Synthesis and biological evaluation of tricyclic matrinic derivatives as a class of novel anti-HCV agents

Background 12N-benzyl matrinic acid analogues had been identified to be a novel scaffold of anti-HCV agents with a specific mechanism, and the representative compound 1 demonstrated a moderate anti-HCV activity. The intensive structure–activity relationship of this kind of compounds is explored so as to obtain anti-HCV candidates with good druglike nature. Results Taking compound 1 as the lead, 32 compounds (of which 27 were novel) with diverse structures on the 11-side chain, including methyl matrinate, matrinol, matrinic butane, (Z)-methyl Δβγ-matrinic crotonate derivatives were synthesized and evaluated for their anti-HCV activities. Among all the compounds, matrinol 7a demonstrated potential potency with a greatly improved SI value of 136. Pharmacokinetic studies of 7a showed the potential for oral administration that would allow further in vivo safety studies. The free hydroxyl arm in 7a made it possible to prepare pro-drugs for the potential in the treatment of HCV infection. Conclusions 27 novel 12N-substituted matrinol derivatives were prepared. The SAR study indicated that the introduction of electron-donating substitutions on the benzene ring was helpful for the anti-HCV activity, and the unsaturated 11-side chain might not be favorable for the activity. This study provided powerful information on further strategic optimization and development of this kind of compounds into a novel family of anti-HCV agents. Graphical abstract Matrinol derivatives as a class of novel anti-HCV agents


Background
Currently, at least 130-150 million people worldwide have been infected with hepatitis C virus (HCV) [1]. Each year, 3-4 million people are newly infected and HCV-related liver complications kill estimated 700,000 people annually [1,2]. In recent years, new direct acting antivirals (DAAs) specifically targeting HCV proteins have made a great breakthrough to HCV treatment, and NS3/4A HCV protease inhibitors telaprevir, boceprevir and simeprevir, NS5A inhibitors asunaprevir and ledipasvir, NS5B polymerase inhibitors sofosbuvir and dasabuvir have been approved by FDA for the HCV treatment successively since 2011 [3]. To deal with the springing up of drug resistance challenges [4][5][6], multiple of DAA combinations have been developed [7][8][9]. Therefore, it is still imperative to develop new anti-HCV agents with novel structure skeleton or mechanism of action as a new component to DAA combination.
In our earlier studies, 12N-benzyl matrinic acid analogues had been successfully identified to be a novel class of anti-HCV agents from matrine, a natural product extracted from traditional Chinese herb. The representative compound, 12N-4-methoxylbenzyl matrinic acid (1, Fig. 1) was identified to be active against HCV with a novel mechanism targeting on host protein Hsc70 and demonstrated a moderate anti-HCV activity with SI over 22 [10,11]. The special tricyclic flexible scaffold and Open Access *Correspondence: wangyanxiang@imb.pumc.edu.cn; songdanqingsdq@hotmail.com † Sheng Tang and Zong-Gen Peng equally contributed to this work Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China appealing druglike of compound 1 strongly provoked our interesting to continuously explore the structure-activity relationship (SAR) of this kind of compounds, in an effort to discover novel anti-HCV candidates which could be used in the combination with current DAA.
In the present study, as illustrated in Fig. 1, taking 1 as the lead, SAR studies were further conducted with the variations of the 11-side chain and diverse substituents on 12N-atom. Therefore, series of novel methyl matrinate, matrinol, matrinic butane, 1′, 1′-dialkyl matrinol, methyl (Z)-Δ βγ -matrinic crotonate and (Z)-Δ βγ -matrinic crotonl derivatives were designed, synthesized and evaluated for their in vitro anti-HCV activities as well as the in vivo pharmacokinetic (PK) and safety profile of the representative compounds.

Anti-HCV activity and SAR analysis of matrinol derivatives
All the target compounds were evaluated for their anti-HCV activities (EC 50 ) and cytotoxicities (CC 50 ) in human Huh7.5 cells using specific real-time RT-PCR assay, as described earlier [11]. As an important indicator, the selectivity index (SI) was calculated as a ratio of CC 50 to EC 50 . Anti-HCV ability of a given compound was estimated by combining its EC 50 with SI values. Totally 32 compounds were gathered, and their structures and anti-HCV effects were shown in Table 1. SAR investigation was initiated with the variation of carboxylic acid group, by which 7 methyl matrinates (2, 6a-f) and 13 matrinols (3a-d and 7a-i) were generated. As depicted in Table 1 Table 1 SAR of all the targeted compounds for anti-HCV activity in Huh7.5

cells
Tela telaprevir a Cytotoxic concentration required to inhibit Huh7.5 cell growth by 50% matrinates exerted higher activities than the lead 1 by showing lower EC 50 values and higher SI values of over 50. In particular, 12N-4-fluorobenzyl 2, 4-methylbenzyl 6b, 4-vinylbenzyl 6c and 2,4-difluorobenzyl 6d displayed potent anti-HCV activities with EC 50 values ranging from 1.61 to 2.09 µM, which were over 20 times more potent than that of 1. It appeared that the electron-donating substitutions on the benzene ring were more favorable than the electron-withdrawing groups in the methyl matrinate series.
Then, SAR investigation was focused on the influence of the structural type of the 11-side chain while the 12N-benzyl/pyridylmethyl substitution was retained. In the first round, matrinic butane (9), five 1′, 1′-dialkyl substituted matrinols (10a-e) were designed and synthesized. Among them, benzyl derived analogues (9, 10a-d) exhibited promising anti-HCV activities with low micro molar EC 50 values ranging from 0.23 to 11.70 μM, as well as limited toxicity with CC 50 between 12.3 and 155.8 µM, while the 12N-pyrid-4-ylmethyl derivative 10e showed a high EC 50 value of 80.38 µM. The results indicated that 11-butane or 1′,1′-dialkyl butanol chain might not be helpful for the activity.
In the second round, to further examine the influence of saturation of 11-side chain on the activity, double bond was introduced to the β,γ position of the butyl acid chain, and the corresponding methyl Δ βγ -matrinic crotonates (13a-c) and crotonyl alcohols (14a-b and 20) with 4-methoxyl, 4-fluoro, 4-nitrobenzyl substitution on the 12N atom were generated respectively. As described in Table 1, most compounds afforded very weak potencies with SI values between 10.0-24.3, inferring that the unsaturated side-chain might not be favorable for the HCV activity.

PK study
Based on above, methyl matrinates and matrinols exhibited the most potent anti-HCV activities, however, methyl matrinates might not possess favorable PK profiles in vivo owing to the exposed metabolically labile ester group. Therefore, two representative matrinols 7a and 7b were chosen to examine their PK parameters in SD rats at the single dosage of 25 mg kg −1 via oral route. As indicated in Table 2 and Fig. 2, both of them showed acceptable PK profiles with the areas under the curve (AUCs) of 1.58 and 2.36 μM·h and the half-times of 4.69 h and 3.39 h respectively, indicating reasonable stabilities in vivo. Meanwhile, the results demonstrated that the concentration of compounds 7a and 7b showed a significant difference at 2 h, owing to different dissolution rate at that time in vivo.

Acute toxicity study
The acute toxicity tests of 7a and 7b were performed in Kunming mice. Each compound was given orally in a single-dosing experiment at 250, 500, 750 or 1000 mg kg −1 , respectively. The mice were closely monitored for 7 days. As indicated in Table 3, the LD 50 values for 7a and 7b were 708 and 392 mg kg −1 , respectively, therefore, 7a seemed to be more promising as a parent drug from a safety prospective.

Instruments
Unless otherwise noted, all commercial reagents and solvents were obtained from the commercial provider and used without further purification. Melting points (mp) were obtained with CXM-300 melting point apparatus and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 400 (400/101 MHz

General procedures for methyl 12N-substituted matrinate derivatives 6a-f
Matrine (5.0 g, 20.0 mmol) was added to 5 N NaOH in water (30 mL), and the reaction mixture was refluxed for 9 h, cooled in an ice bath and then acidified with HCl (2 N) to pH 6-7. The solvent was removed in vacuo and the residue was dissolved with 2 N HCl in methanol and then heated at refluxing for 2 h. The solvent methanol was removed under reduced pressure to give crude 5 (5.5 g, yield 77%), which was applied directly in the next step without further purification.
To a stirred solution of 5 (10.0 mmol) and K 2 CO 3 (35.0 mmol) in chloroethane (50 mL), the substituted benzyl halide (10 mmol) was added. The reaction mixture was stirred at room temperature for 5-8 h until TLC analysis showed completion of the reaction. Water (20 mL) was added to the mixture and the organic phase was separated and dried with anhydrous Na 2 SO 4 , concentrated, and the gained residue was purified by flash column chromatography on silica gel with CH 2 Cl 2 / CH 3 OH as the eluent to afford the title compounds.

Methyl 12N-(4-methoxybenzyl)matrinate dihydrochloride (6a)
The title compound was prepared from 5 and 4-methoxybenzyl bromide in the same manner as described above followed by an acidification with 2 N hydrochloride/ ether (10 mL

Methyl 12N-(4-methylbenzyl)matrinate (6b)
The title compound was prepared from 5 and 4-methylbenzyl bromide in the same manner as described in the general procedures.

General procedures for 12N-substituted matrinol derivativess 7a-e
A solution of LiAlH 4 (12 mmol) in anhydrous THF (20 mL) was added to the solution of compound 6 (10 mmol) in anhydrous THF (3 mL) in an ice bath, the mixture solution was then stirred at room temperature for 30 min before the reaction was quenched with acetone. Saturated ammonium chloride (2 mL) was then added and the mixture was stirred for 30 min, and the precipitation was filtered off. The solvent was evaporated, and the residue was purified by flash column chromatography on silica gel with CH 2 Cl 2 /CH 3 OH as the eluent or followed by an acidification with 2 N hydrochloride/ether (10 mL) to afford target compounds.

General procedures for 12N-substituted matrinol derivatives 7f-i
To a stirred solution of 5 (5.0 mmol) and K 2 CO 3 (17.0 mmol) in dichloroethane (50 mL), substituted pyridylmethyl halide or phenylcarbamic chloride (5 mmol) was added. The reaction mixture was stirred at room temperature for 8 h until TLC analysis showed completion of the reaction. Water (20 mL) was added to the mixture and the organic phase was separated and dried with anhydrous Na 2 SO 4 , concentrated. To a solution of the gained residue in anhydrous THF (3 mL) in an ice bath, a solution of LiAlH 4 (6 mmol) in anhydrous THF (10 mL) was added, the mixture solution was stirred at room temperature for 30 min before the reaction was quenched with acetone. The saturated ammonium chloride (2 mL) was then added and the mixture was stirred for 30 min, and the precipitation was filtered off. Then the solvent was evaporated, and the residue was purified by flash column chromatography on silica gel with CH 2 Cl 2 /CH 3 OH as the eluent to afford the target compounds.

Synthesis of 12N-4-methoxybenzyl matrinic butane 9
To a solution of 7a (5 mmol) in anhydrous CH 2 Cl 2 (20 mL), TsCl (5 mmol), TEA (10 mmol) and dimethylamino pyridine (0.5 mmol) were added and stirred at room temperature until the TLC showed completion of the reaction. The solution was washed successively by water (10 mL), saturated ammonium chloride solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain crude 8. To a solution of the crude 8 in anhydrous THF, a solution of LiAlH 4 (6 mmol) in anhydrous THF was added in an ice bath, then the mixture was stirred at room temperature for 30 min, the reaction was then quenched with acetone, 2 ml saturated ammonium chloride was added and stirred for 30 min, and the precipitation was filtrated. The gained residue was purified by flash column chromatography on silica gel with CH 2 Cl 2 /CH 3 OH as the eluent to afford the title compound 9 as a yellow solid.

General procedures for 1′,1′-dialkyl-12N-substituted matrinol derivatives 10a-e
To a solution of compound 6 (5 mmol) in anhydrous THF (10 mL), a solution of 2 N alkylmagnesium chloride in THF (25 mmol) was added in an ice bath, and the mixture solution was heated at refluxing for 2 h. After reaction completed, the reaction was quenched with a solution of saturated aqueous ammonium chloride (2 mL). The residue was purified by flash column chromatography on silica gel with CH 2 Cl 2 /CH 3 OH as the eluent followed by the acidification with 2 N hydrochloride/ ether (3 mL) to afford the title compounds.

1′,1′-Dimethyl-12N-(4-pyridylmethyl)matrinol (10e)
The title compound was prepared from 6f and methylmagnesium chloride using the same method as described above without acidification. Compound 11 (1.0 g, 3.0 mmol) was dissolved in 2 N MeOH/HCl (30 mL), and the reaction mixture was refluxed for 2 h. Compound 12 was obtained by evaporation and used in the next reaction without further purification. Anhydrous K 2 CO 3 (3.5 equiv) and substituted benzyl bromide (1.5 equiv) were added to a solution of compound 12 in acetonitrile (30 mL), and the reaction solution was then stirred at room temperature until TLC analysis showed completion of the reaction. The reaction mixture was filtered, and the filtrate was washed by water and brine, dried with anhydrous Na 2 SO 4 , filtrated, and concentrated to afford crude compound 13. The title compounds were obtained by purifying with flash column chromatography on silica gel with dichloromethane and methanol as the eluent.