powder; IR (KBr) 2974, 2930, 1789, 1775, 1717, 1677, 1497, 1418, 1392, 1195, 1092, 966, 912, 718 cm^-1; 1H NMR (DMSO-d6) δ: 1.23-1.28 (t, 3H, CH3), 2.11 (m, 1H, H-4´), 2.55-2.57 (m, 1H, H-5´), 2.79-2.91 (m, 1H, H-4´), 2.93-3.02 (m, 1H, H-5´ CO), 3.41-3.47 (q, 2H, CH2), 3.59-3.63 (t, 2H, N-CH2CH2), 3.77-3.82 (t, 2H, N-CH2), 5.22-5.28 (m, 1H, H3´), 7.91-7.93 (m, 4H, H aryl.); 13C NMR (DMSO-d6) δ 13.65 (CH3), 21.92 (C4´), 30.72 (C5´), 36.08 (NCH2), 39.58 (NCH2 CH2S), 44.07 (CH2), 48.92 (C3´), 123.45, 123.52 (C4, C7), 131.22 (C3a, C7a), 134.97, 135.03 (C5, C6), 167.16,167.25 (C1, C3), 169.67 ( C2´), 172.89 ( C6´), 195.87 (C=S). EIMS m/z=433 [M+]. Anal. Calc. for C20H23N3O4S2: C, 55.41; H, 5.34; N, 9.69; S, 14.76. Found: C, 55.28; H, 5.29; N, 9.53; S, 14.71.

[3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopiperidin-1-yl] propyl diethyldithiocarbamate (5): Yield (83 %), m.p. 117°C, off white powder; IR (KBr) 3098, 2986, 2917, 1787, 1773, 1716, 1682, 1391, 1198, 1158, 1113, 1090, 727 cm^-1; 1H NMR (DMSO-d6) δ: 1.24-1.28 (t, 3H, CH3), 1.91 (m, 2H, N-CH2CH2 ), 2.09-2.13 (m, 1H, H-4´), 2.55-2.64 (m, 1H, H-5´), 2.78-2.83 (m, 1H, H-4´), 2.95-3.00 (m, 1H, H-5´ CO), 3.37-3.44 (m, 2H, N-CH2CH2CH2), 3.63-3.65 (q, 2H, CH2), 3.97-4.05 (m, 2H, N-CH2), 5.23-5.28 (m, 1H, H3´), 7.88-7.93 (m, 4H, H aryl.);13C NMR (DMSO-d6) δ 13.70 (CH3), 21.95 (C4´), 27.85 (N-CH2CH2), 30.15 (C5´), 33.45 (N-CH2CH2CH2), 41.75 (N-CH2), 44.25 (CH2), 48.90 (C3´), 123.22, 123.31 (C4, C7), 131.28 (C3a, C7a), 134.80, 134.89 (C5, C6), 167.35, 167.46 (C1, C3), 169.85 ( C2´), 172.95 ( C6´), 195.95 (C=S). EIMS m/z=448 [M+]. Anal. Calc. for C21H25N3O4S2: C, 56.36; H, 5.63; N, 9.39; S, 14.33. Found: C, 56.19; H, 5.49; N, 9.22; S, 14.22.

Anticancer Screening

In vitro study: Thalidomide and its new dithiocarbamate derivatives have been tested for their cytotoxic activity against human hepato carcinoma (HEPG-2), lung carcinoma (A-549), and breast adenocarcinoma (MCF-7) cell line in the National Cancer Institute, Cairo University. Potential cytotoxicity of compounds was tested using the described method [18-21].

The cytotoxic evaluation of all compounds (0, 5, 12.5, 25 and 50 μg/ml) against normal cells was carried out to explore the toxicity and selectivity of the tested compounds. Fetal bovine serum was obtained from (GIBCO, UK). The cells were cultured in RPMI-1640 medium with 10 % fetal bovine serum. Antibiotics (penicillin 100 units/ml and streptomycin 100 mg/ml) were added at 37 ºC in a 5 % CO2 incubator. The cells were seeded in a 96-well plate at a density of 1.0 x104 cells/well at 37°C For 48 hrs. under 5 % CO2. After incubation, the cells were treated with different concentration of compounds and incubated for 24 hrs. The medium was discarded. Fix with 10 % trichloroacetic acid (TCA) 150 mL/well for 1 h at 4°C. Wash by water 3 times (TCA reduce SRB protein binding). Wells were stained by SRB 70 ml/well for 10 min. at room temperature with 0.4 % 70 mL/well (keep in dark place). Then washed with acetic acid 1 % to remove unbound dye (end point: colorless drain age). The plates were subjected to air drying for 24 hrs. The dye was solubilized with 50 ml/well of 10 mM tris base (PH 7.4) for 5 min. on a shaker at 1600 rpm. The optical density (OD) of each well was measured at 570 nm with an ELISA microplate reader (EXL 800 USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100 and The IC50 values were calculated using the Microsoft Excel. The data were recorded and analyzed to estimate the effects of the tested compounds on cell viability and growth; IC50 values for the tested compounds are reported in below table, three independent experiments for each concentration were performed.

In vivo study: Lung cancer was processed in rats by using Diethylnitrosamine (rats injected intraperitoneal with Diethyl nitrosamine (DEN) (70 mg/2 ml/kg body weight/rat, once a week for eight weeks). Then rats were treated by compounds through intraperitoneal injection daily for 6 weeks then rats were scarified, and lung tissue was preserved in formalin and processed in an automated tissue processor. The processing consisted of an initial 2 step fixation and dehydration. Fixation comprising tissue immersion in 10 % buffered formalin for 48 hrs, followed by removal of fixative in distilled water for 30 minutes. Dehydration was then carried out by running the tissues through a graded series of alcohol (70 %, 90 %, and 100 %). The tissue was initially exposed to 70 % alcohol for 120 minutes followed by 90 % alcohol for 90 minutes and then two cycles of absolute alcohol, each for one hour. Dehydration was then followed by clearing the samples in several changes of xylene. It consisted of tissue immersion for an hour in a mixture comprising 50 % alcohol and 50 % xylene, followed by pure xylene for one and a half hour. Samples were then impregnated with molten paraffin wax, then embedded and blocked out. Paraffin sections (4–5 um) were stained with hematoxylin and eosin. Stained sections were examined for pathological lesions in the lungs of the examined rats.

Conclusions

A two-step synthesis of novel thalidomide dithiocarbamate analogs (4, 5) was evaluated. These new analogs were found to possess anticancer activity against human lung cancer with significant difference. Moreover, these analogs possess improved physicochemical properties, therefore; they may be formulated as inhalers for lung cancer treatment.

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