Design, Synthesis and Antiproliferative Evaluation of Novel Disulfides Containing 1,3,4-Thiadiazole Moiety

Shuai Zhang; Xiao-jia Liu; Rui Tang; Hai-xin Wang; Hai-ying Liu; Yu-ming Liu; Bao-quan Chen

doi:10.1248/cpb.c17-00485

Abstract

A series of novel disulfides containing 1,3,4-thiadiazole moiety were designed, synthesized, and the structures of all products were identified by spectral data (IR, NMR, and high resolution (HR)-MS). Their in vitro antiproliferative activities were evaluated using 2-(2-methoxy-4-nitro-phenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfopheyl)-2H-tetrazolium monosodium salt (CCK-8) assay against human cancer cell lines, A549 (human lung cancer cell), HeLa (human cervical cancer cell), SMMC-7721 (human liver cancer cell) and normal cell lines L929. The bioassay results indicated that most of the tested compounds 6a–k, 7a–k and 8a–k exhibited antiproliferation with different degrees, and some compounds showed better effects than positive control 5-fluorouracil (5-FU) against various cancer cell lines. Among these compounds, compound 6e exhibited the most potent inhibitory activity against A549 cells with IC₅₀ value of 3.62 µM. Compounds 6i, 7a, 7g, 8a and 8b showed significantly antiproliferative activities against HeLa cells with IC₅₀ values of 3.88, 3.76, 3.59, 3.38 and 3.12 µM, respectively. Compounds 6a, 7a and 8a owned high antiproliferative activities against SMMC-7721 cells with IC₅₀ values of 2.54, 2.69 and 2.31 µM, respectively. Furthermore, all of the tested compounds showed weak cytotoxic effect against the normal cell lines L929. Based on the preliminary results, the substituent groups are vital for improving the potency and selectivity of this class of compounds.

Cancer has become one of the most terrible diseases around the world because of its low cure and high mortality.^1,2) It is predicted that the number of new cases could further rise to 19.3 million by 2025.^3,4) Despite significant progress has been achieved in anticancer therapy, identification of safe and effective anticancer agents are still one of the challenges in the field of drug discovery.

1,3,4-Thiadiazoles are an important class of heterocyclic bioactive compounds with a broad range of biological activities such as antioxidant,^5,6) antibacterial,^7,8) antidepressant,^9,10) antidiabetic,^11,12) antifungal,^13,14) anticonvulsant,^15,16) anti-inflammatory,^17,18) anticholinesterase,¹⁹⁾ antileishmanial,²⁰⁾ inhibition of human protoporphyrinogen oxidase.²¹⁾ In particular, a large number of 1,3,4-thiadiazole derivatives display interesting antitumour activities in vitro and in vivo conditions.^22–26) Therefore, the importance of 1,3,4-thiadiazole in the fields of medicinal chemistry has received much attention in the past few decades.²⁷⁾ On the other hand, disulfide derivatives are also acknowledged to possess a wide spectrum of bioactivities because the disulfide group makes up the core structure of numerous biologically active compounds with many types of biological activities including antibacterial,^28,29) anti-human immunodeficiency virus (HIV)-1,³⁰⁾ antitumor,^31–33) and herbicidal properties.^34,35)

Particularly, the antitumor character of disulfide derivatives attracts great interests from medicinal chemists in recent years. Moreover, the metabolic evaluation of the most promising compound is further performed to reveal that disulfide bond can be stable in human plasma over 8 h, indicating a good prospect of these compounds for in vitro antitumor activities.³⁶⁾

We previously reported the synthesis of a series of disulfides bearing 1,3,4-oxadiazole moiety and their in vitro antiproliferative activities were evaluated.³⁷⁾ We observed that some compounds showed better effects than positive control 5-fluorouracil against various cancer cell lines. Due to their attractive pharmacological property, in the paper we report the synthesis of disulfides containing 1,3,4-thiadiazole moiety. These compounds were evaluated for their antiproliferative activities against three human cancer cell lines, A549 (human lung cancer cell), HeLa (human cervical cancer cell), SMMC-7721 (human liver cancer cell) and one normal cell line L929 (Mouse fibroblasts) with the ultimate aim of developing novel potent antitumor agents.

Results and Discussion

Chemistry

As depicted in Chart 1, thirty three disulfides containing 1,3,4-thiadiazole moiety were synthesized and reported for the first time. The preparation of S-alkyl-thioisothiourea hydrochloride 2 was carried out by reported literature method.³⁸⁾ The preparation of 2-chloro-N-(substituted-phenyl)-acetamide 4 was carried out by reported literature method.³⁹⁾ The reaction of commercially available 2,5-dimercapto-1,3,4-thiadiazole with compound 4 in the presence of NaOH in water and ethanol at room temperature yielded the desired intermediates 5a–k in 70–81% yields. Finally, the target compounds 6a–k, 7a–k and 8a–k were successfully obtained by the reaction of intermediates 5a–k and compound 2 in the presence of NaHCO₃ in methanol and water at room temperature. All of the novel synthesized compounds 6a–k, 7a–k and 8a–k were purified by silica gel column chromatography and their structures were characterized by IR, ¹H-NMR, ¹³C-NMR and high resolution-electrospray ionization (HR-ESI)-MS.

Chart 1. Synthesis of the Target Compounds 6a–k, 7a–k and 8a–k

Reagents and conditions: (a) conc. HCl, H₂O₂ (30%), 0–5°C, 6 h; (b) CH₂Cl₂–NaOH, r.t. 5–6 h; (c) ethanol, NaOH–H₂O, r.t. 4–5 h; (d) ethanol, NaHCO₃–H₂O, r.t. 6 h.

Biological Activity

The antiproliferative activities of target compounds 6a–k, 7a–k and 8a–k against three human cancer cell lines, A549, HeLa, SMMC-7721 and one normal cell line L929 were evaluated by 2-(2-methoxy-4-nitro-phenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfopheyl)-2H-tetrazolium monosodium salt (CCK-8) assay in vitro, and their IC₅₀ values were calculated and are presented in Table 1. 5-Fluorouracil (5-FU) was used as a positive control.

Table 1. In Vitro Antiproliferative Activities of Compounds 6a–k, 7a–k and 8a–k against Various Cell Lines

Compound	IC₅₀ (µM)^a)
Compound	A549	HeLa	SMMC-7721	L929
6a	12.65±0.35	4.77±0.06	2.54±0.10	18.28±1.26
6b	9.50±0.24	20.89±0.17	8.21±0.30	62.51±2.85
6c	8.02±0.19	6.11±0.12	18.71±1.28	55.17±3.08
6d	8.26±0.20	5.48±0.08	6.08±0.57	7.81±0.03
6e	3.62±0.06	8.16±0.23	3.74±0.12	5.44±0.12
6f	17.63±0.51	8.07±0.45	4.21±0.22	6.72±0.17
6g	8.69±0.27	6.86±0.37	3.00±0.04	9.91±0.37
6h	8.00±0.38	8.23±0.51	4.49±0.34	33.08±2.17
6i	6.53±0.15	3.88±0.11	3.53±0.26	23.50±0.59
6j	25.86±0.81	4.91±0.07	9.78±0.39	34.60±1.20
6k	19.90±0.26	8.04±0.30	6.50±0.45	18.13±0.38
7a	12.76±1.06	3.76±0.13	2.69±0.22	68.79±2.45
7b	8.71±0.28	6.17±0.02	5.26±0.81	69.76±1.95
7c	8.51±0.32	9.62±0.39	15.43±0.73	67.88±2.06
7d	23.27±0.78	8.93±0.45	4.34±0.35	29.77±1.92
7e	7.70±0.13	6.76±0.24	4.32±0.29	13.69±0.56
7f	22.12±0.57	27.36±1.58	18.06±0.18	18.54±1.78
7g	8.17±0.25	3.59±0.12	6.40±0.25	29.14±0.76
7h	7.84±0.30	4.70±0.15	7.29±0.34	38.65±1.68
7i	25.90±1.08	6.85±0.76	7.55±0.13	31.74±0.97
7j	28.49±1.23	16.10±0.81	26.33±1.67	36.51±2.07
7k	12.19±0.36	12.33±1.02	25.40±0.76	38.17±1.76
8a	8.56±0.21	3.38±0.05	2.31±0.27	57.20±2.58
8b	24.42±0.57	3.12±0.32	7.85±0.36	63.51±1.89
8c	26.72±1.08	9.23±0.23	27.09±1.34	29.32±2.67
8d	29.52±0.89	9.82±0.17	9.23±0.32	8.06±0.33
8e	7.71±0.01	5.80±0.14	3.92±0.05	8.70±0.04
8f	25.47±0.91	11.52±0.25	11.19±0.51	7.41±0.12
8g	9.80±0.38	7.22±0.53	22.87±0.47	10.60±0.27
8h	8.71±0.29	6.19±0.18	7.32±0.55	26.70±0.36
8i	17.29±0.21	10.42±0.28	9.29±0.78	32.41±1.73
8j	20.02±0.17	8.44±0.12	25.17±1.23	29.45±0.67
8k	9.31±0.23	8.36±0.32	12.82±0.04	6.99±0.02
5-FU^b)	8.13±0.34	17.21±0.67	5.62±0.28	2.98±0.15

a) IC₅₀ is the drug concentration effective in inhibiting 50% of the cell growth measured by CCK-8 assay. b) Used as a positive control.

As shown in Table 1, some of the tested compounds revealed stronger antiproliferative effect than previous reported compounds against various cancer cell lines.³⁷⁾ All compounds 6a–k, 7a–k and 8a–k showed antiproliferation against four cell lines with certain degrees, and the substituent groups of phenyl ring played important roles in the potency of biologically active compounds. In A549 cells, some compounds exhibited better activities than positive control 5-FU. In particular, compound 6e exhibited the most potent inhibitory activity against A549 cells with IC₅₀ value of 3.62 µM. As compared with compound 6a which has no substituent at the phenyl ring while R₁ is 2-butyl group, except compounds 6f, j and k, the other compounds showed enhanced antiproliferative activities. As compared with compound 7a which has no substituent at the phenyl ring while R₁ is n-butyl group, compounds 7g, h and k carrying electron-donating substituents exhibited increased activities. As compared with compound 8a which has no substituent at the phenyl ring while R₁ is i-butyl group, except compound 8e, electron-donating group substituted derivatives, including 8g, h and k displayed better activities than compounds 8b–f, 8i and j carrying electron-drawing substituents. Especially, while R₁ is the same substituent, 3-nitro substituted derivatives 6e, 7e and 8e displayed higher activities than other compounds. These results indicated that the influence of substituents on antiproliferative activity against A549 cells is complex and unexplained at present. In HeLa cells, except compounds 6b and 7f showed moderate antiproliferative activity with IC₅₀ values of 20.89 and 27.36 µM, respectively, the other compounds showed good antiproliferative effect, and displayed higher activities than positive control 5-FU. Among them, compounds 6i, 7a, g, 8a and b showed significantly biological activities with IC₅₀ values of 3.88, 3.76, 3.59, 3.38 and 3.12 µM, respectively. As compared with compounds 6a, 7a and 8a which have no substituent at the phenyl ring, except compounds 6i, 7g, h and 8b, the other compounds 6b–k, 7b–k and 8b–k all resulted in a decrease in activity. But, no matter R₁ is 2-butyl, n-butyl or i-butyl group, the electronic effects of substituents at the phenyl ring on antiproliferative activity against HeLa cells did not show apparent regularity. In SMMC-7721 cells, compounds 6a, e–i, 7a, b, d, e, 8a and e displayed good proliferation inhibitory activities, the other compounds showed lower antiproliferative effect than positive control 5-FU. As compared with 6a, 7a and 8a which have no substituent at phenyl ring and owned high antiproliferative activities with IC₅₀ values of 2.54, 2.69 and 2.31 µM respectively, the other compounds bearing electron-donating or electron-withdrawing groups, including 6b–k, 7b–k and 8b–k, all exhibited decreased antitumor effects. However, the bioassay results also indicated that the electronic effects of substituents on antitumor activity against SMMC-7721 cells did not show apparent regularity. In L929 cells, all compounds exhibited weak cytotoxic activity, and showed lower cytotoxic effect than positive control 5-FU. Especially noteworthy are compounds 6g, i, 7b and 8a, which showed significant antiproliferative activities against A549, HeLa and SMMC-7721 cells, exhibited weak cytotoxicities against L929 cells. The results suggested that compounds 6g, i, 7b and 8a displayed superior selectivity against the tested cancer cell lines. Moreover, we also found from the results that the 2-butyl substituted derivatives showed better biological activities than n-butyl and i-butyl substituted derivatives on the whole. Therefore, it is necessary that the further investigation is carried out through structural transformation for improving the potency and selectivity of this class of compounds.

Conclusion

We successfully prepared a series of novel disulfides containing 1,3,4-thiadiazole moiety, and the newly synthesized compounds were evaluated for their in vitro antiproliferative activities against A549, HeLa, and SMMC-7721 human cancer cell lines and L929 normal cell lines by CCK-8 assay. Some of the compounds inhibited the proliferation better than positive control 5-FU. In particular, compound 6e exhibited the most potent inhibitory activity against A549 cells with IC₅₀ value of 3.62 µM. Compounds 6i, 7a, g, 8a and b displayed significantly antiproliferative activities against HeLa cells with IC₅₀ values of 3.88, 3.76, 3.59, 3.38 and 3.12 µM respectively. Compounds 6a, 7a and 8a owned high antiproliferative activities against SMMC-7721 cells with IC₅₀ values of 2.54, 2.69 and 2.31 µM respectively. In addition, all the tested compounds showed low cytotoxic effect against the normal cell lines L929. Further structural optimization and mechanism studies on compounds 6a–k, 7a–k and 8a–k are ongoing.

Experimental

Chemistry

All reagents were obtained from commercial suppliers and used without further purification, unless specified. All reactions were monitored by TLC performed on glass packed silica gel GF254 plates. Melting points were determined by an X-6 microscope melting point apparatus and are uncorrected. Infrared spectra were recorded in KBr pellets on a Nicolet Avatar 370 spectrometer. ¹H- and ¹³C-NMR spectra were collected at resonance frequencies of 400 MHz and 100 MHz, respectively. NMR spectra were performed on a Bruker Avance III 400 MHz spectrometer using dimethyl sulfoxide (DMSO)-d₆ as solvent and tetramethylsilane as internal standard. The chemical shifts for ¹H-NMR are reported in ppm from tetramethylsilane (0 ppm) or referenced to the solvent (DMSO-d₆ 2.50) on the δ scale. Chemical shifts (δ) for ¹³C-NMR spectra are referenced to the signals for residual deuterated solvents (DMSO-d₆ 39.5). Multiplicities are reported by the following abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublet), J (coupling constants in hertz). High-resolution mass data were taken with a Waters Xevo G2 QT of mass spectrometer. The products were purified by flash column chromatography using silica gel (200–300 mesh) with indicated solvents.

General Procedure for the Synthesis of Intermediates 5a–k

A solution of sodium hydroxide (6 mmol) in water (20 mL) was added to a mixture of 2,5-dimercapto-1,3,4-thiadiazole (6 mmol), appropriate 2-chloro-N-(substituted-phenyl)-acetamide 4 (5 mmol) and ethanol (10 mL). The mixture was stirred for 4–5 h at room temperature (the end of reaction was monitored by TLC), after completion, the mixture was poured into water. The resulting precipitate was collected by filtration, washed well with water and further purified by recrystallization from ethanol to afford target compounds.

2-Sulfhydryl-5-[(phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5a)

White solid; Yield 76.7%; mp 136.5–138.7°C; IR (KBr) cm⁻¹: 3273, 2529, 1663, 1538, 1441, 757; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.59 (s, 1H, SH), 10.34 (s, 1H, NH), 7.57 (d, 2H, Ar-H, J=8.1 Hz), 7.33 (t, 2H, Ar-H, J=7.6 Hz), 7.08 (t, 1H, Ar-H, J=7.3 Hz), 4.17 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₁₀N₃OS₃ [M+H]⁺ 284.0.

2-Sulfhydryl-5-[(2-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5b)

White solid; Yield 75.9%; mp 132.7–134.6°C; IR (KBr) cm⁻¹: 3339, 2546, 1653, 1542, 1442, 752; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.63 (s, 1H, SH), 9.91 (s, 1H, NH), 7.72 (d, 1H, Ar-H, J=7.9 Hz), 7.51 (d, 1H, Ar-H, J=7.9 Hz), 7.35 (t, 1H, Ar-H, J=7.5 Hz), 7.22 (t, 1H, Ar-H, J=7.5 Hz), 4.22 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₃OS₃Cl [M+H]⁺ 318.3.

2-Sulfhydryl-5-[(3-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5c)

White solid; Yield 73.2%; mp 157.7–159.8°C; IR (KBr) cm⁻¹: 3362, 2529, 1645, 1549, 1429, 778; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.60 (s, 1H, SH), 10.61 (s, 1H, NH), 7.78 (s, 1H, Ar-H), 7.44 (d, 1H, Ar-H, J=8.4 Hz), 7.36 (t, 1H, Ar-H, J=8.0 Hz), 7.15 (d, 1H, Ar-H, J=7.8 Hz), 4.18 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₃OS₃Cl [M+H]⁺ 318.3.

2-Sulfhydryl-5-[(4-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5d)

White solid; Yield 80.7%; mp 164.8–166.4°C; IR (KBr) cm⁻¹: 3242, 2522, 1658, 1542, 1490, 744; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.59 (s, 1H, SH), 10.49 (s, 1H, NH), 7.60 (d, 2H, Ar-H, J=8.8 Hz), 7.38 (t, 2H, Ar-H, J=8.8 Hz), 4.18 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₃OS₃Cl [M+H]⁺ 318.3.

2-Sulfhydryl-5-[(3-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5e)

Yellow solid; Yield 70.3%; mp 180.3–182.6°C; IR (KBr) cm⁻¹: 3339, 2548, 1681, 1548, 1432, 736; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.60 (s, 1H, SH), 10.85 (s, 1H, NH), 8.59 (s, 1H, Ar-H), 7.95 (d, 1H, Ar-H, J=7.9 Hz), 7.89 (d, 1H, Ar-H, J=8.2 Hz), 7.64 (t, 1H, Ar-H, J=8.2 Hz), 4.22 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₄O₃S₃ [M+H]⁺ 329.0.

2-Sulfhydryl-5-[(4-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5f)

Yellow solid; Yield 73.8%; mp 194.8–196.8°C; IR (KBr) cm⁻¹: 3323, 2541, 1696, 156, 1497, 751; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.61 (s, 1H, SH), 10.97 (s, 1H, NH), 8.24 (d, 2H, Ar-H, J=8.9 Hz), 7.82 (d, 2H, Ar-H, J=8.9 Hz), 4.22 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₄O₃S₃ [M+H]⁺ 329.0.

2-Sulfhydryl-5-[(2-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5g)

White solid; Yield 78.9%; mp 145.2–146.7°C; IR (KBr) cm⁻¹: 3302, 2540, 1668, 1543, 1489, 741; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.61 (s, 1H, SH), 9.58 (s, 1H, NH), 7.94 (d, 1H, Ar-H, J=8.0 Hz), 7.05–7.12 (m, 2H, Ar-H), 6.92 (t, 1H, Ar-H, J=7.1 Hz), 4.23 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃); MS-ESI (m/z): C₁₁H₁₂N₃O₂S₃ [M+H]⁺ 314.0.

2-Sulfhydryl-5-[(4-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5h)

White solid; Yield 74.5%; mp 157.2–158.5°C; IR (KBr) cm⁻¹: 3296, 2549, 1662, 1545, 1489, 721; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.60 (s, 1H, SH), 10.20 (s, 1H, NH), 7.47 (d, 2H, Ar-H, J=9.0 Hz), 6.90 (d, 2H, Ar-H, J=9.0 Hz), 3.84 (s, 2H, CH₂), 3.72 (s, 3H, OCH₃); MS-ESI (m/z): C₁₀H₉N₄O₃S₃ [M+H]⁺ 314.0.

2-Sulfhydryl-5-[(4-fluorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5i)

White solid; Yield 72.1%; mp 170.2–172.1°C; IR (KBr) cm⁻¹: 3249, 2524, 1658, 1553, 1510, 715; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.60 (s, 1H, SH), 10.40 (s, 1H, NH), 7.57–7.60 (d, 2H, Ar-H, J=9.0 Hz), 7.17 (t, 2H, Ar-H, J=8.8 Hz), 4.16 (s, 2H, CH₂); MS-ESI (m/z): C₁₀H₉N₃OS₃F [M+H]⁺ 302.0.

2-Sulfhydryl-5-[(4-trifluoromethylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5j)

White solid; Yield 70.8%; mp 171.5–172.2°C; IR (KBr) cm⁻¹: 3440, 2539, 1670, 1546, 1489, 715; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.60 (s, 1H, SH), 10.71 (s, 1H, NH), 7.78 (d, 2H, Ar-H, J=8.4 Hz), 7.70 (d, 2H, Ar-H, J=8.4 Hz), 4.23 (s, 2H, CH₂); MS-ESI (m/z): C₁₁H₉N₃OS₃F₃ [M+H]⁺ 352.0.

2-Sulfhydryl-5-[(4-methylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (5k)

White solid; Yield 77.6%; mp 178.5–180.2°C; IR (KBr) cm⁻¹: 3268, 2532, 1654, 1538, 1495, 719; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 14.59 (s, 1H, SH), 10.25 (s, 1H, NH), 7.45 (d, 2H, Ar-H, J=8.3 Hz), 7.13 (d, 2H, Ar-H, J=8.2 Hz), 4.14 (s, 2H, CH₂), 2.26 (s, 3H, CH₃); MS-ESI (m/z): C₁₁H₁₂N₃OS₃ [M+H]⁺ 298.0.

General Procedure for the Synthesis of Compounds 6a–k, 7a–k and 8a–k

S-Alkyl-thioisothiourea hydrochloride 2 (2.2 mmol) and 2-sulfhydryl-5-(arylaminoformoxyl methylthio)-1,3,4-thiadiazol 5a–k (2.0 mmol) were dissolved in 2 mL water and 15 mL ethanol. A solution of NaHCO₃ (3.0 mmol) in 4 mL water was added dropwise with vigorous stirring at room temperature. The mixture was stirred for additional 6h. The insoluble solid was collected and purified by silica gel column chromatography with petroleum ether–ethyl acetate (10 : 1, volume ratio) as eluent to afford the desired products.

2-(2-Butyldisulfanyl)-5-[(phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6a)

Yellow oily liquid; Yield 75.6%; IR (KBr) cm⁻¹: 3446, 1684, 1558, 1507, 755, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.37 (s, 1H, NH), 7.57 (d, 2H, Ar-H, J=7.8 Hz), 7.33 (t, 2H, Ar-H, J=7.7 Hz), 7.08 (t, 1H, Ar-H, J=7.4 Hz), 4.29 (s, 2H, CH₂), 3.12–3.21 (m, 1H, CH), 1.51–1.71 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.8 Hz), 0.92 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.27, 167.01, 165.49, 139.13, 129.28 (2C), 124.12, 119.66 (2C), 49.37, 38.82, 28.75, 20.13, 11.59; HR-MS (ESI): m/z 438.2515 [M+Na]⁺ (Calcd for C₁₄H₁₇N₃OS₄Na 438.2505).

2-(2-Butyldisulfanyl)-5-[(2-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6b)

Yellow oily liquid; Yield 72.8%; IR (KBr) cm⁻¹: 3447, 1699, 1588, 1523, 753, 526; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.95 (s, 1H, NH), 7.74 (d, 1H, Ar-H, J=8.0 Hz), 7.51 (d, 1H, Ar-H, J=8.0 Hz), 7.34 (t, 1H, Ar-H, J=8.4 Hz), 7.21 (t, 1H, Ar-H, J=7.5 Hz), 4.36 (s, 2H, CH₂), 3.13–3.21 (m, 1H, CH), 1.52–1.72 (m, 2H, CH₂), 1.30 (d, 3H, CH₃, J=6.8 Hz), 0.93 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.45, 166.85, 166.23, 134.97, 129.96, 127.91, 126.85, 126.43, 125.86, 49.40, 38.34, 28.79, 20.12, 11.59; HR-MS (ESI): m/z 427.9773 [M+Na]⁺ (Calcd for C₁₄H₁₆N₃OS₄NaCl 427.9763).

2-(2-Butyldisulfanyl)-5-[(3-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6c)

Yellow oily liquid; Yield 76.7%; IR (KBr) cm⁻¹: 3447, 1684, 1595, 1541, 779, 526; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.57 (s, 1H, NH), 7.79 (s, 1H, Ar-H), 7.43 (d, 1H, Ar-H, J=8.3 Hz), 7.36 (t, 1H, Ar-H, J=7.9 Hz), 7.14 (d, 1H, Ar-H, J=7.8 Hz), 4.29 (s, 2H, CH₂), 3.12–3.20 (m, 1H, CH), 1.51–1.71 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.8 Hz), 0.93 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.40, 166.74, 165.92, 140.55, 133.69, 130.91, 123.81, 119.16, 118.01, 49.39, 38.76, 28.77, 20.11, 11.57; HR-MS (ESI): m/z 427.9766 [M+Na]⁺ (Calcd for C₁₄H₁₆N₃OS₄NaCl 427.9763).

2-(2-Butyldisulfanyl)-5-[(4-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6d)

White solid; Yield 74.8%; mp 65.1–67.6°C; IR (KBr) cm⁻¹: 3447, 1667, 1541, 1490, 746, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.53 (s, 1H, NH), 7.62 (d, 2H, Ar-H, J=8.7 Hz), 7.33 (d, 2H, Ar-H, J=8.7 Hz), 4.30 (s, 2H, CH₂), 3.04–3.12 (m, 1H, CH), 1.46–1.67 (m, 2H, CH₂), 1.25 (d, 3H, CH₃, J=6.7 Hz), 0.88 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.41, 166.72, 165.62, 138.08, 129.11 (2C), 127.79, 121.14 (2C), 49.38, 38.78, 28.78, 20.09, 11.59; HR-MS (ESI): m/z 405.9958 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(2-Butyldisulfanyl)-5-[(3-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6e)

White solid; Yield 71.7%; mp 93.1–94.5°C; IR (KBr) cm⁻¹: 3439, 1694, 1605, 1525, 741, 509; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.90 (s, 1H, NH), 8.61 (s, 1H, Ar-H), 7.94 (d, 1H, Ar-H, J=8.1 Hz), 7.88 (t, 1H, Ar-H, J=8.0 Hz), 7.63 (d, 1H, Ar-H, J=8.2 Hz), 4.34 (s, 2H, CH₂), 3.11–3.19 (m, 1H, CH), 1.50–1.70 (m, 2H, CH₂), 1.28 (d, 3H, CH₃, J=6.7 Hz), 0.91 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.43, 166.61, 166.31, 148.40, 140.23, 130.64, 125.51, 118.53, 113.71, 49.38, 38.68, 28.75, 20.07, 11.53; HR-MS (ESI): m/z 417.0198 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(2-Butyldisulfanyl)-5-[(4-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6f)

White solid; Yield 75.4%; mp 107.0–108.9°C; IR (KBr) cm⁻¹: 3441, 1680, 1563, 1511, 751, 497; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.98 (s, 1H, NH), 8.24 (d, 2H, Ar-H, J=9.2 Hz), 7.83 (d, 2H, Ar-H, J=9.2 Hz), 4.37 (s, 2H, CH₂), 3.11–3.19 (m, 1H, CH), 1.50–1.70 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.8 Hz), 0.91 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.44, 166.47, 165.11, 145.18, 142.88, 125.28 (2C), 119.28 (2C), 49.39, 38.85, 28.77, 20.04, 11.53; HR-MS (ESI): m/z 417.0197 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(2-Butyldisulfanyl)-5-[(2-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6g)

Yellow oily liquid; Yield 70.1%; IR (KBr) cm⁻¹: 3364, 1687, 1602, 1536, 749, 512; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.62 (s, 1H, NH), 7.97 (d, 1H, Ar-H, J=8.0 Hz), 7.04–7.12 (m, 2H, Ar-H), 6.91 (t, 1H, Ar-H, J=8.1 Hz), 4.35 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃), 3.12–3.21 (m, 1H, CH), 1.51–1.72 (m, 2H, CH₂), 1.30 (d, 3H, CH₃, J=6.8 Hz), 0.92 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.33, 167.16, 165.85, 149.80, 127.42, 125.12, 121.76, 120.77, 111.69, 56.20, 49.38, 38.63, 28.75, 20.12, 11.57; HR-MS (ESI): m/z 402.0448 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(2-Butyldisulfanyl)-5-[(4-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6h)

Yellow oily liquid; Yield 74.8%; IR (KBr) cm⁻¹: 3437, 1665, 1549, 1512, 745, 521; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.25 (s, 1H, NH), 7.48 (d, 2H, Ar-H, J=8.8 Hz), 6.89 (d, 2H, Ar-H, J=8.9 Hz), 4.25 (s, 2H, CH₂), 3.72 (s, 3H, OCH₃), 3.12–3.20 (m, 1H, CH), 1.51–1.71 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.7 Hz), 0.92 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.25, 167.08, 164.96, 155.98, 132.27, 121.21 (2C), 114.41 (2C), 55.64, 49.36, 38.74, 28.75, 20.12, 11.58; HR-MS (ESI): m/z 402.0440 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(2-Butyldisulfanyl)-5-[(4-fluorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6i)

White solid; Yield 73.6%; mp 67.6–68.6°C; IR (KBr) cm⁻¹: 3444, 1666, 1558, 1508, 745, 515; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.44 (s, 1H, NH), 7.59 (dd, 2H, Ar-H, J₁=5.0 Hz, J₂=9.0 Hz), 7.17 (d, 2H, Ar-H, J=8.8 Hz), 4.28 (s, 2H, CH₂), 3.12–3.21 (m, 1H, CH), 1.51–1.74 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.7 Hz), 0.92 (t, 3H, CH₃, J=7.32 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.33, 166.85, 165.40, 158.68 (d, J_C–F=239.1 Hz), 135.53 (d, J_C–F=2.3 Hz), 121.43 (d, 2C, J_C–F=7.8 Hz), 115.79 (d, 2C, J_C–F=22.1 Hz), 49.38, 38.73, 28.76, 20.08, 11.56; HR-MS (ESI): m/z 390.0245 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄F 390.0238).

2-(2-Butyldisulfanyl)-5-[(4-trifluoromethylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6j)

White solid; Yield 74.5%; mp 82.7–84.1°C; IR (KBr) cm⁻¹: 3446, 1676, 1606, 1543, 709, 506; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.75 (s, 1H, NH), 7.79 (d, 2H, Ar-H, J=8.5 Hz), 7.69 (d, 2H, Ar-H, J=8.6 Hz), 4.34 (s, 2H, CH₂), 3.10–3.19 (m, 1H, CH), 1.49–1.70 (m, 2H, CH₂), 1.28 (d, 3H, CH₃, J=6.7 Hz), 0.91 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.41, 166.64, 166.16, 142.66, 126.45 (q, 2C, J_C–F=3.5 Hz), 124.70 (q, J_C–F=269.8 Hz), 124.24 (q, 2C, J_C–F=31.9 Hz), 119.52, 49.35, 38.82, 28.74, 19.99, 11.45; HR-MS (ESI): m/z 440.0216 [M+H]⁺ (Calcd for C₁₅H₁₇N₃OS₄F₃ 440.0207).

2-(2-Butyldisulfanyl)-5-[(4-methylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (6k)

White solid; Yield 72.1%; mp 82.7–84.1°C; IR (KBr) cm⁻¹: 3442, 1668, 1610, 1542, 747, 510; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.30 (s, 1H, NH), 7.45 (d, 2H, Ar-H, J=8.2 Hz), 7.13 (d, 2H, Ar-H, J=8.1 Hz), 4.27 (s, 2H, CH₂), 3.12–3.20 (m, 1H, CH), 2.25 (s, 3H, CH₃), 1.51–1.71 (m, 2H, CH₂), 1.29 (d, 3H, CH₃, J=6.8 Hz), 0.92 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 172.26, 167.01, 165.20, 136.66, 133.06, 129.62 (2C), 119.67 (2C), 49.38, 38.85, 28.77, 20.92, 20.11, 11.58; HR-MS (ESI): m/z 386.0498 [M+H]⁺ (Calcd for C₁₅H₂₀N₃OS₄ 386.0489).

2-(n-Butyldisulfanyl)-5-[(phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7a)

White solid; Yield 72.3%; mp 89.7–91.5°C; IR (KBr) cm⁻¹: 3447, 1681, 1600, 1542, 753, 502; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.38 (s, 1H, NH), 7.57 (d, 2H, Ar-H, J=7.72 Hz), 7.33 (t, 2H, Ar-H, J=7.6 Hz), 7.08 (t, 1H, Ar-H, J=7.4 Hz), 4.29 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.61–1.68 (m, 2H, CH₂), 1.32–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.65, 167.05, 165.46, 139.18, 129.21 (2C), 124.06, 119.67 (2C), 38.93, 38.90, 30.90, 21.37, 13.87; HR-MS (ESI): m/z 372.0346 [M+H]⁺ (Calcd for C₁₄H₁₈N₃OS₄ 372.0333).

2-(n-Butyldisulfanyl)-5-[(2-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7b)

White solid; Yield 71.9%; mp 68.8–69.7°C; IR (KBr) cm⁻¹: 3446, 1684, 1558, 1507, 757, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.95 (s, 1H, NH), 7.74 (d, 1H, Ar-H, J=7.1 Hz), 7.51 (d, 1H, Ar-H, J=8.0 Hz), 7.35 (t, 1H, Ar-H, J=8.6 Hz), 7.22 (t, 1H, Ar-H, J=6.6 Hz), 4.36 (s, 2H, CH₂), 3.01 (t, 2H, CH₂, J=7.2 Hz), 1.61–1.69 (m, 2H, CH₂), 1.32–1.41 (m, 2H, CH₂), 0.87 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.81, 167.02, 166.26, 134.95, 129.99, 127.95, 126.95, 126.53, 126.00, 38.85, 38.31, 30.87, 21.31, 13.86; HR-MS (ESI): m/z 405.9955 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(n-Butyldisulfanyl)-5-[(3-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7c)

White solid; Yield 73.7%; mp 80.6–81.8°C; IR (KBr) cm⁻¹: 3446, 1683, 1594, 1541, 779, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.57 (s, 1H, NH), 7.79 (s, 1H, Ar-H), 7.43 (d, 1H, Ar-H, J=8.3 Hz), 7.36 (t, 1H, Ar-H, J=7.9 Hz), 7.15 (d, 1H, Ar-H, J=7.8 Hz), 4.30 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.61–1.68 (m, 2H, CH₂), 1.32–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.72, 166.95, 165.97, 140.55, 133.66, 130.99, 123.84, 119.14, 118.04, 38.85, 38.72, 30.86, 21.29, 13.85; HR-MS (ESI): m/z 405.9955 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(n-Butyldisulfanyl)-5-[(4-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7d)

White solid; Yield 73.6%; mp 102.7–103.4°C; IR (KBr) cm⁻¹: 3447, 1663, 1569, 1541, 745, 510; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.51 (s, 1H, NH), 7.61 (d, 2H, Ar-H, J=8.8 Hz), 7.39 (d, 2H, Ar-H, J=8.8 Hz), 4.29 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.61–1.68 (m, 2H, CH₂), 1.32–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.70, 166.91, 165.64, 138.07, 129.13 (2C), 127.80, 121.18 (2C), 38.91, 38.80, 30.88, 21.35, 13.85; HR-MS (ESI): m/z 405.9955 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(n-Butyldisulfanyl)-5-[(3-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7e)

White solid; Yield 72.8%; mp 44.9–46.3°C; IR (KBr) cm⁻¹: 3426, 1683, 1614, 1527, 737, 511; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.89 (s, 1H, NH), 8.61 (s, 1H, Ar-H), 7.94 (d, 1H, Ar-H, J=8.0 Hz), 7.89 (d, 1H, Ar-H, J=8.1 Hz), 7.63 (t, 1H, Ar-H, J=8.2 Hz), 4.34 (s, 2H, CH₂), 2.98 (t, 2H, CH₂, J=7.2 Hz), 1.59–1.66 (m, 2H, CH₂), 1.30–1.39 (m, 2H, CH₂), 0.85 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 166.41, 166.35, 165.23, 148.40, 140.22, 130.67, 125.54, 118.56, 113.72, 38.88, 38.61, 30.85, 21.29, 13.80; HR-MS (ESI): m/z 417.0197 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(n-Butyldisulfanyl)-5-[(4-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7f)

White solid; Yield 73.2%; mp 118.9–121.7°C; IR (KBr) cm⁻¹: 3442, 1686, 1566, 1504, 751, 498; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 11.0 (s, 1H, NH), 8.25 (d, 2H, Ar-H, J=9.2 Hz), 7.83 (d, 2H, Ar-H, J=9.2 Hz), 4.37 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.60–1.67 (m, 2H, CH₂), 1.31–1.40 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.77, 166.71, 166.57, 145.21, 142.92, 125.40 (2C), 119.34 (2C), 38.87, 38.81, 30.86, 21.31, 13.82; HR-MS (ESI): m/z 417.0194 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(n-Butyldisulfanyl)-5-[(2-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7g)

Yellow oily liquid; Yield 72.6%; IR (KBr) cm⁻¹: 3402, 1687, 1602, 1536, 749, 513; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.63 (s, 1H, NH), 7.97 (d, 1H, Ar-H, J=8.0 Hz), 7.04–7.12 (m, 2H, Ar-H), 6.91 (t, 1H, Ar-H, J=8.1 Hz), 4.35 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.61–1.68 (m, 2H, CH₂), 1.32–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.69, 167.28, 165.87, 149.79, 127.44, 125.10, 121.75, 120.76, 111.67, 56.19, 38.84, 38.63, 30.86, 21.30, 13.85; HR-MS (ESI): m/z 402.0454 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(n-Butyldisulfanyl)-5-[(4-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7h)

White solid; Yield 69.7%; mp 92.0–93.6°C; IR (KBr) cm⁻¹: 3430, 1674, 1602, 1512, 828, 521; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.25 (s, 1H, NH), 7.48 (d, 2H, Ar-H, J=8.9 Hz), 6.90 (t, 2H, Ar-H, J=8.9 Hz), 4.25 (s, 2H, CH₂), 3.72 (s, 3H, OCH₃), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.60–1.68 (m, 2H, CH₂), 1.31–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.64, 167.12, 164.94, 155.99, 132.30, 121.23 (2C), 114.34 (2C), 55.59, 38.92, 38.81, 30.90, 21.37, 13.85; HR-MS (ESI): m/z 402.0452 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(n-Butyldisulfanyl)-5-[(4-fluorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7i)

White solid; Yield 74.3%; mp 101.7–103.0°C; IR (KBr) cm⁻¹: 3430, 1664, 1558, 1509, 838, 518; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.45 (s, 1H, NH), 7.59 (dd, 2H, Ar-H, J₁=5.0 Hz, J₂=9.0 Hz), 7.17 (d, 2H, Ar-H, J=8.8 Hz), 4.28 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.60–1.68 (m, 2H, CH₂), 1.31–1.40 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.70, 166.94, 165.38, 158.70 (d, J_C–F=239.2 Hz), 135.51 (d, J_C–F=2.0 Hz), 121.43 (d, 2C, J_C–F=7.7 Hz), 115.74 (d, 2C, J_C–F=22.2 Hz), 38.92, 38.75, 30.88, 21.35, 13.81; HR-MS (ESI): m/z 390.0252 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄F 390.0238).

2-(n-Butyldisulfanyl)-5-[(4-trifluoromethylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (7j)

White solid; Yield 71.2%; mp 105.9–107.0°C; IR (KBr) cm⁻¹: 344, 1684, 1608, 1543, 840, 506; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.76 (s, 1H, NH), 7.79 (d, 2H, Ar-H, J=8.6 Hz), 7.7 (d, 2H, Ar-H, J=8.6 Hz), 4.34 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 1.60–1.67 (m, 2H, CH₂), 1.31–1.40 (m, 2H, CH₂), 0.85 (t, 3H, CH₃, J=7.4 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.72, 166.91, 166.23, 142.67, 126.57 (q, 2C, J_C–F=3.6 Hz), 124.74 (q, J_C–F=269.7 Hz), 124.18 (q, 2C, J_C–F=31.8 Hz), 119.56, 38.83, 38.78, 30.84, 21.27, 13.78; HR-MS (ESI): m/z 440.0217 [M+H]⁺ (Calcd for C₁₅H₁₇N₃OS₄F₃ 440.0207).

2-(n-Butyldisulfanyl)-5-[(4-methylphenylcarbamoyl)-methylhio]-1,3,4-thiadiazole (7k)

White solid; Yield 70.7%; mp 79.7–82.0°C; IR (KBr) cm⁻¹: 3450, 1677, 1605, 1544, 817, 506, 7.46 (d, 2H, Ar-H, J=8.3 Hz), 7.13 (d, 2H, Ar-H, J=8.2 Hz), 4.27 (s, 2H, CH₂), 3.0 (t, 2H, CH₂, J=7.2 Hz), 2.25 (s, 3H, CH₃), 1.60–1.68 (m, 2H, CH₂), 1.31–1.41 (m, 2H, CH₂), 0.86 (t, 3H, CH₃, J=7.3 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.60, 167.14, 165.21, 136.67, 133.06, 129.62 (2C), 119.66 (2C), 39.01, 38.87, 30.88, 21.33, 20.91, 13.86; HR-MS (ESI): m/z 386.0498 [M+H]⁺ (Calcd for C₁₅H₂₀N₃OS₄ 386.0489).

2-(i-Butyldisulfanyl)-5-[(phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8a)

White solid; Yield 73.6%; mp 90.8–92.1°C; IR (KBr) cm⁻¹: 3447, 1682, 1600, 1541, 751, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.38 (s, 1H, NH), 7.57 (d, 2H, Ar-H, J=8.0 Hz), 7.33 (t, 2H, Ar-H, J=7.6 Hz), 7.08 (t, 1H, Ar-H, J=7.3 Hz), 4.30 (s, 2H, CH₂), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.85–1.98 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.44, 167.16, 165.48, 139.16, 129.25 (2C), 124.09, 119.68 (2C), 48.22, 38.87, 28.16, 21.68 (2C); HR-MS (ESI): m/z 372.0349 [M+H]⁺ (Calcd for C₁₄H₁₈N₃OS₄ 372.0333).

2-(i-Butyldisulfanyl)-5-[(2-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8b)

White solid; Yield 70.4%; mp 90.7–91.7°C; IR (KBr) cm⁻¹: 3447, 1685, 1590, 1524, 757, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.95 (s, 1H, NH), 7.75 (d, 1H, Ar-H, J=7.8 Hz), 7.50 (d, 1H, Ar-H, J=7.4 Hz), 7.34 (t, 1H, Ar-H, J=7.3 Hz), 7.21 (t, 1H, Ar-H, J=7.3 Hz), 4.37 (s, 2H, CH₂), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.85–1.99 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.7 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.65, 166.98, 166.21, 135.00, 129.93, 127.87, 126.74, 126.29, 125.70, 48.30, 38.40, 28.20, 21.70 (2C); HR-MS (ESI): m/z 405.9941 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(n-Butyldisulfanyl)-5-[(3-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8c)

White solid; Yield 68.7%; mp 62.8–64.5°C; IR (KBr) cm⁻¹: 3447, 1684, 1593, 1541, 779, 503; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.57 (s, 1H, NH), 7.79 (s, 1H, Ar-H), 7.42 (d, 1H, Ar-H, J=8.3 Hz), 7.36 (t, 1H, Ar-H, J=8.0 Hz), 7.14 (d, 1H, Ar-H, J=7.9 Hz), 4.30 (s, 2H, CH₂), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.85–1.98 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.7 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.54, 166.98, 165.95, 140.55, 133.67, 130.97, 123.83, 119.15, 118.04, 48.20, 38.74, 28.15, 21.67 (2C); HR-MS (ESI): m/z 405.9958 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(i-Butyldisulfanyl)-5-[(4-chlorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8d)

White solid; Yield 69.8%; mp 101.6–103.5°C; IR (KBr) cm⁻¹: 3447, 1663, 1558, 1541, 836, 510; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.54 (s, 1H, NH), 7.61 (d, 2H, Ar-H, J=8.8 Hz), 7.38 (d, 2H, Ar-H, J=8.8 Hz), 4.30 (s, 2H, CH₂), 2.89 (d, 2H, CH₂, J=6.8 Hz), 1.83–1.97 (m, 1H, CH), 0.95 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.55, 167.06, 165.72, 138.09, 129.23 (2C), 127.71, 121.17 (2C), 48.10, 38.71, 28.13, 21.67 (2C); HR-MS (ESI): m/z 405.9950 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄Cl 405.9943).

2-(i-Butyldisulfanyl)-5-[(3-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8e)

White solid; Yield 65%; mp 44.7–46.5°C; IR (KBr) cm⁻¹: 3431, 1692, 1620, 1528, 737, 514; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.89 (s, 1H, NH), 8.61 (s, 1H, Ar-H), 7.95 (d, 1H, Ar-H, J=8.0 Hz), 7.89 (d, 1H, Ar-H, J=7.8 Hz), 7.64 (t, 1H, Ar-H, J=8.2 Hz), 4.35 (s, 2H, CH₂), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.84–1.98 (m, 1H, CH), 0.95 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.61, 166.84, 166.34, 148.41, 140.22, 130.68, 125.54, 118.56, 113.72, 48.21, 38.65, 28.14, 21.62 (2C); HR-MS (ESI): m/z 417.0195 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(i-Butyldisulfanyl)-5-[(4-nitro-phenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8f)

White solid; Yield 71.8%; mp 120.9–122.7°C; IR (KBr) cm⁻¹: 3441, 1687, 1615, 1504, 751, 497; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.98 (s, 1H, NH), 8.24 (d, 2H, Ar-H, J=9.2 Hz), 7.83 (d, 2H, Ar-H, J=9.2 Hz), 4.37 (s, 2H, CH₂), 2.90 (d, 2H, CH₂, J=6.8 Hz), 1.84–1.97 (m, 1H, CH), 0.95 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.60, 166.75, 166.54, 145.20, 142.91, 125.39 (2C), 119.34 (2C), 48.21, 38.81, 28.15, 21.63 (2C); HR-MS (ESI): m/z 417.0187 [M+H]⁺ (Calcd for C₁₄H₁₇N₄O₃S₄ 417.0183).

2-(i-Butyldisulfanyl)-5-[(2-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8g)

White solid; Yield 65.3%; mp 51.1–53.0°C; IR (KBr) cm⁻¹: 3374, 1675, 1602, 1529, 746, 512; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 9.62 (s, 1H, NH), 7.97 (d, 1H, Ar-H, J=7.32 Hz), 7.05–7.12 (m, 2H, Ar-H), 6.91 (t, 1H, Ar-H, J=8.1 Hz), 4.35 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃), 2.92 (d, 2H, CH₂, J=6.8 Hz), 1.86–1.99 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.7 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.52, 167.28, 165.83, 149.70, 127.51, 125.02, 121.64, 120.76, 111.60, 56.17, 48.24, 38.68, 28.17, 21.67 (2C); HR-MS (ESI): m/z 402.0449 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(i-Butyldisulfanyl)-5-[(4-methoxyphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8h)

White solid; Yield 70.5%; mp 95.0–95.9°C; IR (KBr) cm⁻¹: 3431, 1673, 1603, 1511, 829, 522; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.25 (s, 1H, NH), 7.48 (d, 2H, Ar-H, J=8.8 Hz), 6.90 (t, 2H, Ar-H, J=8.9 Hz), 4.26 (s, 2H, CH₂), 3.72 (s, 3H, OCH₃), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.85–1.98 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.14, 167.19, 164.94, 155.98, 132.30, 121.23 (2C), 114.37 (2C), 55.61, 48.23, 38.80, 28.17, 21.67 (2C); HR-MS (ESI): m/z 402.0443 [M+H]⁺ (Calcd for C₁₅H₂₀N₃O₂S₄ 402.0438).

2-(i-Butyldisulfanyl)-5-[(4-fluorophenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8i)

White solid; Yield 75.6%; mp 89.9–91.3°C; IR (KBr) cm⁻¹: 3431, 1680, 1557, 1500, 834, 515; ¹H-NMR 400 MHz, DMSO-d₆): δ (ppm): 10.45 (s, 1H, NH), 7.59 (dd, 2H, Ar-H, J₁=5.0 Hz, J₂=9.2 Hz), 7.17 (d, 2H, Ar-H, J=8.8 Hz), 4.29 (s, 2H, CH₂), 2.91 (d, 2H, CH₂, J=6.8 Hz), 1.85–1.98 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆): δ (ppm): 171.50, 167.04, 165.40, 158.68 (d, J_C–F=239.0 Hz), 135.53 (d, J_C–F=2.1 Hz), 121.44 (d, 2C, J_C–F=7.7 Hz), 115.79 (d, 2C, J_C–F=22.1 Hz), 48.22, 38.74, 28.16, 21.65 (2C); HR-MS (ESI): m/z 390.0246 [M+H]⁺ (Calcd for C₁₄H₁₇N₃OS₄F 390.0238).

2-(i-Butyldisulfanyl)-5-[(4-trifluoromethylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8j)

White solid; Yield 73.6%; mp 94.3–96.3°C; IR (KBr) cm⁻¹: 3429, 1685, 1606, 1544, 840, 506; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.76 (s, 1H, NH), 7.79 (d, 2H, Ar-H, J=8.6 Hz), 7.7 (d, 2H, Ar-H, J=8.6 Hz), 4.34 (s, 2H, CH₂), 2.90 (d, 2H, CH₂, J=6.8 Hz), 1.84–1.97 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.55, 166.97, 166.25, 142.67, 126.61 (q, 2C, J_C–F=3.7 Hz), 124.75 (q, J_C–F=269.7 Hz), 124.16 (q, 2C, J_C–F=31.9 Hz), 119.56, 48.15, 38.77, 28.13, 21.63 (2C); HR-MS (ESI): m/z 440.0204 [M+H]⁺ (Calcd for C₁₅H₁₇N₃OS₄F₃ 440.0207).

2-(i-Butyldisulfanyl)-5-[(4-methylphenylcarbamoyl)-methylthio]-1,3,4-thiadiazole (8k)

White solid; Yield 70.9%; mp 72.5–74.3°C; IR (KBr) cm⁻¹: 3440, 1677, 1602, 1542, 817, 507; ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 10.30 (s, 1H, NH), 7.46 (d, 2H, Ar-H, J=8.2 Hz), 7.12 (d, 2H, Ar-H, J=8.2 Hz), 4.27 (s, 2H, CH₂), 2.90 (d, 2H, CH₂, J=6.8 Hz), 2.25 (s, 3H, CH₃), 1.85–1.98 (m, 1H, CH), 0.96 (d, 6H, CH₃, J=6.6 Hz); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 171.42, 167.19, 165.20, 136.67, 133.06, 129.62 (2C), 119.67 (2C), 48.21, 38.86, 28.16, 21.67 (2C); HR-MS (ESI): m/z 386.0496 [M+H]⁺ (Calcd for C₁₅H₂₀N₃OS₄ 386.0489).

Cell Culture and CCK-8 Bioassay

The following established in vitro human cancer cell lines and normal cell lines were used: A549, HeLa, SMMC-7721 and L929, which were obtained from Tumor Cell Resources Bank, Chinese Academy of Medical Sciences. Cell counting kit-8 was from Dojindo (Japan).

Compounds 6a–k, 7a–k and 8a–k, and 5-FU were respectively dissolved in DMSO to make stock solutions at a concentration of 1.0×10⁻² mol/L. During the experiment, cell culture medium RPMI-1640 was used to dilute the stock solution to the desired concentration. Cells in the exponential phase were seeded in 96-well culture plates at the confluence of 1×10⁴ cells/well, kept in 37°C, 5% CO₂ incubator for 24 h. Replaced the medium containing different concentrations of compounds in fresh medium, at 37°C, 5% CO₂ incubator for 48 h. Then added 90 µL of fresh medium and 10 µL of CCK-8 into culture plates and cultivated at the same conditions for 1h. The sample cell was added into 96-well microplate reader and read the plate at 450 nm, recorded the absorbance value (optical density (OD)). Cell viability was calculated from the mean values for three wells using the following formula:

The IC₅₀ value is defined as the concentration that causes a 50% cell proliferation inhibition.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (20971097) and Tianjin Municipal Natural Science Foundation (13JCYBJC24500) for financial support.

Conflict of Interest

The authors declare no conflict of interest.

References

1) Ren F., Zhong Y., Mai X., Liao Y. J., Liu C., Feng L. H., Sun W., Zen W. B., Liu W. M., Liu J., Jin N., Chem. Pharm. Bull., 62, 898–905 (2014).
2) Anand P., Kunnumakkara A. B., Sundaram C., Harikumar K. B., Tharakan S. T., Lai O. S., Sung B., Aggarwal B. B., Pharm. Res., 25, 2097–2116 (2008).
3) Li Z., Su H., Yu W., Li X., Cheng H., Liu M., Pang X., Zou X., Org. Biomol. Chem., 14, 277–287 (2016).
4) Hamam F., Food Nutr. Sci., 5, 2257–2264 (2014).
5) Hamama W. S., Gouda M. A., Badr M. H., Zoorob H. H., Med. Chem. Res., 22, 3556–3565 (2013).
6) Khan I., Ali S., Hameed S., Rama N. H., Hussain M. T., Wadood A., Uddin R., Ul-Haq Z., Khan A., Ali S., Choudhary M. R., Eur. J. Med. Chem., 45, 5200–5207 (2010).
7) Patel H., Mishra L., Noolvi M., Karpoormath R., Cameotra S. S., Arch. Pharm. Chem. Life Sci., 347, 668–684 (2014).
8) Chandrakantha B., Isloor A. M., Shetty P., Fun H. K., Hegde G., Eur. J. Med. Chem., 71, 316–323 (2014).
9) Yusuf M., Khan R. A., Ahmed B., Bioorg. Med. Chem., 16, 8029–8034 (2008).
10) Clerici F., Pocar D., Guido M., Loche A., Perlini V., Brufani M., J. Med. Chem., 44, 931–936 (2001).
11) Pattn S. R., Kittur B. S., Sastry B. S., Jadav S. G., Thakur D. K., Madamwar S. A., Shinde H. V., Indian J. Chem., 50B, 615–618 (2011).
12) Lee J., Lee S. H., Seo H. J., Son E. J., Lee S. H., Jung M. E., Lee M. W., Han H. K., Kim J., Kang J., Lee J., Bioorg. Med. Chem., 18, 2178–2194 (2010).
13) Güzeldemirci N. U., Satana D., Küçükbasmacı O., J. Enzyme Inhib. Med. Chem., 28, 968–973 (2013).
14) Wang C. X., Song H. L., Liu W. Q., Xu C. L., Chem. Res. Chin. Univ., 32, 615–620 (2016).
15) Harish K. P., Mohana K. N., Mallesha L., Russ. J. Bioorganic Chem., 40, 97–105 (2014).
16) Siddiqui N., Ahuja P., Malik S., Arya S. K., Arch. Pharm. Chem. Life Sci., 346, 819–831 (2013).
17) Dekhane D. V., Pawar S. S., Gupta S., Shingare M. S., Patil C. R., Thore S. N., Bioorg. Med. Chem. Lett., 21, 6527–6532 (2011).
18) Amir M., Kumar H., Javed S. A., Arch. Pharm. Chem. Life Sci., 340, 577–585 (2007).
19) Skrzypek A., Matysiak J., Karpińska M. M., Niewiadomy A., J. Enzyme Inhib. Med. Chem., 28, 816–823 (2013).
20) Tahghighi A., Emami S., Razmi S., Marznaki F. R., Ardestani S. K., Dastmalchi S., Kobarfard F., Shafiee A., Foroumadi A., J. Enzyme Inhib. Med. Chem., 28, 843–852 (2013).
21) Zuo Y., Yang S. G., Jiang L. L., Hao G. F., Wang Z. F., Wu Z., Xi Q. Y., Yang G. F., Bioorg. Med. Chem., 20, 296–304 (2012).
22) Hosseinzadeh L., Khorand A., Aliabadi A., Arch. Pharm. Chem. Life Sci., 346, 812–818 (2013).
23) Zhao H. C., Shi Y. P., Liu Y. M., Li C. W., Xuan L. N., Wang P., Zhang K., Chen B. Q., Bioorg. Med. Chem. Lett., 23, 6577–6579 (2013).
24) Shi Y. J., Song X. J., Li X., Ye T. H., Xiong Y., Yu L. T., Chem. Pharm. Bull., 61, 1099–1104 (2013).
25) Revelant G., Gadais C., Mathieu V., Kirsch G., Hesse S., Bioorg. Med. Chem. Lett., 24, 2724–2727 (2014).
26) Gomha S. M., Kheder N. A., Abdelhamid A. O., Mabkhot Y. N., Molecules, 21, 1532–1539 (2016).
27) Li Y., Geng J., Liu Y., Yu S., Zhao G., ChemMedChem, 8, 27–41 (2013).
28) Park H. B., Kwon H. C., Lee C. H., Yang H. O., J. Nat. Prod., 72, 248–252 (2009).
29) Turos E., Revell K. D., Ramaraju P., Gergeres D. A., Greenhalgh K., Young A., Sathyanarayan N., Dickey S., Lim D., Alhamadsheh M. M., Reynolds K., Bioorg. Med. Chem., 16, 6501–6508 (2008).
30) Cesarini S., Spallarossa A., Ranise A., Schenone S., Bruno O., La Colla P., Casula L., Collu G., Sanna G., Loddo R., Bioorg. Med. Chem., 16, 6353–6363 (2008).
31) Ryu E. K., Choe Y. S., Byun S. S., Lee K. H., Chi D. Y., Choi Y., Kim B. T., Bioorg. Med. Chem., 12, 859–864 (2004).
32) Hunter R., Kaschula C. H., Parker I. M., Caira M. R., Richards P., Travis S., Taute F., Qwebani T., Bioorg. Med. Chem. Lett., 18, 5277–5279 (2008).
33) DiRaimondo T. R., Plugis N. M., Jin X., Khosla C. J., Med. Chem., 56, 1301–1310 (2013).
34) Li Z. S., Wang W. M., Lu W., Niu C. W., Li Y. H., Li Z. M., Wang J. G., Bioorg. Med. Chem. Lett., 23, 3723–3727 (2013).
35) Shang J., Wang W. M., Li Y. H., Song H. B., Li Z. M., Wang J. G., J. Agric. Food Chem., 60, 8286–8293 (2012).
36) Zhu S. J., Ying H. Z., Wu Y., Qiu N., Liu T., Yang B., RSC Adv., 5, 103172–103183 (2015).
37) Zhao J. J., Wang X. F., Li B. L., Zhang R. L., Li B., Liu Y. M., Li C. W., Liu J. B., Chen B. Q., Bioorg. Med. Chem. Lett., 26, 4414–4416 (2016).
38) Sirakawa K., Aki O., Tsujikawa T., Tsuda T., Chem. Pharm. Bull., 18, 235–242 (1970).
39) Baraldi P. G., Preti D., Tabrizi M. A., Fruttarolo F., Saponaro G., Baraldi S., Romagnoli R., Moorman A. R., Gessi S., Varani K., Borea P. A., Bioorg. Med. Chem., 15, 2514–2527 (2007).

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