Abstracts

ARTICLE

Improved synthesis of 1,3,4-thiadiazolium-2-phenylamines using microwave and ultrasound irradiation and investigation of their cytotoxic activity

Camilla Moretto dos Reis^I; Juliana Echevarria-Lima^II; Amanda Fraga Miranda^I; Aurea Echevarria^I,* * e-mail: echevarr@ufrrj.br

^IDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, 23890-000 Seropédica-RJ, Brazil

^IIDepartamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro-RJ, Brazil

ABSTRACT

A new and efficient synthesis of eight 1,3,4-thiadiazolium-2-phenylamine derivatives (1-8, where 8 is novel in the literature) was performed using thionyl chloride or trimethylsilyl chloride as catalysts under microwave or ultrasound irradiation. The target compounds were obtained in good yields and remarkably short times, 5 min under microwave irradiation and 10 min under ultrasound irradiation, where compared to traditional methodology (24 to 48 h at room temperature standing). The best yields were obtained using the microwave irradiation and, in general way, using thionyl chloride instead trimethylsilyl chloride. The cytotoxicity against K562 human leukemia and Daudi lymphoma lines was evaluated and showed promising results from the 4-phenyl-5-(4'-nitro-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative.

Keywords: mesoionic heterocycle compounds, microwave, ultrasound, cytotoxic activity

RESUMO

Uma nova e eficiente síntese de oito derivados da classe 1,3,4-tiadiazolio-2-fenilaminas (1-8, sendo o derivado 8 inédito na literatura) foi realizada utilizando cloreto de tionila ou cloreto de trimetilsilano como catalisadores sob irradiação de microondas ou ultra-som. Os compostos alvos foram obtidos em bons rendimentos e em tempo extraordinariamente curtos, 5 min sob irradiação de microondas e 10 min sob irradiação de ultra-som, quando comparados com a metodologia tradicional (24 a 48 h em repouso a temperatura ambiente). Os melhores rendimentos foram obtidos usando irradiação de microondas e, de maneira geral, usando o cloreto de tionila ao invés de cloreto de trimetilsilano. A citotoxicidade foi avaliada frente à linhagem de leucemia humana K562 e do linfoma Daudi, apresentando resultados promissores para o derivado cloreto de 4-fenil-5-(4'-nitro-estiril)-1,3,4-tiadiazolio-2-fenilamina.

Introduction

Mesoionic compounds are a special class of heterocycles with potential therapeutic applications due to their unique chemical properties. They possess a betaine-like character with a partial positive charge on the heterocyclic ring that is balanced by a negative charge located on an exocyclic atom or group.^1,2 The large separation between the charged regions leads to large dipole moments of about 4-5 D.^3,4 These properties suggest the possibility of interacting with biomolecules such as proteins and DNA. Additionally, the overall neutral character of these compounds enables them to cross biological membranes. Different classes of mesoionic compounds have demonstrated a wide range of biological activities such as anti-inflammatory and analgesic,⁵ antiparasitic,^6,7 antibacterial,^8,9 antiplatelet, fibrinolytic, thrombolytic and broncholytic effects,¹⁰ as well as anticancer potency.^11-17 The promising therapeutic applications have led us to study these interesting compounds in our laboratory. We have previously described the survival enhancement of Ehrlich and Sarcoma 180 tumor-bearing mice treated with 4-phenyl-5-(4'-X-styryl)-1,3,5-thiadiazolium-2-phenylamine chlorides.¹² Respiratory chain inhibition, transmembrane potential collapse, and ATPase activity in intact rat liver mitochondria were observed when the 4-phenyl-5-(4'X-styryl)-1,3,5-thiadiazolium-2-phenylamine chlorides were used.^13,14 In addition, Senff-Ribeiro et al.¹⁵ showed that 4-phenyl-5-(4'-nitro-styryl)-1,3,5-thiadiazolium-2-phenylamine chloride displays an important antitumor activity against murine melanoma B16F10. This compound was cytotoxic in vitro, decreasing cell viability and proliferation, and was able to inhibit tumor growth by ca. 85% in vivo as well. Additionally, it decreased the viability and proliferation of cell lines of human melanoma (MEL-85, SK-MEL, A2058 and MEWO).^15,16 To contribute to the understanding of the molecular pathways involved in the pharmacological activities of mesoionic compounds, we have recently described the effects of 4-phenyl-5-(4'-nitro-styryl)-1,3,5-thiadiazolium-2-phenylamine chloride on lipid peroxidation in rat liver mitochondria, submitochondrial particles and phosphatidylcholine, as well as its ability to scavenge radicals.¹⁷

In addition, our group has investigated new, more efficient and cleaner synthetic methods for the preparation of these mesoionic compounds,¹⁸ especially due to important environmental concerns with the current synthesis.^19,20 Therefore, the synthesis of the 1,3,4-thiadiazolium-2-phenylamine class of compounds was revisited using alternative energy sources to obtain better yields and lower reaction times.

Towards that end, this work presents the synthesis of 4-phenyl-5-(4'-X-phenyl)-1,3,5-thiadiazolium-2-phenylamine chlorides, where X = H (1), 4'-OCH₃ (2), 4'-NO₂ (3) and 3',4'-OCH₂O (4) (referred to as the benzaldehyde series), and 4-phenyl-5-(4'-X-styryl)-1,3,5-thiadiazolium-2-phenylamine chlorides, where X=H(5), 2'-OCH₃ (6), 4'-NO₂ (7) and 4'-N(CH₃)₂ (8) (the cinnamaldehyde series), by two new alternative methods: using microwave irradiation and an ultrasound bath. Furthermore, derivatives of 1, 3 and 7 were assayed against K562 human leukemia and Daudi lymphoma lines.

Experimental

Chemistry

Melting points were determined with a Meltemp II apparatus and were uncorrected. Infrared spectra (KBr pellets) were recorded on a Perkin-Elmer 1605 FT-IR spectrophotometer. ¹H and ¹³C NMR spectra were obtained on a Bruker AC200 (200 and 50.3 MHz) spectrometer with TMS as the internal reference and DMSO-d₆ as the solvent. The microwave and ultrasound bath assisted organic reactions were performed in a Panasonic Piccilo-NN-S45BH (a domestic oven) and an Ultra-cleaner 700, respectively.

General procedure for preparation of mesoionic chlorides (1-8)

Method A

Using microwave irradiation. The aldehyde (0.35 mmol) and 1,4-diphenyl-thiosemicarbazide (0.35 mmol) were mixed in the presence of a Lewis acid (TMS-Cl or SOCl₂) and a few drops of 1,4-dioxane. The resulting mixture was submitted to microwave irradiation for full 5 min in open vessel. After this continuous time, the mixture was added to 1,4-dioxane and left to stand overnight at room temperature. The obtained solid was filtered, washed with ice-cold water and 1,4-dioxane, and recrystallized from chloroform:ethanol (60:40, v/v).

Method B

Using ultrasound bath irradiation. The aldehyde (0.35 mmol) and 1,4-diphenyl-thiosemicarbazide (0.35 mmol) were mixed in the presence of a Lewis acid (TMS-Cl or SOCl₂) and 1,4 dioxane as solvent. The resulting mixture was submitted to ultrasound bath irradiation for 10 min. The solution containing the reaction mixture was left to stand overnight. The formed precipitate was treated in a similar way as described above.

Table 1 shows the yields obtained for 1-8 using the method A and method B, and the characterization data^6,12,21 for all compounds are in Supplementary Information.

Cell culture

The human erythroleukemia K562 and Burkett lymphoma Daudi cell lines were maintained in culture medium RPMI 1640 (Sigma) supplemented with β-mercaptoethanol 50 µmol L^-1, HEPES 25 mmol L^-1 (Sigma), penicillin 60 mg L^-1 (Sigma), streptomycin 100 mg L^-1 (Sigma) and 10% (v/v) fetal calf serum (Gibco, Life Technologies). They were incubated at 37 ºC in a humidified atmosphere of air and 5% CO₂. The cell line was passaged twice a week at a concentration of 1 × 10⁴ cell mL^-1.

Cytotoxic assays

1 × 10⁴ cell mL^-1 were seeded on a 96-well cell culture plate. The cells were incubated with or without a mesoionic compound 1, 3 or 7 at a concentration of 3.125-50 µmol L^-1 each. They were incubated for 24 h, 48 h, 72 h, 96 h and 7 days at 37 ºC with 5% CO₂. After, cell viability was assessed by adding 20 µL cell^-1 of MTT (5 mg mL^-1; 3-[4,5-dimethylthiazol--2-yl]-2,5-diphenyl tetrazolium bromide, Sigma) as described earlier.²² The plates were read on a microplate reader (Sunrise-Basic TECAN) at a wavelength of 490 nm. The results are represented as the mean ± SD of 5 determinations.

Analysis of the cell cycle

1 × 10⁴ cell mL^-1 were seeded on a 96-well cell culture plate. The cells were incubated with or without mesoionic compound 7 at a concentration of 3.125-50 µmol L^-1 each. They were incubated for 24 h at 37 ºC with 5% CO₂. After, the cells were collected and washed in Hank's buffer. The cell cycle was assessed by adding 200 µL of cell cycle solution (5 mg mL^-1). The cells were then analyzed by flow cytometry. The samples were excited at 488 nm with an argon laser; fluorescence emission was detected at 585 nm (FL-2). Ten thousand cells were acquired. All flow cytometry analyses were accomplished with WinMDI, version 2.9.

Statistical analysis

The values are given as a mean ± SD. The statistical significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni's t-test using Prism 5 software. P values of less than 0.05 were considered significant.

Results and Discussion

Chemistry

Eight mesoionic chloride salts were synthesized by two different methods, microwave irradiation and an ultrasound bath, aimed at reduction of the reaction time and adaptation to the principles of Green Chemistry.²³ The convergent strategy for mesoionic chloride salts synthesis included the reaction of 1,4-diphenyl-thiosemicarbazide with the appropriate aldehyde in the presence of a Lewis acid and 1,4-dioxane as solvent. In methods, two different Lewis acids, SOCl₂ and trimethylsilyl chloride (TMS-Cl), were investigated separately (Scheme 1). When compared to the traditional method,^7,24 the use of an aldehyde and a Lewis acid enables the elimination of a step involving a carboxylic acid and the subsequent acid chloride preparation, as well as a reduction of the time required to obtain the target compounds from 24-48 h standing at room temperature, after 8 h of stirring, to 5 min under microwave irradiation or 10 min under ultrasound irradiation.

The first method consisted of submitting a mixture of 1,4-diphenyl-thiosemicarbazide, the appropriate aldehyde, SOCl₂ (three equivalents) or TMS-Cl (two equiv.) and a few drops of 1,4-dioxane to microwave irradiation (MW) for 5 min. Subsequently, an organic solvent was added, and the mixture was allowed to stand overnight, after which time the precipitate was filtered and washed with ice-cold water to afford the target compounds. In the second method, using similar conditions as above, the reagents were dissolved in 1,4-dioxane and put in the ultrasound bath for 10 min, and the mixture was allowed to stand overnight. The isolation of the target compounds was performed in the same manner as for the microwave experiment. Scheme 1 shows the synthetic route of the two methods used in this work. In both methods, the mesoionic salts were obtained in good yields and high purity (Table 1). When microwave irradiation was employed, the yields were in the range of 41-95% when TMS-Cl was used and 90-98% when SOCl₂ was used. Using the ultrasound bath method, the respective yields for TMS-Cl and SOCl₂ were 66-98% and 72-87%. Thus, the best results were observed using SOCl₂ as the catalyst under microwave irradiation conditions.

The mesoionic chloride structures 1-8 were fully characterized by ¹H and ¹³C NMR and IR spectroscopies. The ¹H and ¹³C chemical shifts (δ) were assigned based on literature data^6,12,21,25 and were consistent with the structures proposed.

Cytotoxicity activity

In Brazil, leukemia represents about 2% of the total cancer cases and affects adults, the elderly and young individuals.²⁶ Cases of leukemia are classified as acute or chronic and, for lymphoid or myeloid, according to the origin of the cell type. The treatment of leukemia is performed using different combinations of chemotherapy drugs; however, the index of remission remains unsatisfactory. The lymphoma cell line Daudi and myeloid cell line K562 were used for studying the effects of the synthesized mesoionic compounds on leukemic cells. The cytotoxic activities of the 1, 3 and 7 were assayed in vitro using MTT.²² Compounds 1 and 3 did not alter the viability of the lymphoma cell line Daudi in 72 h of culture.

The positive results obtained with 7 (the unsaturated series compound with an NO₂ moiety), led us to assay it in 24 h, 48 h, 72 h, 96 h and 7 days incubations with Daudi cell cultures. The compound showed significant cytotoxic activity after 24 h. Moreover, this mesoionic derivative decreased the cellular viability in a concentration-dependent manner (Figure 1A and B). Similar results were obtained in the human erythroleukemia cell line K562. We observed that the mesoionic compound 7 reduced the cellular viability in a concentration-dependent manner for this cell line as well. The compound showed significant cytotoxic activity beginning at 24 h and continuously growing over time (Figure 1C and D).

The IC₅₀ values were determined in µmol L^-1 in at least three independent experiments using 7 at concentrations in the range of 5-25 µmol L^-1. The compound 7 was incubated for 24 h, 48 h, 72 h, 96 h, and 7 days with cultures of Daudi (lymphoid origin) and K562 cells (myeloid cells). When the results with the different cell lines were compared, they showed similar sensitivities for the first 72 h; however, the myeloid cells demonstrated more sensitivity after a long exposure time (7 days), as shown in Table 2.

To understand the mechanism of the cytotoxicity exhibited by the mesoionic compound 7 on the Daudi and K562 cells, they were incubated for 24 h with 7 at a concentration of 3.25-50 µmol L^-1. The capacity of this compound to induce apoptosis in Daudi and K562 cells was analyzed with a cell cycle assay. The results showed an increase in hypodiploid cells, which was correlated with the cytotoxicity results (Figure 2). This suggests that the mesoionic 7 induces cell death by apoptosis.

Conclusions

In summary, the new synthetic methods using microwave irradiation and an ultrasound bath provided the products 1-8, where 8 is novel in the literature, in shorter reaction times and higher yields when compared to the traditional method. SOCl₂ proved to be a better Lewis acid than TMS-Cl, and microwave irradiation led to a more efficient reaction when compared to the ultrasound bath. In addition to improving the synthetic route, these methods adhere to the principles of Green Chemistry. Furthermore, the 4'-nitro-styryl derivative has shown to be cytotoxic against Daudi lymphoma and K562 leukemic myeloid cells in preliminary in vitro studies of cytotoxicity.

Supplementary Information

Supplementary information with characterization data for all compounds (IR, ¹H and ¹³C NMR data) and the corresponding spectra for compound 8 are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for financial support and fellowships.

Submitted: November 6, 2010

Published online: May 3, 2011

Supplementary Information

Characterization data

4,5-Diphenyl-1,3,4-thiadiazolium-2-phenylamine chloride(1)

Pale yellow solid; mp 356-358 ºC (lit.²¹ mp 358 ºC); IR (KBr) ν_max/cm^-1 3419, 3047, 2723, 1569, 1542, 1494, 1444, 1319, 755, 690; ¹H NMR CDCl₃) δ 12.50 (s, N-H), 7.08-7.72 (m, H_arom); ¹³C NMR (DMSO-d₆δ 163.84 (C-5), 160.48 (C-2), 138.56 (C-14), 138.28 (C-10), 138.00 (C-6), 137.46 (C-17), 135.80 (C-13), 134.07 (C-11), 132.98 (C-16), 131.19 (C-8), 130.19 (C-12), 129.88 (C-15), 129.61 (C-9), 118.33 (C-7).

4-Phenyl-5-(4'-methoxy-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (2)

Yellow solid; mp 242-243 ºC (lit.²¹ mp 238 ºC); IR (KBr) ν_max/cm^-1 3425, 3048, 2935, 2665, 1602, 1542, 1448, 1567, 1309, 1175, 1024, 827, 752, 692; ¹H NMR CDCl₃) δ 12.90 (s, N-H), 7.70 (d, H-12,H-12'), 6.90 (d, H-11, H-11'), 7.63-7.08 (m, H_arom), 3.86 (s, CH₃O); ¹³C NMR (DMSO-d₆) δ 163.00 (C-5), 159.48 (C-2), 138.34 (C-14), 137.74 (C-6), 131.89 (C-10), 131.07 (C-17), 129.40 (C-12), 129.73 (C-16), 125.79 (C-8), 126.10 (C-9, C-13), 123.64 (C-11), 118.18 (C-15), 114.69 (C-7), 55.54 (OCH₃).

4-Phenyl-5-(4'-nitrophenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (3)

Orange solid; mp 138-139 ºC (lit.²¹ mp 138-139 ºC); IR (KBr) ν_max/cm^-1 3434, 3043, 2721, 1573, 1537, 1454, 1348, 854, 746, 694; ¹H NMR CDCl₃) δ 12.75 (s, N-H), 8.21 (d, H-12,H-12'), 8.16 (d, H-11, H-11'), 7.71-7.00 (m, H_arom); ¹³C NMR (DMSO-d₆) δ 163.84 (C-5), 159.20 (C-2), 137.95 (C-13), 136.95 (C-10), 131.43 (C-14), 131.25 (C-6), 129.46 (C-17), 128.85 (C-16), 126.00 (C-9), 125.67 (C-8), 124.90 (C-12), 123.79 (C-11), 123.33 (C-15), 118.24 (C-7).

4-Phenyl-5-(3',4'-methylenedioxyphenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (4)

Pale yellow solid; mp 290-293 ºC (lit.⁶mp 286-287 ºC); IR (KBr) ν_max/cm^-1 3438, 3050, 2902, 2642, 1604, 1540, 1450, 1567, 1311, 1243, 1105, 1037, 754, 694; ¹H NMR CDCl₃) δ 11.76 (s, N-H), 7.60-7.20 (m, H_arom), 7.17 (d, H-11'), 7.10 (d, H-12'), 7.02 (d, H-11), 6.08 (s, OCH₂O); ¹³C NMR (DMSO-d₆) δ 163.05 (C-5), 160.05 (C-2), 150.70 (C-12), 148.00 (C-13), 140.50 (C-14), 138.80 (C-6), 133.45 (C-17), 130.95 (C-8), 129.75 (C-16), 126.13 (C-11'), 127.57 (C-15), 127.42 (C-9), 117.56 (C-7), 115.12 (C-10), 115.46 (C-11), 110.42 (C-12'), 103.86 (OCH₂O).

4-Phenyl-5-styryl-1,3,4-thiadiazolium-2-phenylamine chloride (5)

Yellow solid; mp 264-265 ºC (lit.¹² mp 266-267 ºC); IR (KBr) ν_max/cm^-1 3432, 3056, 2670, 1604, 1567, 1538, 1498, 1448, 1330, 954, 746, 690; ¹H NMR CDCl₃) δ 12.58 (s, N-H), 7.95 (d, H_α), 7.19-7.83 (m, H_arom), 7.06 (d, a H_β); ¹³C NMR (DMSO-d₆) δ 164.90 (C-5), 160.30 (C-2),149.84 (C-α), 138.00 (C-14), 137.82 (C-6), 135.35 (C-10), 135.90 (C-17), 134.89 (C-13), 132.30 (C-11), 131.95 (C-16), 133.64 (C-8), 130.56 (C-12), 129.20 (C-15), 127.28 (C-9), 120.79 (C-7), 115.67 (C-β).

4-Phenyl-5-(2'-methoxy-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (6)

Yellow solid; mp 198-200 ºC (lit.¹² mp 195-196 ºC); IR (KBr) ν_max/cm^-1 3423, 3054, 2925, 2786, 1604, 1594, 1540, 1444, 1565, 1295, 1249, 1110, 1031, 966, 836, 754, 690; ¹H NMR CDCl₃) δ 11.90 (s, N-H), 7.92 (d, H_α), 7.84-6.90 (m, H_arom), 6.97 (d, H_β), 3.81 (s, CH₃O); ¹³C NMR (DMSO-d₆) δ 162.29 (C-11), 159.15 (C-5), 158.57 (C-2), 144.58 (C-α), 138.26 (C-14), 136.87 (C-6), 133.94 (C-11'), 131.70 (C-13), 130.51 (C-17), 130.07 (C-16), 128.89 (C-8), 125.56 (C-15), 124.04 (C-9), 122.11 (C-10), 118.89 (C-7), 111.40 (C-β), 55.46 (OCH₃).

4-Phenyl-5-(4'-nitro-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (7)

Orange solid; mp 234-236 ºC (lit.¹² mp 232-233 ºC); IR (KBr) ν_max/cm^-1 3432, 3048, 2721, 1616, 1571, 1519, 1450, 1342, 956, 840, 746, 688; ¹H NMR CDCl₃) δ 12.74 (s, N-H), 8.33 (d, H-12, H-12'), 8.25 (d, H_α), 8.04 (d, H-11, H-11'), 7.96-7.39 (m, H_arom), 7.33 (d, H_β); ¹³C NMR (DMSO-d₆) δ 165.10 (C-5), 162.38 (C-2), 151.00 (C-13), 147.55 (C-α), 142.99 (C-10), 140.20 (C-14), 139.05 (C-6), 132.92 (C-17), 131.98 (C-16), 130.50 (C-8), 129.68 (C-11), 127.20 (C-15), 126.33 (C-9), 125.25 (C-12), 117.71 (C-7), 114.81 (C-β).

4-Phenyl -5- (4 ' -dimethylamino -styryl) -1,3,4-thiadiazolium-2-phenylamine chloride (8)

Purple solid; mp 201-203 ºC; IR (KBr) ν_max/cm^-1 : 3425, 3062, 2923, 2852, 1612, 1565, 1577, 1525, 1444, 1378, 1268, 941, 808, 750, 692; ¹H NMR CDCl₃) δ 12.39 (s, N-H), 7.85 (d, H_α), 7.81 (d, H-12, H-12'), 7.73 (d, H-11, H-11'), 7.71-6.71 (m, H_arom), 6.70 (d, H_β), 3.02 (m, N(CH₃)₂); ¹³C NMR (DMSO-d₆) δ 163.27 (C-13), 157.14 (C-5), 153.01 (C-2), 149.47 (C-α), 138.81 (C-14), 137.15 (C-6), 131.76 (C-11, 11'), 131.20 (C-17), 130.12 (C-16), 129.33 (C-8), 126.16 (C-15), 123.66 (C-9), 121.08 (C-10), 118.30 (C-7), 111.88 (C-12, 12'), 103.81 (C-β), 39.75 (N(CH₃)₂); Elemental analysis: Found: C, 66.38; H, 5.15; N, 12.94. Calc. for C₂₄H₂₃N₄SCl: C, 66.27; H, 5.33; N, 12.88%.

References

Same as cited in the Article

IR, ¹³ C NMR, DEPT-Q and ¹ H, spectra of compound 8

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