Inhibition of the Replication of Different Strains of Chikungunya Virus

Article Cite This: ACS Infect. Dis. 2018, 4, 605−619

pubs.acs.org/journal/aidcbc

Inhibition of the Replication of Different Strains of Chikungunya Virus by 3‑Aryl-[1,2,3]triazolo[4,5‑d]pyrimidin-7(6H)‑ones Asier Gómez-SanJuan,# Ana-María Gamo,# Leen Delang,† Alfonso Pérez-Sánchez,# Siti Naqiah Amrun,§ Rana Abdelnabi,† Sofie Jacobs,† Eva-María Priego,# María-José Camarasa,# Dirk Jochmans,† Pieter Leyssen,† Lisa F. P. Ng,§ Gilles Querat,∥ Johan Neyts,† and María-Jesús Pérez-Pérez*,# #

Instituto de Química Médica, IQM, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain KU Leuven−University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium § Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, 04-06 Immunos, Singapore 138648, Singapore ∥ UMR “Émergence des Pathologies Virales” (EPV: Aix-Marseille Univ−IRD 190−Inserm 1207−EHESP−IHU Méditerranée Infection), 27 Bd Jean Moulin, 13005 Marseille, France

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†

S Supporting Information *

ABSTRACT: The re-emergence of chikungunya virus (CHIKV) is a serious global health threat. CHIKV is an alphavirus that is transmitted to humans by Aedes mosquitoes; therefore, their wide distribution significantly contributes to the globalization of the disease. Unfortunately, no effective antiviral drugs are available. We have identified a series of 3aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication. New series of compounds have now been synthesized with the aim to improve their physicochemical properties and to potentiate the inhibitory activity against different CHIKV strains. Among these newly synthesized compounds modified at position 3 of the aryl ring, tetrahydropyranyl and N-t-butylpiperidine carboxamide derivatives have shown to elicit potent antiviral activity against different clinically relevant CHIKV isolates with 50% effective concentration (EC50) values ranging from 0.30 to 4.5 μM in Vero cells, as well as anti-CHIKV activity in human skin fibroblasts (EC50 = 0.1 μM), a clinically relevant cell system for CHIKV infection. KEYWORDS: triazolopyrimidines, chikungunya virus, physicochemical properties

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genotype has been identified in the outbreaks in the Caribbean regions.9 Chikungunya is thus considered an important reemerging global public health problem in countries where the distribution of the Aedes mosquito vectors continues to expand, particularly in the tropical and subtropical areas.3,10−12 No vaccine or antiviral drug is available to fight against chikungunya infection.13 The use of chloroquine, the antimalarial drug, was evaluated in clinical trials during the outbreak in La Reunion.14 Despite the activity of the compound in cell culture, the benefits of this drug for CHIKF treatment were not clear. Traditional broad-spectrum antivirals such as ribavirin, mycophenolic acid, or interferon have also been used but their effectiveness has not been confirmed.15 Thus, cell-based assays have been set up to identify compounds that may inhibit CHIKV replication.13,16

hikungunya virus (CHIKV) is a positive single-stranded RNA enveloped virus belonging to the alphavirus genus and Togaviridae family. CHIKV is transmitted to humans through the bites of Aedes mosquitoes, particularly Ae. aegypti and Ae. albopictus, and causes chikungunya fever (CHIKF) characterized by chronic and incapacitating arthralgia.1−3 Although mortality is low, symptoms can persist for months or years, and this has important social and economic consequences. In some cases, complications afflicting neonates and the elderly have been reported, and recent outbreaks are also associated with more severe forms of CHIKF involving neurological complications.4 Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia, and more recently the Americas.5 Since 2007, autochthonous chikungunya outbreaks in Italy and France have been documented, the latest one this year.6 There are three different CHIKV genotypes: West African, Asian, and East/Central/South African (ECSA).7,8 The ECSA genotype has been associated with epidemics in the Indian Ocean countries while the Asian © 2018 American Chemical Society

Special Issue: Drug Discovery for Global Health Received: November 7, 2017 Published: February 6, 2018 605

DOI: 10.1021/acsinfecdis.7b00219 ACS Infect. Dis. 2018, 4, 605−619

ACS Infectious Diseases

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Figure 1. Previously identified [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as potent inhibitors of CHIKV replication.

Indeed, in the last three to four years, an increasing number of inhibitors have been described either from natural products such as flavonoids17 and diterpenes18 or from synthetic compounds.19−22 Interestingly, some of the compounds reported to block infection affect host factors, such as berberine.23 Through a collaborative project, we identified the triazolopyrimidines, exemplified by compounds 1 and 2 (Figure 1), as selective inhibitors of CHIKV replication.24 Structure−activity relationship studies performed with a wider series of compounds24,25 revealed that the [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one substituted with a methyl (1) or preferentially an ethyl (2) at position 5 was crucial for antiCHIKV activity together with a meta-substituted aryl ring linked at position 3 of the triazole. As recently reported,25 the oximes 3 and 4 (Figure 1) have 50% effective concentration (EC50) values in the very low μM range suggesting that the substituent at position 3 of the aryl moiety can be significantly enlarged without compromising the antiviral activity. On the basis of the anti-CHIKV activity of the isopropyl derivative 2, it was considered that enlarging the isopropyl substituent into a six membered ring to increase the fraction of sp3 atoms26 and further incorporating a heteroatom might maintain the antiviral activity and improve the physicochemical properties of the new series, as indicated by their theoretical octanol/water partition coefficient (cLogP), topological surface area (tPSA), and solubility values (see Table S1), thus paving the way for compounds with a better profile toward potential “in vivo” testing.

Scheme 1. Synthesis of [1,2,3]Triazolo[4,5-d]pyrimidin7(6H)-ones with a Six-Membered Ring as Substituent at the Aryl Ringa

Reagents and conditions: (a) Cs2CO3, DMF, 80 °C, 2 h (7a, 22% yield; 7b, 46% yield; 7c, 55% yield); (b) cyanoacetamide, NaH 60%, DMF, 0 °C to rt, 1 h (8a, 74% yield; 8b, 87% yield; 8c, 88% yield). (c) For 9a and 9b: EtCO2Et, tBuOK, dioxane, MW, 100 °C, 1 h (9a, 83% yield; 9b, 85% yield); for 9c: EtCO2tBu, tBuOK, dioxane, MW, 100 °C, 1 h (9c, 74%); (d) TFA, DCM, rt, 2 h, (9d, quantitative yield).

a

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RESULTS AND DISCUSSION Chemistry. The synthesis of the first series of six-membered ring derivatives is shown in Scheme 1. Reaction of 3azidophenol (5)27 with the mesylates 6a,28 6b,29 or 629 in the presence of Cs2CO3 afforded the corresponding arylazides 7a−c in moderate yields. The modest yield of the substitution products is due to the competing elimination reaction of the mesylate, as reported for similar analogues.30 Attempts to minimize the elimination reaction by using different bases (K2CO3, Cs2CO3, or NaH), testing different temperatures (from rt to 80 °C), or modifying the dilution were not successful. The best results were obtained using Cs2CO3 as the base and heating at 80 °C for 2 h. Then, reaction of the arylazides 7a−c with cyanoacetamide in the presence of NaH afforded the 5-amino-1H-1,2,3-triazole-4-carboxamides 8a−c in excellent yields. Finally, reaction of 8a and 8b with ethylpropionate or reaction of 8c with tert-butylpropionate in the presence of tBuOK in dioxane afforded the triazolopyrimidines 9a−c. Removal of the Boc protecting group in 9c by treatment with a 5% solution of TFA in DCM for 3 h at room temperature afforded quantitatively the free piperidyl derivative 9d. When these compounds (9a−d) were tested for antiCHIKV activity in cell culture, compound 9b with a

tetrahydropyranyl substituent and compound 9c with a Bocprotected piperidyl showed very potent antiviral activity with EC50 values in the low μM range. The antiviral activity of the N-Boc derivative 9c was particularly attractive since Bocremoval and further acylation reaction on the piperidyl might lead to novel substitutions. Before that and in an effort to further improve cLogP and tPSA values (Table S1), the piperidyl ring was replaced by a piperazinyl and a carbonyl group, instead of an oxygen, was introduced as the linker with the aryl ring. Thus, reaction of 3-azidobenzoic acid31 (10, Scheme 2) with tert-butyl piperazine-1-carboxylate, catalyzed by HCTU in the presence of DIPEA, in DMF/DCM at rt for 2 h afforded the coupling product 11 in 77% yield. Further reaction of this azide with cyanoacetamide in the presence of NaH afforded the aminocarboxamide 12 that was treated with tertbutyl propionate under basic conditions and MW irradiation to obtain 13 (22% yield for the two steps). This compound was 8fold less active than 9c (as it will be later discussed in the Biological Results section), so our efforts were turned back to the piperidyl derivatives. Thus, compound 9c was used as a synthon to introduce novel substitutions at the N atom of the piperidine. Removal of the Boc protecting group by treatment with 5% TFA followed 606



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Scheme 2. Synthesis of the N-Boc Piperazine Derivativea

50% effective concentration (EC50) and the 90% effective concentration (EC90), indicating the concentration of compound required to inhibit the virus-induced CPE by 50% and 90%, respectively. The effect of the compounds on noninfected cells is expressed as CC50, which corresponds to 50% cytotoxic concentration or the concentration that reduces the overall metabolic activity of uninfected cells to 50% (as determined by microscopic observation). Compounds 9a−d behaved very differently against CHIKV replication. While compounds 9b and 9c were very potent compounds with EC50 and EC90 values in the low μM range (Table 1), compounds 9a and 9d were inactive. In order to explain the lack of activity of the 9d compared to its oxygen counterpart 9b, it can be argued that under the physiological conditions the N atom of the free piperidyl might be charged (see Maps of Electrostatic Potential section). Such a positive charge may hamper the correct diffusion through membranes or imply a significant desolvation penalty to reach the target binding site. On the other hand and concerning the tert-butyl carbamate 9c, it is interesting to mention that carbamates are present in many approved drugs, as recently reviewed,32 and in particular, tert-butyl carbamates of piperidine derivatives have been reported to accomplish drug-like properties.33 Compound 13 with a piperazine tert-butyl carbamate had an EC50 value of 27 μM, 8-fold less active than 9c, suggesting that the incorporation of the piperazine was detrimental for antiCHIKV activity. On the other hand, other carbamates (14a−e) or urea (15a−h) derivatives of piperidine showed significant anti-CHIKV activity with EC50 values between 1.5 and 14 μM, although in some cases this was accompanied with significant cytotoxicity as measured by microscopic scoring. Only compounds 15d and 15h were almost inactive against CHIKV replication. It is also interesting to mention that the incorporation of a pyridine at the distal site (compounds 17a and 17b, Table 1) led to poorly active or inactive compounds, further stressing that the incorporation of groups that may be protonated at physiological pH is detrimental for activity. In order to determine whether the cytotoxicity observed with carbamates and ureas was compound-dependent or classdependent, for a few selected compounds with the best EC50 values (9c, 14a, and 15e), additional experiments were performed to measure cytotoxicity in other cell lines (Vero E6, BHK, HeLa Rh, or Huh7) either microscopically or by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium in the presence of phenazine methosulfate (MTS/PMS) colorimetric method. The obtained data are shown in Table 2. Only compound 9c showed CC50 values between 50 and 120 μM, while compounds 14a and 15e were not toxic at concentrations of 220 μM in the tested cell lines, the only exception being the more sensitive Huh7 cells. Thus, from the new series of triazolopyrimidines reported here, compound 9b, with a tetrahydropyranyl substituent, and compounds 9c, 14a, and 15e, with a piperidine substituent functionalized as a carbamate (9c and 14a) or an urea (15e), represent potent and selective anti-CHIKV inhibitors. Evaluation of Anti-CHIKV Activity against Different Clinical Isolates. Selected compounds from this series were evaluated for antiviral activity against several CHIKV clinical isolates (Table 3), including strains obtained from the outbreaks in Italy (Venturini 2008), Africa (Congo 2011), La Réunion Island (Opy 2006), and the Caribbean Americas (St Martin 2013 and EFS-1, Martinique 2013). It should be noted that all these compounds showed antiviral activity at the low

a

Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, HCTU, DIPEA, DMF/DCM, rt 2 h (11, 77% yield); (b) cyanoacetamide, NaH 60%, DMF anh., 0 °C to rt, 1 h (12, 84% yield); (c) EtCOOtBu, tBuOK, dioxane, MW, 100 °C, 60 min (13, 26% yield)

by reaction with different chloroformates in DCM in the presence of Et3N and DMAP (cat) at 0 °C afforded compounds 14a−e (Scheme 3). In a similar way, a number of ureas were synthesized (compounds 15a−h, Scheme 3) by reaction of the free amine after Boc-removal with a variety of isocyanates, in DCM/DMF in the presence of Et3N, or alternatively by reaction of the free amine with triphosgene and further reaction with a second amine. The conditions and yields obtained for each of these compounds are specified in Scheme 3. Amides and sulfonamides as substituents of the N of the piperazine were also synthesized (Scheme 4). Thus, Bocremoval in 9c followed by reaction with methyl 4-chloro-4oxobutanoate afforded the N-acyl derivative 16 (30% yield). Alternatively, reaction of 9c with TFA followed by reaction of the free amine with nicotinic or isonicotinic acid catalyzed by HATU and DIPEA afforded compounds 17a and 17b in 47% and 50% yield, respectively. Finally, reaction of the free amine with 2-propanesulfonyl chloride afforded the sulfonamide 18 in 67% yield. Biological Results. Evaluation of Anti-CHIKV Activity and Cytotoxicity. The synthesized compounds were evaluated for their potential to inhibit the CHIKV-induced cytopathogenic effect (CPE) in Vero cells (Table 1). Chloroquine and our previously reported hits (compounds 1 and 2) were included as reference compounds. The antiviral activity is expressed as the 607



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Scheme 3. Synthesis of Analogues of Carbamates and Ureas Related to Compound 9ca

Reagents and conditions: (a) (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) ROCOCl, Et3N, DMAP, DCM, 0 °C to rt (14a, 30% yield; 14b, 60% yield; 14c, 83% yield; 14d, 20% yield; 14e, 18% yield); (b) for 15a, 15f, and 15h: (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) triphosgene, RNH2, DCM/ DMF, DIPEA, rt, 30 min (15a, 54% yield; 15f, 24% yield; 15h, 26% yield); (c) for 15b, 15c, 15d, 15e, and 15g: (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) RNCO, DCM/DMF, Et3N, 0 °C to rt (15b, 73% yield; 15c, 71% yield; 15d, 25% yield; 15e, 78% yield; 15g, 61% yield) a

Scheme 4. Synthesis of Other Acyl and Sulfonamide Derivatives Related to Compound 9ca

Reagents and conditions: (a) (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) methyl 4-chloro-4-oxobutanoate, Et3N, DMAP, DCM, 0 °C to rt (16, 30% yield); (b) (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) nicotinic acid or isonicitinic acid, PyBOP, DIPEA, DMF/DCM, rt, 2 h (17a, 28% yield; 17b, 50% yield); (c) (i) TFA 5% in DCM, rt, 2 h (quant.); (ii) 2-propanesulfonyl chloride, Et3N, DCM/DMF, 0 °C to rt, 1 h (18, 67% yield). a

μM or sub-μM range, well below their CC50 in Vero cells. In all cases, the most potent inhibition was observed against the African Congo strain with EC50 values as low as 0.30 to 0.60 μM for compounds 9b, 14a, and 15e. In addition, these compounds showed lower EC50 values against St Martin strains than the reference compounds 1 and 2, so it can be concluded

that the new derivatives have a wider profile against different CHIKV strains than previous series. Evaluation of Anti-CHIKV Activity in Human Skin Fibroblasts. Next, the anti-CHIKV activity of some selected triazolopyrimidines was determined in human skin fibroblasts, which is a clinically more relevant system as the skin is the first 608



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different CHIKV strain (CHIKV-SGP11) in fibroblasts providing an EC50 value of 0.78 ± 0.07 and 0.20 ± 0.20 μM, respectively. Moreover, in this cellular system, significant antiviral activity was also found for 9b and 15e against the O’nyong’nyong virus (ONNV), the closest analogue to CHIKV among alphaviruses,7 with EC50 values of 0.66 ± 0.07 and 0.42 ± 0.07 μM, respectively. Evaluation against CHIKV Strains Resistant to Compound 1. In order to determine if the new series of compounds behaved similarly to our initially reported compounds, some of the here described compounds were tested against a CHIKV strain that was selected under the selective pressure of compound 1. This strain is characterized by a single mutation in the nonstructural protein 1 (nsP1) gene (P34S).36 The CHIKV nsP1 is the main enzyme involved in the capping of the viral RNA. The importance of the P34S mutation for the resistance phenotype had been previously confirmed by reverse genetics.36 T-705 (favipiravir), a CHIKV inhibitor with a different mechanism of action,37 was included as a negative control. As shown in Table 4, all triazolopyrimidine compounds were less active against the CHIKV variant resistant against compound 1, albeit not to the same extent. Compound 9b behaved similarly to compound 2 (54- and 64-fold resistance, respectively), whereas compounds 9c and 15e showed lower levels of cross-resistance. The observed difference in fold resistance levels could be due to the difference in the selectivity indexes: compounds 1, 9c, and 15e have lower selectivity indexes than compounds 2 and 9b; their toxicity might limit their resistance indexes. Maps of Electrostatic Potential. In an attempt to explain the differences shown in the antiviral activity of these compounds, molecular electrostatic potential (MEP) maps were calculated for selected compounds (9b, 9c, 9d, and 15e). The geometry of the compounds was first optimized by means of the quantumchemistry software Jaguar (implemented in the Schrödinger Suite), and MEP maps were calculated on the basis of the optimized structures using DelPhi. Results are shown in Figure S1. As expected, MEP maps corresponding to the triazolopyrimidinone core and the aromatic ring, present in all the ligands, showed a common pattern. The major differences were observed at the distal part for compound 9d compared to the other three compounds, where a significant positive map was located around the protonated piperidine ring for compound 9d. This may partially account for the different behavior in terms of antiviral activity between compound 9d and the other three compounds. On the other hand, the urea and carbamate derivatives, 9c and 15e, respectively, both with a tert-butyl distal substituent, showed similar positive and negative MEPs. Microsomal Stability Studies and Preliminary Pharmacokinetics. As indicated in the introduction, one of the objectives of the new series of triazolopyrimidines was to improve their

Table 1. Antiviral Evaluation of the Triazolopyrimidines against CHIKV Strain 899 in Vero Cellsa compound

EC50 (μM)b

EC90 (μM)c

CC50 (μM)d

selectivity index

1 2 9a 9b 9c 9d 13 14a 14b 14c 14d 14e 15a 15b 15c 15d 15e 15f 15g 15h 16 17a 17b 18 chloroquine

19 ± 2e 2.6 ± 1.0e >49 1.2 ± 0.009 3.2 ± 1.0 236 27 ± 11 1.5 ± 0.3 2.5 ± 0.3 2.8 ± 0.2 4.2 ± 0.4 2.9 ± 0.7 7.4 ± 4.1 14 ± 2.2 7.3 ± 1.5 129 ± 79 8.9 ± 6.1 5.1 ± 0.2 2.0 ± 0.3 66 ± 28 26 ± 9.7 >224 34 11 ± 3.4 11 ± 7

38 ± 16e 8.8 ± 6.0e >49 3.4 8.2 ± 1.5 >241 >82 3.1 ± 1.3 >8.2 >20 >8.3 19 >13 >23 >106 >243 11 ± 4.1 35 ± 13 >46 >221 89 >224 >129 >22 21 ± 18

116 ± 60 167 ± 94 >49 84 ± 19 59 ± 23 241 82 ± 3 >220 8.2 ± 0.2 20 ± 6 8.3 ± 0.5 45 ± 15 13 ± 0.9 >220 106 >243 201 ± 26 135 ± 41 46 ± 2.8 >221 >220 >224 129 22 89 ± 28

6.1 64 naf 70 18 1.0 3.0 >147 3.3 7.1 2.0 16 1.8 >16 15 >1.9 23 26 23 >3.4 >8.5 naf 3.8 2.0 8.1

All data are mean values ± standard deviation for at least three independent experiments. b50% effective concentration or concentration required to protect 50% of the cells against the cytopathic effect of the virus. c90% effective concentration or concentration required to protect 90% of the cells against the cytopathic effect of the virus. d50% cytotoxic concentration or concentration that reduces the viability of uninfected cells to 50%. Measured by microscopic scoring. eData as reported in ref 24. fn.a.: not applicable. a

organ supporting viral replication upon the bite of an infected mosquito.34,35 BJ (CRL-2522) fibroblastic cells were infected with Gluc-tagged CHIKV-LR2006 OPY1, and the antiviral effect of selected compounds was determined. In this model, our reference compounds 1 and 2 had EC50 values of 1.10 ± 0.38 and 0.72 ± 0.56 μM, respectively. Interestingly, compound 9b showed a more potent antiviral activity with an EC50 value of 0.12 ± 0.09 μM. Also, compound 15e was significantly active in this assay with an EC50 of 0.55 ± 0.38 μM. It is relevant to mention that in all cases the EC50 values were observed to be in the low μM or sub-μM range while cell viability was not affected at concentrations two to three logs higher (CC50 > 100 μM). Compounds 9b and 15e were also tested against a

Table 2. Determination of Cytotoxicity of Selected Compounds in Different Cell Lines CC50 (μM)a cell type Vero A Vero E6 BHK HeLa Rh Huh7 a

9c 74 84 115 75 60

± ± ± ± ±

0.9 1.8 6 9 0.3

CC50 (μM)b

14a

15e

>220 >220 >220 >220 78 ± 5

>220 >220 >220 >220 152 ± 9

9c 59 60 57 72 48

± ± ± ± ±

23 2 23 9 2

14a

15e

>220 >220 >220 >220 91 ± 2

201 ± 26 >220 >220 >220 137 ± 23

Cytotoxicity determined by MTS. bCytotoxicity determined microscopically. 609



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Table 3. Antiviral Evaluation of Selected Triazolopyrimidines against Different Laboratory Strains and Clinical Isolates of CHIKV in Vero Cells comp

CC50 (μM)a

CHIKV Congo EC50 (μM)

CHIKV Venturini EC50 (μM)

CHIKV OPy EC50 (μM)

CHIKV St Martin EC50 (μM)

CHIKV St Martin EFS EC50 (μM)

1 2 9b 9c 14a 15e

>300 >300 >100 >100 >300c >300

6.35 ± 0.05b 0.75 ± 0.35b 0.3 ± 0.0 1 ± 0.1 0.35 ± 0.5 1.0 ± 0.4

26 ± 2b 1.43 ± 0.01b 1.65 ± 0.5 2.6 ± 1.1 1.2 ± 0.2 2.9 ± 0.4

28 2.6 ± 0.5 5.2 11.5 3.4 ± 1.2 5.4 ± 2.4

23.9 ± 0.5b 2.9 ± 0.05b 2 ± 0.3 NDd 1.2 ± 0.5 3 ± 2.5

55.3 13 ± 6 4.5 ± 0.5 8.7 ± 1.7 3.7 ± 1.1 4.4 ± 3

a

50% cytotoxic concentration or concentration that reduces the overall metabolic activity of uninfected cells to 50%. Measured by CellTiter blue read-out. bReported in ref 24. cPoorly soluble. dND: Not determined.

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CONCLUSIONS On the basis of the identification of 3-aryl-[1,2,3]triazolo[4,5d]pyrimidin-7(6H)-ones as selective anti-CHIKV agents, exemplified by compounds 1 and 2, additional series of compounds were synthesized and evaluated, resulting in the identification of novel derivatives that inhibit CHIKV replication in the very low μM or sub-μM range. Among them, the tetrahydropyranyl derivative 9b or the N-tert-butyl piperidine carboxamide 15e have demonstrated to be potent and selective inhibitors of a variety of clinical CHIKV strains (Venturini, Congo, St Martin) with EC50 values ranging from 0.30 to 4.5 μM. Both compounds also showed potent antiviral activity against two CHIKV strains in human skin fibroblasts, which is a more relevant biological system since the skin is the primary infection site after the mosquito bite. Preliminary pharmacokinetics (PK) studies demonstrated that compound 9b has a reasonable Cmax although further improvement of the half-life is recommended before performing “in vivo” proof-ofconcept experiments in an animal model of CHIKV infection. Compounds 9b, 9c, and 15e showed a significant level of cross resistance when tested against a CHIKV strain selected under the pressure of the prototype triazolopyrimidine 1, and a higher level of cross resistance was obtained for compound 9b that was also the less toxic.

Table 4. In Vitro Anti-CHIKV Activity of Triazolopyrimidine Compounds against WT and against a Resistant CHIKV Strain Selected under the Pressure of the Prototype Triazolopyrimidine 1 EC50 (μM)a T-705 1 2 9b 9c 15e

WT CHIKV

resistant CHIKV strain to 1

± ± ± ± ± ±

12 ± 6 >116b >167b 65 ± 40 35 ± 29 23 ± 14

29 19 2.6 1.2 3.2 8.9

21 2 1 0.01 1.0 6.1

Data shown are average values ± SD of at least two independent experiments. bSimilar data were reported in ref 36.

a

physicochemical properties in order to identify a potential inhibitor for “in vivo” experiments. Among the newly synthesized derivatives, compounds 9b and 15e showed the best inhibition of CHIKV replication in the different systems assayed. Thus, in silico ADME predictions were performed for both compounds using QikProp program (Schrödinger Release 2015-4, Schrödinger, LLC, New York, NY, 2015). The obtained values for the minimum energy conformer of 9b and 15e (Table S2) in terms of calculated Caco permeability (QPPCaco), calculated binding to human albumin (QPlogKHsa), and percentage of human oral absorption indicated superior properties for 9b compared to 15e. Thus, compound 9b was selected for experimental determinations. Microsomal stability studies, in the absence of cofactors, indicated perfect stability while in the presence of cofactors the t1/2 was around 24 min and the clearance value was around 230 μL/min/mg. Data are shown in Table S3, together with reference compounds (diclofenac and imipramine). Interestingly, the values obtained for compound 9b were 3-fold better than those obtained with compound 2. As a follow-up to these results, compound 9b was selected for a preliminary pharmacokinetics study in male BALB/c mice. The compound was administered as a single dose by intraperitoneal and subcutaneous route at 10 mg/kg body weight. The data obtained are compiled in Table S4. The Cmax (1168 and 806 ng/mL for intraperitoneal and subcutaneous dosing, respectively) were acceptable, but the half-lives (0.34 and 0.47 h, respectively) were short, indicating that the compound is well absorbed and distributed but that it is also easily metabolized and/or excreted.

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METHODS Chemistry Procedures. Melting points were obtained on a Mettler Toledo M170 apparatus and are uncorrected. The elemental analysis was performed with a Heraus CHN-ORAPID instrument. The elemental compositions of the compounds agreed to within ±0.4 of the calculated values. For all the tested compounds, satisfactory elemental analysis was obtained supporting greater than 95% purity. Electrospray mass spectra were measured on a quadrupole mass spectrometer equipped with an electrospray source (HewlettPackard, LC/MS HP 1100). 1H and 13C NMR spectra were recorded on a Varian INNOVA-300 operating at 300 MHz (1H) and 75 MHz (13C), respectively, and a Varian INNOVA400 operating at 400 MHz (1H) and 101 MHz (13C), respectively. Analytical TLC was performed on silica gel 60 F254 (Merck) precoated plates (0.2 mm). Spots were detected under UV light (254 nm) and/or charring with ninhydrin. Separations on silica gel were performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a ChromatotronR (Kiesegel 60 F254 gipshaltig (MercK)), with layer thicknesses of 1 and 2 mm and flow rates of 4 or 8 mL/min, respectively. Flash column chromatography was performed with silica gel 60 (230−400 mesh) (Merck). 610



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were dried over anhydrous Na2SO4, filtered, and evaporated. The residue was purified as indicated in each case. 5-Amino-1-(3-(cyclohexyloxy)phenyl)-1H-1,2,3-triazole-4carboxamide (8a). Following the general procedure A, to a solution of cyanoacetamide (13 mg, 0.15 mmol) in anhydrous DMF (1.0 mL) at 0 °C, NaH (60% mineral oil, 10 mg, 0.25 mmol) was slowly added, followed by the addition of a solution of 7a (30 mg, 0.14 mmol) in anhydrous DMF (1.0 mL). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:2) to yield 31 mg (74%) of 8a as a white solid. Mp: 166−168 °C. MS (ES, positive mode): m/z 302 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.26−1.60 (m, 6H, Cy), 1.64−1.79 (m, 2H, Cy), 1.88−2.00 (m, 2H, Cy), 4.45 (m, 1H, OCH), 6.36 (br s, 2H, NH2), 7.04−7.15 (m, 3H, Ar), 7.22 (br s, 1H, CONH2a(b)), 7.48 (m, 1H, Ar), 7.61 (br s, 1H, CONH2b(a)). 5-Amino-1-(3-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)-1H1,2,3-triazole-4-carboxamide (8b). Following the general procedure A, to a solution of cyanoacetamide (49 mg, 0.58 mmol) in anhydrous DMF (1.3 mL) at 0 °C, NaH (60% mineral oil, 38 mg, 0.93 mmol) was slowly added, followed by the addition of a solution of 7b (115 mg, 0.52 mmol) in anhydrous DMF (1.3 mL). After workup, the solid was resuspended in diethyl ether to yield 138 mg (87%) of 8b as a white solid. Mp: 171−173 °C. MS (ES, positive mode): m/z 304 (M + H)+. 1H NMR (300 MHz, DMSO-d6) δ 1.55−1.67 (m, 2H, H-3′a(b), H-5′a(b)), 1.97−2.02 (m, 2H, H-3′b(a), H5′b(a)), 3.45−3.53 (m, 2H, H-2′a(b), H-6′a(b)), 3.82−3.89 (m, 2H, H-2′b(a), H-6′b(a)), 4.69 (m, 1H, OCH), 6.37 (br s, 2H, NH2), 7.11−7.17 (m, 3H, Ar), 7.23 (br s, 1H, CONH2a(b)), 7.50 (t, J = 8.1 Hz, 1H, Ar), 7.62 (br s, 1H, CONH2b(a)). tert-Butyl 4-(3-(5-amino-4-carbamoyl-1H-1,2,3-triazol-1yl)phenoxy)piperidine-1-carboxylate (8c). To a solution of cyanoacetamide (110 mg, 1.30 mmol) in anhydrous DMF (3.25 mL) at 0 °C, NaH (60% mineral oil, 50 mg, 2.10 mmol) was slowly added. After 30 min at this temperature, a solution of 7c (370 mg, 1.16 mmol) in anhydrous DMF (3 mL) was added. The reaction mixture was stirred for 30 min at 0 °C and for an additional 30 min at rt. The mixture was concentrated to dryness. The residue was dissolved in DCM (10 mL) and washed with water (10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (dichloromethane/methanol, 95:5) to yield 460 mg (88%) of 8c as a white solid. MS (ES, positive mode): m/z 403 (M + H)+. 1H NMR (300 MHz, DMSO-d6) δ 1.40 (s, 9H, (CH3)3), 1.55 (m, 2H, CH2CH2N), 1.93 (m, 2H, CH2CH2N), 3.19 (m, 2H, CH2CH2N), 3.66 (m, 2H, CH2CH2N), 4.67 (m, 1H, CHO), 6.37 (br s, 2H, NH2), 7.11−7.17 (m, 3H, Ar), 7.22 (br s, 1H, CONH2), 7.49 (t, J = 8.0 Hz, 1H, Ar), 7.62 (br s, 1H, CONH2). General Procedure for the Synthesis of [1,2,3]Triazolo[4,5d]pyrimidin-7-ones (General Procedure B). A microwave vial was charged with the corresponding carboxamide (1.0 mmol), t BuOK (1 M in THF, 4.0 mmol), and ethyl propionate (4.0 mmol) in anhydrous dioxane (10 mL). The reaction mixture was heated in a microwave reactor at 100 °C for 1 h. The mixture was concentrated to dryness. The crude obtained was dissolved in dichloromethane (10 mL) and washed with a saturated solution of NaHCO3 (3 × 10 mL). The combined aqueous phases were extracted with dichloromethane (3 × 10 mL). The combined organic phases were dried over anhydrous

Microwave reactions were performed using the Biotage Initiator 2.0 single-mode cavity instrument from Biotage (Uppsala). Experiments were carried out in sealed microwave process vials utilizing the standard absorbance level (400 W maximum power). The temperature was measured with an IR sensor on the outside of the reaction vessel. 1-Azido-3-(cyclohexyloxy)benzene (7a). To a solution of 527 (85 mg, 0.63 mmol) in anhydrous DMF (1 mL) under argon atmosphere, Cs2CO3 (246 mg, 0.76 mmol) was added. The reaction mixture was stirred at rt for 5 min. Then, a solution of 6a28 (224 mg, 0.8 mmol) in anhydrous DMF (1.2 mL) was added dropwise, and the reaction was heated at 80 °C for 2 h. After workup, the residue was purified by CCTLC (hexane) to yield 30 mg (22%) of 7a as colorless oil. 1H NMR (400 MHz, CDCl3) δ 1.23−1.45 (m, 3H, Cy), 1.46−1.63 (m, 3H, Cy), 1.73−1.88 (m, 2H, Cy), 1.92−2.02 (m, 2H, Cy), 4.23 (tt, J = 8.9, 3.8 Hz, 1H, OCH), 6.55 (t, J = 2.2 Hz, 1H, Ar), 6.61 (ddd, J = 7.9, 2.1, 0.8 Hz, 1H, Ar), 6.68 (ddd, J = 8.3, 2.4, 0.8 Hz, 1H, Ar), 7.22 (t, J = 8.1 Hz, 1H, Ar). 4-(3-Azidophenoxy)tetrahydro-2H-pyran (7b). To a solution of 527 (188 mg, 1.39 mmol) in anhydrous DMF (3 mL) under argon atmosphere, Cs2CO3 (553 mg, 1.7 mmol) was added. The reaction mixture was stirred at rt for 5 min. Then, a solution of 6b29 (250 mg, 1.39 mmol) in anhydrous DMF (4.2 mL) was added dropwise, and the reaction was heated at 80 °C for 2 h. After workup, the residue was purified by flash chromatography (hexane/ethyl acetate, 8:2) to yield 140 mg (46%) of 7b as yellow oil. MS (ES, positive mode): m/z 637 (2M + H)+. 1H NMR (300 MHz, CDCl3) δ 1.69−1.88 (m, 2H, H-3′a(b), H-5′a(b)), 1.94−2.10 (m, 2H, H-3′b(a), H-5′b(a)), 3.47− 3.66 (m, 2H, H-2′a(b), H-6′a(b)), 3.87−4.15 (m, 2H, H-2′b(a), H6′b(a)), 4.48 (m, 1H, OCH), 6.56 (t, J = 2.2 Hz, 1H, Ar), 6.58− 6.78 (m, 2H, Ar), 7.28 (t, J = 8.1 Hz, 1H, Ar). tert-Butyl 4-(3-azidophenoxy)piperidine-1-carboxylate (7c). To a solution of 527 (405 mg, 3 mmol) in anhydrous DMF (9 mL), Cs2CO3 (1.17 g) was added, and the mixture was stirred for 5 min at rt. A solution of 6c29 (830 mg, 3 mmol) in anhydrous DMF (3 mL) was added dropwise. The reaction mixture was heated at 80 °C for 2 h. Then, a saturated solution of NH4Cl (10 mL) was added, and the solution was extracted with ethyl acetate (3 × 10 mL). The organic phase was washed with NaOH (1 M, 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (hexane/ethyl acetate, 90:10) to yield 509 mg (55%) of 7c as yellow oil. MS (ES, positive mode): m/ z 638 (2M + H)+. 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 9H, (CH3)3), 1.74 (m, 2H, CH2CH2N), 1.91 (m, 2H, CH2CH2N), 3.34 (m, 2H, CH2CH2N), 3.68 (m, 2H, CH2CH2N), 4.45 (m, 1H, CHO), 6.55 (pt, J = 2.2 Hz, 1H, Ar), 6.62−6.69 (m, 2H, Ar), 7.24 (pt, J = 8.1 Hz, 1H, Ar). General Procedure for the Synthesis of 5-Amino-1H-1,2,3triazole-4-carboxamides (General Procedure A). To a solution of cyanoacetamide (1.1 mmol) in anhydrous DMF (3 mL) at 0 °C, NaH (60% mineral oil, 1.8 mmol) was slowly added. After 30 min at this temperature, a solution of the corresponding azide (1.0 mmol) in anhydrous DMF (3 mL) was added. The reaction mixture was stirred for 30 min at 0 °C and an additional 30 min at rt. The mixture was concentrated to dryness. The crude obtained was dissolved in dichloromethane/methanol (9:1) (10 mL) and washed with H2O (3 × 10 mL). The combined aqueous phases were extracted with dichloromethane (3 × 10 mL). The combined organic phases 611



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Na2SO4, filtered, and evaporated. The residue was purified as indicated in each case. 3-(3-(Cyclohexyloxy)phenyl)-5-ethyl-3,6-dihydro-7H[1,2,3]triazolo[4,5-d]pyrimidin-7-one (9a). Following the general procedure B, a microwave vial was charged with 8a (30 mg, 0.10 mmol), tBuOK (1 M in THF, 0.4 mL, 0.4 mmol), and ethyl propionate (46 μL, 0.4 mmol) in anhydrous dioxane (1.0 mL). After workup, the residue was purified by flash chromatography (dichloromethane/methanol, 95:5) to yield 28 mg (83%) of 9a as a white solid. Mp: 262−264 °C. MS (ES, positive mode): 340 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.28−1.62 (m, 6H, Cy), 1.67−1.82 (m, 2H, Cy), 1.89−2.07 (m, 2H, Cy), 2.72 (q, J = 7.5 Hz, CH2CH3), 4.42 (m, 1H, OCH), 7.09 (ddd, J = 8.2, 2.5 Hz, 1H, Ar), 7.52 (t, J = 8.1 Hz, 1H, Ar), 7.58 (m, 1H, Ar), 7.67 (t, J = 2.2 Hz, 1H, Ar), 12.72 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.1 (CH2CH3), 23.2 (Cy), 25.1 (Cy), 27.7 (CH2CH3), 31.2 (Cy), 74.9 (OCH), 108.8, 113.6, 116.5 (Ar), 129.0 (C-7a), 130.7, 136.4 (Ar), 148.6 (C-3a), 155.9, 157.9 (C-5, Ar), 164.6 (C-7). Anal. calcd for (C18H21N5O2 0.5H2O): C, 62.05; H, 6.36; N, 20.10. Found: C, 62.54; H, 6.28; N, 19.84. 5-Ethyl-3-(3-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)-3,6dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (9b). Following the general procedure B, a microwave vial was charged with 8b (100 mg, 0.33 mmol), tBuOK (1 M in THF, 1.3 mL, 1.3 mmol), and ethyl propionate (160 μL, 1.3 mmol) in anhydrous dioxane (3.3 mL). After workup, the residue was purified by flash chromatography (dichloromethane/methanol, 95:5) to yield 95 mg (85%) of 9b as a white solid. Mp: 237− 239 °C. MS (ES, positive mode): 342 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.58−1.70 (m, 2H, H-3′a(b), H-5′a(b)), 1.99−2.08 (m, 2H, H3′b(a), H-5′b(a)), 2.72 (q, J = 7.5 Hz, CH2CH3), 3.49 (ddd, J = 11.9, 9.5, 2.7 Hz, 2H, H-2′a(b), H-6′a(b)), 3.87 (dt, J = 11.7, 4.3 Hz, 2H, H-2′b(a), H-6′b(a)), 4.68 (m, 1H, OCH), 7.15 (ddd, J = 8.2, 2.5, 1.0 Hz, 1H, Ar), 7.54 (t, J = 8.1 Hz, 1H, Ar), 7.61 (ddd, J = 8.1, 1.9, 1.0 Hz, 1H, Ar), 7.70 (t, J = 2.2 Hz, 1H, Ar), 12.72 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.1 (CH2CH3), 27.7 (CH2CH3), 31.6 (C-3′, C-5′), 64.5 (C-2′, C6’), 71.9 (OCH), 109.1, 114.0, 116.5 (Ar), 128.9 (C-7a), 130.7, 136.4 (Ar), 148.6 (C-3a), 155.9, 157.5 (C-5, Ar), 164.6 (C-7). Anal. calcd for (C17H19N5O3): C, 59.81; H, 5.61; N, 20.52. Found: C, 59.67; H, 5.64; N, 20.31. tert-Butyl 4-(3-(5-ethyl-7-oxo-triazolo[4,5-d]pyrimidin-3yl)phenoxy)piperidine-1-carboxylate (9c). A microwave vial was charged with 8c (360 mg, 0.9 mmol), tBuOK 1 M in THF (3.6 mL, 3.6 mmol), and tert-butyl propionate (0.54 mL, 3.6 mmol) in anhydrous dioxane (9 mL). The reaction mixture was heated in a microwave reactor at 100 °C for 1 h. The mixture was concentrated to dryness. Water was added (15 mL), and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (dichloromethane/methanol, 95:5) to yield 296 mg (74%) of 9c as a white solid. Mp: 210−212 °C. MS (ES, positive mode): m/z 441 (M + H)+, 881 (2M + H)+. 1H NMR (300 MHz, DMSO-d6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.40 (s, 9H, (CH3)3), 1.59 (m, 2H, CH2CH2N), 1.70 (m, 2H, CH2CH2N), 2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.19 (m, 2H, CH2CH2N), 3.69 (m, 2H, CH2CH2N), 4.67 (m, 1H, OCH), 7.16 (m, 1H, Ar), 7.55 (pt, J = 8.0 Hz, 1H, Ar), 7.60−7.64 (m, 1H, Ar), 7.69 (pt, J = 2.1 Hz, 1H, Ar), 12.70 (br

s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.7 (CH2CH3), 28.4 (CH2CH3), 28.7 (C(CH3)3), 30.9 (C-3′, C5′) 40.8 (C-2′, C-6’), 73.1 (C-4’), 79.3 (C(CH3)3), 110.0, 114.8, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.0 (CArN), 149.3 (C-3a), 154.6, 156.5, 158.2 (NCOO, C-5, CArO), 165.2 (C-7). Anal. Calc. for C22H28N6O4: C, 59.99; H, 6.41; N, 19.08. Found: C, 59.77; H, 6.41; N, 18.89. Trifluoroacetate Salt of 5-ethyl-3-(3(piperidine-4-yloxy)phenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one (9d). A solution of 9c (35 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h and concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and concentrated to dryness. This procedure was repeated several times until a permanent weight was obtained (quantitative yield). A small portion was crystallized in MeOH/Et2O. Mp: 186−188 °C. MS (ES, positive mode): m/z 341 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.24 (t, J = 7.5 Hz, 3H, CH2CH3), 1.80−1.95 (m, 2H, H-3′a(b), H-5′a(b)), 2.10−2.20 (m, 2H, H-3′b(a), H-5′b(a)), 2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.11−3.13 (m, 2H, H-2′a(b), H-6′a(b)), 3.27−3.30 (m, 2H, H-2′b(a), H-6′b(a)), 4.78 (m, 1H, H-4′), 7.20 (dd, J = 8.3, 2.5 Hz, 1H, Ar), 7.58 (pt, J = 8.1 Hz, 1H, Ar), 7.68 (m, 1H, Ar), 7.69 (pt, J = 2.2 Hz, 1H, Ar), 8.64 (br s, 2H, NH2), 12.76 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 27.8 (C-3′, C-5′), 28.4 (CH2CH3), 41.3 (C-2′, C-6′), 70.1 (C-4′), 110.2, 115.3, 117.0, 135.1 (Ar), 129.6 (C-7a), 137.1 (CArN), 149.3 (C-3a), 156.5, 157.8 (C-5, CArO), 165.3 (C-7). Anal. Calc. for C17H20N6O2·TFA·0.5H2O: C, 49.24; H, 4.79; N, 18.14. Found: C, 49.42; H, 4.93; N, 18.26. tert-Butyl 4-(3-azidobenzoyl)piperazine-1-carboxylate (11). A solution of tert-butyl piperazine-1-carboxylate (0.14 g, 0.74 mmol), DIPEA (0.26 mL, 1.47 mmol) and HCTU (0.46 g, 0.88 mmol) in anhydrous dichloromethane (3 mL) was stirred at rt for 20 min. A solution of 3-azidobenzoic acid 1031 (0.12 g, 0.74 mmol) in anhydrous dichloromethane/DMF (9:1) (2 mL) was added, and the reaction mixture was stirred at rt for 2 h. The reaction mixture was dissolved in dichloromethane (10 mL) and washed with a 5% aqueous solution of citric acid (5 mL), a saturated aqueous solution of NaHCO3 (5 mL), and brine (5 mL). The combined organic phase was dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified by CCTLC (dichloromethane/methanol, 10:0.2) to yield 0.19 g (77%) of 11 as yellow solid. Mp: 103−105 °C. MS (ES, positive mode): m/z 332 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.39 (s, 9H, C(CH3)3), 3.26−3.39 (m, 6H, H-2′, H-6′, H-3′a(b), H-5′a(b)), 3.55−3.57 (m, 2H, H-3′b(a), H-5′b(a)), 7.11 (t, J = 1.8 Hz, 1H, Ar), 7.22−7.16 (m, 2H, Ar), 7.46 (t, J = 7.8 Hz, 1H, Ar). tert-Butyl 4-(3-(5-amino-4-carbamoyl-1H-1,2,3-triazol-1yl)benzoyl)piperazine-1-carboxylate (12). Following general procedure A, to a solution of cyanoacetamide (43 mg, 0.52 mmol) in anhydrous DMF (2.0 mL), NaH (60% mineral oil, 34 mg, 0.84 mmol) was slowly added at 0 °C, followed by the addition of a solution of 11 (0.15 g, 0.47 mmol) in anhydrous DMF (1.5 mL). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 0.16 g (84%) of 12 as white solid. Mp: 125−127 °C. MS (ES, positive mode): m/z 416 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.39 (s, 9H, C(CH3)3), 3.35−3.40 (m, 6H, H-2′, H-6′, H-3′a(b), H-5′a(b)), 3.57−3.59 (m, 2H, H-3′b(a), H-5′b(a)), 6.43 (s, 2H, NH2), 7.22 (s, 1H, CONH2a(b)), 7.54−7.56 (m, 1H, Ar), 7.59− 612



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164.8 (C-7). Anal. Calc. for C24H24N6O4: C, 62.60; H, 5.25; N, 18.25. Found: C, 62.24, H, 5.28; N, 17.88. Benzyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxylate (14b). Following the general procedure C, a solution of 9c (36 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (40 μL, 0.28 mmol), DMAP (5 mg, 0.04 mmol), and benzylchloroformate (22 μL, 0.15 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 23 mg (60%) of 14b as a white solid. Mp: 115−117 °C. MS (ES, positive mode): m/z 475 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.57− 1.65 (m, 2H, H-3′a(b), H-5′a(b)), 1.95−2.01 (m, 2H, H-3′b(a), H5′b(a)), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 3.24−3.29 (m, 2H, H-2′a(b), H-6′a(b)), 3.71−3.77 (m, 2H, H-2′b(a), H-6′b(a)), 4.68 (m, 1H, H-4′), 5.07 (s, 2H, OCH2), 7.15 (d, J = 8.1 Hz, 1H, Ar), 7.28−7.38 (m, 5H, Ar), 7.55 (pt, J = 8.2 Hz, 1H, Ar), 7.60 (d, J = 8.1 Hz, 1H, Ar), 7.68 (pt, J = 2.2 Hz, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.7 (CH 2 CH 3 ), 28.4 (CH 2 CH 3 ), 41.5 (C-2′, C-6′), 66.9 (OCH2), 72.9 (C-4′), 110.0, 114.9, 117.2, 128.2, 128.5, 129.6, 131.5 (Ar), 129.1 (C-7a), 137.1, 137.7 (CArC, CArN), 149.3 (C-3a), 155.1, 156.5, 158.2 (NCOO, C-5, CArO), 165.3 (C-7). Anal. Calc. for C25H26N6O4·H2O: C, 60.97; H, 5.73; N, 17.06. Found: C, 61.38; H, 5.52; N, 16.92. Allyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxylate (14c). Following the general procedure C, a solution of 9c (35 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (40 μL, 0.28 mmol), DMAP (5 mg, 0.04 mmol), and allylchloroformate (16 μL, 0.15 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 28 mg (83%) of 14c as a white solid. Mp: 182−184 °C. MS (ES, positive mode): m/z 425 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.22 (t, J = 7.5 Hz, 3H, CH2CH3), 1.56−1.63 (m, 2H, H-3′a(b), H-5′a(b)), 1.93−2.00 (m, 2H, H-3′b(a), H-5′b(a)), 2.69 (q, J = 7.5 Hz, 2H, CH2CH3), 3.20−3.27 (m, 2H, H-2′a(b), H-6′a(b)), 3.68−3.74 (m, 2H, H-2′b(a), H-6′b(a)), 4.50 (dt, J = 5.2, 1.6 Hz, 2H, OCH2), 4.68 (m, 1H, H-4′), 5.16 (dq, J = 10.5, 1.5 Hz, 1H, CHCH2(cis)), 5.25 (dq, J = 17.2, 1.7 Hz, 1H, CHCH2(trans)), 5.90 (ddt, J = 17.3, 10.5, 5.2 Hz, 1H, CH CH2), 7.13 (dd, J = 8.2, 2.5 Hz, 1H, Ar), 7.52 (pt, J = 8.1 Hz, 1H, Ar), 7.59 (dd, J = 8.0, 1.9 Hz, 1H, Ar), 7.67 (pt, J = 2.1 Hz, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSOd6) δ 11.5 (CH2CH3), 28.2 (CH2CH3), 30.6 (C-3′, C-5′) 41.1 (C-2′, C-6′), 65.6 (OCH2), 72.6 (C-4′), 109.7, 114.6, 116.9, 117.4, 131.2 (Ar, CHCH2), 129.4 (C-7a), 133.9 (CH CH2), 136.8 (CArN), 149.1 (C-3a), 154.6, 156.3, 157.9 (NCOO, C-5, C Ar O), 165.0 (C-7). Anal. Calc. for C21H24N6O4·H2O: C, 57.00; H, 5.92; N, 18.99. Found: C, 56.71; H, 6.20; N, 18.77. Isobutyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxylate (14d). Following the general procedure C, a solution of 9c (42 mg, 0.09 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (40 μL, 0.28 mmol), DMAP (5 mg, 0.04 mmol), and isobutylchloroformate (19 μL, 0.15 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 8 mg (20%) of 14d as a white solid. Mp: 110−112 °C. MS (ES, positive mode): m/z 441 (M + H)+. 1H NMR (400 MHz,

7.60 (m, 1H, Ar), 7.61 (s, 1H, CONH2b(a)), 7.66−7.67 (m, 2H, Ar). tert-Butyl 4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)benzoyl)piperazine-1-carboxylate (13). Following general procedure B, a microwave vial was charged with 12 (0.14 g, 0.035 mmol), tBuOK (1 M in THF, 0.13 mL, 0.13 mmol), and tert-butyl propionate (19 μL, 0.13 mmol) in anhydrous dioxane (1.0 mL). After workup, the residue was purified by CCTLC (hexane/ethyl acetate, 1:9) to yield 42 mg (26%) of 13 as white amorphous solid. MS (ES, positive mode): m/z 454 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.26 (t, J = 7.5 Hz, 3H, CH2CH3),1.41 (s, 9H, C(CH3)3), 2.74 (q, J = 7.5 Hz, 2H, CH2CH3), 3.40−3.43 (m, 6H, H-2′, H-6′, H-3′a(b), H-5′a(b)), 3.62−3.64 (m, 2H, H-3′b(a), H-5′b(a)), 7.60 (d, J = 7.7 Hz, 1H, Ar), 7.76 (t, J = 7.9 Hz, 1H, Ar), 8.08−8.20 (m, 2H, Ar), 12.76 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.7 (CH2CH3), 28.3 (CH2CH3), 28.5 (C(CH3)3), 42.0, 43.5 (C-2′, C-6′), 41.9, 47.5 (C-3′, C5′), 79.7 (C(CH3)3), 120.9, 123.4, 127.9, 130.6 (Ar), 129.4 (C7a), 135.7 (CArCO), 137.5 (CArN), 149.3 (C-3a), 154.3, 156.3 (C-5, COC(CH3)3), 165.3, 168.3 (CArCON, C-7). Anal. calcd for (C22H27N7O4): C, 58.27; H, 6.00; N, 21.62. Found: C, 58.19; H, 6.20; N, 21.22. General Procedure for the Synthesis of Carbamates Derived from 5-ethyl-3-(3-(piperidin-4-yloxy)phenyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (14a−e). General Procedure C. A solution of 9c (1.0 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h. The mixture was concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and concentrated to dryness. This procedure was repeated several times until a permanent weight was obtained. This obtained yellow solid was dissolved in anhydrous dichloromethane (1 mL), and triethylamine (3.0 mmol), DMAP (0.4 mmol), and the appropriate chloroformate (1.5 mmol) in anhydrous DCM (1 mL) were added at 0 °C. After 3 h of stirring at rt, the reaction was concentrated to dryness. A saturated solution of NaCl (10 mL) was added, and the mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography to yield the corresponding carbamates 14a−e. Phenyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxylate (14a). Following the general procedure C, a solution of 9c (44 mg, 0.1 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (42 μL, 0.3 mmol), DMAP (5 mg, 0.04 mmol), and phenylchloroformate (19 μL, 0.15 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 14 mg (30%) of 14a as a white solid. MS (ES, positive mode): m/z 461 (M + H)+. Mp > 225 °C (decomp). 1H NMR (400 MHz, DMSO-d6) δ 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.71−1.73 (m, 2H, H-3′a(b), H-5′a(b)), 2.05−2.08 (m, 2H, H-3′b(a), H-5′b(a)), 2.70 (q, J = 7.5 Hz, 2H, CH2CH3), 3.26−3.48 (m, 2H, H-2′a(b), H-6′a(b)), 3.75−3.89 (m, 2H, H-2′b(a), H-6′b(a)), 4.75 (tt, J = 7.8, 3.6 Hz, 1H, H-4′), 7.10−7.12 (m, 2H, Ar), 7.15−7.21 (m, 2H, Ar), 7.34−7.38 (m, 2H, Ar), 7.54 (pt, J = 8.1 Hz, 1H, Ar), 7.61 (m, 1H, Ar), 7.70 (pt, J = 2.2 Hz, 1H, Ar), 12.71 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.2 (CH2CH3), 27.9 (CH2CH3), 40.2 (C-2′, C-6′), 72.1 (C-4′), 109.3, 114.2, 116.5, 122.0, 125.2, 129.3, 130.8 (Ar), 129.0 (C-7a), 136.5 (CArN), 148.8 (C-3a), 150.5, 151.2, 153.0, 157.5 (NCOO, C-5, CArO), 613



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DMSO-d6) δ 0.87 (d, J = 6.7 Hz, 6H, CH(CH3)3), 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.55−1.64 (m, 2H, H-3′a(b), H-5′a(b)), 1.85 (hept, J = 6.7 Hz, 1H, CH(CH3)3), 1.90−2.00 (m, 2H, H3′b(a), H-5′b(a)), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 3.22−3.24 (m, 2H, H-2′a(b), H-6′a(b)), 3.69−3.73 (m, 2H, H-2′b(a), H6′b(a)), 3.78 (d, J = 6.6 Hz, 2H, OCH2), 4.68 (m, 1H, H-4′), 7.14 (dd, J = 8.3, 2.5 Hz, 1H, Ar), 7.53 (pt, J = 8.1 Hz, 1H, Ar), 7.61 (d, J = 8.1 Hz, 1H, Ar), 7.68 (pt, J = 2.2 Hz, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 19.5 (CH(CH3)2), 28.2, 28.5 (CH2CH3, CH(CH3)2), 41.4 (C-2′, C-6′), 71.4 (OCH2), 73.0 (C-4′), 109.9, 114.8, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN), 149.4 (C-3a), 155.3, 156.8, 158.2 (NCOO, C-5, CArO), 165.4 (C-7). Anal. Calc. for C22H28N6O4·H2O: C, 57.63; H, 6.60; N, 18.33. Found: C, 57.95; H, 6.24; N, 17.97. Tetrahydro-2H-pyran-4-yl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxylate (14e). Following the general procedure C, a solution of 9c (36 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (40 μL, 0.28 mmol), DMAP (5 mg, 0.04 mmol), and tetrahydro-2H-pyran-4-yl chloroformate38 (25 mg, 0.15 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 8 mg (18%) of 14e as a white solid. Mp: 174−176 °C. MS (ES, positive mode): m/ z 469 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.8 Hz, 3H, CH2CH3), 1.49−1.65 (m, 4H, H-3′a(b), H-5′a(b), H3′′a(b), H-5′′a(b)), 1.82−1.88 (m, 2H, H-3′′b(a), H-5′′b(a)), 1.97− 2.02 (m, 2H, H-3′b(a), H-5′b(a)), 2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.27 (m, 2H, H-2′a(b), H-6′a(b)), 3.46 (ddd, J = 11.6, 8.6, 3.1 Hz, 2H, H-2′′a(b), H-6′′a(b)), 3.67−3.85 (m, 4H, H2′b(a), H-6′b(a), H-2′′b(a), H-6′′b(a)), 4.68−4.77 (m, 2H, H-4′, H4′′), 7.16 (dd, J = 8.3, 2.5 Hz, 1H, Ar), 7.56 (pt, J = 8.1 Hz, 1H, Ar), 7.63 (d, J = 8.0 Hz, 1H, Ar), 7.70 (pt, J = 2.2 Hz, 1H, Ar), 12.73 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.1 (CH2CH3), 27.7 (CH2CH3), 31.8 (C-3′′, C-5′′), 40.7 (C-2′, C6′), 64.4 (C-2′′, C-6′′), 69.4 (C-4′′), 72.3 (C-4′), 109.3, 114.2, 116.5, 130.8 (Ar), 128.9 (C-7a), 136.4 (CArN), 148.7 (C-3a), 153.9, 155.8, 157.5 (NCOO, C-5, CArO), 164.6 (C-7). Anal. Calc. for C23H28N6O5·H2O: C, 56.78; H, 6.22; N, 17.27. Found: C, 57.06; H, 6.19; N, 17.18. General Procedures (D and E) for the Synthesis of Ureas Derived from 5-ethyl-3-(3-(piperidin-4-yloxy)phenyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (15a−h). General Procedure D. A solution of 9c (1.0 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h and concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and concentrated to dryness. This procedure was repeated several times until a permanent weight was obtained. A solution containing this yellow solid, the corresponding amine (1.0 mmol), and DIPEA (2.2 mmol) in anhydrous dichloromethane (3.5 mL) or dichloromethane/ DMF (9:1, 3.5 mL) was treated with a solution of triphosgene (0.37 mmol) in dichloromethane (0.5 mL). The reaction mixture was stirred at rt for 30 min. A saturated aqueous solution of NaHCO3 (5 mL) was added, and the mixture was extracted with dichloromethane (10 mL). The organic phase was washed with brine (5 mL), dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified as indicated in each case. General Procedure E. A solution of 9c (1.0 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h and concentrated to dryness. The residue obtained was treated with

dichloromethane/hexane and concentrated to dryness. This procedure was repeated several times until a permanent weight was obtained. A solution containing this yellow solid and Et3N (2.1 mmol) in anhydrous dichloromethane (10 mL) or dichloromethane/DMF (9:1, 10 mL) was stirred at 0 °C for 30 min. The corresponding isocyanate (1.5 mmol) was added at 0 °C, and the reaction mixture was stirred at rt for 1 h. A saturated aqueous solution of NH4Cl (5 mL) was added, and the mixture was extracted with dichloromethane (2 × 10 mL). The combined organic phase was dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified as indicated in each case. 4-(3-(5-Ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)-N-phenylpiperidine-1-carboxamide (15a). Following the general procedure D, a solution of 9c (18 mg, 0.04 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with aniline (4 μL, 0.04 mmol), DIPEA (16 μL, 0.09 mmol), and triphosgene (4.50 mg, 0.02 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 10 mg (54%) of 15a as a white solid. Mp: 259−261 °C. MS (ES, positive mode): m/z 460 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.24 (t, J = 7.5 Hz, 3H, CH2CH3), 1.59−1.68 (m, 2H, H-3′a(b), H-5′a(b)), 1.99−2.05 (m, 2H, H-3′b(a), H-5′b(a)), 2.72 (q, J = 7.5 Hz, 2H, CH2CH3), 3.24−3.33 (m, 2H, H-2′a(b), H-6′a(b)), 3.81−3.87 (m, 2H, H-2′b(a), H-6′b(a)), 4.70 (m, 1H, H-4′), 6.91 (t, J = 7.3 Hz, 1H, Ar), 7.15−7.23 (m, 3H, Ar), 7.42−7.45 (m, 2H, Ar), 7.55 (t, J = 8.1 Hz, 1H, Ar), 7.61 (m, 1H, Ar), 7.70 (s, 1H, Ar), 8.55 (br s, 1H, ArNHCO), 12.72 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.4 (CH2CH3), 31.1 (C-3′, C-5′), 41.8 (C-2′, C-6′), 73.4 (C-4′), 109.9, 114.8, 117.2, 120.3, 122.3, 128.9, 131.4 (Ar), 129.6 (C7a), 137.1 (CArN), 141.2 (CArN) 149.3 (C-3a), 155.6, 156.5, 158.2 (ArNHCO, C-5, CArO), 165.2 (C-7). Anal. calcd for (C24H25N7O3. 0.5H2O): C, 61.53; H, 5.59; N, 20.93. Found: C, 61.44; H, 5.82; N, 20.88. 4-(3-(5-Ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)-N-(4-methoxyphenyl)piperidine-1carboxamide (15b). Following the general procedure E, a solution of 9c (37 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with 4-methoxyphenyl isocyanate (17 μL, 0.13 mmol) and Et3N (25 μL, 018 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 30 mg (73%) of 15b as a white solid. Mp: 229−231 °C. MS (ES, positive mode): m/z 490 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.57− 1.65 (m, 2H, H-3′a(b), H-5′a(b)), 1.98−2.02 (m, 2H, H-3′b(a), H5′b(a)), 2.70 (q, J = 7.5 Hz, 2H, CH2CH3), 3.20−3.26 (m, 2H, H-2′a(b), H-6′a(b)), 3.66 (s, 3H, OCH3), 3.78−3.84 (m, 2H, H2′b(a), H-6′b(a)), 4.68 (m, 1H, H-4′), 6.78 (d, J = 9.2 Hz, 2H, Ar), 7.14 (d, J = 8.3 Hz, 1H, Ar), 7.30 (d, J = 8.3 Hz, 2H, Ar), 7.53 (d, J = 8.1 Hz, 1H, Ar), 7.59 (d, J = 8.1 Hz, 1H, Ar), 7.69 (s, 1H, Ar), 8.37 (br s, 1H, ArNHCO), 12.70 (br s, 1H, NH). 13 C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.4 (CH2CH3), 31.1 (C-3′, C-5′), 41.8 (C-2′, C-6′), 55.8 (OCH3), 73.4 (C-4′), 109.9, 114.1, 114.8, 117.2, 122.3, 131.4 (Ar), 129.6 (C-7a), 134.2 (CArN), 137.1 (CArN), 149.3 (C-3a), 155.1, 155.8, 156.6 (ArNHCO, C-5, CArO), 158.3 (CArO), 165.2 (C7). Anal. calcd for (C25H27N7O4·0.5H2O): C, 60.23; H, 5.66; N, 19.67. Found: C, 59.91; H, 5.57; N, 19.29. N-Benzyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxa614



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mide (15c). Following the general procedure E, a solution of 9c (36 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with benzyl isocyanate (15 μL, 0.12 mmol) and Et3N (24 μL, 017 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 28 mg (71%) of 15c as a white solid. Mp: 217−219 °C. MS (ES, positive mode): m/z 474 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.22 (t, J = 7.5 Hz, 3H, CH2CH3), 1.49−1.57 (m, 2H, H-3′a(b), H-5′a(b)), 1.94−1.97 (m, 2H, H-3′b(a), H-5′b(a)), 2.69 (q, J = 7.5 Hz, 2H, CH2CH3), 3.09−3.16 (m, 2H, H-2′a(b), H-6′a(b)), 3.70−3.76 (m, 2H, H-2′b(a), H-6′b(a)), 4.21 (d. J = 5.6 Hz, 2H, CH2Ph), 4.64 (m, 1H, H-4′), 7.10−7.29 (m, 7H, Ar, BnNHCO), 7.52 (t, J = 8.1 Hz, 1H, Ar), 7.58 (s, 1H, Ar), 7.67 (m, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.4 (CH2CH3), 31.1 (C-3′, C-5′), 41.6 (C-2′, C6′), 44.2 (CH2Ph), 73.5 (C-4′), 109.8, 114.8, 117.2, 127.1, 127.6, 128.8, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN), 141.2 (CArCH2) 149.3 (C-3a), 156.5, 157.9, 158.2 (BnNHCO, C-5, CArO), 165.2 (C-7). Anal. calcd for (C25H27N7O3·2H2O): C, 58.93; H, 6.13; N, 19.24. Found: C, 58.62; H, 6.00; N, 18.87. N-Ethyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxamide (15d). Following the general procedure E, a solution of 9c (72 mg, 0.16 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with ethyl isocyanate (20 μL, 0.24 mmol) and Et3N (48 μL, 0.34 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 16 mg (25%) of 15d as a white solid. Mp: > 255 °C (decomp.). MS (ES, positive mode): m/z 412 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 0.99 (t, J = 7.1 Hz, 3H, NHCH2CH3), 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.47−1.56 (m, 2H, H-3′a(b), H-5′a(b)), 1.91−1.96 (m, 2H, H-3′b(a), H5′b(a)), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.96−3.10 (m, 4H, H-2′a(b), H-6′a(b), NHCH2CH3), 3.65−3.71 (m, 2H, H-2′b(a), H-6′b(a)), 4.63 (m, 1H, H-4′), 6.49 (t, J = 5.4 Hz, 1H, EtNHCO), 7.13 (m, 1H, Ar), 7.53 (t, J = 8.1 Hz, 1H, Ar), 7.59 (m, 1H, Ar), 7.67 (s, 1H, Ar), 12.71 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 16.3 (NHCH2CH3), 28.4 (CH2CH3), 31.0 (C-3′, C-5′), 35.6 (NHCH2CH3), 41.5 (C-2′, C-6′), 73.6 (C-4′), 109.8, 114.7, 117.2, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN) 149.3 (C-3a), 156.5, 157.9, 158.3 (EtNHCO, C-5, C ArO), 165.2 (C-7). Anal. calcd for (C20H25N7O3·H2O): C, 55.93; H, 6.34; N, 22.83. Found: C, 56.37; H, 6.10; N, 22.48. N-(tert-Butyl)-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxamide (15e). Following the general procedure E, a solution of 9c (39 mg, 0.09 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with tert-butyl isocyanate (15 μL, 0.13 mmol) and Et3N (25 μL, 018 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 30 mg (78%) of 15e as a white solid. Mp: 223−225 °C. MS (ES, positive mode): m/z 440 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.20−1.24 (m, 3H, CH2CH3), 1.22 (s, 9H, C(CH3)3), 1.46−1.55 (m, 2H, H-3′a(b), H-5′a(b)), 1.89−1.94 (m, 2H, H-3′b(a), H-5′b(a)), 2.69 (q, J = 7.5 Hz, 2H, CH2CH3), 2.99−3.05 (m, 2H, H-2′a(b), H6′a(b)), 3.64−3.67 (m, 2H, H-2′b(a), H-6′b(a)), 4.60 (m, 1H, H4′), 5.80 (s, 1H, tBuNHCO), 7.11 (m, 1H, Ar), 7.51 (t, J = 8.1 Hz, 1H, Ar), 7.56 (m, 1H, Ar), 7.66 (s, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.4 (CH2CH3), 29.9 (C(CH3)3), 31.1 (C-3′, C-5′), 41.8 (C-

2′, C-6′), 50.6 (C(CH3)3), 73.7 (C-4′), 109.8, 114.7, 117.4, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN) 149.3 (C-3a), 156.5, 157.6, 158.3 (tBuNHCO, C-5, CArO), 165.2 (C-7). Anal. calcd for (C22H29N7O3): C, 60.12; H, 6.65; N, 22.31. Found: C, 59.89; H, 6.61; N, 21.91. N-Cyclohexyl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxamide (15f). Following the general procedure D, a solution of 9c (42 mg, 0.09 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with cyclohexylamine (11 μL, 0.09 mmol), DIPEA (36 μL, 0.21 mmol), and triphosgene (10 mg, 0.03 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 10 mg (24%) of 15f as a white solid. Mp: 245−247 °C. MS (ES, positive mode): m/z 466 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.01−1.20 (m, 5H, Cy), 1.23 (t, J = 7.5 Hz, 3H, CH2CH3), 1.48−1.57 (m, 3H, Cy, H-3′a(b), H-5′a(b)), 1.64− 1.74 (m, 4H, Cy), 1.90−1.97 (m, 2H, H-3′b(a), H-5′b(a)), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 3.06−3.10 (m, 2H, H-2′a(b), H6′a(b)), 3.36 (m, 1H, Cy), 3.67−3.72 (m, 2H, H-2′b(a), H-6′b(a)), 4.62 (m, 1H, H-4′), 6.19 (d, J = 2.2 Hz, 1H, CyNHCO), 7.12 (m, 1H, Ar), 7.53 (t, J = 8.5 Hz, 1H, Ar), 7.60 (m, 1H, Ar), 7.67 (s, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 25.8 (Cy), 26.1 (Cy), 28.4 (CH2CH3), 31.0 (C-3′, C-5′), 33.8 (Cy), 41.6 (C-2′, C-6′), 49.9 (Cy), 73.6 (C-4′), 109.8, 114.7, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN), 149.3 (C-3a), 156.5, 157.7, 158.2 (CyNHCO, C-5, CArO), 165.2 (C-7). Anal. calcd for (C24H31N7O3·2H2O): C, 57.47; H, 7.03; N, 19.55. Found: C, 57.39; H, 6.67; N, 19.21. N-(1S,3R,5S)-Adamantan-1-yl-4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidine-1-carboxamide (15g). Following the general procedure E, a solution of 9c (37 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with adamantyl isocyanate (22 mg, 0.13 mmol) and Et3N (25 μL, 018 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 26 mg (61%) of 15g as a white solid. Mp: 228−230 °C. MS (ES, positive mode): m/z 518 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.22 (t, J = 7.5 Hz, 3H, CH2CH3), 1.46− 1.60 (m, 9H, H-Ad, H-3′a(b), H-5′a(b)), 1.88−1.97 (m, 13H, HAd, H-3′b(a), H-5′b(a)), 2.69 (q, J = 7.5 Hz, 2H, CH2CH3), 2.98−3.04 (m, 2H, H-2′a(b), H-6′a(b)), 3.63−3.66 (m, 2H, H2′b(a), H-6′b(a)), 4.59 (m, 1H, H-4′), 5.68 (br s, 1H, NH-Ad), 7.11 (m, 1H, Ar), 7.51 (t, J = 8.1 Hz, 1H, Ar), 7.58 (m, 1H, Ar), 7.66 (s, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.4 (CH2CH3), 29.7 (C-Ad), 31.0 (C-3′, C-5′), 41.9 (C-2′, C-6′), 42.3 (C-Ad), 51.0 (C-Ad), 73.7 (C-4′), 109.8, 114.7, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN) 149.3 (C-3a), 155.6, 157.2, 158.3 (AdNHCO, C5, CArO), 165.3 (C-7). Anal. calcd for (C28H35N7O3·0.5H2O): C, 63.86; H, 6.89; N, 18.62. Found: C, 64.22; H, 6.86; N, 18.19. 5-Ethyl-3-(3-((1-(morpholine-4-carbonyl)piperidin-4-yl)oxy)phenyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin7-one (15h). Following the general procedure D, a solution of 9c (40 mg, 0.09 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with morpholine (8 μL, 0.09 mmol), DIPEA (35 μL, 0.20 mmol), and triphosgene (10 mg, 0.03 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 12 mg (26%) of 15h as a white solid. Mp: 238−240 °C. MS (ES, positive mode): m/z 454 (M + H)+. 1H NMR (400 MHz, 615



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DMSO-d6) δ 1.22 (t, J = 7.5 Hz, 3H, CH2CH3), 1.57−1.64 (m, 2H, H-3′a(b), H-5′a(b)), 1.94−2.04 (m, 2H, H-3′b(a), H-5′b(a)), 2.69 (q, J = 7.5 Hz, 2H, CH2CH3), 3.01−3.07 (m, 2H, H-2′a(b), H-6′a(b)), 3.09−3.12 (m, 4H, H-3′′, H-5′′), 3.42−3.48 (m, 2H, H-2′b(a), H-6′b(a)), 3.52−3.54 (m, 4H, H-2′′, H-6′′), 4.64 (m, 1H, H-4′), 7.12 (m, 1H, Ar), 7.52 (t, J = 8.1 Hz, 1H, Ar), 7.59 (m, 1H, Ar), 7.67 (s, 1H, Ar), 12.70 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 28.5 (CH2CH3), 30.9 (C-3′, C-5′), 44.1 (C-2′, C-6′), 47.7 (C-3′′, C-5′′), 66.6 (C-2′′, C-6′′), 73.4 (C-4′), 109.9, 114.7, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN) 149.4 (C-3a), 156.7, 158.2 (C-5, C Ar O), 163.7, 165.4 (NCON, C-7). Anal. calcd for (C22H27N7O4·H2O): C, 56.04; H, 6.20; N, 20.79. Found: C, 56.10; H, 6.01; N, 20.46. Methyl 4-(4-(3-(5-ethyl-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)phenoxy)piperidin-1-yl)-4-oxobutanoate (16). Following the general procedure C, a solution of 9c (36 mg, 0.08 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt, followed by reaction with triethylamine (40 μL, 0.28 mmol), DMAP (5 mg, 0.04 mmol), and methyl 4chloro-4-oxobutanoate (40 μL, 0.3 mmol). After workup, the residue was purified by CCTLC (dichloromethane/methanol, 10:1) to yield 14 mg (30%) of 16 as a white solid. MS (ES, positive mode): m/z 455 (M + H)+. Mp: 86−88 °C. 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.55 (m, 1H, H-3′/H-5′), 1.67 (m, 1H, H-3′/H-5′), 1.94−2.06 (m, 2H, H-3′/H-5′), 2.43−2.48 (m, 2H, CH2COO), 2.62 (m, 2H, CH2CON), 2.73 (q, J = 7,5 Hz, 2H, CH2CH3), 3.23−3.38 (m, 2H, H-2′/H-6′), 3.57 (s, 3H, CH3O), 3.76 (m, 1H, H-2′/ H-6′), 3.88 (m, 1H, H-2′/H-6′), 4.74 (tt, J = 7.9, 3.7 Hz, 1H, H-4′), 7.17 (dd, J = 8.3, 1.1 Hz, 1H, Ar), 7.56 (pt, J = 8.1 Hz, 1H, Ar), 7.63 (dd, J = 8.2, 1.2 Hz, 1H, Ar), 7.71 (pt, J = 2.2 Hz, 1H, Ar). 13C NMR (101 MHz, DMSO-d6) δ 11.2 (CH2CH3), 27.3 (CH2CH3), 27.8 (CH2CON), 28.6, 28.8 (C-3′, C-5′), 30.8 (CH2COO), 41.9 (C-2′, C-6′), 51.3 (CH3O), 72.4 (C-4′), 109.3, 114.2, 116.5, 130.8 (Ar), 129.0 (C-7a), 136.4 (CArN), 148.7 (C-3a), 156.0, 157.5 (C-5, CArO), 164.7 (C-7), 169.2 (CH2CON), 173.1 (CH2COO). Anal. Calc. for C22H26N6O5· 2H2O: C, 53.87; H, 6.16; N, 17.13. Found: C, 54.29; H, 5.90; N, 16.85. 5-Ethyl-3-(3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3,6dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (17a). A solution of 9c (54 mg, 0.12 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h and then concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and evaporated. This procedure was repeated several times to obtain a yellow solid, which was used without further purification. A solution of this yellow solid, DIPEA (43 μL, 0.25 mmol), PyBOP (96 mg, 0.18 mmol), and nicotinic acid (15 mg, 0.12 mmol) in anhydrous dichloromethane/DMF (9:1, 1.0 mL) was stirred at rt for 2 h. The reaction mixture was diluted with dichloromethane (10 mL) and washed with an aqueous solution of citric acid (0.1 M), with a saturated aqueous solution of NaHCO3 (10 mL) solution, and with brine (10 mL). The combined organic phases were dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified by CCTLC (dichloromethane/methanol/Et3N 10:0.2:0.2) to yield 15 mg (28%) of 17a as a white amorphous solid. MS (ES, positive mode): m/z 446 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.74−1.76 (m, 2H, H-3′a(b), H-5′a(b)), 2.04−2.08 (m, 2H, H-3′b(a), H-5′b(a)), 2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.34−3.38 (m, 2H, H-2′/H-6′), 3.53 (m, 1H, H-2′/

H-6′), 4.02 (m, 1H, H-2′/H-6′), 4.80 (m, 1H, H-4′), 7.19 (m, 1H, Ar), 7.49 (dd, J = 7.8, 4.9 Hz, 1H, Ar), 7.57 (t, J = 8.1 Hz, 1H, Ar), 7.64 (m, 1H, Ar), 7.72 (pt, J = 2.1 Hz, 1H, Ar), 7.87 (dt, J = 7.9, 1.9 Hz, 1H, Ar), 8.64−8.66 (m, 2H, Ar), 12.72 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.6 (CH2CH3), 28.2 (CH2CH3), 39.2, 44.8 (C-2′, C-6′), 72.7 (C-4′), 109.9, 114.7, 116.9, 124.0, 131.2, 135.1, 147.9, 150.8 (Ar), 129.4 (C-7a), 132.4 (CArCO), 136.9 (CArN), 149.1 (C3a), 156.3, 157.9 (C-5, CArO), 165.1 (C-7), 167.3 (CArCO). Anal. calcd for (C23H23N7O3·0.5H2O): C, 60.78; H, 5.32; N, 21.57. Found: C, 60.57; H, 5.46; N, 21.38. 5-Ethyl-3-(3-((1-isonicotinoylpiperidin-4-yl)oxy)phenyl)3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (17b). A solution of 9c (53 mg, 0.12 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h and then concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and evaporated. This procedure was repeated several times to obtain a yellow solid, which was used without further purification. A solution of this yellow solid, DIPEA (42 μL, 0.24 mmol), HATU (69 mg, 0.18 mmol), and isonicotinic acid (15 mg, 0.12 mmol) in anhydrous DMF (1.0 mL) was stirred at rt for 2 h. The reaction mixture was diluted with dichloromethane (10 mL) and washed with an aqueous solution of citric acid (0.1M), with a saturated aqueous solution of NaHCO3 (10 mL) solution and brine (10 mL). The combined organic phases were dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified by CCTLC (dichloromethane/methanol/Et3N 10:0.2:0.2) to yield 37 mg (50%) of 17b as a white amorphous solid. MS (ES, positive mode): m/z 446 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.5 Hz, 3H, CH2CH3), 1.69−1.79 (m, 2H, H-3′/H5′), 1.99 (m, 1H, H-3′/H-5′), 2.12 (m, 1H, H-3′/H-5′), 2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.28 (m, 1H, H-2′/H-6′), 3.46− 3.53 (m, 2H, H-2′/H-6′), 4.01 (m, 1H, H-2′/H-6′), 4.80 (m, 1H, H-4′), 7.19 (m, 1H, Ar), 7.42−7.44 (m, 2H, Ar), 7.57 (t, J = 8.1 Hz, 1H, Ar), 7.64 (m, 1H, Ar), 7.71 (pt, J = 2.1 Hz, 1H, Ar), 8.67−8.68 (m, 2H, Ar), 12.72 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.6 (CH2CH3), 28.2 (CH2CH3), 38.9, 44.4 (C-2′, C-6′), 72.7 (C-4′), 109.9, 114.7, 116.9, 121.5, 131.2, 150.5 (Ar), 129.4 (C-7a), 136.9 (CArN), 144.1 (CArCO), 149.1 (C-3a), 156.3, 157.9 (C-5, CArO), 165.0 (C-7), 167.2 (CArCO). Anal. calcd for (C23H23N7O3·0.5H2O): C, 60.78; H, 5.32; N, 21.57. Found: C, 60.63; H, 5.47; N, 21.31. 5-Ethyl-3-(3-((1-(isopropylsulfonyl)piperidin-4-yl)oxy)phenyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (18). A solution of 9c (30 mg, 0.07 mmol) in TFA (5% in dichloromethane) (2 mL) was stirred at rt for 2 h. The mixture was concentrated to dryness. The residue obtained was treated with dichloromethane/hexane and concentrated to dryness. This procedure was repeated several times to obtain a yellow solid, which was used without further purification. A solution of this yellow solid and Et3N (20 μL, 0.14 mmol) in anhydrous dichloromethane/DMF (9:1, 1.0 mL) was stirred at 0 °C for 30 min. 2-Propanesulfonyl chloride (11 μL, 0.10 mmol) was added at 0 °C, and the reaction mixture was stirred at rt for 1 h. A saturated aqueous solution of NH4Cl (5 mL) was added, and the mixture was extracted with dichloromethane (2 × 5 mL). The combined organic phase was dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified by CCTLC (dichloromethane/methanol, 10:0.5) to yield 20 mg (67%) of 18 as a white solid. Mp: 245−247 °C. MS (ES, positive mode): m/z 447 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.20− 1.25 (m, 9H, CH2CH3, CH(CH3)2), 1.65−1.73 (m, 2H, H616



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3′a(b), H-5′a(b)), 1.98−2.05 (m, 2H, H-3′b(a), H-5′b(a)), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 3.19−3.25 (m, 2H, H-2′a(b), H-6′a(b)), 3.33 (m, 1H, CH(CH3)2), 3.47−3.53 (m, 2H, H-2′b(a), H6′b(a)), 4.68 (m, 1H, H-4′), 7.15 (m, 1H, Ar), 7.54 (t, J = 8.1 Hz, 1H, Ar), 7.61 (m, 1H, Ar), 7.68 (s, 1H, Ar), 12.71 (br s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 11.8 (CH2CH3), 17.1 (CH(CH3)2), 28.4 (CH2CH3), 31.3 (C-3′, C-5′), 43.5 (C2′, C-6′), 52.5 (CH(CH3)2), 72.4 (C-4′), 110.0, 114.9, 117.1, 131.4 (Ar), 129.6 (C-7a), 137.1 (CArN), 149.3 (C-3a), 156.5, 158.0 (C-5, C A r O), 165.2 (C-7). Anal. calcd for (C20H26N6O4S): C, 53.80; H, 5.87; N, 18.82; S, 7.18. Found: C, 53.50; H, 5.93; N, 18.38; S, 7.11. Virus Strains and Cells. Chikungunya virus (CHIKV) Indian Ocean strain 899 (Genbank FJ959103.1) was generously provided by Prof. C. Drosten (University of Bonn, Germany). CHIKV La Reunion Island strain LR2006_OPY1 (Genbank DQ443544.2) and the clinical isolates Venturini (Italy 2008), Congo 95 (2011), St Martin (2013), and Martinique EFS-1 were used in EPV in Marseille and are freely available from the European Virus Archive (https://www.european-virus-archive. com/). All viruses were propagated in African green monkey kidney cells [Vero cells (ATCC CCL-81) or Vero E6 (ATCC CRL-1586)]. CHIKV isolates from Singapore (SGP11) and Reunion Island (LR2006 OPY1) and O’nyong’nyong virus (ONNV) infectious clones were tagged with Gaussia luciferase (Gluc) from marine copepod Gaussia princeps at a site in between the nonstructural and structural genes. Viral stocks of infectious clones were produced in Vero E6 cells, tittered by plaque assays, and stored at −80 °C. Vero cells were maintained in cell growth medium composed of minimum essential medium (MEM Rega-3, Gibco, Belgium) supplemented with 10% fetal bovine serum (FBS, Integro, The Netherlands), 1% L-glutamine (Gibco), and 1% sodium bicarbonate (Gibco). The antiviral assays were performed in the same medium but supplemented with 2% (instead of 10%) FBS. The E6 subclones of Vero cells were maintained in Eagles MEM (Gibco) supplemented with antibiotics, 1% glutamine, 1% nonessential amino-acids (Gibco), and 7.5% FCS. Plating prior to infection and antiviral assays were done in the same medium but with reduced serum concentration (2.5%). CRL2522 human skin fibroblasts were maintained in Dulbecco’s Modified Eagle Medium (HyClone), with 10% FBS (HyClone), and 1% penicillin/streptomycin (Gibco). Antiviral assays with CRL-2522 cells were performed in serum-free DMEM. All cell cultures were maintained at 37 °C in an atmosphere of 5% CO2 and 95−99% humidity. Antiviral Assays. CPE-Inhibition Assay. Vero cells were seeded in 96-well tissue culture plates (Becton Dickinson, Aalst, Belgium) at a density of 2.5 × 104 cells/well in 100 μL of 2% FBS medium and were allowed to adhere overnight. Next, a compound dilution series was prepared in the medium on top of the cells (100 μL) after which the cultures were infected with 0.01 MOI of CHIKV 899 inoculum or 0.01 MOI of a triazolopyrimidine-resistant CHIKV variant in 100 μL of 2% FBS medium. The starting concentration was 100 μg/mL for all compounds tested. Each assay was performed in 3-fold in the same test, and assays were repeated three times independently to assess for interexperiment variability. On day 5 postinfection (p.i.), the plates were processed using the MTS/PMS method as described by the manufacturer (Promega, The Netherlands). The 50% effective concentration (EC50), which is defined as the compound concentration that is required to inhibit virus-

induced cytopathic effect by 50%, was determined using logarithmic interpolation. The fold resistance value is defined as the ratio of the EC50 value of the resistant CHIKV strain and the EC50 value of the wild-type CHIKV strain (=EC50 resistant CHIKV/EC50 WT CHIKV). Virus Yield Assay. The antiviral activity of the best compounds was validated in an assay for CHIKV OPY 1, Venturini, Congo, Martinique EFS-1, and St. Martin, as previously described.25 Antiviral Activity Assay with CRL-2522. The assay was performed as previously described.22 CRL-2522 cells were seeded at a density of 5 × 103 cells/well in 96-well microtiter plates (Corning). After overnight incubation, cells were infected with the Gluc-tagged viruses at MOI 1. Virus overlay was removed, and cells were treated with serial dilutions of compounds (ranging from 0.001 to 500 μM) prepared in medium in triplicates. After 24 h, 50 μL of conditioned medium was removed and transferred to a new plate, followed by the addition of 50 μL of 20 μM Xenolight RediJect coelenterazine h (PerkinElmer) diluted in PBS with 5 mM NaCl. Luminescent signals were immediately read by the GloMax Multi-Detection System (Promega) at 1 s integration time. Cytotoxicity Assays. For the Vero A, Vero E6, BHK, HeLa RH, and Huh-7 cells, the cytotoxic and cytostatic effects of the compounds were evaluated in uninfected cells by means of microscopic scoring or by the MTS/PMS method. Vero cells were seeded in 96-well tissue culture plates (Becton Dickinson, Aalst, Belgium) at a density of 2.5 × 104 cells/well in 100 μL of 2% FBS medium and were allowed to adhere overnight. Next, a compound dilution series was prepared in the medium on top of the cells. Following 5 days of incubation, the plates were checked microscopically for alterations of the cell or monolayer morphology. After microscopic inspection, the plates were processed using the MTS/PMS method as described by the manufacturer (Promega, The Netherlands). The 50% cytostatic/cytotoxic concentration (CC50; i.e., the concentration of compound that reduces cell viability by 50%) was calculated using logarithmic interpolation. Cytotoxicity in Vero E6 Cells. Cytotoxicities were assayed by measuring the cell viabilities in the same culture settings along antiviral activities in parallel P96 wells plates. 2-Fold serial dilutions, starting with 100 or 300 μM, in duplicates or triplicates, were added to 25−100 μL of Vero E6 cells that had been plated the day before. At day two, the medium was removed and replaced by 70 μL of fresh medium containing 10 μL of CellTiter blue reagent (Promega) and incubated for 90 min at 37 °C. Cell viabilities were measured as resorufin fluorescence readings on a plate reader (Tecan Infinite M200 Pro). Cytotoxicity in CRL-2522 Cells. The cell viability assay in CRL-2522 cells was previously described.22 Briefly, cells were seeded on 96-well microtiter plates (Corning) before being treated with serial dilutions of compounds (ranging from 0.001 to 500 μM) prepared in medium. After 24 h, cell viability assays using CellTiter-Glo Reagent (Promega) were performed according to the manufacturer’s protocol. Luminescent signals were detected by the GloMax Multi-Detection System (Promega) after 1 h of incubation. Maps of Electrostatic Potential. The geometries of compounds 9b, 9c, 9d, and 15e were optimized using Jaguar39 and the B3LYP/6-31G** basis set. Electrostatic potentials were computed by solving the linearized Poisson−Boltzmann equation by a finite difference method as implemented in 617



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DelPhi software (v.4).40,41 Cubic grids with 1.0 Å resolution were centered on each molecule, leaving a separation of 10 Å between any solute atom and the borders of the box, and the atomic charges were distributed onto the grid points. ESP charges and AMBER radii were used. The boundary between the solvent [treated as a high-dielectric medium (€ = 80)] and the ligands [considered a low-dielectric medium (€ = 2)] was defined by the solvent-accessible surfaces using a probe radius of 1.4 Å. Microsomal Stability Studies. These studies have been performed by Anthem Biosciences according to their protocols using diclofenac and imipramine as reference compounds. Pharmacokinetic Studies. These studies have been performed by Anthem Biosciences according to their protocols, using a single dose at 10 mg/kg body weight by subcutaneous or intraperitoneal administration to male Balb/c mice. In Silico ADME Calculations. Calculations were computed with the Schrödinger Molecular Modeling Suite (Schrödinger Release 2015-4, Schrödinger, LLC, New York, NY, 2015). The 3D structures of 9b and 15e were generated using Maestro (version 10.4,) and energy minimizations were carried out using Macromodel (version 9.9). Local minimum energy structures of each compound were used as input for ADME studies with QikProp.

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ACKNOWLEDGMENTS We thank Caroline Collard and Nick Verstraeten for their excellent technical assistance in the acquisition of the antiviral data. This work has been supported by grants from MINECO/ FEDER SAF2015-64629-C2-1-R and by European Union FP7 Program under SILVER grant agreement no. 260644.

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ABBREVIATIONS ADME, absorption, distribution, metabolism, and excretion; CC50, 50% cytotoxic concentration or the concentration that reduces the overall metabolic activity of uninfected cells to 50%; CHIKF, chikungunya fever; CHIKV, chikungunya virus; cLogP, theoretical octanol/water partition coefficient; CPE, cytopathic effect; EC50, 50% effective concentration; EC90, 90% effective concentration; ECSA, East/Central/South African genotypes; MTS/PMS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium in the presence of phenazine methosulfate; ONNV, O’nyong’nyong virus; PK, pharmacokinetics; QPPCaco, calculated Caco permeability; QPlogKHsa, calculated binding to human albumin; tPSA, topological surface area

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.7b00219. Figure S1: MEP maps of compounds 9b, 9c, 9d, and 15e; Table S1: predicted cLogP, tPSA, and solubility values for the proposed compounds; Table S2: in silico rapid ADME predictions performed for compounds 9b and 15e using QikProp program (Schrödinger) and range of recommended values for oral drugs; Table S3: in vitro microsomal stability in mouse liver microsomes; Table S4: mean PK parameters after single dose intraperitoneal and subcutaneous administration of 9b in male BALB/c mice at 10 mg/kg body weight (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*Phone: 34 91 2587516. Fax: 34 91 5644853. E-mail: [email protected]. ORCID

Eva-María Priego: 0000-0001-9470-4508 María-Jesús Pérez-Pérez: 0000-0003-1336-7760 Author Contributions

A.G.-S.J., A.M.-G., and A.P.-S. have performed the synthesis of the compounds, supervised by M.-J.P.-P. E.-M.P. has performed the theoretical calculations. M.-J.C. and M.-J.P.-P. have designed the synthesis. L.D., S.J., R.A., S.N.A., L.F.P.N., and G.Q. performed the in vitro infection assays and cytotoxicity studies. D.J. and P.L. supervised the antiviral assays. M.-J.P.-P., J.N., L.D., and G.Q. have written the paper. Notes

The authors declare no competing financial interest. 618



Article

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Inhibition of the Replication of Different Strains of Chikungunya Virus

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