molecules Article
Discovery of Matrinic Thiadiazole Derivatives as a Novel Family of Anti-Liver Fibrosis Agents via Repression of the TGFβ/Smad Pathway Tianyu Niu † , Weixiao Niu † , Yunyang Bao, Ting Liu, Danqing Song, Yinghong Li * and Hongwei He * Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
[email protected] (T.N.);
[email protected] (W.N.);
[email protected] (Y.B.);
[email protected] (T.L.);
[email protected] (D.S.) * Correspondence:
[email protected] (Y.L.);
[email protected] (H.H.); Tel.:+86-10-6303-3012 (Y.L.); +86-10-8316-6673 (H.H.) † These authors contributed equally to this work. Received: 14 June 2018; Accepted: 30 June 2018; Published: 5 July 2018
Abstract: A series of novel matrinic thiadiazole derivatives were designed, synthesized and evaluated for their inhibitory effect on COL1A1 promotor. The SAR indicated that: (i) the introduction of a thiadiazole on the 11-side chain was beneficial for activity; (ii) a 12-N-benzyl moiety was favorable for activity. Among them, compound 6n displayed a high activity with an inhibitory rate of 39.7% at a concentration of 40 µM. It also effectively inhibited the expression of two representative collagen proteins (COL1A1 and α-SMA) on both the mRNA and protein levels and showed a high safety profile in vivo, indicating its great promise as an anti-liver fibrosis agent. Further study indicated that it might repress hepatic fibrogenesis via the TGFβ/Smad pathway. This study provided powerful information for further strategic optimization and the top compound 6n was selected for further study as an ideal liver fibrosis lead for next investigation. Keywords: liver fibrosis; matrinic; thiadiazole; structure-activity relationship; COL1A1; TGFβ/Smad pathway
1. Introduction Liver fibrosis is a general stage in the process of cirrhosis, which is characterized by the excessive accumulation of extracellular matrix (ECM) and abnormal hyperplasia of intrahepatic connective tissue [1]. Fibrillation is the scarring response to chronic liver injury [2]. It can be controlled in the early phase after effective treatment, but most often, fibrous septa, pseudolobules or nodules would form and eventually develop into liver cirrhosis. Unfortunately, to date, treatments for liver fibrosis are quite limited and therefore, there is still an urgent need to develop new effective anti-liver fibrosis candidates. The activation and proliferation of hepatic stellate cells (HSCs) leads to the excess production and abnormal deposition of ECM components [3,4], as indicated in the cytological basis for liver fibrosis [5].Transforming growth factor β1 (TGFβ1) is closely related to liver fibrosis, and it is one of the most important fibrotic cytokines known so far [6]. It promotes the production of liver fibrosis mainly by activating HSCs and up-regulating the secretion of collagen, especially collagen type I (COL1) and α-smooth muscle actin (α-SMA). Since the transcription of key gene collagen type I α1 chain (COL1A1) for liver fibrosis could be activated by TGFβ1 through TGFβ/Smad pathway [7], a high-throughput drug screening cell model based on COL1A1 promotor was established earlier in our
Molecules 2018, 23, 1644; doi:10.3390/molecules23071644
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our group, in which the activity of COL1A1 promotor and luciferase reporter gene could be elevated by TGFβ1, and inhibited by candidate agents [8]. group, in which the activity of COL1A1 promotor and luciferase reporter gene could be elevated by Our group has been the[8]. discovery of innovative candidates agents with novel TGFβ1, and inhibited by working candidate on agents structureOur skeletons Chinese natural products such matrine (MT) [9–11].agents Sincewith MT is an antigroup from has been working on the discovery ofas innovative candidates novel liverstructure fibrosis monomer [12], Chinese the matrinic acid compound constructed in our lab MT [9,10,13] skeletons from natural products such library as matrine (MT) [9–11]. Since is an was anti-liver fibrosis monomer [12], the matrinic acid compound library constructed in our lab [9,10,13] screened by the high-throughput screening model based on COL1A1 promotor as mentioned above, wasepigallocatechin screened by the high-throughput model based on COL1A1 mentioned taking gallate (EGCG)screening as the positive control [14]. To promotor our greatasdelight, the hit above, taking epigallocatechin gallate (EGCG) as the positive control [14]. To our great delight, the hit compound 12-N-p-methyl benzenesulfonyl matrinic acid (1, Figure 1) [15] had been identified to compound 12-N-p-methyl benzenesulfonyl matrinic acid (1, had been identified to displayof 40 display a reasonable inhibitory effect against COL1A1 atFigure a rate1)of[15] 18.5% at the concentration a reasonable inhibitory effect against COL1A1 at a rate of 18.5% at the concentration of 40 µM. It was μM. It was also worth mentioning that compound 1 possesses a special tricyclic flexible scaffold, and also worth mentioning that compound 1 possesses a special tricyclic flexible scaffold, and appealing appealing druglike characteristics. These studies prompted our interest in continuing our structuredruglike characteristics. These studies prompted our interest in continuing our structure-activity activity relationship (SAR) study of this compound class taking compound 1 as the lead, in an effort relationship (SAR) study of this compound class taking compound 1 as the lead, in an effort to develop to develop and discover novel anti-liver fibrosis candidates. and discover novel anti-liver fibrosis candidates.
Figure 1. Chemicalstructures structures of andand the lead 1, and structure strategy. Figure 1. Chemical ofmatrine matrine the compound lead compound 1, and modification structure modification strategy. The 1,3,4-thiadiazole group is recognized as a bioisostere of the carboxyl group [16], and also a privileged chemical scaffold [17–19], therefore,as inathe present study, thecarboxyl carboxylgroup was replaced with The 1,3,4-thiadiazole group is recognized bioisostere of the [16], and also a 1,3,4-thiadiazole group on the 11-side chain, whereby a series of novel tricyclic matrinic thiadiazole privileged chemical scaffold [17–19], therefore, in the present study, the carboxyl was replaced with derivatives was generated evaluated their anti-COL1A1 Herein,matrinic we describe the 1,3,4-thiadiazole group on theand 11-side chain,for whereby a series of activity. novel tricyclic thiadiazole synthesis of 19 novel thiadiazole derivatives, SAR analysis, as well as in vivo safety profile and primary derivatives was generated and evaluated for their anti-COL1A1 activity. Herein, we describe the mechanism of action of key compounds.
synthesis of 19 novel thiadiazole derivatives, SAR analysis, as well as in vivo safety profile and primary mechanism of action of key compounds. 2. Results and Discussion 2.1. Chemistry 2. Results and Discussion The semi-synthetic routes of all target compounds were outlined in Scheme 1, taking MT as the
2.1. Chemistry starting material. 12-N-substituted matrinic acids 1, 5a–d were obtained in high yields of 42–66% by a four-step procedure including esterification, were 12-N substitution hydrolysis instead The semi-synthetic routes ofhydrolysis, all target compounds outlined in and Scheme 1, taking MT of as the the original diphenyldiazomethane protection method [9,15]. Then a series of matrinic thiadiazole starting material. 12-N-substituted matrinic acids 1, 5a–d were obtained in high yields of 42–66% by derivatives 6a–o were obtained by the cyclization of 1 or 5a–d with thiosemicarbazide or various of a four-step procedure including hydrolysis, esterification, 12-N substitution and hydrolysis instead N-substituted thiosemicarbazides in the presence of phosphorus pentoxide/methanesulfonic acid (1:5) of theinoriginal protection method [9,15]. Then a series of matrinic thiadiazole yields ofdiphenyldiazomethane 28–58% [20,21]. The acylation of 6a, 6f, 6i and 6l in triethylamine generated compounds derivatives 6a–o were obtainedThe bydesired the cyclization of 1 or 5a–d with thiosemicarbazide or various of 7a–d with yields of 93–96%. products were purified with flash column chromatography N-substituted in the presence of phosphorus pentoxide/methanesulfonic acid on silica gelthiosemicarbazides using dichloromethane/methanol or petroleum/ethyl acetate as eluents.
(1:5) in yields of 28–58% [20,21]. The acylation of 6a, 6f, 6i and 6l in triethylamine generated compounds 7a–d with yields of 93–96%. The desired products were purified with flash column chromatography on silica gel using dichloromethane/methanol or petroleum/ethyl acetate as eluents.
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Scheme 1. Reagents conditions:(a) (a)5 5NNNaOH, NaOH, reflux, reflux, 99h;h;1010NNHCl, pHpH = 3–5; (b) (b) 2 N2MeOH/HCl, Scheme 1. Reagents andand conditions: HCl, = 3–5; N MeOH/HCl, reflux, 2 h; (c) Substituted benzensulfonyl chloride or benzyl bromide, TEA or K CO , CH Cl 2 3 or K22CO 2 or reflux, 2 h; (c) Substituted benzensulfonyl chloride or benzyl bromide, TEA 3, MeCN, CH2Cl2 or r.t., 6–8 (d) 5h; N NaOH, reflux, 6 h; 10 N HCl,6pH 7–7.5; P2 OpH (1:5),(e) thiosemicarbazide, 5 /MsOH MeCN, r.t.,h;6–8 (d) 5 N NaOH, reflux, h; =10 N (e) HCl, = 7–7.5; P2O5/MsOH (1:5), 70 ◦ C, 8 h; (f) Acetyl chloride, TEA, THF, r.t., 0.5 h. thiosemicarbazide, 70 °C, 8 h; (f) Acetyl chloride, TEA, THF, r.t., 0.5 h.
2.2. Inhibition of COL1A1 Promotor in Human Hepatic Stellate LX-2 Cells by Target Compounds
2.2. Inhibition of COL1A1 Promotor in Human Hepatic Stellate LX-2 Cells by Target Compounds A single luciferase reporter gene detection model was applied to screen the inhibitory A single luciferase reporter gene detection model was applied to screen the inhibitory effects effects towards COL1A1 promotor of all target compounds in human hepatic stellate LX-2 towards promotor of compounds in human hepatic LX-2 controls. cells at the cells COL1A1 at the concentration of all 40 target µM, taking EGCG and compound 1 as stellate the positive concentration of monoclone 40 μM, taking and simultaneously compound 1 with as the positive The LX2-COL The LX2-COL cell lineEGCG was treated TGFβ1 and a controls. target compound [13]. monoclone cell line was treated simultaneously with TGFβ1 and a target compound [13]. The cytotoxicity was determined using MTT assay in HepG2 cells. The structures and inhibitory effects The (%) of allwas target compoundsusing are shown Tablein 1. HepG2 cells. The structures and inhibitory effects cytotoxicity determined MTTinassay Taking 1 as the lead, the carboxyl group was (%) of all target compounds are shown in Table 1. retained, and the SAR investigation was initiated with the 1variation of 12-N by which 4 matrinicand acidthe derivatives 5a–d were was obtained. Taking as the lead, the substitution, carboxyl group was retained, SAR investigation initiated As depicted in Table 1, the replacement of the methyl group on the benzene ring with a trifluoromethyl with the variation of 12-N substitution, by which 4 matrinic acid derivatives 5a–d were obtained. As group (compound 5a) or the replacement of a benznesulfonyl with a benzyl motif (compounds 5b–d) depicted in Table 1, the replacement of the methyl group on the benzene ring with a trifluoromethyl caused a significant decrease in activity. group (compound 5a) or the replacement of a benznesulfonyl with a benzyl motif (compounds 5b– The carboxyl group at the end of the 11-side chain was then replaced by its 1,3,4-thiadiazole d) caused a significant decrease in activity. bioisostere, and a series of target matrinic thiadiazole compounds was prepared and evaluated. The carboxyl group at the end of the 11-side chain and wasfive then replaced by its 1,3,4-thiadiazole To start, the 12-N-p-methylbenzenesulfonyl was retained, matrinic aminothiadiazoles 6a–e bioisostere, and a series of target matrinichigher thiadiazole compounds was prepared were obtained. Compound 6a showed inhibitory effects than 6b–d and theand leadevaluated. 1, which To start,indicated the 12-N-p-methylbenzenesulfonyl and five matrinic aminothiadiazoles that 5-amino-1,3,4-thiadiazole was motifretained, was a beneficial group, and the introduction of an6a–e alkyl group on the amino group higher was notinhibitory helpful for effects activitythan in this series. In the the meantime, wereextra obtained. Compound 6a showed 6b–d and lead 1, which the introduction of a phenyl group into the amino (compound 6e) resulted in an obvious increase indicated that 5-amino-1,3,4-thiadiazole motif was a beneficial group, and the introduction of aninextra that thewas aminothiadiazole motif was more beneficial, result the alkylactivity. group It onwas theindicated amino group not helpful for activity in this series. which In themight meantime, from the smaller spatial hindrance. Then, the 12-N-substituent was replaced with a p-trifluoromethyl introduction of a phenyl group into the amino (compound 6e) resulted in an obvious increase in benzenesulfonyl moiety to prepare target compounds 6f–h, which displayed decreased activities in activity. It was indicated that the aminothiadiazole motif was more beneficial, which might result varied degrees, as anticipated. from the smaller spatial hindrance. Then, the 12-N-substituent was replaced with a p-trifluoromethyl Next, the aminothiadiazole motif was retained and the 12-N-benzenesulfonyl was replaced with a benzenesulfonyl moiety to prepare target compounds 6f–h, which displayed decreased activities in 12-N-benzyl group to generate the corresponding compounds 6i–o. To our great delight, most of them varied degrees, as anticipated. showed a significant improvement in activity, and compounds 6i, 6l and 6n gave inspiring inhibitory Next,ofthe aminothiadiazole motif was retained and thehigher 12-N-benzenesulfonyl wasand replaced rates 47.8%, 38.7% and 39.7%, respectively, significantly than that of 1 (18.5%) EGCGwith a 12-N-benzyl group to generate compounds 6i–o. To our great delight, most of (27.5%). These results suggestedthe thatcorresponding benzyl was a favorable 12-N substitution.
them showed a significant improvement in activity, and compounds 6i, 6l and 6n gave inspiring inhibitory rates of 47.8%, 38.7% and 39.7%, respectively, significantly higher than that of 1 (18.5%) and EGCG (27.5%). These results suggested that benzyl was a favorable 12-N substitution. Finally, an acyl group was condensed to the amino group of 6a, 6f, 6i and 6l to generate 7a–d,
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Finally, an acyl 6a group condensed to the amino group of7c 6a,and 6f,7d 6i displayed and 6l to generate 7a–d, amino counterparts and was 6f, while the 12-N-benzyl compounds lower activity respectively, and 12-N-benzenesulfonyl compounds 7a and 7b displayed higher activity than their than their counterparts 6i and 6l. amino counterparts 6a and 6f,6a, while compounds 7c and 7d displayed lower activity Five target compounds 6e, the 6i, 12-N-benzyl 6l and 6n with the highest activity were selected as the than their counterparts 6i and 6l. representative compounds to further evaluate their cytotoxicities. As indicated in Table 1, Five target compounds 6e, 6i, high 6l and 6n with the with highest were selected as the compounds 6a, 6i, 6l and 6n 6a, displayed cellular safety the activity concentration of half cellular representative to 320 further theirto cytotoxicities. As indicated in 6e Table 1, compounds toxicity (CC50) compounds value of over μM,evaluate comparable that of 1, while compound displayed a high 6a, 6i, 6l and 6n displayed high cellular safety with the concentration of half cellular toxicity (CC50 ) cellular toxicity with a CC50 value of over 82.6 μM, indicating that the introduction of a benzylamino value over 320 µM, comparable to that ofto1,awhile displayed a high cellular toxicity to theof thiadiazole motif might contribute high compound cytotoxicity,6ewhich might result from its high with a CC value of over 82.6 µM, indicating that the introduction of a benzylamino to the thiadiazole 50 lipophilicity. motif might contribute to a high cytotoxicity, which might result from its high lipophilicity. Table 1. Inhibition of COL1A1 promotor of target compounds and cytotoxicity of key compounds. Table 1. Inhibition of COL1A1 promotor of target compounds and cytotoxicity of key compounds.
a a b b Code R11 R Inhibition Percentage CC Code R R22 Inhibition Percentage CC (μM) 5050(µM) 11 p-CH 18.5% 1.1% >320 p-CH33C C66H H44SO SO22 -18.5% ±± 1.1% >320 p-CF33C C66H H44SO SO22 -−3.7% ± ±3.0% NT 5a5a p-CF −3.7% 3.0% NTc c c 5b C H CH − 33.6% ± 10.6% NT 5b C66H55CH22 −33.6% ± 10.6% NT c 5c p-CH3 C6 H4 CH2 −116.2% ± 45.7% NT c c 5c5d p-CH 3C6H4CH2 −116.2% ± 45.7% NT p-BrC6 H4 CH2 6.4%± 0.9% NT c 5d6a p-BrC 6 H 4 CH 2 6.4%± 0.9% NT c p-CH3 C6 H4 SO2 NH2 21.2% ± 10.4% >320 c p-CH33C C66H H44SO SO22 NHCH 12.8% NT 6a6b p-CH NH2 3 21.2%±±7.3% 10.4% >320 c 6c p-CH C H SO NHCH(CH ) − 84.2% ± 63.1% NT 6b p-CH33C66H44SO22 NHCH33 2 12.8% ± 7.3% NT c 6d p-CH C H SO N(CH3 )2 −36.7% ± 17.3% NT c c 6c p-CH33C66H44SO22 NHCH(CH 3)2 −84.2% ± 63.1% NT 6e p-CH3 C6 H4 SO2 NHPh 69.0% ± 13.5% 82.55 6d6f p-CH N(CH 17.3% NTc c p-CF33C C66H H44SO SO22 NH2 3)2 −−36.7% 19.0% ±±7.8% NT c 6e6g p-CH NHPh3 69.0% 13.5% 82.55 p-CF33C66H44SO22 NHCH 6.1% ±± 0.8% NT p-CF33C C66H H44SO SO22 N(CH −35.2% ± ±2.1% NT 6f6h p-CF NH32)2 −19.0% 7.8% NTc c C63H CH NH2 3 47.8% ± ±29.0% >320 2 2 6g6i p-CF C56H 4SO NHCH 6.1% 0.8% NT c 6j C6 H5 CH2 NHCH3 29.5% ± 6.1% NT c c 6h6k p-CF 3C6H4SO2 N(CH3)2 −35.2% ± 2.1% NT C6 H5 CH2 N(CH3 )2 15.2% ± 7.1% NT c 6i6l C 6 H 5 CH 2 NH 2 47.8% ± 29.0% >320 p-CH3 C6 H4 CH2 NH2 38.7% ± 4.4% >320 p-CH H4 CH NHCH33 −29.6% ± ±13.6% NT 6j6m C6H 2 NHCH 29.5% 6.1% NTc c 3 C56CH 2 6n p-CH C H CH N(CH ) 39.7% ± 12.3% >320 3 56CH 42 2 6k C6H N(CH33)22 15.2% ± 7.1% NT c 6o p-BrC6 H4 CH2 NH2 7.8% ± 3.0% NT c 6l p-CH3C6H4CH2 NH2 38.7% ± 4.4% >320 7a p-CH3 C6 H4 SO2 NHCOCH3 33.9% ± 9.9% NT c 6m7b p-CH 3C6H4CH2 NHCH 3 −29.6% ± 13.6% NTc c p-CF3 C6 H4 SO2 NHCOCH3 −6.0% ± 5.0% NT c 6n7c p-CH C56H 4CH N(CH3)23 39.7%± 12.3% >320 C63H CH NHCOCH 12.9% ± 10.6% NT 2 2 c 7d p-CH C H CH NHCOCH 27.0% ± 9.5% NT 3 6H 6 4CH 4 22 6o p-BrC NH2 3 7.8% ± 3.0% NT c EGCG 27.5% ±± 7.9% - c 7a p-CH3C-6H4SO2 NHCOCH 3 33.9% 9.9% NT DMSO 2.9% ± 0 7b p-CF3C6H4SO2 NHCOCH3 −6.0% ± 5.0% NT c a The c 7c inhibition rateCof 6H 5CH2 NHCOCH 12.9% ±concentration 10.6% NT COL1A1 promotor’s activity in 3LX2 cells; b Cytotoxic required to inhibit c Not tested. HepG2 cells cell growth by 50%; 7d p-CH3C6H4CH2 NHCOCH3 27.0% ± 9.5% NT c EGCG 27.5% ± 7.9% 2.3. Inhibition Effects of COL1A1 on RNA and Protein Levels by Key Compounds DMSO 2.9% ± 0 a The inhibition b Next, compounds 6i, 6l and 6n were chosen as the key compounds to verify their inhibitory rate of COL1A1 promotor’s activity in LX2 cells; Cytotoxic concentration required c Not tested. effects COL1A1. LX-2 cells by were stimulated with TGFβ1 (2 ng/mL), and then treated with to against inhibit HepG2 cells cell growth 50%; compounds 6i, 6l and 6n (80 µM) respectively, taking 1 as the control. Their anti-COL1A1 effects 2.3. Inhibition Effects of COL1A1 on real-time RNA and Protein Levels by Key As Compounds were evaluated on RNA level by PCR amplification. indicated in Figure 2A, all three compounds 6i, 6l and 6n6i,could significantly reduce as COL1A1 level with the inhibitory rates of Next, compounds 6l and 6n were chosen the keymRNA compounds to verify their inhibitory
effects against COL1A1. LX-2 cells were stimulated with TGFβ1 (2 ng/mL), and then treated with compounds 6i, 6l and 6n (80 μM) respectively, taking 1 as the control. Their anti-COL1A1 effects
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wereMolecules evaluated onx RNA level by real-time PCR amplification. As indicated in Figure 2A,5 all three 2018, 23, 23, Molecules 2018, 1644 5 of of 15 16 compounds 6i, 6l and 6n could significantly reduce COL1A1 mRNA level with the inhibitory rates were evaluated RNA respectively, level by real-time PCR amplification. As1indicated in varying Figure 2A, all threeThen of 46.1%,58.2% andon 78.4%, prevailing over that of (41.9%) to degrees. 46.1%,58.2% and 78.4%, respectively, prevailing over that of 1 (41.9%) to varying degrees. Then their compounds 6i, 6l and 6n could significantly reduce COL1A1 mRNA level with the inhibitory rates their anti-COL1A1 effects were investigated on the protein level by western blot assays. As indicated anti-COL1A1 effects were investigated on the protein level by western blot assays. As indicated in of 46.1%,58.2% respectively, prevailing of 1 (41.9%) to level varying degrees. Then in Figure 3A, 6i, 6l and and78.4%, 6n could significantly reduceover thethat COL1A1 protein with inhibition rates Figure 3A, 6i, 6l and 6n could reduce the COL1A1 level inhibition rates their anti-COL1A1 effects were significantly investigated on the protein level byprotein western blotwith As indicated of 65.0%, 99.3% and 47.6%, respectively, significantly higher than that of 1 assays. (16.2%). These results of 65.0%, 3A, 99.3% and 47.6%, respectively, significantly higher thanprotein that oflevel 1 (16.2%). These results in Figure 6i, 6l and 6n could significantly reduce the COL1A1 with inhibition rates suggested thatthat these matrinic thiadiazole compounds couldeffectively effectively reduce COL1A1 on both the suggested these matrinic thiadiazole compounds could reduce COL1A1 on both the of 65.0%, 99.3% and 47.6%, respectively, significantly higher than that of 1 (16.2%). These results mRNA andand protein levels. mRNA protein suggested that theselevels. matrinic thiadiazole compounds could effectively reduce COL1A1 on both the mRNA and protein levels.
Figure 2. Effects of compounds 6i, inhibiting A)COL1A1, COL1A1, α-SMA mRNA levels in LX-2 Figure compounds 6i,6l6l 6land and6n 6ninhibiting inhibiting(A) A) COL1A1, B)B) α-SMA mRNA levels in LX-2 2. Effects of compounds 6i, and 6n (B) α-SMA mRNA levels cells.cells. DataData were analyzed byby Real-time presented asthe the mean ± SEM, < 0.05 as compared were analyzed by Real-timePCR PCRand andpresented presentedas as the mean as analyzed Real-time PCR and mean ±± SEM, (#) (#) p
