Synthesis and evaluation of novel potent HCV NS5A inhibitors

Abstract

Judicious modifications to the structure of the previously reported HCV NS5A inhibitor1, resulted in more potent anti-HCV compounds with similar and in some cases improved toxicity profiles. The synthesis of nineteen new NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. For the most potent compounds chemical stability, stability in liver microsomes and inhibition of relevantCYP450 enzymes is also presented.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease, liver transplantation and is the most common blood-borne infection in developed countries.¹ The prevalence of HCV infection ranges from 1–5% in most developed countries and is estimated to be 5-fold greater than HIV infection.¹ Until recently, the only approved drugs for the treatment of HCV were pegylated interferon-α (IFN) and ribavirin (RBV), both of which are poorly tolerated and have limited efficacy, with less than 50% response rates among individuals infected with the most common virus genotype (1b).² The FDA approval, in May 2011, of the protease inhibitors (PI) boceprevir (Victrelis) and telaprevir (Incivek) which created a lot of excitement however, their real impact on standard of care (SOC) remains unclear as sustained virologic response (SVR) for genotype 1 HCV is still only about 70 to 80% when administered with IFN and RBV.³

Efforts toward improved HCV treatment include the development of antiviral agents that inhibit HCV via new targets such as the viral NS5A protein. Despite significant gaps in the understanding of the role of NS5A in the virus replication cycle, it is known that it plays an integral role in a number of host and/or pathogen events such as RNA replication, assembly of viral particles and host immune response.⁴ NS5A phosphokinase is phosphorylated by cellular protein kinases and the phosphorylation sites are conserved among the various HCV genotypes.^5,6Recently, the NS5A inhibitor BMS-790052 (Figure 1) was discovered and displayed in vitro potency ((EC₅₀) of ≤ 50 pM)⁷ that translated to rapid viral load decline (≈ 3.3 log₁₀) after a single 100 mg dose with no signs of toxicity.⁸ While compound 1 has established NS5A as a valid target for effective clinical treatment of HCV, it also provided a foundation for the design of second-generation inhibitors that could be more resistant to selection of mutant virus with reduced potential cytotoxic liabilities. This lead us to dissect the symmetrical inhibitor 1 into four major structural zones: zone I (biphenyl linker zone) has already been studied extensively by other research groups; zone II (imidazole portion) despite claims of small modifications no significant exploration of this area has been disclosed; zone III (proline moiety), minor modifications such as fluorination or cyclopropanation have been reported; zone IV (amino acid and its “cap”) has been extensively studied.^8,9

Figure 1.

Chemical structure of BMS-790052

In consideration of novelty and retention of potency, we initially chose to focus on both zones II and III. Indeed, the lack of significant exploration of zone II along with the ease of halogenation of the imidazole ring, which would allow for the efficient preparation of more complex structures using transition metal catalyzed cross coupling reactions, made zone II ideal for investigation. Similarly, zone III has not been extensively explored while readily available proline analogs would allow for extensive modifications from well known organic transformations. Herein we wish to report our preliminary structure activity relationship (SAR) efforts for zones II and III.

The biphenyl core of the targeted compounds was prepared according to general Scheme 1. Diketone 1 was first converted to the α, α′-dibromodiketone using Br₂ in CH₂Cl₂ then coupled with N-Boc protected proline derivatives A¹⁰ in the presence of DIPEA. Refluxing the corresponding ester intermediates with ammonium acetate in toluene allowed for the formation of the imidazole ring and lead to the versatile Boc protected intermediates 3a-c in 63% to 75%yields.

Scheme 1.

Reagents and conditions: (a) Br₂, CH₂Cl₂, rt, overnight; (b) (i) A, DIPEA, rt, 5 h; (ii) NH₄OAc, toluene, 100-110 °C, 15-20 h.

Exploration of 4-substituted imidazole derivatives, as shown in Scheme 2, marked the beginning of our synthetic efforts and initial SAR studies. Bromination of the 4-position of the imidazoles of 3a was accomplished using N-bromosuccinimide (NBS) in dichloromethane (52% yield). After removal of Boc protection with 6N HCl, the resulting amine was coupled with N-methylcarbamate protected valine using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxybenzotriazole (HOBt) in the presence of N, N-diisopropylethylamine (DIPEA). The palladium catalyzed cross coupling reaction of 5a under Stille conditions¹¹ resulted in 4-aryl and 4-heteroaryl compounds 6-8 in moderate yields providing the first in this zone II series of aryl substituted imidazoles.

Scheme 2.

Reagents and conditions: (a) X = Br, I: NXS, CH₂Cl₂, rt, 30 min; X = Cl: NCS, 40 °C, 15 h; X = F, NFSI, NaHCO₃, CH₂Cl₂/acetone (1:1), 50 °C. (b) 6 N HCl, CH₃OH, 50 °C, 5 h; (c) HOOC-CH[CH(CH₃)₂]-NHCOOCH₃, EDC, HOBt, DIPEA, 0 °C – rt, 15 h; (d) R-Sn(Bu)₃, Pd(PPh₃)₂Cl₂, dioxane, 80 °C, overnight.

Introduction of thiophene, furan or phenyl at the 4-position of the imidazole lead to a noticeable decrease of potency toward HCV replication (EC₅₀ of 100, 26 and 200 pM respectively) when compared to 1. Evaluation of the brominated intermediate 5a for anti-replication activity versus HCV proved surprising as it displayed an EC₅₀ of 5.9 pM and EC₉₀ of 14 pM; thus compound 5a was equipotent to BMS-790052 at the EC₅₀ and EC₉₀. This unexpected result prompted us to investigate the influence of other halogens at the 4-position of the imidazole ring. Iodine and chlorine were thus introduced on the imidazole ring of compound 3a using N-iodosuccinimide (NIS) and N-chlorosuccinimide (NCS) respectively while fluorine was introduced by careful reaction with N-fluorobenzenesulfonimide (NFSI) and NaHCO₃. It is noteworthy that this fluorination reaction leads to the formation of both the difluorinated derivative 4d and the monofluorinated derivative 4e. As above, simple Boc deprotection under acidic conditions followed by the coupling of N-methylcarbamate protected valine in the presence of HOBt and DIPEA resulted in the dihalogeneated compounds 5b-d and monofluoro derivative 5e. Remarkably the bis-iodo, bis-chloro and bis-fluoro compounds 5b, 5c and 5d exhibited an EC₅₀ of 6.4 ± 3.0, 5.5 ± 1.9 and 6.8 ± 3.3 pM respectively which, as in the case of the bis-bromo compound, are equipotent to the reference compound 1. The EC₉₀ values for these three were all close to 10 pM representing a modest improvement relative to the reference compound 1. In addition, all of the bis-halogen compounds 5c and 5d exhibited increased toxicity in primary human lymphocytes, although they were non-toxic at concentrations up to 100 μM in Vero cells. These results suggest that a small modification in this region (Zone II) such as introduction of a halogen is well tolerated but introduction of larger groups such as thiophene, furane and phenyl are detrimental to the HCV replicon activity.

Zone III modifications (Scheme 3) included introduction of various triazoles using copper catalyzed alkyne azide cycloadditions.¹² Azido intermediates 3b and 3c were first deprotected under acidic conditions then coupled with N-methylcarbamate protected valine in presence of HOBt and DIPEA to give compounds 9-10 in respectively 64 and 59% yields. Finally, treatment of 9-10 with various alkenes in the presence of CuSO₄ and sodium ascorbate afforded the 1,4-triazolo derivatives 11-14 in good yields. Like for Zone II, introduction of bulky modifications such as 1,4-substituted triazoles on the proline moiety (Zone III) considerably decreased potencies. On the other hand, introduction of a small group such an azido group seems to be well tolerated. Indeed, (4-R)-azido compound 10 with an EC₅₀ = 9.5 pM appeared as potent as reference compound 1 without displaying any signs of toxicity up to 100 μM in PBM, CEM and Vero cells. Interestingly, (4-S)-azido compound 9 appeared slightly less active (EC₅₀ = 23 pM) and showed toxicities in PBM and CEM cells (IC₅₀ = 20.3 and 18.4 μM respectively).

Scheme 3.

Reagents and conditions: (a) 6 N HCl, CH₃OH, 50 °C, 5 h (b) HOOC-CH[CH(CH₃)₂]-NHCOOCH₃, EDC, HOBt, DIPEA, 0 °C, rt, overnight. (c) alkyne, sodium ascorbate, CuSO₄.5H₂O, t-BuOH/H₂O (1:1), rt, overnight.

Based on these results, a combination of our best modifications in both zone II and III was also studied. Thus, (4-S)- and (4-azido compounds 9 and 10 were halogenated at the R)- azido compounds 9 and 10 were halogenated at the 4-position of the imidazoles rings by using either NBS or NCS in CH₂Cl₂ (Scheme 4) and all the synthesized compounds displayed an EC₅₀ ≤ 10 pM. Interestingly, compound 17 showed an EC₅₀ of 2.6 pM with no toxicity toward Vero cells up to 100 μM.

Scheme 4.

Reagents and conditions: (a) X = Br: NBS, CH₂Cl₂, rt, 0.5-1 h; X = Cl: NCS, CH₂Cl₂, 50 °C, 4-24 h.

Among all the molecules synthesized, the most promising compounds 5a-d were further investigated. First of all, their stability at pH 3 and 7 in phosphate buffer was evaluated. After 48 h at 20°C, LC/MS analysis at time 0, 1, 8, 24 and 48 h revealed no evidence of degradation of these compounds. Next stability in human liver microsomes was established (Table 2). Compounds 5a-d (1 μM) were incubated with human liver microsomes in potassium phosphate buffer and 1 mg/mL microsomal protein. Samples were collected at 5, 15, 30 and 60 min, analyzed by LC-MS/MS and half-life (t_1/2) and intrinsic clearance (CL_int) for each compound were calculated based on the elimination rate constant (k). Interestingly, compounds 5b and 5d show higher metabolic stability and therefore lower clearance (CL_int< 8.6) compared with BMS-790052.

Table 2. Half-life and intrinsic clearance values in human liver microsomes of compounds 5a-d and BMS-790052.

Cmpd	Elimination rate constant (k)	t1/2 (min)	CLint (μL/min/mg protein)
5a	0.01	67.3	10.3
5b	0.0058	119.5	5.8
5c	0.018	38.5	18.0
5d	0.0056	123.8	5.6
BMS-790052	0.012	56.3	12.3

Drug-drug interactions have become an important issue in modern health care. It is now apparent that many drug-drug interactions can be explained by alterations in the metabolic lenzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (CYP450) enzymes being affected by one or more of the drugs present in a given individual. Furthermore, drug interactions can be a result of inhibition or induction of CYP450 enzymes. The potential drug-drug interaction liabilities of these compounds were investigated by a CYP450 reversible inhibition assay. Compounds 5b and 5d did not appear to inhibit the major CYP450 enzymes at the IC₉₀ level and had a favorable CYP profile, which is suggestive of no potential drug-drug interactions (Table 3). Due to the large therapeutic window, the inhibition observed for 3A4 (in the micromolar range) is expected not to be clinically relevant at therapeutic doses.

Table 3. Cytochrome P450 inhibition data for compounds 5b, 5d and BMS-790052^a.

	CYP1A2 (μM)		CYP3A4 (μM)		CYP2D6 (μM)		CYP2C9 (μM)
Cmpd	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90
5b	> 100	> 100	13.8 ± 11.2	> 100	> 100	> 100	81.6 ± 26.1	> 100
5d	> 100	> 100	3.1 ± 2.3	> 100	> 100	> 100	> 100	> 100
BMS-790052	> 100	> 100	7.2 ± 1.7	> 100	> 100	> 100	59.5 ± 13.6	> 100

^aMean ± SD of at least two independent assays; IC₅₀ = 50% inhibitory concentration. The following positive controls were used (IC₅₀, μM): α -naphthoflavone for 1A2 (> 93% at 3 μM); ketoconazole for 3A4 (> 93% at 10 μM); PH-053 (proprietary) for 2D6 (> 70% at 100 μM); sulfaphenazole for 2C9 (> 90% at 10 μM).

Initial resistant virus selection studies using 5b and BMS-790052 were performed and mutations identified by population sequencing. Mutations observed for BMS-790052 included L31V, which was previously reported,¹³ in addition to several unreported mutations, which we will disclose in full detail in subsequent publications. Compound 5b selected for several different mutations including Y93H, which was previously reported as an NS5A resistance mutation.¹³

In conclusion, extremely potent new NS5A inhibitors (picomolar activity) with a wide therapeutic window (> 10⁴) are disclosed. Among all the compounds prepared 5b and 5d exhibited improved liver microsome stability compared to reference compound BMS-790052 and no inhibition of major human CYP enzymes was exhibited at therapeutically relevant concentrations. In addition, these two halogenated compounds displayed improved stability toward human liver microsomes versus BMS-790052 while maintaining comparable anti-HCV replication activity. Finally, further mutation selection studies are currently being investigated in our laboratory and will be the subject of future publications.

Table 1. Chemical Structure, Anti-HCV Activity and Cytotoxicity of Compounds 5-19.

Cmpd	X	X′	Y	Y′	Z	Z′	HCV Replicon (pM)		Cytotoxicity, CC50 (μM)*
							EC50	EC90	PBM	CEM	Vero
BMS-790052	H	H	H	H	H	H	4.2 ± 1.5	20 ± 7.5	19	9.6	21
5b	Cl	Cl	H	H	H	H	6.4 ± 3.0	11.4 ± 4.8	2.8	12	≥ 100
5a	Br	Br	H	H	H	H	5.9 ± 3.9	14 ± 8.3	2.9	5.7	84
5c	I	I	H	H	H	H	5.5 ± 1.9	11 ± 2.2	4.7	35	> 100
5d	F	F	H	H	H	H	6.8 ± 3.3	21.2 ± 11.0	19	3.0	> 100
5e	H	F	H	H	H	H	14.4 ±4.0	52.5 ±11.1	16	2.8	83
6	Ph	Ph	H	H	H	H	200	700	> 100	37.4	> 100
7			H	H	H	H	26	97	49.2	2.9	>100
8			H	H	H	H	100	300	44.6	> 100	> 100
9	H	H	N3	N3	H	H	23	35.2	20.3	18.4	> 100
10	H	H	H	H	N3	N3	18.4 ± 12.6	54.2 ± 34.1	> 100	≥ 100	> 100
11	H	H	H	H			2700	9200	> 100	> 100	> 100
12	H	H	H	H			11.8	42.8	6.6	10.7	> 100
13	H	H			H	H	757 102	> 104	96	> 100	> 100
14	H	H			H	H	> 104	> 104	> 100	> 100	> 100
15	Cl	Cl	H	H	N3	N3	4.7	9.6	7.8	3.8	> 100
16	Cl	H	H	H	N3	N3	6.3	29.5	9.7	31	> 100
17	Br	Br	H	H	N3	N3	2.6	21.6	19.8	10.8	> 100
18	Cl	Cl	N3	N3	H	H	11.2	74.6	3.3	4.0	98.1
19	Br	Br	N3	N3	H	H	8.3	55.3	3.4	4.5	> 100

^*All compounds had a CC₅₀ of > 1 μM in Huh-7 cells