Synthesis and evaluation of non-dimeric HCV NS5A inhibitors

Abstract

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI - 90,000).

According to the World Health Organization, HCV has infected an estimated 170 million individuals worldwide and more than three million people contract HCV each year.¹ Of the six major HCV genotypes known, type 1a/b accounts for approximately 70% of all infections in the US, Europe, China and Japan.² Although often asymptomatic, it can progress to chronic hepatitis leading to liver cirrhosis and in many cases hepatocellular carcinoma. Until recently, HCV therapy, utilizing interferon/ribavirin (IFN/RBV), was effective in only 40-60% of patients and was often associated with significant side effects. In May 2011, two HCV protease inhibitors, Incivek and Victrelis, were approved by the US FDA for use in combination with the standard of care (IFN/RBV) and provided a sustained virologic response (SVR) of about 70 to 80% in genotype 1 HCV-infected subjects.³ However, these drugs have limitations that include the need of response-guided therapy and the management of severe skin rashes and anemia.

In the search for new therapeutic agents interfering with HCV replication, non-structural (NS) viral proteins have been primarily targeted including NS2, NS3 (protease), NS4A, NS4B, NS5B (polymerase) and NS5A. NS5A is a large phosphoprotein (49kDa) required for HCV replication and particle assembly. Although its ultimate role in the HCV replication cycle is not fully understood,⁴ it has been shown to be a required component of the HCV RNA replication and several molecules have been recently identified as potent inhibitors. For example, BMS-790052 has subnanomolar EC₅₀ values against genotype 1 replicons and a phase I clinical trial, in patients chronically infected with HCV, produced a 3.3 log₁₀ reduction in mean viral load 24 h after administration of a single 100 mg dose.⁵ Unfortunately, for both subtypes 1a and 1b, treatment with BMS-790052 quickly lead to the emergence of several drug-induced mutations.

We hypothesized that lower molecular weight unsymmetrical compounds could be developed to have greater activity toward known NS5A mutations and therefore, a higher barrier to resistance (Figure 1). As part of our SAR study, modifications were combined on various positions of the monodentate NS5A scaffold (A, B, C and D) and served as probes for 3D-exploration of the NS5A binding area (Figure 1). Based on this strategy, we have developed a second generation of unsymmetrical, still highly potent, NS5A inhibitors.

Figure 1.

Structures of BMS-790052 and targeted monodentate compounds.

Modifications on the various targeted positions have been achieved through general Scheme 1 by adapting chemistry previously described.⁶ Commercially available phenyl methyl ketones 1 were brominated in moderate to excellent yields then condensed with N-Boc protected proline in presence of DIPEA to afford compounds 2. Further, cyclization of the keto-ester of 2 to the corresponding imidzole was accomplished in presence of ammonium acetate followed by Boc deprotection under acidic conditions gave compounds 3. Modification of Part C and D was achieved by reaction of 3 with various protected amino acids under traditional peptidic coupling conditions and generated the analogs 4a-o. Secondary modifications of Part A were explored using Pd-catalyzed cross coupling between the bromo compound 4b and various substituted phenyl boronic acids (Scheme 2, compounds 6a-l) but also by alkylation of amino derivative 8 obtained by hydrogenation of azido compound 4c (Scheme 3, compounds 9a-b). Because we recently determined that halogenation of BMS-790052‘ imidazole ring using NBS, NCS or NIS lead to highly potent compounds exhibiting excellent overall profiles,⁷ we restricted our efforts on Part B to the preparation of compounds 5a-c and 7 (Scheme 1 and 2).

Scheme 1.

Reagents and conditions: (a) Br₂, CH₂Cl₂, rt, overnight, 70-95%; (b) Boc-Pro-OH, DIPEA, CH₃CN, rt, 5 h; (c) NH₄OAc, toluene, 95 °C, 15-20 h, 62-72% 2 steps; (d) 6N HCl, CH₃OH, 50 °C; (e) R⁴-(CO)NH-CHR³-COOH, DIPEA, HOBt, EDC, CH₃CN, 0 °C to rt, 15 h, 45-65% 2 steps; (f) NXS, CH₂Cl₂, rt, 0.5-15 h, 42-65% (X = Br, Cl, I).

Scheme 2.

Reagents and conditions: a) R⁵-Ph-B(OH)₂, PdCl₂(PPh₃)₂, Na₂CO₃, THF, rt, 50-77%; (b) NCS, CH₂Cl₂, rt, 15 h, 58%.

Scheme 3.

Reagents and conditions: (a) H₂, Pd/C, MeOH, rt, 15 h, 91%; (b) MeOC(O)Cl, pyridine, rt or MeSO₂Cl, pyridine, rt, 15 h, 60-75%.

Overall, 36 monodentate compounds were evaluated in an in vitro HCV replicon assay and their potential toxic effects assessed in multiple cell lines (Table 1), including: Huh-7 (a human hepatocellular carcinoma cell line), primary human hepatocytes, Vero (African green monkey kidney epithelial cells), and PBM (primary human peripheral blood mononuclear cells). While the first simple truncated compound 4a and modified derivatives 4b and 4d-o did not show any activity against HCV in the replicon system when tested up to 10 μM (Table 1), functionalization of the phenyl ring with an azido group lead to compound 4c, exhibiting a median effective concentration (EC₅₀) of 8 nM against HCV with no apparent toxicity in four different cell lines. Interestingly, halogenation of 4c on the imidazole moiety afforded even more potent compounds, including bromo derivative 5a that displayed an EC₅₀ of 0.7 nM. Elongation of compound 4a by addition of a second phenyl ring lead to compound 4j that displayed an EC₅₀ of 300 nM. Interestingly, substitution of this second ring with a 4-CF₃- or 4-OH-group lead to compounds 6c and 6e displaying EC₅₀ values of 4.6 and 5 nM, respectively. However, unlike for compound 4c bearing only one phenyl ring, halogenation of the imidazole ring of compound 6c led to loss of anti-HCV activity (compound 7).

Table 1.

Structures, Anti-HCV Activity and Cytotoxicity of BMS-790052 and compounds 4a-o, 5a-c, 6a-l, 7, 8, 9a-b.

Cmpd	R1	R2	R3	R4	HCV Replicon in Huh-7 cells		Cytotoxicity, CC50 (μM)a
					EC50 (nM)	EC90 (nM)	PBM	CEM	Vero
BMS	N/Ab	N/A	N/A	N/A	0.01	0.03	19	9.6	21
4a	H	H	iPr	OMe	>1000	N/A	>100	>100	>100
4b	4-Br	H	iPr	OMe	>100	N/A	>100	48	50
4c	4-N3	H	iPr	OMe	8	52	>100	>100	>100
4d	4-CN	H	iPr	OMe	>100	N/A	>100	33	98
4e	4-NO2	H	iPr	OMe	>100	N/A	>100	>100	>100
4f	4-CF3O	H	iPr	OMe	>100	N/A	49	30	>100
4g	4-CF3	H	iPr	OMe	>100	N/A	>100	26	>100
4h	4-OMe	H	iPr	OMe	>1000	N/A	>100	>100	>100
4i	4-Me	H	iPr	OMe	>1000	N/A	>100	>100	>100
4j	4-Ph	H	iPr	OMe	300	900	14	18	70
4k	4-Ph	H	iBu	OMe	>100	>1000	16	25	61
4l	4-Ph	H	Me	OtBu	>1000	N/A	16	13	11
4m	4-Ph	H	allyl	Me	>1000	N/A	100	21	73
4n	2,4-diCl	H	iPr	OMe	1000	N/A	56	15	>100
4o	4-PhSO2-	H	iPr	OMe	>100	N/A	>100	>100	>100
5a	4-N3	Br	iPr	OMe	0.7	2.9	35	16	52
5b	4-N3	Cl	iPr	OMe	4	14	94	12	>100
5c	4-N3	I	iPr	OMe	2	3	64.2	14.2	>100
6a	4-CN-Ph	H	iPr	OMe	>100	N/A	>100	33	98
6b	4-Me-Ph	H	iPr	OMe	>100	N/A	90	14	16
6c	4-CF3-Ph	H	iPr	OMe	4.6	9.6	>100	14	12
6d	2-CF3-Ph	H	iPr	OMe	>100	N/A	24	13	75
6e	3-CF3-Ph	H	iPr	OMe	>100	N/A	44	12	32
6d	4-CF3O-Ph	H	iPr	OMe	>100	N/A	11	10	10
6e	4-HO-Ph	H	iPr	OMe	5	17	70	13	55
6f	3-OH-Ph	H	iPr	OMe	>1000	N/A	>100	50	>100
6g	4-MeO-Ph	H	iPr	OMe	>100	N/A	85	13	30
6h	4-NH2C(O)-Ph	H	iPr	OMe	21	76	>100	>100	>100
6i	4-MeOOC-Ph	H	iPr	OMe	>100	N/A	14	17	61
6j	4-HO-CH2-Ph	H	iPr	OMe	2	9	>100	59	67
6k	4-MeSO2-Ph	H	iPr	OMe	>100	N/A	>100	6	54
6l	4-N3-Ph	H	iPr	OMe	>100	N/A	37	11	11
7	4-CF3-Ph	Cl	iPr	OMe	>100	N/A	11	10	10
8	4-NH2	H	iPr	OMe	200	300	>100	>100	>100
9a	4-NHCO2Me	H	iPr	OMe	>100	N/A	>100	>100	>100
9b	4-NHSO2Me	H	iPr	OMe	>100	N/A	>100	13	>100

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

^bN/A, not applied.

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 enzymes 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 the most active compounds were investigated by a CYP450 reversible inhibition assay. Compounds 4c, 5a, 6c and 6j did not appear to inhibit the major CYP450 enzymes at the IC₉₀ level and had a favorable CYP profile, which is suggestive of no drug-drug interactions (Table 2). Due to the large therapeutic window, the CYP inhibitions observed in the micromolar ranges are expected not to be clinically relevant at therapeutic doses.

Table 2.

Cytochrome P450 inhibition data for compounds 4c, 5a, 6c, 6j and BMS-790052^a

	CYP1A2 (μM)		CYP3A4 (μM)		CYP2D6 (μM)		CYP2C9 (μM)
Cmpd	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90
4c	> 100	> 100	5.1 ± 0.6	96.1	> 100	> 100	> 100	> 100
5a	80.9 ± 21.1	> 100	1.5 ± 1.6	46.1 ± 23.8	50.5 ± 6.8	> 100	50.5 ± 19.7	> 100
6c	82.9 ± 13.4	> 100	6.5 ± 0.6	> 100	62.1 ± 32.9	> 100	9.1 ± 1.9	> 100
6j	56.7 ± 12.4	> 100	6.9 ± 2.1	> 100	> 100	> 100	31.2 ± 26.0	> 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).

In conclusion, extremely potent non-dimeric NS5A inhibitors (picomolar activity) with a wide therapeutic window (> 10⁴) have been discovered. Out of all compounds prepared, compound 5a appeared to be the most promising with an EC₅₀ of 0.7 nM and no toxicity or inhibition of major human CYP enzymes at therapeutically relevant concentrations. In vitro resistance profile of compound 5a was established by mutation selection in HCV subgenomic replicon containing Huh-7 cells. After 2 months exposure, Y93H and Q30E were among the selected resistant virus, similar to those observed with BMS-790052 treatment, which confirmed that this monodentate compound acts as an NS5A inhibitor. Through this work, we demonstrated for the first time that, a bidentate structure (i.e. BMS-790052) was not a sine qua non condition for a molecule to inhibit HCV NS5A.