key: cord-329078-gnnis7pl
authors: Musella, Simona; di Sarno, Veronica; Ciaglia, Tania; Sala, Marina; Spensiero, Antonia; Scala, Maria Carmina; Ostacolo, Carmine; Andrei, Graciela; Balzarini, Jan; Snoeck, Robert; Novellino, Ettore; Campiglia, Pietro; Bertamino, Alessia; Gomez-Monterrey, Isabel M.
title: Identification of an indol-based derivative as potent and selective varicella zoster virus (VZV) inhibitor
date: 2016-11-29
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2016.09.014
sha: 
doc_id: 329078
cord_uid: gnnis7pl

We report the synthesis and antiviral activity of a new family of non-nucleoside antivirals, derived from the indole nucleus. Modifications of this template through Mannich and Friedel-Crafts reactions, coupled with nucleophilic displacement and reductive aminations led to 23 final derivatives, which were pharmacologically tested. Tryptamine derivative 17a was found to have a selective inhibitory activity against human varicella zoster virus (VZV) replication in vitro, being inactive against a variety of other DNA and RNA viruses. A structure-activity relationship (SAR) study showed that the presence of a biphenyl ethyl moiety and the acetylation at the amino group of tryptamine are a prerequisite for anti-VZV activity. The novel compound shows the same activity against thymidine kinase (TK)-competent (TK(+)) and TK-deficient (TK(−)) VZV strains, pointing to a novel mechanism of antiviral action.

Varicella zoster virus (VZV) is a ubiquitous and highly infectious human virus that belongs to the herpesviridae family. It is classified within the group of a-herpesviruses which also includes herpes simplex virus (HSV) [1] . A VZV primary infection leads to acute varicella or "chickenpox", while reactivation of latent virus, established in cranial nerve and dorsal root ganglia, causes herpes zoster (shingles). The course of varicella is generally benign in immune-competent children, but can cause severe morbidity and mortality in adults and in immune-compromised individuals [2] . Complications of herpes zoster in immunecompetent hosts include post-herpetic neuralgia (PHN), a persistent pain syndrome, which is the most challenging complication particularly in older individuals [2, 3] . Central nervous system (CNS) complications can follow both primary infection and reactivation of VZV [4, 5] . The most serious manifestations arise when VZV invades the spinal cord or cerebral arteries after reactivation of the virus, causing diseases such as myelitis and focal vasculopathies [2, 4, 5] . Other neurological complications of herpes zoster include motor neuropathy, particularly in patients with zoster ophthalmicus [6, 7] . In patients with the acquired immune deficiency syndrome (AIDS), transplant recipients, and cancer patients, VZV infection can be associated with severe acute retinal necrosis (ARN), a disease with poor prognosis [8, 9] . The outcomes of varicella and herpes zoster have been dramatically improved by the development of safe and effective antiviral drugs with potent activity against VZV [10] . Three oral guanine-based antivirals are approved worldwide for the treatment of VZV-associated diseases: acyclovir, valacyclovir, and famciclovir [11] . The thymidine analog brivudin has been licensed for the therapy of herpes zoster in some European and Central American countries [12] . These drugs are (a) nucleoside analogs that after predominant phosphorylation by the virus-encoded thymidine kinases (TKs), act as competitive inhibitors of the viral DNA polymerase or alternate substrates to the natural triphosphates, inhibiting DNA replication [13] . Other anti-VZV nucleoside inhibitors such as the stearyl/valyl diester valomaciclovir and the valyl-ester prodrug of the bicyclic nucleoside analog (BCNA) FV100 are under clinical investigation [14e17].

One of the limitations of the use of nucleoside derivatives is the emergence of single and multiple drug resistance which could be partially avoided with the use of non nucleoside compounds [18, 19] . A drug of choice for treatment of acyclovir-resistant VZV disease is foscarnet, a direct inhibitor of viral DNA polymerase that is not dependent on viral TK for activation [20e22] . A number of small molecules have been identified and reported as potent and selective VZV inhibitors with different mechanisms of action. Some examples are the 4-oxo-dihydroquinoline [23] and 4-oxo-dihydrothieno [2,3-b] pyridine derivatives [24] as inhibitors of the viral DNA polymerases, the oxadiazolephenyl derivative (ASP2151) as a helicase-primase inhibitor [25] , and N-a-methylbenzyl-N 0 -arylthiourea derivatives that interfere with the function of the viral ORF54 protein, impairing morphogenesis of the capsid [26, 27] . Finally, a series of 4-benzyloxy-g-sultone derivatives has been also reported as non-selective VZV inhibitors with unknown mechanism of action [28] . Given the difficulty of identifying initial hit compounds in this field, where the synthesized compounds are in primis subject to a cellular screening, we considered of interest to use a privileged scaffold as effective starting point in the search for anti-VZV ligands [29] . Indole and its bioisosteres, as privileged scaffolds, represent one of the most important structural motifs in drug discovery [30e32], and it is widely used in antiviral research [32, 33] . Arbidol [34] and delavirdine [35] , are examples of marketed indole-containing antiviral drugs, whereas Panobinostat (LBH589) [36] , being a HDAC (histone deacetylase) inhibitor, is actively undergoing clinical evaluation against human immunodeficiency virus (HIV) type 1 (See Fig. 1 ).

However the use of the indole-based structures in the research of anti-herpes virus agents is rather unusual. Hence we explored the minimum structural requirements for anti-VZV activity starting from this easily derivatizable scaffold. Two small libraries were synthesized based on substituted indoles (A, B) and tryptamines (C). Their cytotoxic and antiviral activity was then evaluated using cellular assays. Some interesting structure-activity relationships were evidenced regarding the N-1 and C-3 substituents.

Compounds 4a-d were prepared starting from indole 1 according to Scheme 1. N-1 alkylation of 1 with propyl iodide or 4phenylbenzyl iodide in DCM/DMF using NaH as base, gave the corresponding intermediates 2 and 3, respectively. The 3-acyl derivative 4a was obtained from 2 by Friedel-Crafts acylation, using 4chlorobenzoyl chloride and AlCl3 in acetonitrile (32% yield). Functionalization of 2 and 3 through a Mannich reaction, using formaldehyde and piperidine/or biphenyl ethyl amine, and TFA as catalyst, led to 3-methylamine derivatives 4b-d (25e38% yield).

The 5-substituted indole derivatives (8a-c) were synthesized using the indole-5-carboxyaldehyde (5) and the 5-aminoindole (9) as starting material and following the two-synthetic strategy indicated in Scheme 2. 5 was first N-alkylated (6) and then subjected to a Mannich reaction to obtain the corresponding aldehydes 7a and 7b, as described above. Treatment of these intermediates with 4-chloro aniline and sodium triacetoxyborohydride in reductive amination conditions, led to final products 8a and 8b in 24% and 30% yield, respectively. On the other hand, reaction of 9 with benzoic acid using HOBt/HBTU as coupling agents gave N-(1Hindol-5-yl)benzamide 10 in 63% yield. N-alkylation of 10 with npropyl iodide followed by Mannich reaction with formaldehyde and piperidine afforded final compound 8c.

The tryptamine-based derivatives 15, 16a-i were prepared following Scheme 3. N-alkylation of 3-(2-bromoethyl)-1H-indole (12) with methyl iodide or 4-phenylbenzyl iodide led to derivatives 13 and 14 in 67% and 61% yield, respectively. Nucleophilic displacement of the bromine atom in these intermediates by different commercially available amines was performed under microwave conditions, using palladium acetate as catalyst, obtaining the compounds 15, 16a-i in 38e75% yield [37] .

Treatment of compounds 16a and 16d-i with acetyl chloride gave the corresponding acetyl derivatives 17a and 17d-i (42e58% yield).

Analogously, treatment of 16a with different aromatic and aliphatic acyl chlorides gave the corresponding acylated compounds 18a-c (48e57% yield). The carbamoyl derivative 18d was obtained in 58% yield by reaction of 16a with di-tert-butyl dicarbonate in DCM/TEA (See Scheme 4). The acyl derivatives 17, except 17d and 17e, and 18 were obtained as a mixture of rotamers due to the atropisomerism of the amide function, as confirmed by ROESY experiments, which showed exchange cross-peaks between the hydrogen signals of the two conformers (as examples see NMR spectra of compounds 17f and 17 d in Supporting Information) [38, 39] .

First, compounds 4, 8, and 15, 16 and 17a were evaluated for their ability to inhibit the replication of VZV in human embryonic lung (HEL) cells, and the results were compared to those obtained for the reference compounds acyclovir and brivudin ( Table 1 ). The antiviral activity was expressed as EC50, being the compound concentration required to reduce virus-plaque formation (VZV) by 50%.

As shown in Table 1 , the N-propyl-3-acyl (4a) and N-propyl-3piperidine methyl (4b) derivatives do not displayed antiviral or cytotoxic activities at 100 mM, while the substitution of piperidine with CC50 values of 1.7 and 6.9 mM, respectively. Compounds 8 a-c, derivatized at the C-5 indole position, proved less cytotoxic, CC50 z 10e30 mM, but were also ineffective as inhibitors of VZVinduced plaque formation at similar concentrations.

The most interesting results were obtained for the tryptamine derivatives. In this series, the presence of a methyl group at the Nindole position lead to weaker cytotoxic derivatives (16a-d versus 15, CC50 20e41 versus 7.3 mM), while the acylation of the amine group (17a) also reduces cytotoxicity (CC50 ≈ 31e39 mM) but with an inhibitory effect on the anti-VZV activity. Tryptamine derivative 17a was indeed able to inhibit replication of TKþ and TK-VZV strains with EC50 values in the range of 1.7e3.6 mM ( Table 1 ). The potency of 17a against OKA strain (EC50 ¼ 2.5e3.6 mM) was found comparable to that of the reference drug acyclovir (EC50 ¼ 1.9e2.1 mM) but two orders of magnitude lower than that of brivudin (EC50 ¼ 0.02e0.03 mM). However, the activity of this compound against thymidine kinase-deficient VZV strains, both 07-1 and YS-R, was maintained in the micromolar range with EC50 ¼ 1.7e3.1 mM being, at least, one order of magnitude more active than acyclovir (EC50 ¼ 33e103 mM) and brivudin (EC50 ¼ 26e165 mM).

The activity of these compounds against other members of the Herpesviridae family was tested in HEL cell cultures. As shown in Table 2 none of these compounds was found active either towards cytomegalovirus (HCMV, AD-169 strain and Davis strain) or against herpes simplex virus-1 (HSV-1; KOS and thymidine kinasedeficient acyclovir-resistant) (HSV-1/TK-KOS ACVr strains) or against herpes simplex virus-2 (HSV-2; G strain).

The compounds were not active against other DNA viruses such as vaccinia virus, adenovirus-2, and feline herpesvirus. All these compounds also lacked inhibitory activity against a broad variety of RNA viruses, including HIV-1 and HIV-2, feline coronavirus, vesicular stomatitis virus, Coxsackie virus B4 and respiratory syncytial virus, para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, influenza A virus (H1N1 and H3N2 subtypes) and influenza B virus (data not shown).

To further explore the SAR within the tryptamine series, we next prepared a second set of 17a analogs featuring a) a modified aromatic moiety linking the acetamide group (compounds 17 d-i) and b) a modified acyl group (compounds 18a-d).

As shown in Table 3 , substitution of biphenyl ethyl moiety by the more rigid naphthyl (compounds 17d, 17e) or their superior analog naphthylmethyl groups (17f, 17g), as well as, the introduction of a biphenylmethyl or phenyloxybenzyl moieties (17h, 17i) led to a complete loss of the antiviral activity. However, compounds containing 1-naphtyl (17e) or 1-naphtyl methyl (17g) groups or the inferior analog biphenylmethyl group (17h) were less cytotoxic. On the other hand, substitution of the acylmethyl group by bulkier alkyl groups such as cyclohexyl, morpholinethyl and tert-butyloxy groups (compound 18a, 18c, 18d) led to an annihilation of antiviral activity. In this series of compounds, only the benzoyl derivative 18b somewhat maintains the antiviral activity at an EC 50 of 20 mM, displaying also a reduced cytotoxic activity (MMC >100 mM).

The identification of a new family of non-nucleoside anti-VZV agents based upon the indole template has been reported. We have shown that the substitution on the tryptamine moiety strongly influences the activity/toxicity of these compounds. In particular, the concomitant presence of a biphenylethyl and a methyl(phenyl) carboxy group at the amino group of tryptamine is a requisite for antiviral activity. In fact, the N-biphenylethyl acetamide derivative 17a displayed an interesting inhibition against VZV and was endowed with a selectivity of 10e20 (ratio CC50/EC50). It must be also considered that 17a showed similar activity against TKþ and TK-VZV strains, thus indicating a mechanism of action independent from the virus-encoded thymidine kinase. Additionally, this compound is able to exert antiviral activity exclusively on VZV, being totally inactive against other members of Herpesviridae family as well as against a wide variety of RNA virus strains suggesting a selective action on a specific antiviral target untargeted by the currently existing compounds. The possible mechanism of action of this novel class of compounds will be further investigated.

Reagents, starting materials, and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used as received. Reactions were carried out with magnetic stirring in round-bottomed flasks unless otherwise noted. Moisture-sensitive reactions were conducted in oven-dried glassware under a positive pressure of dry nitrogen, using pre-dried, freshly distilled solvents. Microwave assisted reactions were performed in a Biotage Initiator þ reactor. Analytical thin layer chromatography (TLC) was performed on pre-coated glass silica gel plates 60 (F254, 0.25 mm, VWR International). Purifications were performed by flash column chromatography on silica gel (230e400 mesh, Merck Millipore). NMR spectra were recorded on Varian Mercury-400 apparatus. 1HNMR and 13C NMR spectra were recorded with a Varian-400 spectrometer, operating at 400 and 100 MHz, respectively. Chemical shifts are reported in spectrometer. Combustion microanalyses were performed on a Carlo Erba CNH 1106 analyzer, and were within 0.4% of calculated values and confirmed >95% purity for the final products.

4.1.1. General procedure for the synthesis of N-alkylated indole intermediates (2, 3, 6, 11, 13 and 14) Indole (1, 1.0 eq) or 1H-indole-5-carbaldehyde (5, 1.0 eq), or N-(1H-indol-5-yl)benzamide (10, 1.0 eq), or 3-(2-Bromoethyl)indole (13, 1.0 eq.) were dissolved in a mixture of anhydrous DCM/DMF (2/1 v/v) under magnetic stirring and the temperature was set to 0 C. To this solution, 1.5 equivalents of NaH were added portionwise and the mixture was allowed to react for 30 min. Then, 1.5 equivalents of alkyl iodide [methyl iodide, or n-propyl iodide, or 4-(phenyl)iodomethylbenzene] in DCM were added dropwise and the reaction was warmed to room temperature and maintained under stirring for further 12 h. The reaction was then quenched by a 10% aqueous solution of citric acid and washed with brine. The organic layer was separated, dried over anhydrous Na2SO4, filtered and evaporated in vacuo. Crude products were purified by column chromatography using n-hexane/ethyl acetate (4:1 v:v) as mobile phase. N-alkylated compounds were obtained in 75% yield (derivative 2); 80% yield (derivatives 3 and 13); 67% yield (derivative 6); 55% yield (derivative 11) and 50% yield (derivative 14).

To a solution of 1-propyl-1H-indole (2) in dry CH3CN was added aluminium trichloride (15 eq) and benzoyl chloride (10 eq), the reaction was stirred at room temperature overnight. The resulting mixture was dried in vacuo and reconstituted in DCM. The organic phase was washed with water (3 Â 50 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography using n-hexane/ethyl acetate (3/2) as mobile phase. Compound 4a was obtained in 32% yield.

A solution of formaldehyde (2 eq.), trifluoroacetic acid (2 eq.) and 2 equivalents of the proper amine (piperidine or bifenylethilamine) was added portionwise to a solution of the intermediate 2 or 3 (1 eq.) in DCM. The mixture was stirred for 3 h at room temperature, then was washed with water (3 Â 50 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography using DCM/MeOH (9:1 v/v) as mobile phase. Compounds 4b-d were obtained in 25%e38% yield.

The intermediate 7a or 7b (1.0 eq) was dissolved in a solution of DCM:CH3COOH (5:1 v/v) at room temperature. To this solution 2.0 equivalents of 4-chloroaniline was added and the mixture was warmed to reflux for 1.5 h. Then, 1.8 equivalents of sodium triacetoxyborohydride were added portionwise and the mixture was allowed to reflux for further 3e5 h. After cooling to room temperature, NaOH 1 N was added. The organic phase was separated and extracted one more time with the alkaline solution. Then it was dried over Na2SO4, filtered and concentrated in vacuo. The crude products were purified by column chromatography using mixtures of DCM/MeOH as eluent giving desired compounds 8a and 8b in 24% and 30% yield respectively.

To a solution of 5-amino indole (9, 1.0 eq) in dichloromethane/ DMF (20 mL/5 mL) were successively added benzoic acid (1.1 eq), HBTU (1.2 eq), HOBt (1.2 eq), and DIEA (2.4 eq), and stirring was continued at room temperature for 24 h. Afterward, the reaction mixture was diluted with dichloromethane (20 mL), and the resulting solution was washed successively with 10% citric acid (2 Â 25 mL), 10% NaHCO3 (2 Â 25 mL), and water (2 Â 25 mL), dried over Na2SO4, and evaporated to dryness. Intermediate 10 was obtained after flash chromatography using n-hexane/ethyl acetate 2/1 as ratio with 63% yield. Substitution with propyl group of indolic N1 was carried out using the same conditions described elsewhere, to give compound 11 with 44% yield. Mannich reaction performed on position 3 of intermediate 11 follows the procedure previously described yielded final compound 12 in 24% yield.

4.1.6. General procedure for the synthesis of derivatives 15, and 16a-i

One equivalent of intermediate 14a or 14b was dissolved in THF and 1.5 eq of the proper amine, 1.5 eq of TEA, 1.5 eq of NaI and 0.3 eq of (CH3COO)2Pd were added to this solution. The reaction was conducted under mW, at 100 C, for 20 min. The resulting mixture was filtered through Celite, dried in vacuo and reconstituted in DCM. The organic phase was washed with water (3 Â 50 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography using DCM/MeOH (9:1 v/v) as mobile phase giving derivative 15 in 38% yield and intermediates 16a-i in 55e75% yield.

4.1.7. General procedure for the synthesis of derivatives 17a-i and 18a-c

One equivalent of intermediate 16a-i was dissolved in DCM and 1.5 eq of the proper acyl chloride and 1.5 eq of TEA, were added to the solution. The reaction was conducted for 20 min at room temperature. The organic phase was washed with water (3 Â 50 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography using n-hexane/ethyl acetate (2:1 v/v) as mobile phase. Final derivatives 17a-i and 18ad were obtained as an atropisomer mixtures in 42e58% yield.

To a mixture of 16a (1.0 eq.) in DCM, (Boc)2O (3.0 eq.) was added followed by TEA (1.5 eq.). The mixture was allowed to stir at room temperature for 12 h. Afterward, the reaction mixture was diluted with dichloromethane (20 mL), and the resulting solution was washed with 10% citric acid (2 Â 25 mL). The organic phase was dried over Na2SO4, and evaporated to dryness. Compound 17 k was obtained as atropisomer mixture after flash chromatography using n-hexane/ethyl acetate 2/1 in 58% yield.

The compounds were evaluated against different herpes viruses, including varicella-zoster virus (VZV) strains Oka and YS, TK À VZV strains 07-1 and YS-R, herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK À ) HSV-1 KOS strain resistant to ACV (ACV r ), herpes simplex virus type 2 (HSV-2) strain G, human 

Cytotoxicity measurements were based on the inhibition of HEL cell growth. HEL cells were seeded at a rate of 5 Â 103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 C, the cell number was determined with a Coulter counter. The 50% cytostatic concentration (CC50) was calculated as the compound concentration required to reduce cell growth by 50% relative to the number of cells in the untreated controls. CC50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Cytotoxicity was expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that causes a microscopically detectable alteration of cell morphology.

This work was supported by grant from the Italian Ministry of Education (MIUR) (PRIN n 2010-11E61J12000210001) . The biological part of this work was supported by the KU Leuven (GOA 15/19 TBA).

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Roscovitine, a cyclindependent kinase inhibitor, prevents replication of varicella-zoster virus

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]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases

ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2

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Identification of small molecule compounds that selectively inhibit varicella-zoster virus replication

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The authors wish to express their gratitude to Mrs. Leentje Persoons, Mrs. Lies Van Den Heurck and Mrs. Ellen De Waegenaere for excellent technical assistance.

Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2016.09. 014. These data include MOL files and InChiKeys of the most important compounds described in this article.