Drug repurposing: Discovery of troxipide analogs as potent antitumor agents.

Drug repurposing plays a vital role in the discovery of undescribed bioactivities in clinical drugs. Based on drug repurposing strategy, we for the first time reported a novel series of troxipide analogs and then evaluated their antiproliferative activity against MCF-7, PC3, MGC-803, and PC9 cancer cell lines and WPMY-1, most of which showed obvious selectivity toward PC-3 over the other three cancer cell lines and WPMY-1. Compound 5q, especially, could effectively inhibit PC3 with an IC50 value of 0.91 μM, which exhibited around 53-fold selectivity toward WPMY-1. Data indicated that 5q effectively inhibited the colony formation, suppressed the cell migration, and induced G1/S phase arrest in PC3 cells. Also, compound 5q induced cell apoptosis by activating the two apoptotic signaling pathways in PC3 cells: death receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway. Compound 5q up-regulated the expression of both pro-apoptotic Bax and P53, while down-regulated anti-apoptotic Bcl-2 expression. Besides, compound 5q significantly increased the expression of cleaved caspase 3/9 and cleaved PARP. Therefore, the successful discovery of compound 5q may further validate the feasibility of this theory, which will encourage researchers to reveal undescribed bioactivities in traditional drugs.

Introduction

The rapid development of technology and enhanced knowledge of human diseases fails to meet therapeutic advances, since the new drug discovery will be subjected to the high attrition, plenty of costs, and slow pace process [1,2]. Drug repurposing, an effective strategy for the discovery of undescribed bioactivities in clinical drugs, has attracted a lot of researchers to devote themselves to seeking novel applications of traditional drugs, which offers many advantages over the development of an entirely novel drug [1,3]. First, the most important advantage of drug repurposing is the lower risk of failure due to the fact that the repurposed drug has been validated safe enough in preclinical models and humans. Second, as most of the preclinical testing and safety assessment has already been completed, the required length of time for novel drug development will be significantly reduced. Third, the costs of the overall development of the repurposing candidate will markedly decrease due to the above two advantages. The proportion of drugs developed based on drug repurposing theory is probably around 75% [4]. Historically, drug repurposing strategy has achieved great success, resulting in a lot of promising candidate drugs, some of which have been used for the treatment of both common and rare diseases [[5], [6], [7], [8]]. As one of the most successful examples of drug repurposing so far, sildenafil citrate, originally developed as an antihypertensive drug by Pfizer, was repurposed for the effective treatment of erectile dysfunction [9]. Thalidomide, originally used for the treatment of sedative in 1957, was infamous for its serious side effect of leading to severe skeletal birth defects in children born to mothers who had taken the drug the first trimester of their pregnancies, which was serendipitously discovered to be effective for the treatment of multiple myeloma [10], subsequently generating some more successful derivatives, such as lenalidomide, with worldwide sales of up to $8.2 billion in 2017 [11]. Based on drug repurposing strategy, dolutegravir and dictegravir, anti-HIV drugs targeting integrase, were developed as the first PA endonuclease inhibitor—Baloxavir marboxil, which was approved in 2018 by Japan for the treatment of influenza A and B virus infections in paediatric and adult patients [12]. Besides, given the pretty severe situation facing the world, there is still no approved effective drug against COVID-19. How to quickly obtain effective compounds for the treatment of COVID-19 remains a challenge. Therefore, drug repurposing, as a fast and effective strategy to reveal new indications of traditional drugs, has been successfully exploited to identify novel application of ‘old’ drugs for the treatment of COVID-19, generating some potent candidates, such as chloroquineor or hydroxychloroquine, Remdesivir, Favipiravir, and Tocilizumab [13,14]. Chloroquine or hydroxychloroquine, clinically used as an antimalarial drug, has been found to effectively inhibit COVID-19 in vitro [15,16]. As an antiviral agent, Remdesivir, initially researched in Ebola virus clinical studies, showed potent inhibitory activity against COVID-19 in vitro [17]. Besides, Favipiravir, previously approved by Japan for the treatment of anti-flu drug, was validated to be safe and effective for the treatment of COVID-19 patients [14]. Tocilizumab, clinically used as an immunosuppressive agent in the clinic, was discovered to be effective for the treatment of COVID-19 patients in vivo [18]. All these discovered drugs need to be subjected to further testing to avoid unexpected side effects.

Troxipide, clinically used in the treatment of gastroesophageal reflux disease, has been reported to have anti-inflammatory properties, which has not been found any reports related to tumor so far [19]. In addition, our previous work has indicated that [1,2,4]triazolo [1,5-a]pyrimidine fragment possesses various pharmaceutical properties [[20], [21], [22], [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors. Eur. J. Med. Chem.. 2019 ">23], [24]], making it promising in drug design. In this work, we performed drug repurposing strategy to design and synthesize a novel series of troxipide derivatives by introducing [1,2,4]triazolo [1,5-a]pyrimidine with different substituents to troxipide, hopefully achieving some promising compounds with interesting bioactivities, leading to the identification of the most potent compound 5q (Fig. 1 ).

Fig. 1

Design of Troxipide analog 5q based on the Drug Repurposing strategy.

Results and discussion

Chemistry

The synthetic routes for the synthesis of compounds 5a-5v. Compounds 4a-4v were obtained by following the previously reported method [23]. As depicted in Scheme 1 , compounds 2a-2s were obtained by reacting 5-amino-4H-1,2,4-triazole-3-thiol with different alkyl halides, followed by refluxing with various β-ketoesters in acetic acid, respectively, giving compounds 3v-3v, which were further subjected to chlorination by POCl3, affording compounds 4a-4v. Finally, compounds 4a-4v reacted with commercially available Troxipide to give compounds 5a-5v.

Scheme 1

Synthesis of compounds 5a-5v.

Biochemical evaluation of troxipide analogs

Initially, the first troxipide analog 5a was obtained by introducing [1,2,4]triazolo [1,5-a]pyrimidines group to troxipide based on drug repurposing strategy. We next screened its antitumor activities against four different kinds of cancer cell lines, namely, MCF-7 (human breast cancer cell line), PC3 (human prostatic carcinoma cell line), MGC-803 (human gastric cancer cell line), and PC9 (human lung cancer cell line) by the MTT assay, taking 5-Fu and troxipide as the controls. Intriguingly, compound 5a displayed acceptable antiproliferative activity toward the four selected cell lines, but less potent than that of 5-Fu, while troxipide showed no any activity (IC50 > 100 μM) (Table 1 ). Encouraged by the interesting result, we next performed further structural modification around the different positions of the [1,2,4]triazolo [1,5-a]pyrimidines group, giving compounds 5a-5v. All these compounds were evaluated for their antitumor efficacy against MCF-7, PC3, MGC-803, and PC9 cancer cell lines, respectively, and 5-Fu was chosen as a positive control. As shown in Table 1, compounds 5a-5v displayed weak to strong inhibitory activity against the selected cancer cells, and it was worth noting that all of these compounds showed obvious selectivity toward PC3 over the other three cancer cell lines, except compound 5s. Therefore, in the following structural modifications, we mainly concentrated on the effect of compounds 5a-5v on PC3.

Table 1

In vitro inhibitory activity of compound 5a-5v against the selected cancer cells.

Compound	R1	R2	R3	IC50 Value (μM)
				MCF-7	PC3	MGC-803	PC9
5a	Image 2	-Me	-H	12.21 ± 0.72	7.86 ± 0.69	28.89 ± 0.84	41.41 ± 1.06
5b	Image 3	-Me	-H	10.45 ± 0.16	4.23 ± 0.35	23.69 ± 0.57	23.38 ± 0.53
5c	Image 4	-Me	-H	5.20 ± 0.18	1.53 ± 0.42	8.57 ± 0.55	33.00 ± 0.48
5d	Image 5	-Me	-H	8.56 ± 0.41	1.87 ± 0.27	10.30 ± 0.52	39.27 ± 0.63
5e	Image 6	-Me	-H	13.35 ± 0.53	8.87 ± 0.46	14.84 ± 0.68	24.24 ± 0.63
5f	Image 7	-Me	-H	24.87 ± 0.06	11.64 ± 0.21	34.59 ± 0.66	>50
5g	Image 8	-Me	-H	6.61 ± 0.42	2.22 ± 0.09	13.19 ± 0.50	>50
5h	Image 9	-Me	-H	16.49 ± 1.22	10.46 ± 1.02	26.38 ± 1.21	>50
5i	Image 10	-Me	-H	>50	25.09 ± 0.34	>50	>50
5j	Image 11	-Me	-H	23.59 ± 0.20	26.10 ± 0.52	32.92 ± 0.47	>50
5k	Image 12	-Me	-H	5.79 ± 0.25	1.89 ± 0.03	14.73 ± 0.67	36.21 ± 0.79
5l	Image 13	-Me	-H	>50	24.35 ± 0.80	>50	>50
5m	Image 14	-Me	-H	17.16 ± 0.71	13.38 ± 0.37	30.37 ± 0.52	>50
5n	Image 15	-Me	-H	12.62 ± 0.42	4.56 ± 0.19	13.13 ± 0.48	45.32 ± 0.73
5o	Image 16	-Me	-H	36.98 ± 0.60	19.79 ± 0.25	35.59 ± 0.41	>50
5p	Image 17	-Me	-H	>50	28.75 ± 0.24	32.06 ± 0.49	>50
5q	Image 18	-Me	-H	11.54 ± 0.18	0.91 ± 0.31	8.21 ± 0.50	34.68 ± 0.67
5r	Image 19	-Me	-H	>50	>50	>50	>50
5s	Image 20	-Me	-H	3.55 ± 0.55	6.30 ± 0.11	4.02 ± 0.48	10.37 ± 0.64
5t	Image 21	-Ethyl	-H	5.74 ± 0.12	2.18 ± 0.07	10.62 ± 0.42	43.48 ± 0.54
5u	Image 22	-Ph	-H	>50	15.81 ± 0.26	28.50 ± 0.54	>50
5v	Image 23	-Me	n-pentane	>50	29.65 ± 0.22	>50	>50
5-Fu	–	–	–	4.55 ± 1.03	5.32 ± 1.17	6.52 ± 1.06	4.12 ± 0.83
Troxipide	–	–	–	>100	>100	>100	>100

Firstly, we focused primarily on the molecular fine-tuning of substituents on the phenyl of compound 5a, generating compounds 5b-5n, most of which displayed moderate to strong inhibitory activity against PC3. Compared to compound 5a and 5-Fu, compounds 5b-5d, 5g, and 5k, bearing electron-withdrawing group at the 4-position of phenyl in compound 5a, showed pretty strong inhibitory activity against PC3 while compound 5h, with pretty strong electron-withdrawing –CF3 group at the 4-position of phenyl, exhibited reduced activity. Whether moving 4-Br to 2-Br or 3-Br of the phenyl in compound 5d, resulting in compound 5e and 5f, led to the decrease of inhibitory activity toward PC3. Similarly, both compounds 5i (2–CF3) and 5j (4–CF3) significantly decreased the inhibitory activity toward PC3 compared with compound 5h (4–CF3). Besides, incorporation of a methoxy group at the 4-position of the phenyl in compound 5a giving compound 5l induced a sharp decrease of activity, while compound 5m, bearing methyl group at the 4-position of the phenyl, exhibited almost comparable activity toward PC3 with compound 5a. Besides, compound 5n, with 2,6-diCl groups at the phenyl in compound 5a, also displayed moderate antitumor activity toward PC3, which was less potent than compound 5c. Taken together, these data suggested that the introduction of moderate electron-withdrawing group at the 4-position of the phenyl in compound 5a was favorable for increasing the antiproliferative activity toward PC3. To further investigate the SARs, we investigated the effect of the number of methylene between sulfydryl and Phenyl on the inhibitory activity, giving compounds 5o-5p. Unfortunately, both of them showed significantly decreased inhibitory activity toward PC3, suggesting the number of methylene between sulfydryl and Phenyl was necessary for the activity. Intriguingly, replacement of the phenyl with bulky group naphthalene in compound 5a resulted in compound 5o, which could significantly inhibit PC3 with an IC50 value of 0.91 μM, about 5.8-fold more potent than 5-Fu. However, replacement of the phenyl group with small alkyl group n-propylene in compound 5a gave compound 5r, which led to significant loss of antiproliferative activity. Besides, replacement of the phenyl group with heterocyclic group benzimidazole in compound 5a, generating compound 5s, exhibited improved anticancer efficacy toward PC3 but less than that of compound 5q, while compound 5s showed much better inhibitory activity toward MCF-7, MGC-803, and PC9 than compound 5a.

Also, we next explored the effect of the size of R2 on the antiproliferative activity toward PC3 by replacing the methyl group at R2 in compound 5c with ethyl or Phenyl group, respectively, producing compounds 5t-5u. Compound 5t showed almost the same inhibitory activity as compound 5c toward PC3 while compound 5u displayed significantly decreased antiproliferative activity, indicating the bulky group such as phenyl group at R2 was not favorable for other for the antitumor activity. To the end, one n-pentane group was also incorporated into the position of R3 in compound 5c to further investigate the SARs, generating compound 5v, which led to a sharp decrease of the antiproliferative activity toward PC3.

Given the encouraging results, troxipide analogs 5a-5v have further tested their possible toxicity toward the WPMY-1 (normal human prostatic stromal myofibroblast cell line), taking 5-Fu as the control. As depicted in Table 2 , compounds 5a-5v displayed moderate to weak toxicity toward WPMY-1. Compound 5q, especially, showed pretty weak inhibitory activity toward WPMY-1 with an IC50 value of 48.15 μM while it could effectively inhibit PC3 (IC50 = 0.91 μM), showing up to 53-fold selectivity, which exhibited obvious advantages over 5-Fu (see Table 2).

Table 2

In vitro inhibitory activity of compound 5a-5v against WPMY-1.

Compound	IC50 (μM)	Compound	IC50 (μM)
	WPMY-1		WPMY-1
5a	44.86 ± 0.69	5m	34.29 ± 0.63
5b	30.22 ± 0.34	5n	28.24 ± 0.35
5c	29.55 ± 0.40	5o	37.44 ± 0.38
5d	31.48 ± 0.17	5p	>50
5e	>50	5q	48.15 ± 0.33
5f	41.55 ± 0.19	5r	>50
5g	32.72 ± 0.43	5s	>50
5h	>50	5t	39.56 ± 0.19
5i	42.27 ± 0.35	5u	45.25 ± 0.72
5j	33.47 ± 0.39	5v	>50
5k	>50	5-Fu	8.32 ± 0.97
5l	36.27 ± 0.87

The effect of compound 5q on colony formation and cell cycle distribution of PC3

Encouraged by the potent antiproliferative activity of compound 5q against PC3, we next evaluated the inhibitory activity of compound 5q against PC3 at 24 h, 48 h, and 72 h, respectively. The results demonstrated that compound 5q significantly inhibited the proliferation of PC3 cells dose- and time-dependently (Fig. 2A). Subsequently, colony formation assay was performed, and we found that cells incubated with 0.125 μM, 0.25 μM, and 0.5 μM compound 5q formed fewer and smaller colonies (Fig. 2B & C). Inducement of cell cycle arrest is an important method of human anti-tumor therapy [25,26]. Therefore, we tested the effect of different concentrations of compound 5q on the cell cycle in PC3 cells using flow cytometry. As shown in Fig. 2D & E, compound 5q significantly increased the G1/S phase population while decreased G2/M content at high concentration in PC3 cells. It has been reported that p27 is a cyclin-dependent kinase inhibitor and its expression level can reflect cell cycle progression [27]. Therefore, we further explored the effect of compound 5q on the expression of p27 by western blotting. As depicted in Fig. 2F & G, compound 5q dose-dependently elevated the expression of p27. The data revealed that the antiproliferative activity of compound 5q toward PC3 cells was associated with the G1/S cell cycle arrest.

Fig. 2

The effect of Compound 5q on colony formation and cell cycle distribution of PC3 (A) The cell viabilities of PC3 cells treated with different concentrations of compound 5q for 24 h, 48 h, and 72 h, respectively by MTT assay. (B, C) The colony formation and absorbance analysis of crystal violet in PC3 cells treated with indicated concentrations of compound 5q for 6 days. (D, E) PC3 cells were treated with compound 5q at the indicated concentrations for 24 h and the cell cycle distributions were then analyzed by flow cytometry. (F, G) The expression and quantitative analysis of p27 protein in PC3 cells treated with compound 5q at the indicated concentrations for 24 h by western blotting. Three independent experiments were performed. All data were expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The effect of compound 5q on apoptosis of PC3 cells

To further explore the cytotoxicity of compound 5q on PC3 cells, we further investigated the mechanism of inhibitory efficacy of compound 5q on PC3 cells. Firstly, Hoechst 33342 staining was carried out to examine the effect of compound 5q on cell apoptosis. As shown in Fig. 3 A & B, PC3 cells treated with compound 5q at the indicated concentrations displayed apoptosis-associated morphologies, such as cell rounding up, chromatin condensation, and formation of apoptotic bodies. Next, Annexin V-FITC/PI apoptosis detection kit was utilized to examine the apoptosis of PC3 cells using flow cytometry. As shown in Fig. 3C & D, treatment of PC3 with compound 5q at different concentrations (1, 2, and 4 μM) dose-dependently led to significant increase of FITC-Annexin V/PI positive population, and especially the treatment of PC3 by compound 5q at 4 μM, the apoptotic percentage was up to 70.7%, which was far higher than the control group (3.5%).

Fig. 3

The effect of compound 5q on apoptosis of PC3 cells and cellular ROS. (A, B) The apoptotic morphological changes (marked with red arrows) and the quantitative analysis of PC3 cells treated with different concentrations of compound 5q for 48h, respectively, by Hoechst 33342 staining. (C, D) The quantitative analysis of PC3 cells treated with compound 5q at the indicated concentrations for 48h, respectively, using Annexin V-FITC/PI double staining through flow cytometry. (E, F) The quantitative analysis of the levels of ROS in PC3 cells treated with different concentrations of compound 5q for 24 h, respectively, by flow cytometry. Three independent experiments were performed. All data were expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

It has been reported that the accumulation of reactive oxygen species (ROS), the main source of mitochondrial metabolism, could induce cellular apoptosis through a mitochondrial-dependent pathway [28]. So we next explored the effect of compound 5q on the level of intracellular ROS by using DCFH-DA. As shown in Fig. 3E & F, compound 5q could effectively increase the accumulation of intracellular ROS in a concentration-dependent manner, which at 4 μM led to 2-fold increase compared with the control. The data indicated that compound 5q could induce the accumulation of ROS which contributed to the apoptosis of PC3 cells.

Apoptosis has been reported to be mainly mediated by the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway [29]. To further investigate the underlying molecular mechanisms of compound 5q on the apoptosis of PC3 cells, we next tested the apoptosis-related proteins affected by compound 5q based on western blotting assay. As shown in Fig. 4 A–D, compound 5q markedly elevated the expression of pro-apoptotic Bax and P53 while anti-apoptotic Bcl-2 expression was down-regulated.

Fig. 4

The effect of compound 5q on apoptosis-related proteins. (A, B, C, D) Expression and quantitative analysis of Bax, Bcl-2, and P53 in PC3 cells treated with compound 5q at the indicated concentrations for 48h. (E, F, G, H) Expression and quantitative analysis of Cleaved-caspase 9/3 and cleaved-PARP in PC3 cells treated with compound 5q at the indicated concentrations for 48h. Three independent experiments were performed. All data were expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group.

Besides, upon apoptotic stimulation, caspase-9, a member of the cysteine aspartic acid protease (caspase) family, performs interaction with cytochrome C and then it mediates activation of caspase-9. Cleaved caspase-9 further processes other caspase members, such as caspase-3, which is responsible for the proteolytic cleavage of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). They all interact to initiate a caspase cascade and eventually leads to cell apoptosis [30]. As shown in Fig. 4A–D, compound 5q significantly increased the expression of cleaved caspase 3/9 and cleaved PARP in a dose-dependent manner. These findings demonstrated that compound 5q could induce the apoptosis of PC3 cells through activating the two apoptotic signaling pathways simultaneously.

The effect of compound 5q on the migration of PC3 cells

Cancer metastasis represents an advanced stage of malignancy and is the leading cause of cancer-related deaths while cell migration is involved in cancer metastasis [31,32]. Inhibition of tumor cell migration is an important strategy for the treatment of various cancers in the clinic. Therefore, we evaluated whether compound 5q affected the inhibition of the migration of PC3 cells. As shown in Fig. 5 A–D, the wound healing assay indicated that compound 5q effectively inhibited the wound healing in a dose-dependent manner, while transwell assay demonstrated that compound 5q could also significantly inhibit the migration of PC3 cells concentration-dependently.

Fig. 5

The effect of compound 5q on the migration ability of PC3 cells. (A, B) Effect and statistical analysis of migration in PC3 cells treated with compound 5q at the indicated concentrations for 48h in wound healing assay. (C, D) Effect and statistical analysis of migration in PC3 cells treated with different concentrations of compound 5q for 48h in transwell assay. Three independent experiments were performed. All data were expressed as the mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to Con group.

Conclusions

In this work, we for the first time reported a novel series of troxipide analogs and then evaluated their antiproliferative activity against MCF-7, PC3, MGC-803, and PC9 cancer cell lines and WPMY-1, most of which showed obvious selectivity toward PC-3 over the other three cancer cell lines and WPMY-1. Among these analogs, compound 5q could effectively inhibit PC3 with an IC50 value of 0.91 μM, which exhibited around 53-fold selectivity toward WPMY-1. Biological studies indicated that 5q could effectively inhibit the colony formation, suppress the cell migration, and induce G1/S phase arrest in PC3 cells. What’s more, compound 5q could induce cell apoptosis by activating the two apoptotic signaling pathways in PC3 cells: death receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway. Compound 5q could up-regulate the expression of both pro-apoptotic Bax and P53, but down-regulate anti-apoptotic Bcl-2 expression. Besides, compound 5q could significantly increase the expression of cleaved caspase 3/9 and cleaved PARP. Therefore, the successful application of drug repurposing strategy may further encourage researchers to reveal undescribed indications in clinical drugs.

Experimental section

General information

Reagents and solvents were purchased from commercial sources and used without further purification. Thin-layer chromatography (TLC) was carried out on glass plates coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized by UV light (254 nm). The products were purified by column chromatography over silica gel (Qingdao Haiyang Chemical Co., 200–300 mesh). Melting points were determined on an X-5 micromelting apparatus and are uncorrected. All the NMR spectra were recorded with a Bruker DPX 400 MHz spectrometer with TMS as the internal standard in CDCl3 or DMSO‑d 6. Chemical shifts are given as δ ppm values relative to TMS. High-resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray ionization (ESI).

The general method for the synthesis of 1a-1k, 2a-2q, and 3a-3q

Compounds 2a-2s, 3a-3v, and 4a-4v were prepared following the previously reported method [21].

The general method for the synthesis of 5a-5v

N-(1-(2-(benzylthio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5a), white solid, yield: 84%. m.p.: 146–148 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 7.4 Hz, 2H), 7.34–7.20 (m, 3H), 7.17 (s, 2H), 5.49 (s, 1H), 4.38 (s, 2H), 4.11 (s, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.33 (d, J = 11.6 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 2.79 (dd, J = 13.9, 7.2 Hz, 2H), 2.17 (s, 3H), 1.96–1.82 (m, 2H), 1.62 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.93, 160.98, 160.36, 158.41, 157.47, 153.02, 140.67, 138.82, 129.78, 129.27, 128.82, 127.49, 105.52, 96.04, 60.58, 56.53, 47.62, 44.79, 44.00, 34.90, 28.95, 23.90, 22.09. HRMS (ESI): m/z calcd for C28H32N6NaO4S (M + Na)+, 571.2103; found, 571.2102.

N-(1-(2-((4-fluorobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5b), white solid, yield: 87%. m.p.: 150–152 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.4 Hz, 1H), 7.51–7.42 (m, 2H), 7.17 (s, 2H), 7.11 (t, J = 8.6 Hz, 2H), 5.49 (s, 1H), 4.38 (s, 2H), 4.13 (d, J = 6.5 Hz, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.7 Hz, 1H), 3.22 (d, J = 12.5 Hz, 1H), 2.80 (dd, J = 20.5, 9.8 Hz, 2H), 2.17 (s, 3H), 1.96–1.84 (m, 2H), 1.72–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.94, 162.92, 160.83, 160.22, 158.31, 157.42, 153.02, 140.69, 135.26, 135.23, 131.24, 131.15, 129.76, 115.64, 115.43, 105.53, 96.09, 60.57, 56.54, 47.54, 44.73, 43.95, 34.00, 28.90, 23.82, 22.01. HRMS (ESI): m/z calcd for C28H31FN6NaO4S (M + Na)+, 589.2009; found, 589.2009.

N-(1-(2-((4-chlorobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5c), white solid, yield: 93%. m.p.: 143–145 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.36 (d, J = 7.4 Hz, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.17 (s, 2H), 5.48 (s, 1H), 4.37 (s, 2H), 4.11 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.33 (d, J = 11.4 Hz, 1H), 3.20 (d, J = 12.5 Hz, 1H), 2.79 (dd, J = 20.7, 9.9 Hz, 2H), 2.17 (s, 3H), 1.95–1.82 (m, 2H), 1.70–1.53 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 160.65, 160.48, 158.51, 157.46, 153.02, 140.67, 138.26, 132.05, 131.09, 129.77, 128.73, 105.52, 96.03, 60.57, 56.53, 47.65, 44.81, 44.01, 34.02, 28.97, 23.95, 22.13. HRMS (ESI): m/z calcd for C28H32ClN6O4S (M + H)+, 583.1894; found, 583.1894.

N-(1-(2-((4-bromobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5d), white solid, yield: 89%. m.p.: 154–157 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.36 (d, J = 7.3 Hz, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.9 Hz, 2H), 7.17 (s, 2H), 5.48 (s, 1H), 4.36 (s, 2H), 4.12 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.6 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 2.80 (dd, J = 20.9, 10.1 Hz, 2H), 2.17 (s, 3H), 1.97–1.83 (m, 2H), 1.71–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.93, 160.67, 160.18, 158.30, 157.39, 153.03, 140.69, 138.67, 131.66, 131.45, 129.75, 120.56, 105.53, 96.12, 60.58, 56.55, 47.53, 44.73, 43.95, 34.08, 28.90, 23.80, 22.01. HRMS (ESI): m/z calcd for C28H32BrN6O4S (M + H)+, 627.1389; found, 627.1390.

N-(1-(2-((2-bromobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5e), white solid, yield: 87%. m.p.: 145–148 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.36 (d, J = 7.4 Hz, 1H), 7.62 (t, J = 9.0 Hz, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.25–7.12 (m, 3H), 5.48 (s, 1H), 4.47 (s, 2H), 4.11 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.37–3.30 (m, 1H), 3.21 (d, J = 12.6 Hz, 1H), 2.79 (dd, J = 21.6, 10.6 Hz, 2H), 2.17 (s, 3H), 1.90 (t, J = 17.0 Hz, 2H), 1.70–1.55 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 160.46, 160.40, 158.47, 157.43, 153.02, 140.67, 137.81, 133.10, 131.64, 129.80, 129.76, 128.31, 124.42, 105.52, 96.08, 60.58, 56.54, 47.60, 44.78, 43.99, 35.54, 28.94, 23.92, 22.08. HRMS (ESI): m/z calcd for C28H32BrN6O4S (M + H)+, 627.1389; found, 627.1389.

N-(1-(2-((3-bromobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5f), white solid, yield: 90%. m.p.: 155–159 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.3 Hz, 1H), 7.65 (s, 1H), 7.48–7.38 (m, 2H), 7.25 (t, J = 7.8 Hz, 1H), 7.17 (s, 2H), 5.49 (s, 1H), 4.38 (s, 2H), 4.12 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.8 Hz, 1H), 3.22 (d, J = 12.5 Hz, 1H), 2.80 (dd, J = 21.7, 10.7 Hz, 2H), 2.17 (s, 3H), 1.95–1.85 (m, 2H), 1.78–1.50 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.94, 160.61, 160.20, 158.32, 157.38, 153.02, 142.06, 140.68, 131.88, 130.94, 130.32, 129.75, 128.35, 121.92, 105.53, 96.15, 60.58, 56.54, 47.51, 44.72, 43.94, 34.05, 28.89, 23.81, 22.00. HRMS (ESI): m/z calcd for C28H32BrN6O4S (M + H)+, 627.1389; found, 627.1387.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((4-nitrobenzyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5g), white solid, yield: 86%. m.p.: 164–166 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.3 Hz, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.17 (s, 2H), 5.49 (s, 1H), 4.38 (s, 2H), 4.12 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.6 Hz, 1H), 3.22 (d, J = 12.5 Hz, 1H), 2.80 (dd, J = 20.8, 10.0 Hz, 2H), 2.17 (s, 3H), 1.95–1.85 (m, 2H), 1.74–1.54 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.93, 160.70, 160.17, 158.28, 157.39, 153.02, 140.69, 138.22, 132.06, 131.09, 129.75, 128.73, 105.53, 96.13, 60.58, 56.54, 47.50, 44.71, 43.93, 34.03, 28.88, 23.79, 21.98. HRMS (ESI): m/z calcd for C28H32N7O6S (M + H)+, 594.2135; found, 594.2134.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((4-(trifluoromethyl)benzyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5h), white solid, yield: 79%. m.p.: 159–162 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.38 (d, J = 7.4 Hz, 1H), 7.65 (s, 4H), 7.18 (s, 2H), 5.48 (s, 1H), 4.47 (s, 2H), 4.13 (d, J = 6.4 Hz, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.7 Hz, 1H), 3.21 (d, J = 12.7 Hz, 1H), 2.80 (dd, J = 19.7, 9.5 Hz, 2H), 2.17 (s, 3H), 1.95–1.84 (m, 2H), 1.65 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 160.47, 160.42, 158.47, 157.43, 153.02, 144.24, 140.68, 129.99, 125.66, 125.62, 105.53, 96.09, 60.56, 56.53, 47.54, 44.72, 43.94, 34.17, 28.89, 23.91, 21.99. HRMS (ESI): m/z calcd for C29H32F3N6O4S (M + H)+, 617.2158; found, 617.2159.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((2-(trifluoromethyl)benzyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5i), white solid, yield: 83%. m.p.: 150–152 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.36 (d, J = 7.3 Hz, 1H), 7.75 (dd, J = 18.3, 7.8 Hz, 2H), 7.62 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.17 (s, 2H), 5.49 (s, 1H), 4.56 (s, 2H), 4.12 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.33 (d, J = 11.6 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 2.79 (dd, J = 21.8, 10.8 Hz, 2H), 2.17 (s, 3H), 1.88 (m, 2H), 1.72–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 164.87, 160.48, 158.52, 157.45, 153.02, 140.67, 137.06, 133.28, 132.20, 129.75, 128.37, 126.47, 126.42, 105.52, 96.13, 60.57, 56.53, 47.53, 44.73, 43.94, 31.56, 28.90, 23.95, 22.02, 21.63. HRMS (ESI): m/z calcd for C29H32F3N6O4S (M + H)+, 617.2158; found, 617.2158.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((3-(trifluoromethyl)benzyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5j), white solid, yield: 87%. m.p.: 161–163 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.4 Hz, 1H), 7.81 (s, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.18 (s, 2H), 5.48 (s, 1H), 4.48 (s, 2H), 4.12 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.33 (d, J = 11.8 Hz, 1H), 3.21 (d, J = 12.6 Hz, 1H), 2.80 (dd, J = 19.1, 8.5 Hz, 2H), 2.17 (s, 3H), 1.90 (t, J = 16.6 Hz, 2H), 1.70–1.57 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 160.50, 160.20, 158.51, 157.44, 153.02, 140.85, 140.67, 133.41, 129.83, 129.76, 125.77, 125.73, 124.18, 124.14, 105.52, 96.06, 60.57, 56.54, 47.55, 44.74, 43.96, 34.12, 28.92, 23.95, 22.03. HRMS (ESI): m/z calcd for C29H32F3N6O4S (M + H)+, 617.2158; found, 617.2156.

N-(1-(2-((4-cyanobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5k), white solid, yield: 92%. m.p.: 147–150 °C. 1H NMR (400 MHz, DMSO‑d 6)) δ 8.38 (d, J = 7.3 Hz, 1H), 7.75 (d, J = 7.7 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.17 (s, 2H), 5.49 (s, 1H), 4.46 (s, 2H), 4.14 (d, J = 6.7 Hz, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.35 (d, J = 11.7 Hz, 1H), 3.23 (d, J = 12.5 Hz, 1H), 2.81 (dd, J = 21.0, 10.1 Hz, 2H), 2.17 (s, 3H), 1.91 (t, J = 15.3 Hz, 2H), 1.74–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.95, 160.40, 160.23, 158.29, 157.37, 153.02, 145.33, 140.69, 132.70, 130.21, 129.72, 119.27, 110.17, 105.52, 96.19, 60.58, 56.54, 47.38, 44.63, 43.87, 34.32, 28.81, 23.79, 21.87. HRMS (ESI): m/z calcd for C29H31N7NaO4S (M + Na)+, 596.2056; found, 596.2057.

3,4,5-trimethoxy-N-(1-(2-((4-methoxybenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5l), white solid, yield: 90%. m.p.: 144–148 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.39 (d, J = 7.3 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.17 (s, 2H), 6.85 (d, J = 8.0 Hz, 2H), 5.51 (s, 1H), 4.33 (s, 2H), 4.14 (d, J = 6.1 Hz, 1H), 3.81 (s, 6H), 3.71 (d, J = 4.3 Hz, 6H), 3.36 (d, J = 11.3 Hz, 1H), 3.24 (d, J = 12.5 Hz, 1H), 2.82 (dd, J = 20.4, 9.9 Hz, 2H), 2.18 (s, 3H), 1.91 (t, J = 15.1 Hz, 2H), 1.74–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.95, 161.24, 159.47, 158.84, 157.76, 157.28, 153.03, 140.70, 130.46, 129.74, 114.25, 105.54, 96.28, 60.58, 56.54, 55.50, 47.37, 44.62, 43.87, 34.46, 28.81, 23.44, 21.85. HRMS (ESI): m/z calcd for C29H35N6O5q (M + H)+, 579.2390; found, 579.2390.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((4-methylbenzyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5m), white solid, yield: 86%. m.p.: 151–154 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.38 (d, J = 7.4 Hz, 1H), 7.29 (d, J = 7.5 Hz, 2H), 7.17 (s, 2H), 7.09 (d, J = 7.6 Hz, 2H), 5.50 (s, 1H), 4.34 (s, 2H), 4.14 (d, J = 6.5 Hz, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.35 (d, J = 9.7 Hz, 1H), 3.22 (d, J = 12.5 Hz, 1H), 2.81 (dd, J = 19.9, 9.6 Hz, 2H), 2.25 (s, 3H), 2.18 (s, 3H), 1.96–1.84 (m, 2H), 1.72–1.55 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.94, 161.12, 159.88, 158.05, 157.37, 153.02, 140.69, 136.66, 135.59, 129.75, 129.38, 129.18, 105.53, 96.16, 60.58, 56.53, 47.46, 44.69, 43.91, 34.71, 28.87, 23.64, 21.94, 21.14. HRMS (ESI): m/z calcd for C29H35N6O4S (M + H)+, 563.2440; found, 563.2438.

N-(1-(2-((2,6-dichlorobenzyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5n), white solid, yield: 76%. m.p.: 140–142 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.36 (d, J = 7.3 Hz, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 8.0 Hz, 1H), 7.17 (s, 2H), 5.50 (s, 1H), 4.66 (s, 2H), 4.11 (s, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 11.6 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 2.79 (dd, J = 21.2, 10.3 Hz, 2H), 2.18 (s, 3H), 1.95–1.85 (m, 2H), 1.73–1.55 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.91, 160.64, 160.48, 158.59, 157.52, 153.01, 140.67, 135.54, 133.21, 130.65, 129.75, 129.18, 105.51, 96.06, 60.57, 56.53, 47.64, 44.79, 44.01, 31.72, 28.95, 24.01, 22.10. HRMS (ESI): m/z calcd for C28H31Cl2N6O4S (M + H)+, 617.1505; found, 617.1504.

3,4,5-trimethoxy-N-(1-(5-methyl-2-(phenethylthio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5o), white solid, yield: 85%. m.p.: 149–152 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.38 (d, J = 7.3 Hz, 1H), 7.29 (m, 4H), 7.24–7.14 (m, 3H), 5.50 (s, 1H), 4.13 (d, J = 6.3 Hz, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.39–3.29 (m, 3H), 3.22 (d, J = 12.5 Hz, 1H), 2.99 (t, J = 7.4 Hz, 2H), 2.80 (dd, J = 20.5, 9.9 Hz, 2H), 2.18 (s, 3H), 1.97–1.82 (m, 2H), 1.73–1.54 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.93, 161.15, 159.89, 158.15, 157.40, 153.02, 140.77, 140.67, 129.77, 129.02, 128.77, 126.69, 105.52, 96.10, 60.58, 56.53, 47.57, 44.76, 43.97, 35.94, 32.22, 28.93, 23.67, 22.04. HRMS (ESI): m/z calcd for C29H35N6O4S (M + H)+, 563.2440; found, 563.2441.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((3-phenylpropyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5p), white solid, yield: 77%. m.p.: 154–156 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.3 Hz, 1H), 7.39–7.05 (m, 7H), 5.48 (s, 1H), 4.13 (d, J = 6.4 Hz, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 10.1 Hz, 1H), 3.22 (d, J = 12.4 Hz, 1H), 3.10 (t, J = 7.0 Hz, 2H), 2.80 (dd, J = 20.4, 9.8 Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.16 (s, 3H), 2.04–1.83 (m, 4H), 1.73–1.54 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.91, 161.20, 159.88, 158.13, 157.38, 153.03, 141.74, 140.69, 129.79, 128.79, 128.77, 126.28, 105.54, 96.06, 60.57, 56.90, 56.54, 47.67, 44.83, 44.02, 34.53, 31.53, 30.46, 28.99, 23.68, 22.14. HRMS (ESI): m/z calcd for C30H37N6O4S (M + H)+, 577.2597; found, 577.2597.

3,4,5-trimethoxy-N-(1-(5-methyl-2-((naphthalen-1-ylmethyl)thio)- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)benzamide (5q), white solid, yield: 88%. m.p.: 174–176 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.4 Hz, 1H), 8.18 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.70–7.48 (m, 3H), 7.42 (t, J = 7.6 Hz, 1H), 7.17 (s, 2H), 5.51 (s, 1H), 4.89 (s, 2H), 4.12 (d, J = 6.1 Hz, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.34 (d, J = 9.7 Hz, 1H), 3.20 (d, J = 12.6 Hz, 1H), 2.79 (t, J = 11.1 Hz, 2H), 2.19 (s, 3H), 1.90 (t, J = 18.3 Hz, 2H), 1.72–1.53 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.93, 161.08, 160.29, 158.37, 157.47, 153.02, 140.67, 133.94, 131.52, 129.75, 129.10, 128.48, 127.86, 126.79, 126.38, 125.93, 124.35, 105.52, 96.13, 60.57, 56.53, 47.54, 44.74, 43.95, 32.89, 28.90, 23.86, 22.01. HRMS (ESI): m/z calcd for C32H35N6O4S (M + H)+, 599.2440; found, 599.2439.

N-(1-(2-(allylthio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5r), white solid, yield: 82%. m.p.: 138–140 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.37 (d, J = 7.4 Hz, 1H), 7.17 (s, 2H), 5.97 (m, 1H), 5.48 (s, 1H), 5.26 (d, J = 17.0 Hz, 1H), 5.06 (d, J = 10.0 Hz, 1H), 4.12 (d, J = 6.6 Hz, 1H), 3.82 (s, 6H), 3.78 (d, J = 6.8 Hz, 2H), 3.70 (s, 3H), 3.34 (d, J = 9.5 Hz, 1H), 3.22 (d, J = 12.6 Hz, 1H), 2.80 (dd, J = 21.9, 10.7 Hz, 2H), 2.16 (s, 3H), 1.96–1.85 (m, 2H), 1.71–1.56 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.92, 160.69, 160.32, 158.40, 157.47, 153.02, 140.66, 134.87, 129.79, 117.87, 105.51, 95.98, 60.57, 56.53, 47.64, 44.81, 44.01, 33.62, 28.98, 23.88, 22.12. HRMS (ESI): m/z calcd for C24H31N6O4S (M + H)+, 499.2127; found, 499.2126.

N-(1-(2-(((1H-benzo[d]imidazole-2-yl)methyl)thio)-5-methyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5s), white solid, yield: 84%. m.p.: 174–177 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.39 (d, J = 7.4 Hz, 1H), 7.52 (dd, J = 5.2, 3.3 Hz, 2H), 7.24–7.10 (m, 4H), 5.52 (s, 1H), 4.62 (s, 2H), 4.16 (d, J = 6.5 Hz, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.40 (d, J = 9.3 Hz, 1H), 3.27 (d, J = 12.5 Hz, 1H), 2.85 (dd, J = 19.9, 9.6 Hz, 2H), 2.18 (s, 3H), 1.92 (dd, J = 18.5, 11.5 Hz, 2H), 1.81–1.54 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.97, 161.21, 160.49, 158.77, 157.67, 153.02, 151.92, 140.67, 129.79, 122.27, 105.53, 95.99, 60.58, 56.54, 47.54, 44.80, 43.92, 28.96, 28.52, 24.18, 22.04. HRMS (ESI): m/z calcd for C29H33N8O4S (M + H)+, 589.2345; found, 589.2344.

N-(1-(2-((4-chlorobenzyl)thio)-5-ethyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5t), white solid, yield: 89%. m.p.: 162–166 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.38 (d, J = 7.3 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.17 (s, 2H), 5.50 (s, 1H), 4.38 (s, 2H), 4.13 (s, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.35 (d, J = 11.6 Hz, 1H), 3.23 (d, J = 12.6 Hz, 1H), 2.81 (dd, J = 20.6, 10.0 Hz, 2H), 2.44 (m, 2H), 1.91 (t, J = 15.3 Hz, 2H), 1.74–1.54 (m, 2H), 1.16 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 165.95, 165.18, 160.79, 158.33, 157.65, 153.03, 140.70, 138.23, 132.06, 131.07, 129.74, 128.74, 105.54, 94.84, 60.58, 56.54, 47.40, 44.63, 43.89, 34.02, 30.36, 28.81, 21.86, 13.77. HRMS (ESI): m/z calcd for C29H34ClN6O4S (M + H)+, 597.2051; found, 597.2050.

N-(1-(2-((4-chlorobenzyl)thio)-5-phenyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5u), white solid, yield: 86%. m.p.: 170–172 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.39 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 7.4 Hz, 2H), 7.54–7.30 (m, 7H), 7.18 (s, 2H), 6.15 (s, 1H), 4.43 (s, 2H), 4.17 (d, J = 6.3 Hz, 1H), 3.82 (s, 6H), 3.70 (s, 3H), 3.26 (d, J = 12.4 Hz, 2H), 2.84 (m, 2H), 1.92 (t, J = 12.2 Hz, 2H), 1.78–1.55 (m, 2H). 13C NMR (100 MHz, DMSO‑d 6) δ 166.01, 161.29, 159.27, 158.81, 158.05, 153.04, 140.75, 139.46, 138.28, 132.07, 131.11, 129.66, 129.38, 128.77, 127.18, 105.56, 93.15, 60.59, 56.56, 46.97, 44.32, 43.67, 34.02, 28.55, 21.45. HRMS (ESI): m/z calcd for C33H34ClN6O4S (M + H)+, 645.2051; found, 645.2050.

N-(1-(2-((4-chlorobenzyl)thio)-5-methyl-6-pentyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-yl)piperidin-3-yl)-3,4,5-trimethoxybenzamide (5v), white solid, yield: 85%. m.p.: 167–169 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 8.33 (d, J = 7.4 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 7.17 (s, 2H), 4.38 (s, 2H), 4.08 (s, 1H), 3.81 (s, 6H), 3.70 (s, 3H), 3.30 (d, J = 10.3 Hz, 1H), 3.17 (d, J = 12.4 Hz, 1H), 2.75 (dd, J = 20.4, 9.8 Hz, 2H), 2.46–2.36 (m, 2H), 2.24 (s, 3H), 1.88 (dd, J = 27.9, 12.3 Hz, 2H), 1.62 (m, 2H), 1.44–1.22 (m, 6H), 0.86 (t, J = 6.3 Hz, 3H). 13C NMR (100 MHz, DMSO‑d 6) δ 165.86, 160.88, 157.11, 155.54, 153.01, 140.63, 138.22, 132.07, 131.08, 129.84, 128.73, 107.48, 105.50, 60.56, 56.52, 48.10, 45.11, 44.25, 34.06, 31.82, 29.23, 29.11, 25.96, 22.59, 21.28, 14.45. HRMS (ESI): m/z calcd for C33H42ClN6O4S (M + H)+, 653.2677; found, 653.2676.

Cell culture

Human prostatic carcinoma cell line PC3, human breast adenocarcinoma cell line MCF-7, human gastric carcinoma cell line MGC-803, human lung carcinoma cell line PC9, normal human prostatic cell line WPMY-1 were all purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. They were cultured in RPMI-1640 and DMEM complete medium (Solarbio, China) at 37 °C in a 5% CO2 humidified atmosphere.

MTT assay

Briefly, cells were incubated with serial concentrations of the compounds for 24 h, 48 h and 72 h, respectively. 20 μl MTT solution was added to each well and then the plates were incubated for another 4 h. Finally, absorbance values were measured by an enzyme-linked immunosorbent assay reader (BioTek, USA) at 490 nm. The cell viability and IC50 were calculated by SPSS20 software.

Colony formation assay

Cells were seeded into 6-well plates at a concentration of 800 cells per well. Then they were respectively incubated with 0.125 μM, 0.25 μM and 0.5 μM compound 5q for 6 days. After that, cells were stained by 0.1% crystal violet solution at room temperature for 30min and then photographed. Subsequently, the crystal violet crystals were dissolved in 75% ethanol and the absorbance values were measured by an enzyme-linked immunosorbent assay reader at 595 nm.

Cell cycle assay

PC3 cells seeded in 6-well plates at a concentration of 4 × 105 cells per well were respectively incubated with 1 μM, 2 μM and 4 μM compound 5q for 24h. Subsequently, cells were harvested and stained in 500 μl Propidium Iodide solution (PI 1 mg/ml, RNase A 10 mg/ml, Solarbio) at room temperature away from light for 30 min. Staining cells were detected and analyzed by flow cytometry (BD Bioscience, USA).

Hoechst 33342 staining

Cells were incubated with indicated concentrations of compound 5q in 12-well plate for 48h. Then cells were stained by Hoechst 33342 buffer (10 μg/ml, Solarbio) for 20 min in the dark. The morphological changes and nuclear fragmentation were imaged by an inverted fluorescence microscope (Nikon Eclipse TE 2000-S).

ROS assay

Cells seeded in 6-well plates were incubated with indicated concentrations of compound 5q for 24 h. Then cells were washed in PBS and stained by 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime) at 37 °C for 20 min in the dark. Then cells were collected and detected by flow cytometry.

Cell apoptosis assay

Cells plated in 6-well plates were respectively incubated with 1 μM, 2 μM and 4 μM compound 5q for 48h. Then cells were collected and stained by 200 μl binding buffer including 2 μl Annexin V-FITC and 2 μl PI (Beyotime) for 10 min at room temperature in the dark. Lastly, flow cytometry was used to detect the apoptotic cells.

Migration assay

For the wound healing assay, when PC3 cells seeded in 6-well plates reached to the density of 90%, they were scraped off using a sterile yellow pipette tip and photographed. Subsequently, cells were incubated with 0.25 μM, 0.5 μM and 1 μM compound 5q for 48h, and then each well was photographed again.

For the transwell assay, 600 μl fresh medium with 20% FBS were added to the substrate of the 24-well plates. 2 × 104 cells per well were seeded and incubated with indicated concentrations of compound 5q in the upper chambers for 48h. Then these chambers were stained by 0.1% crystal violet solution and photographed.

Western blotting analysis

Cells incubated with indicated concentrations of compound 5q for 24 h or 48 h were collected with trypsin and total proteins were extracted in lysis buffer. BCA protein assay kit (Solarbio) was used to determine the concentration of the total proteins. Then these samples were detected by standard protocol of Western blot as described before.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.