Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus

Bioorganic & Medicinal Chemistry Letters 27 (2017) 2201–2206

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Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus Euan A.F. Fordyce a,⇑, Daniel W. Brookes b, Claire Lise-Ciana a, Matthew S. Coates b, S. Fraser Hunt a, Kazuhiro Ito b,⇑, John King-Underwood c, Stuart T. Onions a, Guillaume F. Parra a, Garth Rapeport b, Vladimir Sherbukhin a, Jennifer A. Stockwell a, Peter Strong b, Jennifer C. Thomas a, John Murray b a b c

Sygnature Discovery Ltd., BioCity, Nottingham NG1 1GF, United Kingdom Pulmocide Ltd., Imperial BioIncubator, Level 1 Bessemer Building (RSM), Imperial College, London SW7 2BP, United Kingdom CompChem Resource, Old Cottage Hospital, Homend, Ledbury, Herefordshire HR8 1ED, United Kingdom

a r t i c l e

i n f o

Article history: Received 24 January 2017 Revised 15 March 2017 Accepted 20 March 2017 Available online 23 March 2017 Keywords: RSV Polymerase inhibitor Antiviral

a b s t r a c t The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use. Ó 2017 Elsevier Ltd. All rights reserved.

Human respiratory syncytial virus (hRSV) is the leading cause of lower respiratory tract infections (LRTIs) in infants and young children,1 and a significant pathogen in both the elderly and in immunocompromised patients.2 An estimated 33.8 million cases of RSV-associated LRTIs in children under the age of five were reported worldwide in 2005, of which 3.4 million (10%) were admitted to hospital and between 66,000 and 199,000 patients subsequently died.3 High-risk groups include premature infants and those with a low birth weight, HIV-infected children and those with compromised immune systems. In addition to the immediate health implications, there is also a strong correlation between severe RSV infection and the development of asthma during childhood.4 Not only is RSV a significant cause of mortality in at-risk populations, it represents a major financial burden since the cost of hospitalisations is considered to exceed $1 billion annually.2a Human RSV viruses are classified into two major groups, A and B, each comprising of several distinct subgroups.5 Viruses of the RSV A strain have generally been regarded as those responsible for the majority of clinical disease and are reported to produce ⇑ Corresponding authors. E-mail addresses: [email protected] (E.A.F. Fordyce), [email protected] (K. Ito). http://dx.doi.org/10.1016/j.bmcl.2017.03.053 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

more symptomatic pathology.6 However, in a number of recent outbreaks strains of the RSV B sub-group were found to be more prevalent in the afflicted populations.7 Currently, only two drugs are approved for the treatment of RSV infection: virazoleÒ, an aerosol formulation comprising ribavirin, and the humanised monoclonal antibody, palivizumab (SynagisÒ). Due to high cost the use of palivizumab is restricted to immunoprophylaxis in high-risk, paediatric patients.8 Concerns with efficacy and toxicity have limited the adoption of ribavirin as a first line therapy against RSV.9 The lack of a safe, effective treatment for RSV and the clear unmet medical need, has spurred interest from both pharmaceutical companies and academic research groups. This has resulted in a small number of compounds progressing into clinical trials, of which GS-580610 and ALS-817611 (Fig. 1) have advanced the furthest. The orally bioavailable, fusion protein inhibitor GS-5806 is currently undergoing efficacy and safety studies in hospitalised adults.12 Whilst this compound has activity against a broad spectrum of RSV A and B clinical isolates,10c evidence is emerging of treatment induced viral mutations which reduce its efficacy.13 Although the clinical significance of these mutations is unknown, these results raise the likelihood of resistance emerging to this compound. The nucleoside prodrug ALS8176 is a polymerase inhibitor which is also undergoing Phase 2

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for delivering a safe and effective treatment for this respiratory virus. In this letter, we report that the replacement of the cyclopropyl amide, a key pharmacophore of AZ-27 and a common structural feature present in virtually all of the analogues previously disclosed, with a heteroaromatic group, providing a novel series of highly potent inhibitors of RSV in vitro. In addition, it will be revealed that the resulting family of compounds manifested physicochemical properties that are desirable in topical pulmonary agents, and that an exemplary structure was effective at inhibiting RSV replication in a rodent model of infection. We have previously shown that replacement of the cyclopropyl amide, present in AZ-27, with a suitably functionalised phenylamido group, increased activity towards RSV A2 virions whilst maintaining that against the B-WST strain (Table 1, Entries 1 and 2).18 In conjunction with efforts to optimise the aromatic amide analogues, we explored the possibility of replacing the secondary amido group altogether; a change not previously demonstrated

Fig. 1. Selected small molecule RSV inhibitors in development.

clinical trials.14 This agent was effective at inhibiting virus replication, having an IC50 value of 0.26 mM in an RSV replicon assay.11a A number of non-nucleoside polymerase inhibitors have also been developed,15 amongst which YM-53403 (Fig. 1) was one of the first to emerge.16 This therapeutic, discovered through screening of Yamanouchi’s in-house compound collection, is a potent replication inhibitor of RSV viruses belonging to the A group. Forced mutation studies using the RSV Long strain indicated that YM-53403 targets the viral RNA polymerase L-protein, thereby inhibiting viral genome replication and transcription of mRNA. Further optimisation of the pharmacophore by Yu et al. led to the discovery of AZ-27 (Fig. 2).17 This agent shows good activity against both subtypes of the pathogen, exhibiting EC50 values of 0.01 mM and 1.0 mM respectively for RSV-A2 and B-Washington (WST). It was also found to be selective for RSV, showing no inhibition against metapneumovirus, influenza A virus, human rhinovirus, or cytomegalovirus. Given its high molecular weight and lipophilicity, further studies were undertaken to assess the lung retention and distribution properties of AZ-27 following intra-tracheal dosing.17a These experiments revealed that AZ-27 was not retained to a significant extent in either lung tissue or in BALF but was, instead, rapidly absorbed into the systemic circulation. These results make it unlikely that AZ-27 is a suitable candidate for development as an inhaled anti-viral agent, as it is likely to be rapidly and widely distributed post administration and therefore insufficiently long acting. The identification of an alternative compound, specifically designed for topical delivery to the lung, was adopted as a strategy

Fig. 2. Structure of AZ-27.

Table 1 Antiviral activity and cytotoxicity for compounds 1a–g.a

R

S O N N N

Entry

Compound

O

O N H

R

IC50/nM

CC50/nM

A2

B-WST

1

1a

2.2

286

6410

2

1b

0.5

283

>14,900

3

1c

56

>1500

>14,900

4

1d

1.5

1021

>15,000

5

1e

0.8

59.8

>14,900

6

1f

7.6

844

>14,900

7

1g

13.1

>1495

>14,900

a IC50: the concentration of compound that reduced by 50% the cytopathic effect (CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from 2 experiments.

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in this series. Initial attempts at supplanting this functionality with traditional amide bioisosteres proved unsuccessful in delivering compounds with the necessary levels of activity (results not shown). Non-classical bioisosteres such as benzoxazole also had a similar effect, resulting in significantly diminished potencies at both viral strains (Table 1, Entry 3). Inhibitory activity towards the A2 subtype was restored to a level similar to that exhibited by the phenyl thiophenecarboxamide 1b on generating the indole derivative 1d (Table 1, Entry 4), although this homologue was still 5-fold weaker towards the B-WST strain. Encouragingly the corresponding benzimidazole 1e displayed increased activity against both sub-types, exhibiting IC50 values of 0.8 and 60 nM for the A2 and B-WST strains respectively (Table 1, Entry 5). Incorporation of additional heteroatoms into the fused phenyl ring, as in the case of the 5-azabenzimidazole 1f and the purine derivative 1g (Table 1, Entries 6 and 7), had a deleterious effect. These two analogues were >10-folder weaker against both subtypes of the virus than was the benzimidazole 1e. Compounds listed in Table 1 (inter alia) were also tested for non-specific, mammalian cell toxicity in the same HEp2 cell line used in the phenotypic screens. It was notable that no toxicity was observed for the new compounds 1b–g, at concentrations up to at least 10 mM. In contrast the previously disclosed cyclopropylamide 1a, was detectably cytotoxic in this assay (CC50 value = 6410 nM). We next explored the influence of incorporating a diverse range of functional groups into the benzimidazole ring system (Table 2). These results reveal that small lipophilic groups, such as simple alkyl substituents and halogen atoms, provided the most potent analogues. More polar groups such as sulfones, carboxamides and carboxylic acids had a detrimental effect; the resulting compounds showed either modest potency or were completely inactive (results not disclosed). The position in the benzimidazole nucleus, chosen to install the substituent, also proved significant. Those

analogues substituted at C-4 were found to be more active than those derivatised at C-5 (cf. the regioisomeric compound pairs: 2b/2c, 2e/2f and 2g/2h; Table 2). In an effort to selectively increase activity against the B serotype, modifications to the pyridine nucleus of the nicotinamide domain were investigated next. It was quickly determined that methylation at both C-5 and C-6 increased potency against the Bstrain by about 10-fold, resulting in IC50 values of 5 and 13 nM respectively (Table 3 entries 3 and 4).19 Crucially this improvement was achieved without being detrimental to activity at the A2-subtype. In contrast methylation at C-4 had the opposite effect: the resulting regioisomer 3a being essentially inactive towards the Bstrain virus (Table 3, entry 2). This difference in biological activity is probably due to a forced conformational change resulting from steric repulsion that distorts the preferred dihedral angle, normally adopted in the bioactive conformation, between the amide group and pyridine ring of the nicotinamide motif. A deformation of this kind would be expected to exert a profound change on the spatial orientation of the spirocyclic ether, which is a key binding feature of these molecules. The alkyl substituent at C-5 can be increased to an ethyl radical with minimal effect (Table 3, entry 7). However, the introduction of even an isopropyl group resulted in a significant drop in activity, particularly towards the B-WST strain (Table 3, entry 8). Modifying the nature of the substituent revealed that not only its volume but the resulting electron density of the pyridine ring system are important determinants of biological activity. For example, the incorporation of strongly electron withdrawing functionality, such as a C-6 trifluoromethyl group, diminished the ability of the compound thus generated to inhibit RSV B-WST induced CPE to at least the micromolar level (Table 3, entry 5; IC50 value >1300 nM). Having adopted the C-5 methyl-substituted nicotinamide as the most preferred ‘southern’ pharmacophoric group, additional

Table 3 Antiviral activity and cytotoxicity for compounds 3a–g.a Table 2 Antiviral activity and cytotoxicity for compounds 2a–h.a

N

NH

S O N N

O

O

N R

1

R

3

N H

R2 Entry

Compound

R

IC50/nM A2

1 2 3 4 5 6 7 8 9

1e 2a 2b 2c 2d 2e 2f 2g 2h

H 4-F 4-Cl 5-Cl 4-Me 4-CF3 5-CF3 4-CN 5-CN

0.8 1.6 0.8 1.6 0.6 0.6 3.1 0.6 0.5

CC50/nM Entry

B-WST 59.8 90.9 30.7 161 52.9 38.8 >1361 69.0 162

>14,900 >14,600 >14,200 >14,200 >14,600 >13,600 >13,600 >14,400 >14,400

a IC50: the concentration of compound that reduced by 50% the cytopathic effect (CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from 2 experiments.

1 2 3 4 5 6 7 8

Compound

1e 3a 3b 3c 3d 3e 3f 3g

R1

H H H Me CF3 MeO H H

R2

H H Me H H H Et i Pr

R3

H Me H H H H H H

IC50/nM A2

B-WST

0.8 – 0.5 0.3 7.3 0.5 0.7 5.7

59.8 >1469 5.2 13.1 >1360 37.8 13.3 >1410

CC50/nM

>14,900 – >14,600 >14,600 >13,600 >14,300 >14,300 >14,100

a IC50: the concentration of compound that reduced by 50% the cytopathic effect (CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from 2 experiments.

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Table 4 Antiviral activity and cytotoxicity for compounds 4a–e.a

R

S O N N N

O

O N H

Me

Entry

Compound

R

IC50/nM

CC50/nM

A2

B-WST

1

3c

0.8

5.2

>14,600

2

4a

1.4

2.1

>14,300

3

4b

1.8

14.4

>14,600

4

4c

0.7

8.1

>14,600

5

4d

1.6

6.2

>15,100

6

4e

0.7

3.3

>15,500

a IC50: the concentration of compound that reduced by 50% the cytopathic effect (CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from 2 experiments.

changes to the benzimidazole motif were examined. A relatively conservative change: the introduction of fluorine at C-4 gave rise to compound 4a that was very nearly equipotent at both viral

strains of interest (Table 4, entry 2). Next we explored the significance of the position of the nitrogen atoms within the five-membered heterocycle. As can be seen, imidazolopyridine 4b and pyrazolopyridine 4c (Table 4, entries 3 and 4) are both potent inhibitors of the RSV A2 and B-WST strains, exhibiting activities comparable to that of the benzimidazole 3c. These results reveal that disruption of viral pathogenicity is not clearly linked to any single, readily identifiable feature of these heteroaromatic nuclei, such as their relative basicity, or even to the presence, or absence, of an NAH in this particular pharmacophoric domain. Furthermore, it was found that the bicyclic ring system could be truncated to furnish the imidazoles 4d and 4e (Table 4, entries 5 and 6), both of which show a remarkably similar activity profile to the parent benzimidazole 3c. In order to verify the specific target of this series of analogues, we isolated and characterised forced mutants of the virus. RSV A2 virus was repeatedly passaged in HEp-2 cells in the presence of increasing concentrations of compound 4a (escalating from 0.001 to 3.125 lg/mL) and after six passages an escape mutant was isolated. When its RSV genome was sequenced the mutant virus showed one amino acid substitution had occurred with high frequency: that of tyrosine 1631 to histidine (Y1631H), at a locus in the RSV L-gene. This mutation was also elicited by both YM5340316 and AZ-27,17 suggesting that compound 4a shares at least one common target and binds in a similar fashion to these two agents. Furthermore, compound 4a demonstrated potent and concentration dependent inhibition of the expression of luciferase driven by a minigenome construct comprising of the L, M2-1, N and P proteins of RSV A2 (IC50: 2.0 nM). This provides direct evidence that compound 4a inhibits virus gene transcription/replication by the RSV A2 derived ribonucleoprotein (RNP) complex. In addition to enhancing potency, the structural modifications reported above were designed to provide compounds of high molecular weight and lipophilicity, thereby ensuring they possessed low oral bioavailability. Desirable properties for their development as topical agents also include physical attributes such as a well-defined, thermodynamically stable, polymorphic form.20 Compound 4a, for instance, was obtained as a white, crystalline solid with a melting point of 349 °C, making it a potentially attractive development candidate as an inhaled antiviral. It was therefore selected for profiling in vivo. A variety of animal models of RSV infection have been developed over the past four decades.21 For the present study cotton rats (Sigmodon hispidus) were chosen as they are susceptible to infection with non-adapted, human RSV and because the resulting infection closely resembles human pathology.22 Male cotton rats

Fig. 3. The effects of intranasal treatment with Compound 4a on RSV A Long viral titre in lung from RSV A Long infected cotton rats.a,b

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Fig. 4. The effects of intranasal treatment with Compound 4a on RSV A Long NS-1 gene expression in lung from RSV A Long infected cotton rats.a

were treated with compound 4a, administered intranasally as an aqueous suspension, at doses of 0, 0.33, 1.0 and 3.3 mg/mL (50 mL/animal), four hours prior to inoculation with hRSV/A/long and then again on days 1, 2 and 3 post infection. Four days after RSV challenge, the animals were euthanised and the lungs removed. The left lobe was used for viral titration via a plaque assay and the lingular lobe for RSV/A/long NS-1 reverse transcription quantitative polymerase chain reaction (RT-qPCR). As can be seen below (Fig. 3) compound 4a demonstrated potent dosedependent inhibition of viral titre in lung homogenates. Most notably, viral titre levels were suppressed to below the limit of detection at the highest dose screened (<2.3 log PFU/lung). In addition, the compound displayed a dose dependent inhibition of RSV NS1 gene transcripts (Fig. 4). These results demonstrated that the benzimidazole 4a is effective at inhibiting viral replication in vitro and demonstrates impressive efficacy when administered

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topically, in a rodent model of RSV infection. Therefore these data indicate that the problem identified by Yu et al.,17a namely that poor lung retention would prevent the identification of an inhalable antiviral from their test series, has been overcome. Syntheses of compound 4a and of the benzimidazole congeners have been disclosed previously.23 The remaining heteroaromatic analogues, represented by generic structure 9, were constructed from the aminoester derivative 518 (Scheme 1). Acid-mediated decarboxylation of the ester yielded the corresponding thienobenzazepine 6, which was sequentially N-acylated with 4-nitrobenzoyl chloride and the thiophene nucleus then halogenated regioselectively, at C-2, by treatment with bromine. Reduction of the nitrobenzene was followed by acylation of the aniline so derived, with a chloronicotinic acid derivative. Installation of the spirocyclic amine was achieved by a subsequent SNAr addition to provide intermediates of formula 8. The key thiophene heterocycle CAC bond was formed either by a palladium-mediated cross-coupling of the bromothiophenes 8 with a boronic acid or ester; or alternatively, by conversion, in situ, to the corresponding boronic ester followed by coupling with a suitable heteroaryl halide. In conclusion, we have discovered and report several novel families of C-2 aryl benzothienoazepines that are highly potent RSV polymerase inhibitors, including examples that show low nanomolar activity against both RSV A and B viral subtypes. A fluorinated analogue 4a, derived from this series, is effective at suppressing viral replication when administered in a rodent model of RSV infection. The results obtained suggest that compounds belonging to one or more of the new chemotypes described herein have the potential to provide superior anti-RSV agents than those currently available for clinical use.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Scheme 1. Synthetic routes for the preparation of non-benzimidazole series. Reagents and conditions: (a) H2SO4, H2O, reflux 94%; (b) 4-nitrobenzoyl chloride, pyridine, RT 88%; (c) Br2, CHCl3, 0 °C 96%; (d) Fe, NH4Cl, EtOH:H2O (15:4), reflux 96%; (e) chloronicotinic acid, (COCl)2, DMF, RT then aniline, pyridine, RT; (f) 7-oxa-2-azaspiro[3.5]nonane hemioxalate, K2CO3, NMP, 130 °C; (g) Het-B(OR)2, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C; (h) B2Pin2, PdCl2[P(Cy)3]2, KOAc, 1,4-dioxane, 95 °C then Het-Br, Pd(PPh3)4, Na2CO3, 90 °C.

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Acknowledgements The authors would like to thank Sigmovir Biosystems, Inc (Rockville, MD) for performing the relevant in vivo studies, and BEI Resource (Manassas, VA) for providing RSV L, P, M2-1 and N protein expression plasmids.

11.

12. 13. 14. 15.

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