Discovery, Synthesis and Biological Evaluation of a Novel Group of Selective Inhibitors of Filoviral Entry

Abstract

Herein, we report the development of an anti-filoviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the Ebola and Marburg glycoprotein (GP)-mediated infection of human cells. Using Ebola Zaire GP-pseudotyped HIV particles bearing a luciferase reporter gene and 293T cells, a library of 237 small molecules was screened for inhibition of GP-mediated viral entry. From this assay, lead compound 8a was identified as a selective inhibitor of filoviral entry with an IC₅₀ of 30 μM. In order to analyze functional group requirements for efficacy, a structure-activity relationship analysis of this 3,5-disubstituted isoxazole was then conducted with 56 isoxazole and triazole derivatives prepared using “click” chemistry. This study revealed that while the isoxazole ring can be replaced by a triazole system, the 5-(diethylamino)acetamido substituent found in 8a is required for inhibition of viral-cell entry. Variation of the 3-aryl substituent provided a number of more potent anti-viral agents with IC₅₀ values ranging to 2.5 μM. Lead compound 8a and three of its derivatives were also found to block the Marburg glycoprotein (GP)-mediated infection of human cells.

Introduction

The Ebola and Marburg viruses are enveloped, non-segmented, single-stranded RNA viruses and among the most dangerous pathogens known. They abruptly emerge in Central Africa, spread rapidly, cause epidemics of severe hemorrhagic fever with unmatched mortality rates (up to 90%) and, as such, are classified as biosafety level 4 agents.¹ Since their respective identification in 1967 and 1976, there have been more than 2300 reported cases of Ebola and Marburg hemorrhagic fever, one-half of which occurred in the last decade. In addition to its effect on humans, there is compelling evidence that Ebola has simultaneously contributed to the catastrophic decline in the western gorilla population in regions of west-central Africa.²

Despite recent progress towards the production of vaccines³ and neutralizing monoclonal antibodies⁴ for the prophylaxis of the Ebola and Marburg viruses, it may never be entirely practical, or for that matter, desirable, to inoculate large portions of the population against these emerging pathogens. For that reason and given the growing concern that members of the family Filoviridae might be employed as agents of bioterrorism,⁵ the concurrent identification and development of antiviral agents that can substantially mitigate the effects of filoviral infections is a public health priority.

Notwithstanding an increasing understanding of the pathology of filoviruses, only a limited number of low-molecular-weight inhibitors of these systems have been discovered, to date.⁶ Existing anti-filoviral agents can be characterized by three general modes of action, including a) impairment of viral mRNA methylation; b) stimulation of innate antiviral mechanisms; and c) prevention of virion entry and/or fusion (Figure 1). With regards to the former group, the carbocyclic adenosine analog 3-deazaadenosine (C-c3Ado, 1)⁷ blocks the cellular enzyme S-adenosylhomocysteine hydrolase (SAH) and inhibits the replication of Ebola Zaire in vitro, with an IC₅₀ of 30 μM.⁸ The activity of this antiviral agent has been attributed to diminished methylation of the 5′ cap of viral mRNA by (guanine-7-)methyltransferase, which impairs the translation of viral transcripts.⁹ Administration of 1 to Ebola-infected mice has also been found to dramatically increase production of IFN-α,¹⁰ which may serve to counteract the virus’s suppression of the innate antiviral response. Unfortunately, compound 1 failed to promote IFN-α production in Ebola-infected monkeys.¹¹ Glycyrrhizinic acid (2), a triterpene glycoside present in licorice extract, has been found to inhibit the replication of a number of viruses, including Marburg and Ebola.¹² In this case, large doses of compound 2 partially protected Ebola-infected mice and slowed the onset of infection, but ultimately did not prevent death. Although the precise mode of action has yet to be delineated, there is evidence that 2 may induce IFN-α production as well as inhibit membrane penetration and uncoating of the Ebola virus.¹³

Figure 1.

Structure of low-molecular weight compounds that display antiviral activity against the Ebola and/or Marburg virus: 3-deazaadenosine (C-c3Ado, 1); glycyrrhizinic acid (2); latrunculin A (3); colchicine (4); balfilomycin A1 (5); CA-074Me (6); and tunicamycin (7).

Since EBOV enters target cells by an endocytic pathway,¹⁴ agents that disrupt the efficient trafficking of internalized vesicles, via the various components of the cytoskeleton, hold the potential to abrogate its entry and fusion into cells. Indeed, latrunculin (3) and colchicine (4), which respectively impairs the formation of microfilaments and microtubules, have been shown to prevent the infection of HeLa cells by Ebola virus GP pseudotypes.¹⁵ Given the importance of low endosome pH during the GP-mediated internalization process, lysosomotropic agents^27,16 which prevent vesicle and endosome acidification are also potential antifiloviral agents. Indeed, HeLa cells pretreated with the bafilomycin A1 (5), an inhibitor of vacuolar ATPase, are resistant to infection by pseudotyped HIV-1 virions.¹⁵ This general observation has been touted as evidence for the existence of an acid-dependant, post-endocytic conformational change in the virus GP,¹⁷ or as has more recently been suggested, that one or more acid-dependant endosomal proteases are involved the infection process. In this regard, the groups of Cunningham¹⁸ and White¹⁹ have independently demonstrated that the proteases, cathepsin B (CatB) and cathepsin L (CatL) play a key role in viral entry by mediating proteolysis of the EBOV GP. Selective inhibitors of the former enzyme, including CA-074Me (6), have been shown to reduce the infectivity of VSV pseudotypes bearing Ebola virus GP. The importance of CatB in the EBOV infection process is further underlined by Goldsmith’s observation¹⁷ that pretreatment of HeLa cells with the CatB N-glycosylation inhibitor tunicamycin (7)²⁰ serves to diminish infection by Ebola GP-pseudotyped HIV virions. Unfortunately, given the demonstrated hypersensitivity of Ebola GP to digestion by other proteases,²¹ such as thermolysin, the clinical prospects for antiviral agents that solely target CatB and CatL are not encouraging. More recently, Davey and co-workers have successfully employed siRNA screening to identify cellular gene products, namely calmodulin kinase 2 and phosphatidylinositol-3-kinase, which play critical roles in the infection of cells by the Zaire Ebola virus.²² Having identified these potential targets for therapeutic intervention, it was found that KN-93²³ and LY294002,²⁴ known inhibitors of these respective enzymes, effectively block infection by both Ebola GP-pseudotyped HIV particles and live Ebola viruses.

While the inherent difficulties associated with handling filoviruses have undoubtedly contributed to the slow pace of the development of potential therapeutic agents, the advent of replication-competent pseudotyped or chimeric viruses,²⁵ which utilize the replication machinery of vesicular stomatitis viruses (VSV),²⁶ murine leukemia viruses (MLV),²⁷ or human immunodeficiency viruses (HIV),²⁸ but package the Ebola and Marburg membrane glycoproteins (GP) on the virion surface offer an opportunity to screen libraries of small molecules for antiviral properties under low level containment (biosafety level 2).²⁹ Furthermore, since this is a cell-based assay, it has an inherent bias against hit compounds, which display cytotoxic properties. While it must be understood that pseudotyped viruses are not the same as wild type filoviruses, the functionality of the surface Ebola glycoprotein in these systems has been shown to closely mimic the native virus with regards to tropism, cell binding and penetration.²⁷ Given the proximal nature of viral fusion and entry during the life cycle of filoviruses, it is this process which appears to merit the greatest attention, vis-à-vis the development of small-molecule antiviral agents.³⁰ Herein, we report the development of an anti-filoviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the GP-mediated entry of Ebola and Marburg viruses into human cells.

Results and Discussion

1. Chemistry

In order to confirm the chemical identity of lead hit compound 8a and also to provide sufficient material for more detailed evaluation of its biological activity, our initial undertaking was to develop an efficient solution-phase synthesis of this small molecule. As shown in Scheme 1, 8a was conveniently prepared through a Huisgen 1,3-dipolar cycloaddition using the one-pot, copper(I)-catalyzed method reported by Fokin.³¹ Thus, p-anisaldehyde (10a) was first converted to the corresponding aldoxime 11,³² via reaction with hydroxylamine in the presence of aqueous sodium hydroxide. Without isolation, this substrate was treated with chloramine-T trihydrate³³ to generate the corresponding nitrile oxide 12, which in the presence of a catalytic amount of copper(I) sulfate and copper powder, underwent [2+3] cycloaddition³⁴ with terminal alkyne 13,³⁵ at ambient temperature, to furnish 3,5-disubstituted isoxazole 8a in good overall yield. Notably, this reaction proceeded with excellent regioselectivity, as evidenced by analysis of the unpurified reaction product mixture by ¹H NMR studies, which indicated the absence of the regioisomeric cycloadduct.

Scheme 1.

One-Pot, Three Step Synthesis of 3,5-Disubstituted Isoxazole 8a.^a

^aReagents and conditions: (a) NH₂OH•HCl, NaOH, t-BuOH-H₂O (1:1), rt, 4 h; (b) TsN(Cl)Na•3H₂O (chloramine-T), rt, 3 min; (c) 13, CuSO₄•5H₂O (3 mol%), Cu powder (6 mol%), NaOH aq., rt, 12 h.

Employing the three step, one-pot sequence detailed in Scheme 1, a library of C-5 substituted analogs of compound 8a, where prepared from the corresponding aryl and aliphatic aldehydes and alkyne 13. In all cases, only a single isoxazole regioisomer was isolated from the reaction mixture (Table 1). In order to evaluate the impact of changes in both the steric and electronic properties of the arene ring, a variety of substitution patterns were examined as well as heterocyclic analogs, as in the case of 8m.

Table 1.

Preparation of C-5 Substituted Analogs of Lead Compound 8a

entry	R	product	entry	R	product
1		8a	10		8j
2		8b	11		8k
3		8c	12		8l
4		8d	13		8m
5		8e	14		8n
6		8f	15	n-Pr	8o
7		8g	16		8p
8		8h	17		8q
9		8i	18		8r

In order to examine the role which the core isoxazole ring of 8a plays in the anti-filoviral activity of this compound, a group of 1,4-disubstituted 1,2,3-triazoles analogs 15 were synthesized through the 1,3-dipolar cycloaddition of aryl and alkyl azides 14 with terminal alkyne 13 (Table 2). In this case, the copper(I) catalyst was generated in situ by the reduction of copper(II) sulfate with sodium ascorbate, as described by Sharpless.³⁶

Table 2.

Preparation of C-1 Substituted, 1,2,3-Triazole Analogs of Compound 8a

entry	R	product	entry	R	product
1		15a	9		15i
2		15b	10		15j
3		15c	11		15k
4		15d	12		15l
5	n-heptyl	15e	13		15m
6		15f	14		15n
7		15g	15		15o
8		15h

In an effort to study the effect of structural variation at and within the 5-(diethylamino)acetamido substituent in 8a, two sub-groups of derivatives of this system were prepared: those bearing a stereogenic center at the α-position and simple amide derivatives derivatives. The first group of derivatives where conveniently accessed through the coupling of 18 (Scheme 2) with a variety of protected amino acid derivatives 19 (Table 3). Common building block 18 in turned was synthesized from cycloadduct 16 through a sequence of O-tosylation, substitution with azide and Staudinger reduction.

Scheme 2.

Preparation of Building Blocks 18a and 18b.^a

^aReagents and conditions: (a) NH₂OH•HCl, NaOH, tert-BuOH, H₂O, rt; (b) chloramine-T, propargyl alcohol, CuSO₄•5H₂O (3 mol%), Cu powder (6 mol%), NaOH aq., rt; (c) TsCl, Et₃N, CH₂Cl₂; (d) NaN₃, DMF; (e) PPh₃, H₂O, THF, 0 °C → rt, 3 h.

Table 3.

Preparation of C-5 Amidomethyl Analogs of Isoxazole 8a

entry	R1	R2	product
1	NHBoc	Bn	20a
2	NH2	Bn	20b a
3	NHCbz	i-Pr	20c
4	NHCbz		20d
5	NHAc	CH2SH	20e
6	NHCbz	H	20f
7	NHBoc	CH2COOBn	20g

^aobtained via Boc deprotection of 20a with TFA.

Unexpectedly, amide coupling of primary amine 18a (R = OMe) with carboxylic acids 19 proved to be sluggish when mediated by carbodiimide reagents, such as EDC, and lead to incomplete conversion. In an alternative approach to improve the overall efficiency of this transformation, it was found that activation of the amino acid derivatives as their corresponding mixed isobutyl anhydrides and subsequent treatment with 18a at low temperature (−40 °C), provided the desired amides 20a-g in good overall yield (Table 3).

While attempts to remove the Cbz protecting group in 20d and 20f under hydrogenolytic conditions, in order to gain the corresponding amine for evaluation and coupling with other amino acids or peptides failed, exposure of these compounds to Pd/C under an atmosphere of H₂ did provide a 1:2 mixture of enaminone 21 and 5-methylisoxazole 22 (Scheme 3). Ultimately, access to free amine 20b was accomplished by treatment of 20a with TFA.

Scheme 3.

Attempted Hydrogenolytic Deprotection of Cbz derivatives 20d and 20f. ^a

^aReagents and conditions: (a) H₂, Pd/C (10 mol%), EtOAc, 3 h, rt.

The second series of aminomethyl derivatives, 20h-n, incorporated various simple alkyl and aryl chains, including methyl, ethyl, isopropyl, phenyl and bromomethyl groups. All compounds in this group were prepared via the coupling of 18a or 18b and the corresponding carboxylic acids to give the desired C-3/C-5 substituted isoxazole products in good to excellent overall yield (Table 4).

Table 4.

Synthesis of C-3/C-5 Substituted Isoxazoles Derivatives of 8a

entry	R1	R2	product
1	MeO	i-BuO	20h a
2	MeO	CH2Br	20i
3	Br	Me	20j
4	Br	Et	20k
5	Br	i-Pr	20l
6	Br	Ph	20m
7	Br	CH2Br	20n

^aPrepared by treatment of 8a with i-BuOCOCl

Other analogs of lead compound 8a, including 4,5-dihydroisoxazole 27, ester derivative 28 and thioamide 29, where prepared by the dipolar cycloaddition of the nitrile oxide generated from oxime 11 (Scheme 4). The respective dipolarophiles in these reactions, 24, 25 and 26, were readily obtained from N,N-diethylglycine.

Scheme 4.

Synthesis of C-5 Amidomethyl Analogs of 8a.^a

^aReagents and conditions: (a) NH₂OH•HCl, NaOH, tert-BuOH, H₂O, rt, 4 h; (b) chloramine-T, rt, 3 min; (c) 24, 25 or 26, CuSO₄•5H₂O (3 mol%), Cu powder (6 mol%), NaOH aq., rt, 12 h.

2. Biology

To produce Ebola virus GP pseudotyped HIV virions, 293T cells were co-transfected with the DNA of wild type Ebola Zaire GP (EboZ GP)²⁸ and the vector pNL4-3.Luc.R-E, which contains the env deficient HIV proviral genome and an intact firefly luciferase reporter gene (luc) inserted into the pNL4-3 nef gene.^37,38 This proviral construct expresses luciferase activity as a marker of viral gene expression and although it carries a deletion within the env coding region, contains all sequences necessary for reverse transcription, vector integration, and expression of the reporter gene. Upon transfection of cells, the HIV vector and the plasmid encoding the EboZ virus envelope protein are coated and expressed, generating GP pseudotyped HIV virions. These particles are assembled, released by cell lysis, and harvested. In order to determine the level of incorporation of wt GP protein into the pseudotyped viruses, Western blot analysis of the transfected 293T cell lysates was employed.

To screen for potential Ebola entry inhibitors, individual compounds (30~60 μM, final concentration) were mixed with Ebola GP pseudotyped HIV and the mixture incubated with the target cells (293T). At 24 and 48 hours post-infection, cell morphology was examined for signs of toxicity using light microscopy. At 48 hours post infection, the cells were lyzed and the level of viral infection analyzed by measuring firefly luciferase enzyme activity using a luminometer. DMSO, the vehicle in which test compounds were dissolved, was used as a background control (final concentration of 0.1~0.2%) and found not to significantly effect infection. Compounds initially identified as viral entry inhibitors were retested in order to confirm their antiviral properties. In order to confirm that the hit compounds displayed specificity for the function of the Ebola glycoprotein, VSV-G-pseudotyped HIV virions, which carry the envelope protein of the vesicular stomatitis virus (VSV), were produced and the effect of upon infectivity also examined. Inhibition of Ebola GP-mediated entry, but not VSV-G suggests that inhibition is specifically effecting Ebola GP-mediated entry rather than the pseudotyped core: the only difference between Ebola GP and VSV-G pseudotype viruses is the envelope protein and viral entry. In contrast, inhibition of both Ebola and VSV-G entry likely suggests one of two possible situations: 1) the compound in question inhibits post-entry replication of the HIV vector; 2) the compound is toxic to cells. In order to exclude the possibility of cell line bias, compounds were screened against both 293T and HeLa cells, for which Ebola GP pseudotyped HIV virions exhibit tropism.¹⁷ Select compounds found to specifically inhibit Ebola cell entry were finally evaluated at a range of concentrations in order to determine the dose-dependent inhibition (IC₅₀).

From an initial screen of an “in-house” library of 237 small molecules, two compounds (8a and 9 Figure 2) were found to decrease infectivity of the Ebola pseudotype virus without apparent side effects on cell morphology and growth. Consequently, these hit compounds were further evaluated for specificity (Figure 3). In comparison to a DMSO control, compound 8a blocked Ebola GP entry by over 50% at concentrations of 30 μM in both 293T and HeLa cells. In contrast, at this concentration 8a did not affect VSV-G mediated cell entry compared to DMSO. Viral infection without added reagents was also examined as a control (NC), and indicated that there was no differences between NC and DMSO (data not shown). These findings suggest that 8a specifically blocks Ebola GP-mediated viral entry independent of cell type. Titration showed that the IC₅₀ of 8a is approximately 30 μM (Figure 4). Furthermore, 8a did not affect VSV-G entry even at 120 μM (data not shown), indicating that the inhibition is specific to Ebola entry and not due to cytotoxicity. In contrast to 8a, similar analysis of hit compound 9 (data not shown) revealed this oxazolidinone derivative to be a non-selective inhibitor of Ebola-GP, Marburg-GP and VSV-G mediated cell entry. In light of these findings, further studies of this compound were not undertaken.

Figure 2.

Using an Ebola Zaire glycoprotein (GP)-pseudotyped HIV virus bearing a luciferase (luc) reporter gene and Human 293T, a library of 237 small molecules was assayed for antiviral activity based on prevention of Ebola GP-mediated viral entry. The luciferase activities of the infected cells were determined as a measure of GP-mediated viral infection. From this assay, compounds 8a and 9 were identified as antiviral agents that inhibit viral cell entry in a dose-dependent manner.

Figure 3.

Inhibition of Ebola GP-mediated infection of 293T and HeLa cells. Shown are the effects of lead compound 8a (final concentration, 30 μM) on the infectivity of Ebola virus GP (Ebola-GP) and VSV-G pseudotyped HIV virions (VSV-G) with 293T (■) and HeLa (□) cell lines. To determine the level of viral infection, 293T and HeLa cells were challenged with pseudotypes in the presence and absence of test compounds. After 48 h, luciferase expression was assessed using the method described in the Experimental Section. Infectivity is expressed as a percentage of luciferase activity, relative to the DMSO control. Values are reported as mean of data from three to five independent experiments, ± standard error.

Figure 4.

Concentration-response curve for DMSO control (■) and compound 8a (◆) (range = 3.75–120 μM), which inhibits Ebola GP-mediated viral entry into 293T cells in a dose dependant manner with an IC₅₀ of 30 μM. After 48 h, luciferase expression was assessed using the method described in the Experimental Section and is expressed as relative light units (RLU). Values are reported as mean of data from three independent experiments.

In order to optimize the anti-viral properties of 8a, fifty-five derivatives of this lead compound were synthesized and evaluated for inhibition of EBOV-GP-mediated viral-cell entry. The results of this investigation are outlined in Tables 5 (isoxazoles) and Table 6 (triazoles). Most notably, a number of the analogs of 8a were found to be significantly more potent inhibitors of cell entry, e.g. movement of the methoxy group on the aryl ring from the para to meta position enhanced the anti-Ebola entry activity (8b). Replacement of the methoxy group with halogens, including iodine, bromine, or chlorine, also improved anti-Ebola entry activity (8k, 8j, and 8l), as did replacement of the para-methoxylphenyl group with a 2-furyl substituent (8m).

Table 5.

Anti-Ebola Activity Data for Lead Compound 8a and its Isoxazole Analogs as Inhibitors of Ebola GP-Mediated Cell Entry.^a

Compound	Structure	Infectivityb	Compound	Structure	Infectivity
8a		56	8q		381
8b		4	8r		4
8c		132	20a		188
8d		30	20b		106
8e		37	20c		220
8f		24	20d		254
8g		160	20e		421
8h		181	20f		148
8i		87	20g		118
8j		6	20h		118
8k		30	20i		121
8l		17	20j		105
8m		77	20k		99
8n		52	20l		116
8o		102	20m		82
8p		59	20n		Kills cells at 60 μM

^aEbola virus GP pseudotyped HIV virions were mixed with compounds prior to infecting 293T cells (final concentration of compounds was 60μM) After 48 h, luciferase expression was assessed using the method described in the Experimental Section.

^bInfectivity is expressed as a percentage of luciferase activity, relative to the DMSO control.

Table 6.

Anti-Ebola Activity Data for Lead Compound 8a and its Triazole Analogs as Inhibitors of Ebola GP-Mediated Cell Entry.^a

Compound	Structure	Infectivityb	Compound	Structure	Infectivity
15a		58	15i		90
15b		4	15j		35
15c		105	15k		3
15d		18	15l		33
15e		60	15m		92
15f		46	15n		19
15g		66	15o		19
15h		111

^aEbola virus GP pseudotyped HIV virions were mixed with compounds prior to infecting 293T cells (final concentration of compounds was 60μM) After 48 h, luciferase expression was assessed using the method described in the Experimental Section.

^bInfectivity is expressed as a percentage of luciferase activity, relative to the DMSO control.

An examination of Table 6 reveals that substitution of the core isoxazole ring in 8a with a triazole system did not adversely impact anti-viral efficacy. Indeed, a number of triazoles, most notably 15b and 15k displayed greater activity than lead compounds 8a. On the basis of these findings, four of the most active derivatives (8b, 8j, 15b, and 15k) were chosen and their IC₅₀ values measured using 293T cells (Figure 5). While the IC₅₀ of 8b was improved to 10 μM, compounds 15b and 15k displayed higher activity of 5 μM. The highest anti-Ebola activity was shown by 8j (IC₅₀ = 2.5 μM). Notably, none of these four compounds displayed inhibition of VSV-G-mediated cell entry (data not shown).

Figure 5.

Concentration-response curve for DMSO control (◆) and compounds 8a (■), 8b (▲), 8j (×), 15b (*) and 15k (○) (range = 3.75-60 μM), which inhibit Ebola GP-mediated viral entry into 293T cells in a dose dependant manner with respective IC₅₀ values of 30, 10, 2.5, 5 μM (see also Tables 5 and 6). To determine the level of infection, 293T cells were challenged with pseudotypes in the presence and absence of test compounds. After 48 h, luciferase expression was assessed using the method described in the Experimental Section. Infectivity is expressed as a percentage of luciferase activity, relative to the DMSO control. Values are reported as mean of data from three to five independent experiments, ± standard error.

Replacement of the aryl p-methoxy substituent with a more polar hydroxyl group lead to a decrease in anti-viral activity as did removal of the removal of the diethylamino group (20j), removal of the ethanamide and diethylamino groups (22), and replacement of the amide with an ester (28).]

Somewhat unexpectedly, a number of compounds, most notably 8q, the p-methyl analog of 8a, and cysteine derivative 20e actually enhanced the infectivity of Ebola virus GP pseudotyped HIV virions with 293T cells up to 4 fold. It should be noted, however, that this enhancement of infectivity was found to be cell type-specific, since 8q and 20e, did not enhance Ebola GP-mediated entry in HeLa cells (data not shown). Interestingly, only derivative 20n displayed appreciable toxicity towards cells: at concentrations of 60 μM and above this compound killed 293T cells, which all other compounds evaluated did not show any obvious effects on cell morphology and growth. The pronounced cytotoxicity of 20n may arise from the presence of the α-bromoamide group, which may act as a biological alkylating agents: notable in this regard is the lack of toxicity displayed by compound 20j, the debrominated derivative of 20-n.

Since an earlier study (LR) suggested that Ebola and Marburg viruses might share a common cellular factor for viral entry,²⁸ lead compound 8a and the three derivatives that displayed improved anti-Ebola activity (8j, 15b, and 15k) were also evaluated for their ability to inhibit Marburg GP-mediated viral entry (Figure 5). Each compound (final concentration 60 μM) was used to block Marburg entry into 3 different cell lines (293T, HeLa and A549). In comparison to a DMSO control, compound 8a blocked 20-40% of Marburg entry while 8j, 15b, and 15b displayed more dramatic blocking of Marburg entry, especially in HeLa and A549 (human lung carcinoma) cells (>90%). Therefore, 8a and three derivatives not only blocked Ebola entry, but also impaired viral entry mediated by Marburg GP independent of cell types. This further supports the hypothesis that Ebola and Marburg viruses share a common cellular factor and a common entry inhibitor could be developed for both viruses.

Conclusions

In summary, we report the development of an anti-filoviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the Ebola and Marburg glycoprotein (GP)-mediated infection of human cells. Using Ebola Zaire GP-pseudotyped HIV particles bearing a luciferase reporter gene and 293T cells, a library of 237 small molecules was screened for inhibition of GP-mediated viral entry. From this assay, lead compound 8a was identified as a selective inhibitor of filoviral entry with an IC₅₀ of 30 μM. In order to analyze functional group requirements for efficacy, a structure-activity relationship analysis of this 3,5-disubstituted isoxazole was then conducted with 56 isoxazole and triazole derivatives prepared using “click” chemistry. This study revealed that while the isoxazole ring can be replaced by a triazole system, the 5-(diethylamino)acetamido substituent found in 8a is required for inhibition of viral-cell entry. Variation of the 3-aryl substituent also provided a number of more potent anti-viral agents with IC₅₀ values ranging to 2.5 μM. Lead compound 8a and three of its derivatives were also found to block the Marburg glycoprotein (GP)-mediated infection of human cells. This group of compounds are not only potential leads in the search for therapeutic anti-filoviral candidates, but may also serve as mechanistic probes in the elucidation of the mechanism of filoviral fusion and entry.

Experimental Section

Chemistry

All non-aqueous reactions were carried out in oven- or flame-dried glassware under an atmosphere of nitrogen, unless otherwise noted. All solvents were reagent grade. Triethylamine was distilled from calcium hydride, under nitrogen, and stored over potassium hydroxide. N,N-Dimethylformamide (DMF) was purchased from a commercial vendor and dried with freshly activated 4 Å molecular sieves prior to use. All products were purified by flash column chromatography using silica gel 60 (mesh 230-400). All other reagents and starting materials, unless otherwise noted, were purchased from commercial vendors and used without further purification. N,N-Diethylglycine and 3-(4-methoxyphenyl)-5-hydroxymethyl isoxazole (16a) were prepared according to procedures reported by Tonellato³⁹ and Fokin,³¹ respectively. All melting points were determined in open Pyrex capillaries with a Thomas Hoover Unimelt melting point apparatus and are uncorrected. IR spectra were recorded as thin films on sodium chloride plates or as suspensions in compressed potassium bromide discs. ¹H and ¹³C spectra were recorded on a Bruker Avance 400 (400 MHz ¹H, 100 MHz ¹³C) or a Bruker Avance 500 (500 MHz ¹H, 125 MHz ¹³C) spectrometer. DEPT 135 and two-dimensional (COSY, HMQC, HMBC) NMR experiments were employed, where appropriate, to aid in the assignment of signals in the ¹H NMR spectra. The purity of the compounds was determined using an analytical HPLC (Agilent 1100 with diode-array detector, Supercosil LC 18 (250 mm × 4.6 mm, 5 micron) acetonitrile (unless otherwise noted), 2.00 mL/min) and was confirmed to be ≥95% for all compounds.

General Synthetic Procedure A. Preparation of 3,5-Disubstituted Isoxazoles, as Exemplified by the Preparation of 2-(Diethylamino)-N-((3-(4-methoxyphenyl)isoxazol-5-yl)methyl)ethanamide (8a)

To a solution of p-anisaldehyde (140 mg, 1.03 mmol, 1.0 equiv) and hydroxylamine hydrochloride (75 mg, 1.07 mmol, 1.05 equiv) in a mixture of t-BuOH and H₂O (1:1, 4 mL) was added 1 M aqueous NaOH (1.1 mL). The reaction mixture was stirred at rt for 4 h, or until thin-layer chromatography indicated consumption of the aldehyde. After oxime formation was complete, chloramine-T (301 mg, 1.07 mmol, 1.05 equiv) was added portionwise over 3 min, followed by CuSO₄β5H₂O (5 mg, 3 mol%) and copper powder (4 mg, 6 mol%). N’-Propargyl-N,N-diethylglycine (13)(182 mg, 1.07 mmol, 1.05 equiv) was then added, the pH of the reaction medium adjusted by the addition of 1 M aqueous NaOH (6 drops), and the mixture stirred at rt for 12 h. The reaction was then quenched with 1 M aqueous NH₄OH (1 ml), extracted with EtOAc (3 × 15 mL) and the combined organic extracts dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by flash chromatography over silica gel (EtOAc) to provide 8a (209 mg, 64%): white solid; mp 77-78 °C; R_f 0.15 (EtOAc); t_R – HPLC: 1.46 min (100 %); FTIR (KBr pellet) υ_max 3336 (br), 2967 (br), 1674, 1612, 1514, 1432, 1254, 1177, 1028, 838 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1 H), 7.71 (d, J = 8.7 Hz, 2 H), 6.95 (d, J = 8.7 Hz, 2 H), 6.43 (s, 1 H), 4.61 (d, J = 6.0 Hz, 2 H), 3.84 (s, 3 H), 3.09 (s, 2 H), 2.56 (q, J = 7.1 Hz, 4 H), 1.02 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 169.3, 162.3, 161.1, 128.3 (2 C), 121.5, 114.4 (2 C), 100.0, 57.4, 55.5, 48.9 (2 C), 34.9, 12.4 (2 C); HRMS-EI calcd for C₁₇H₂₄N₃O₃ [M+H]⁺: 318.1812, found: 318.1802.

General Synthetic Procedure B. Preparation of of N,N-Diethylglycine Derivatives, as Exemplified by the Preparation of N’-Propargyl-N,N-diethylglycine (13)

To a stirred solution of N,N-diethylglycine (312 mg, 2.4 mmol, 1 equiv) and propargylamine (250 μL, 3.8 mmol, 1.6 equiv) in anhydrous CH₂Cl₂ (3.7 mL), at rt was added EDC (818 mg, 4.3 mmol, 1.8 equiv) followed by addition of Et₃N (530 μL, 1.6 equiv). After stirring for 12 h, the reaction was quenched with 1 M aqueous HCl (500 μL) and extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine (2 × 10 mL), dried (Na₂SO₄), concentrated under reduced pressure, and the residue purified by flash chromatography over silica gel (EtOAc/hexanes, 1:3) to afford 13 (488 mg, 52%): yellow oil; R_f 0.25 (EtOAc); IR (film) υ_max 3308 (br), 2970, 2934, 2823, 1673, 1516, 1456, 1426, 1347, 1260, 1205, 1089 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.53 (br s, 1 H), 4.01 (dd, J = 2.4, 5.4 Hz, 2 H), 2.98 (s, 3 H), 2.50 (q, J = 7.1 Hz, 4 H), 2.18 (t, J = 2.4 Hz, 1 H), 0.98 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 79.8, 71.3, 57.4, 48.9 (2 C), 28.6, 12.4 (2 C); HRMS-EI calcd for C₉H₁₆N₂O [M+H]⁺: 96.1072, found: 96.1074.

General Synthetic Procedure C. Preparation of 3,5-Disubstituted Triazoles, as Exemplified by the Preparation of 2-(Diethylamino)-N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15a)

To a solution of 1-azido-4-methoxybenzene (108 mg, 0.72 mmol, 1.0 equiv) in a mixture of t-BuOH and H₂O (1:1, 3 mL) was added N’-propargyl-N,N-diethylglycine (13) (109 mg, 0.72 mmol, 1.0 equiv), followed by addition of 1M aqueous sodium ascorbate (72 μL, 0.1 mmol) and CuSO₄•5H₂O (2.5 mg, 0.02 mmol, 6 mol%). The heterogeneous reaction mixture was stirred vigorously at rt for 12 h. After completion of the reaction, as indicated by thin-layer chromatography, it was diluted with water (15 mL) and extracted with EtOAc (3 × 15 mL). The combined organic extracts were dried (Na₂SO₄), filtered and concentrated under reduced pressure. The crude 1,2,3-triazole was purified by flash chromatography over silica gel (EtOAc) to provide 15a (174 mg, 82%): off-white solid; mp 109-110 °C; R_f 0.10 (EtOAc); t_R – HPLC: 5.4 min (MeOH, 99.6 %); FTIR (KBr pellet) υ_max 3349 (br), 2966 (br), 1672, 1518, 1255, 1043, 833 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.05 (br s, 1 H), 7.89 (s, 1 H), 7.60 (dd, J = 1.9, 5.0 Hz, 1 H), 7.00 (dd, J = 1.9, 5.0 Hz, 1 H), 4.60 (d, J = 6.2 Hz, 2 H), 3.86 (s, 3 H), 3.05 (s, 2 H), 2.53 (q, J = 7.2 Hz, 4 H), 0.99 (t, J = 7.2 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.6, 160.0, 145.6, 130.7, 122.3 (2 C), 120.7, 114.9 (2 C), 57.7, 55.8, 48.9 (2 C), 34.59, 12.4 (2 C); HRMS-EI calcd for C₁₆H₂₃N₅O₂ [M+H]⁺: 318.1930, found: 318.1942.

General Synthetic Procedure D. Preparation of 3,5-Disubstituted Isoxazoles via Amide Coupling, as Exemplified by the Preparation of Benzyl 2-((3-(4-methoxyphenyl)isoxazol-5-yl)methylamino)-2-oxoethylcarbamate (20f)

To a stirred solution of carbobenxyloxyglycine (200 mg, 0.618 mmol, 1 equiv) in anhydrous CH₂Cl₂ (5 mL) under an atmosphere of nitrogen, at −40 °C (MeCN/CO₂), was added isobutyl chloroformate (105 μL, 0.624 mmol, 1.05 equiv) and triethylamine (86 μL, 0.618 mmol, 1 equiv). After stirring for 10 min, the cold bath was removed and reaction mixture was allowed to warm to rt and stirred for 12 h. 3(4-Methoxyphenyl)isoxazol-5-yl)methanamine (18b) (100 mg, 0.618 mmol, 1 equiv) and triethylamine (86 μL, 0.618 mmol, 1 equiv) were then added sequentually. The reaction mixture was stirred for 12 h at rt then quenched with 1M aqueous HCl and the aqueous portion extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried (Na₂SO₄), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography over silica gel (EtOAc/hexanes, 1:4) to provide 20f (332 mg, 88%): white solid; mp 117-119 °C; R_f 0.28 (EtOAc/hexanes, 1:3); t_R – HPLC: 1.46 min (100 %); FTIR (KBr pellet) υ_max 3323, 3285, 1690, 1658, 1548, 1293, 1247, 1165, 1074 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.41 (d, J = 8.7 Hz, 2 H), 8.03 (m, 5 H), 7.65 (d, J = 8.7 Hz, 2 H), 7.27 (s, 1 H), 6.08 (br s, 1 H), 5.96 (s, 2 H), 5.81 (s, 2 H), 4.69 (d, J = 5.9 Hz, 2 H), 4.54 (s, 3 H), 2.86 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 169.7, 166.2, 162.4, 161.3, 156.5, 129.7, 128.7 (2 C), 128.3 (5 C), 121.1, 114.5 (2 C), 102.5, 67.4, 57.2, 55.5, 42.7; HRMS-EI calcd for C₂₁H₂₂N₃O₅ [M+H]⁺: 396.4165, found: 396.4168.

2-(Diethylamino)-N-((3-(3-methoxyphenyl)isoxazol-5-yl)methyl)ethanamide (8b)

Yield 36%: white solid; mp 90-92 °C; R_f 0.10 (EtOAc); t_R – HPLC: 1.47 min (97.6 %); FTIR (KBr pellet) υ_max 3308 (br), 2966, 2926, 1669, 1608, 1506, 1456, 1254, 1158, 1042, 1000 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1 H), 7.35 (m, 3 H), 7.00 (m, 1 H), 6.43 (m, 1 H), 6.48 (s, 2 H), 4.64 (d, J = 6.2 Hz, 2 H), 3.86 (s, 3 H), 3.12 (s, 2 H), 2.60 (q, J = 7.1 Hz, 4 H), 1.05 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.3, 169.6, 162.7, 160.1, 130.2, 130.1, 119.5, 116.4, 111.8, 100.5, 57.4, 48.9 (2 C), 35.0, 29.9, 12.3 (2 C); HRMS-ESI calcd for C₁₇H₂₄N₃O₃ [M+H]⁺: 318.38122, found: 318.18129.

2-(Diethylamino)-N-((3-(4-hydroxyphenyl)isoxazol-5-yl)methyl)ethanamide (8c)

Yield 62%: colorless oil; R_f 0.12 (EtOAc); t_R – HPLC: 1.45 min (99.2 %); IR (film) υ_max 3349 (br), 2967, 2811, 1671, 1517, 1487, 1373, 1203, 1036, 856 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1 H), 7.56 (d, J = 8.7 Hz, 2 H), 6.87 (d, J = 8.7 Hz, 2 H), 6.31 (s, 1H), 4.59 (d, J = 6.2 Hz, 1 H), 3.12 (s, 2 H), 2.59 (q, J = 7.1 Hz, 4 H), 0.91 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 173.5, 168.8, 162.4, 158.5, 128.5 (2 C), 121.0, 116.2 (2 C), 100.5, 57.4, 49.0 (2 C), 35.1, 12.4 (2 C); HRMS-EI calcd for C₁₆H₂₁N₃O₃ [M+H]⁺: 303.3562, found: 303.3568.

2-(Diethylamino)-N-((3-(4-nitrophenyl)isoxazol-5-yl)methyl)ethanamide (8d)

Yield 17%: colorless oil; R_f 0.12 (EtOAc); t_R – HPLC: 1.45 min (100 %); IR (film) υ_max 3338 (br), 2967, 2966, 2853, 1673, 1610, 1522, 1456, 1434, 1229, 1157, 1074, 914, 842 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.32 (d, J = 8.8 Hz, 2H), 8.12 (br s, 1 H), 7.98 (d, J = 8.8 Hz, 2 H), 6.58 (s, 1 H), 4.66 (d, J = 6.3 Hz, 2 H), 3.11 (s, 2 H), 2.58 (q, J = 7.1 Hz, 4 H), 1.04 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 170.9, 161.0, 149.0, 135.2, 127.9 (2C), 124.4 (2 C), 100.6, 57.5, 49.0 (2 C), 34.9, 12.5 (2 C); HRMS-EI calcd for C₁₆H₂₁N₄O₄ [M+H]⁺: 333.3623, found: 333.3628.

2-(Diethylamino)-N-((3-(3-nitrophenyl)isoxazol-5-yl)methyl)ethanamide (8e)

Yield 15%: off-white solid; mp 90-91 °C; R_f 0.11 (EtOAc); t_R – HPLC: 1.45 min (98.8 %); FTIR (KBr pellet) υ_max 3291, 3152, 3054, 2967, 2932, 2805, 1654, 1525, 1498, 1348, 1224, 1045, 984 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.62 (m, 1 H), 8.31 (dd, J = 2.1, 8.2 Hz, 1 H), 8.15 (d, J = 7.9 Hz, 1 H), 8.04 (br s, 1H), 7.66 (t, J = 8.0 Hz, 1 H), 6.59 (s, 1 H), 4.67 (d, J = 6.3 Hz, 2 H), 3.12 (s, 2 H), 2.60 (q, J = 7.1 Hz, 4 H), 1.05 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 170.9, 160.9, 148.8, 132.7, 130.9, 130.3, 124.9, 122.04, 100.4, 57.5, 49.00 (2 C), 35.02, 12.5 (2 C); HRMS-EI calcd for C₁₆H₂₁N₄O₄ [M+H]⁺: 333.3623, found: 333.3629.

2-(Diethylamino)-N-((3-(4-(trifluoromethyl)phenyl)isoxazol-5-yl)methyl)ethanamide (8f)

Yield 58%: colorless oil; R_f 0.11 (EtOAc); t_R – HPLC: 1.47 min (96.8 %); IR (film) υ_max 3308, 2967, 2927, 1672, 1608, 1515, 1354, 1252, 1176, 898, 832 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.19 (s, 1 H), 7.91 (d, J = 8.1 Hz, 2 H), 7.72 (d, J = 8.1 Hz, 2 H), 6.55 (s, 1 H), 4.65 (d, J = 6.2 Hz, 2 H), 3.16 (s, 2 H), 2.63 (q, J = 7.2 Hz, 4 H), 1.07 (t, J = 7.2 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 170.3, 161.6, 132.5, 131.9, 128.7, 127.3 (2 C), 126.1 (2 C), 100.4, 57.3, 48.8 (2 C), 35.0, 29.9, 12.15 (2 C); HRMS-ESI calcd for C₁₇H₂₁F₃N₃O₃[M+H]⁺: 356.15804, found: 356.15787.

N-(3-(3,4-Dimethoxyphenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8g)

Yield 12%: colorless oil; R_f 0.11 (EtOAc); t_R – HPLC: 1.44 min (96.6 %); IR (film) υ_max 3324 (br), 2965, 2933, 1674, 1605, 1516, 1426, 1262, 1206, 1134, 1074, 809 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.02 (br s, 1 H), 7.39 (d, J = 1.9 Hz, 1 H), 7.27 (d, J = 1.9 Hz, 1 H), 6.92 (s, 1H), 6.91 (s, 1 H), 6.46 (s, 1 H), 5.30 (s, 1 H), 4.61 (d, J = 6.2 Hz, 2 H), 3.91 (s, 3 H), 3.90 (s, 3 H), 3.11 (s, 2 H), 2.58 (q, J = 7.0 Hz, 4 H), 2.17 (s, 2 H), 1.04 (t, J = 7.0 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 169.2, 161.2, 151.6, 150.6, 129.1, 127.4, 119.9, 110.9, 109.2, 107.7, 100.0, 57.2, 56.0, 48.7 (2 C), 34.8, 12.2 (2 C); HRMS-EI calcd for C₁₈H₂₆N₃O₄ [M+H]⁺: 348.4167, found: 348.4162.

2-(Diethylamino)-N-((3-phenylisoxazol-5-yl)methyl)ethanamide (8h)

Yield 66%: white solid: mp 118-120 °C; R_f 0.37 (EtOAc); t_R – HPLC: 1.42 min (100 %); FTIR (KBr pellet) υ_max 3331 (br), 2965, 2933, 1709, 1612, 1529, 1432, 1366, 1253, 1175, 1029, 837 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1 H), 7.78 (m, 2 H), 7.45 (m, 3 H), 6.50 (s, 1 H), 4.67 (d, J = 6.2 Hz, 2 H), 3.16 (s, 2 H), 2.63 (q, J = 7.1 Hz, 4 H), 1.07 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 169.6, 162.8, 136.5, 132.2, 130.3, 129.1 (2 C), 127.0 (2 C), 100.4, 57.3, 49.0 (2 C), 35.0, 12.3 (2 C); HRMS-EI calcd for C₁₆H₂₂N₃O₂[M+H]⁺: 288.3648, found: 288.3651.

N-((3-(2-methoxyphenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8i)

Yield 60%: colorless oil; R_f 0.12 (EtOAc); t_R – HPLC: 1.43 min (100 %); IR film υ_max 3308 (br), 2962, 2926, 1669, 1603, 1506, 1456, 1254, 1158, 1042 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.97 (br s, 1 H), 7.85 (dd, J = 1.7, 7.7 Hz, 1 H), 7.40 (dt, J = 1.7, 9.0 Hz, 1 H), 7.02 (t, J = 7.5 Hz, 1 H), 7.0 (d, J = 8.4 Hz, 1 H), 4.63 (d, J = 6.1 Hz, 2 H), 3.86 (s, 3 H), 3.09 (s, 2 H), 2.56 (q, J = 7.1 Hz, 4 H), 1.03 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.4, 168.3, 160.3, 157.3, 131.4, 129.5, 121.3, 121.0, 117.9, 111.6, 103.6, 57.4, 49.0 (2 C), 35.0, 12.4 (2 C); HRMS-ESI calcd for C₁₇H₂₄N₃O₃ [M+H]⁺: 318.18122, found: 318.18129.

N-((3-(4-Iodophenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8j)

Yield 68%: white solid; mp 104-105 °C; R_f 0.21 (EtOAc); t_R – HPLC: 1.45 min (95.4 %); FTIR (KBr pellet) υ_max 3287 (br), 3123, 2968, 2935, 2870, 2819, 1666, 1521, 1425, 1216, 1067, 1027 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.99 (br s, 1H), 7.79 (d, J = 6.5 Hz, 2 H), 7.51 (d, J = 6.5 Hz, 2 H), 6.45 (s, 1 H), 4.63 (d, J = 6.2 Hz, 2 H), 3.08 (s, 2 H), 2.55 (q, J = 7.3 Hz, 4 H), 1.02 (t, J = 7.3 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 170.1, 162.0, 138, 129.5 (2 C), 128.6 (2 C), 100.2, 96.5, 57.5, 49.0 (2 C), 35.0, 12.5 (2 C); HRMS-ESI calcd for C₁₆H₂₁IN₃O₂ [M+H]⁺: 414.06730, found: 414.06654.

N-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8k)

Yield 64%: yellow oil; R_f 0.11 (EtOAc); t_R – HPLC: 1.47 min (98.2 %); IR (film) υ_max 3307 (br), 2966, 2923, 1670, 1508, 1427, 1275, 1072, 830 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.04 (s, 1 H), 7.64 (d, J = 8.4 Hz, 2 H), 7.58 (d, J = 8.4 Hz, 2 H), 6.47 (s, 1 H), 4.63 (d, J = 6.1 Hz, 2 H), 4.29 (s, 2 H), 3.16 (s, 2 H), 2.61 (q, J = 6.9 Hz, 4 H), 0.88 (t, J = 6.9 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.5, 170.0, 161.8, 136.3, 129.5 (2 C), 128.3 (2 C), 127.6, 100.3, 57.4, 49.0 (2 C), 35.0, 12.5 (2 C); HRMS-ESI calcd for C₁₆H₂₁BrN₃O₂ [M+H]⁺: 366.08117, found 366.08056.

N-(3-(4-Chlorophenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8l)

Yield 42%: white solid; mp 81-83 °C; R_f 0.11 (EtOAc); t_R – HPLC: 1.46 min (97.1 %); FTIR (KBr pellet) υ_max 3286 (br), 2969, 1659, 1521, 1424, 1090, 1028, 847, 805 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.01 (s, 1 H), (d, J = 8.4 Hz, 2 H), (d, J = 8.4 Hz, 2 H), 6.47 (s, 2 H), 4.63 (d, J = 6.2 Hz, 2 H), 3.10 (s, 2 H), 2.57 (q, J = 7.1 Hz, 4 H), 1.03 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.6, 170.0, 161.8, 136.3, 129.4 (2 C), 128.3 (2 C), 127.6, 100.3, 57.5, 49.0 (2 C), 35.0, 12.5 (2 C); HRMS-ESI calcd for C₁₆H₂₁ClN₃O₂ [M+H]⁺: 322.13168, found: 322.13131.

2-(Diethylamino)-N-((3-(2-furyl)isoxazol-5-yl)methyl)ethanamide (8m)

Yield 56%: colorless oil; R_f 0.16 (EtOAc); t_R – HPLC: 1.46 min (96.8 %); IR (film) υ_max 3342 (br), 2966 (br), 2928, 1669, 1517, 1455, 1334, 1259, 1207, 1049 (br) cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.08 (br s, 1 H), 7.54 (dd, J = 0.6, 1.2 Hz, 1 H), 6.87 (dd, J = 0.5, 2.8 Hz, 1 H), 6.51 (dd, J = 1.6, 1.8 Hz, 1 H), 6.43 (s, 1 H), 4.61 (d, J = 6.2 Hz, 2 H), 3.15 (s, 2 H), 2.62 (d, J = 7.1 Hz, 4 H), 1.05 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 169.4, 155.1, 144.1, 111.9, 110.6, 99.9, 57.01, 49.0 (2 C), 34.9, 29.9, 28.8, 12.3 (2 C); HRMS-EI calcd for C₁₄H₂₀N₃O₃[M+H]⁺: 278.3269, found: 278.3265.

2-(Diethylamino)-N-((3-(pentafluorophenyl)isoxazol-5-yl)methyl)ethanamide (8n)

Yield 68%: white solid; mp 100-102 °C; R_f 0.39 (EtOAc); t_R – HPLC: 1.42 min (100 %); FTIR (KBr pellet) υ_max 3316, 2973, 2939, 2877, 2823, 1670, 1508, 1423, 1388, 1361, 1211, 1079, 995, 817 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (br s, 1 H), 6.51 (s, 1 H), 4.70 (d, J = 6.2 Hz, 2H), 3.11 (s, 2 H), 2.58 (q, J = 7.0 Hz, 4 H), 1.04 (t, J = 7.0 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.4, 170.2, 161.0, 151.7, 145.8, 143.8, 139.0, 137.0, 103.6, 103.1, 57.2, 48.8 (2 C), 34.7, 12.2 (2 C); HRMS-EI calcd for C₁₆H₁₇F₅N₃O₃ [M+H]⁺: 378.3171, found: 378.3176.

2-(Diethylamino)-N-((3-propylisoxazol-5-yl)methyl)ethanamide (8o)

Yield 46%: colorless oil; R_f 0.11 (EtOAc); t_R – HPLC: 1.47 min (100 %); IR (film) υ_max 3324 (br), 2965, 2933, 1674, 1605, 1516, 1426, 1262, 1206, 1134, 1074, 809 cm⁻¹; ¹H NMR (500 MHz, CDCl ₃) δ 7.82 (br s, 1 H), 6.00 (s, 1 H), 4.55 (d, J = 6.2 Hz, 1 H), 3.10 (s, 2 H), 2.61 (t, J = 7.5 Hz, 2 H), 2.57 (q, J = 7.1 Hz, 4 H), 1.67 (s, 2 H), 1.03 (t, J = 7.1 Hz, 6 H), 0.97 (t, J = 7.4 Hz, 3 H), 1.05 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 168.7, 164.3, 125.5, 101.8, 57.4, 49.0 (2 C), 35.0, 28.2, 21.8, 13.9, 12.4 (2 C); HRMS-EI calcd for C₁₃H₂₄N₃O₂ [M+H]⁺: 254.3486, found: 254.3489.

2-(Diethylamino)-N-((3-(2,6-dimethylphenyl)isoxazol-5-yl)methyl)ethanamide (8p)

Yield 69%: colorless oil; R_f 0.12 (EtOAc); t_R – HPLC: 1.44 min (96.1 %); IR (film) υ_max 3319 (br), 2969 (br), 2932, 2871, 2823, 1676, 1601, 1513, 1456, 1423, 1388, 1355, 1207, 1162 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.00 (br s, 1 H), 7.20 (t, J = 7.9 Hz, 1 H), 7.08 (d, J = 7.9 Hz, 2 H), 6.09 (s, 1 H), 4.66 (d, J = 6.3 Hz, 2 H), 3.08 (s, 2 H), 2.54 (q, J = 7.1 Hz, 4 H), 2.12 (d, J = 2.7 Hz, 6 H), 1.01 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.4, 169.3, 162.3, 137.4 (2 C), 129.2, 128.9 (2 C), 127.6, 103.1 (2 C), 57.4, 49.04 (2 C), 34.9, 20.4, 12.4 (2 C); HRMS-EI calcd for C₁₈H₂₆N₃O₂ [M+H]⁺: 316.2019, found: 316.2010

2-(Diethylamino)-N-((3-(4-methylphenyl)isoxazol-5-yl)methyl)ethanamide (8q)

Yield 50%: yellow oil; R_f 0.17 (EtOAc); t_R – HPLC: 1.43 min (100 %); IR (film) υ_max 3330 (br), 2962, 1674, 1612, 1516, 1432, 1254, 1168, 1026 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.15 (br s, 1H), 7.66 (d, J =7.9 Hz, 2H), 7.24 (d, J = 7.9 Hz, 2H), 6.48 (s, 1H), 4.61 (br s, 1H), 4.11 (q, J = 7.2 Hz, 4H), 2.39 (s, 3H), 1.25 (t, J = 7.2 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 172.3, 169.3, 162.3, 161.3, 137.4 (2 C), 129.2, 128.9 (2 C), 127.6, 102.1, 57.4, 49.04 (2 C), 34.9, 12.4 (2 C); HRMS-EI calcd for C₁₇H₂₄N₃O₂ [M+H]⁺: 302.3914, found: 302.3926.

N-((3-(3-Iodo-4methoxyphenyl)isoxazol-5-yl)methyl)-2-(diethylamino)ethanamide (8r)

Yield 62%: colorless oil; R_f 0.22 (EtOAc); t_R – HPLC: 1.43 min (99.6 %); IR film υ_max 3324 (br), 2960, 2928, 1668, 1605, 1516, 1426, 1262, 1134, 1071 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 2.1 Hz, 1 H), 7.99 (s, 1 H), 7.73 (dd, J = 2.0, 8.4 Hz, 1 H), 6.86 (d, J = 8.5 Hz, 1 H), 6.45 (s, 1 H), 4.61 (d, J = 6.2 Hz, 2 H), 3.93 (s, 3 H), 3.11 (s, 2 H), 2.57 (q, J = 7.1 Hz, 4 H), 1.03 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 169.7, 161.1, 159.6, 138.0, 128.3, 119.5, 111.0, 100.1, 86.4, 57.5, 56.7, 49.0 (2 C), 35.0, 12.5 (2 C); HRMS-EI calcd for C₁₇H₂₃IN₃O₃ [M+H]⁺: 444.0784, found: 444.0789.

2-(Diethylamino)-N-((1-(4-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15b)

Yield 90%: white solid; mp 127-129 °C; R_f 0.19 (MeOH/EtOAc, 1%); t_R – HPLC: 5.3 min (MeOH, 97.2 %); FTIR (KBr pellet) υ_max 3291, 3152, 3054, 2967, 2932, 2805, 1654, 1525, 1498, 1348, 1224, 1045 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (br s, 1 H), 7.97 (s, 1 H), 7.81 (d, J = 8.7 Hz, 2 H), 7.47 (d, J = 8.7 Hz, 2 H), 4.58 (d, J = 6.1 Hz, 2 H), 3.04 (s, 2 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.96 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 146.1, 139.0 (2 C), 136.7, 122.1 (2 C), 120.3, 93.7, 57.5, 48.8 (2 C), 34.5, 12.3 (2 C); HRMS-EI calcd for C₁₅H₂₁IN₅O [M+H]⁺: 414.2646, found: 414.2648

N-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15c)

Yield 55%: white solid; mp 100 °C; R_f 0.61 (EtOAc/hexanes, 1:3); t_R – HPLC: 1.47 min (97.6 %); FTIR (KBr pellet) υ_max 3286 (br), 2972, 1660, 1521, 1424, 1090, 1028 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (br s, 1 H), 7.96 (s, 1 H), 7.66 (d, J = 8.8 Hz, 1 H), 7.49 (d, J = 8.8 Hz, 2 H), 4.59 (d, J = 6.2 Hz, 2 H), 3.04 (s, 1 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 146.2, 135.7, 134.8, 130.1 (2 C), 121.8 (2 C), 120.6, 57.6, 48.9 (2 C), 34.5, 12.4 (2 C); HRMS-EI calcd for C₁₅H₂₁ClN₅O [M+H]⁺: 322.1435, found: 322.1437.

N-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15d)

Yield 86%: yellow solid; mp 116-118 °C; R_f 0.17 (EtOAc); t_R – HPLC: 1.45 min (100 %); FTIR (KBr pellet) υ_max 3288 (br), 3056, 2970, 2817, 1663, 1500, 1349, 1265, 1045 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.03 (br s, 1 H), 7.97 (s, 1 H), 7.62 (m, 4 H), 4.59 (d, J = 6.1 Hz, 2 H), 3.04 (s, 3 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.98 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 146.2, 136.1, 133.1 (2 C), 122.6, 122.0 (2 C), 120.5, 57.6, 48.9 (2 C), 34.5, 12.4 (2 C); HRMS-EI calcd for C₁₅H₂₁BrN₅O [M+H]⁺: 367.2641, found: 367.2643.

2-(Diethylamino)-N-((1-heptyl-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15e)

Yield 59%: white solid; mp 120 °C; R_f 0.10 (EtOAc); t_R – HPLC: 1.47 min (98.8 %); FTIR (KBr pellet) υ_max 3297, 1646, 1544, 1420, 1264, 1221, 1026 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.95 (br s, 1 H), 7.51 (s, 1 H), 4.53-4.56 (m, 1 H), 4.31-4.33 (m, 1 H), 3.04 (s, 2H), 2.55 (q, J = 7.1 Hz, 4 H), 1.88 (m, 2H), 1.33 (m, 9H), 0.99 (t, J = 7.1 Hz, 6 H), 0.87 (t, J = 5.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 145.1, 122.1, 57.6, 50.6, 48.9 (2 C), 34.6, 31.8, 29.9, 28.8, 26.6, 22.7, 14.2, 12.5 (2 C); HRMS-EI calcd for C₁₆H₃₂N₅O [M+H]⁺: 310.2610, found: 310.2604.

2-(Diethylamino)-N-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15f)

Yield 50%: white solid; mp 92 °C; R_f 0.29 (MeOH/EtOAc, 1%); t_R – HPLC: 1.47 min (99.7 %); FTIR (KBr pellet) υ_max 3288 (br), 3153, 3056, 2970, 2933, 2871, 2817, 1663, 1524, 1500, 1349, 1265, 1224 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.03 (br s, 1 H), 7.97 (s, 1 H), 7.60-7.65 (m, 4 H), 4.59 (d, J = 6.1 Hz, 2 H), 3.04 (s, 2 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 146.2, 136.1, 133.1 (2 C), 122.6, 122.0 (2 C), 120.5, 57.6, 48.9 (2 C), 34.5, 12.5 (2 C); HRMS-ESI calcd for C₁₅H₂₁BrN₅O [M+H]⁺: 366.09240, found: 366.09183.

N-((1-(2-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15g)

Yield 42%: yellow oil; R_f 0.13 (MeOH/EtOAc, 1%); t_R – HPLC: 1.46 min (100 %); IR υ_max 3343, 2970, 29.34, 2874, 2821, 1668, 1536, 1355, 1039 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.05 (dd, J = 1.4, 8.1 Hz, 2 H), 7.85 (s, 1 H), 7.78 (dt, J = 1.4, 7.7 Hz, 1 H), 7.68 (dt, J = 1.4, 8.1 Hz, 1 H), 7.59 (dd, J = 1.4, 8.0 Hz, 1 H), 4.63 (d, J = 6.1 Hz, 1 H), 3.05 (s, 2 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 145.8, 144.6, 134.0, 131.0, 130.3, 127.9, 125.7, 124.0, 57.5, 48.9 (2 C), 34.4, 12.4 (2 C); HRMS-EI calcd for C₁₅H₂₁N₆O₃ [M+H]⁺: 333.1675, found: 333.16758.

N-((1-(3-Iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15h)

Yield 87%: yellow solid; mp 77 °C; R_f 0.29 (EtOAc); t_R – HPLC: 1.47 min (100 %); FTIR (KBr pellet) υ_max 3343, 2967, 2933, 1668, 1587, 1519, 1486, 1043 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.1 (t, J = 1.7 Hz, 1 H), 8.03 (br s, 1 H), 7.99 (s, 1 H), 7.75 (d, J = 8.0 Hz, 2 H), 7.70 (dd, J = 1.8, 8.0 Hz, 1 H), 7.24 (t, J = 8.0 Hz, 1 H), 4.60 (d, J = 6.2 Hz, 2 H), 3.05 (s, 2 H), 2.53 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 146.1, 138.2, 137.9, 131.3, 129.4, 120.6, 119.7, 94.6, 57.6, 48.9 (2 C), 34.5, 12.4 (2 C); HRMS-EI calcd for C₁₅H₂₁N₅OI [M+H]⁺: 414.0791, found: 414.0788.

2-(Diethylamino)-N-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15i)

Yield 78%: yellow oil; R_f 0.12 (MeOH/EtOAc, 1%); t_R – HPLC: 1.46 min (97.2 %); IR (film) υ_max 3346, 2967, 2932, 1670, 1609, 1506, 1254, 1157, 1043 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.03 (br s, 1 H), 7.97 (s, 1 H), 7.41 (t, J = 8.2 Hz, 1 H), 7.33 (t, J = 2.2 Hz, 1 H), 7.24 (d, J = 1.4 Hz, 1 H), 6.97 (dd, J = 2.7, 8.2 Hz, 1 H), 4.62 (d, J = 6.2 Hz, 2 H), 3.82 (s, 3 H), 3.06 (d, J = 4.4 Hz, 2H), 2.54 (q, J = 7.1 Hz, 4 H), 1.02 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 160.8, 145.8, 138.2, 130.7, 120.5, 114.9, 112.5, 106.4, 57.6, 55.9, 48.9 (2C), 34.5, 12.5 (2 C); HRMS-EI calcd for C₁₆H₂₄N₅O ₂[M+H]⁺: 318.3941, found: 318.3943.

N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15j)

Yield 80%: white solid; mp 67-69 °C; R_f 0.15 (EtOAc); t_R – HPLC: 1.47 min (99.2 %); FTIR (KBr pellet) υ_max 3342 (br), 2966 (br), 2928, 1669, 1517, 1455, 1334, 1259, 1207, 1049 (br) cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.85 (br s, 1 H), 7.36 (s, 1 H), 7.27 (m, 2 H), 7.18 (m, 1 H), 5.42 (s, 2 H), 4.43 (d, J = 6.1 Hz, 2 H), 2.92 (s, 2 H), 2.41 (q, J = 7.1 Hz, 4 H), 0.87 (t, J = 7.1 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 172.4, 145.5, 134.7, 129.2 (2 C), 128.9, 128.2 (2 C), 122.0, 57.5, 48.8 (2C), 34.4, 29.8, 12.3 (2 C); HRMS-EI calcd for C₁₆H₂₃N₅O [M+H]⁺: 302.1981, found: 302.1983.

2-(Diethylamino)-N-((1-(2,4,6-tribromophenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15k)

Yield 67%: white solid; mp 129-130 °C; R_f 0.22 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.46 min (97.1 %); FTIR (KBr pellet) υ_max 3349 (br), 2967, 2811, 1671, 1517, 1487, 1373, 1203, 1036 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.87 (br s, 1 H), 7.87 (s, 1 H), 7.72 (s, 1 H), 7.53 (s, 1 H), 4.67 (d, J = 1.1 Hz, 1 H), 3.04 (s, 1 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.97 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 145.5 135.4 (2 C), 132.6, 129.1, 125.6, 123.7, 123.4, 57.6, 49.2 (2C), 34.4, 12.6 (2 C); HRMS-ESI calcd for C₁₅H₁₉Br₃N₅O [M+H]⁺: 521.91342, found: 521.91266.

2-(Diethylamino)-N-((1-(2,4,6-trichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15l)

Yield 23%: yellow solid; mp 102-103 °C; R_f 0.74 (EtOAc/hexanes, 1:3); t_R – HPLC: 1.46 min (96.0 %); FTIR (KBr pellet) υ_max 3349 (br), 2967, 2822, 1671, 1521, 1487, 1392, 1203, 1036 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.04 (br s, 1 H), 7.70 (s, 1 H), 7.53 (s, 2 H), 4.67 (d, J = 6.2 Hz, 2 H), 2.99 (s, 2 H), 2.52 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 145.4 (2 C), 137.5, 134,6 (2 C), 129.1, 124.6, 57.7, 49.2 (2 C), 34.4, 29.9, 12.6 (2 C); HRMS-EI calcd for C₁₅H₁₉Cl₃N₅O [M+H]⁺: 390.0655, found: 390.0656.

2-(Diethylamino)-N-((1-(4-methoxy-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15m)

Yield 54%: dark red oil; R_f 0.10 (EtOAc); t_R – HPLC: 1.45 min (99.2 %); IR υ_max 3347, 2967, 2935, 1670, 1540, 1519, 1351, 1284, 1245, 1039 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.03 (br s, 1 H), 7.78 (s, 1 H), 7.56 (d, J = 2.4 Hz, 1 H), 7.46 (d, J = 8.0 Hz, 1 H), 7.25 (m, 1 H), 7.23 (d, J = 2.7 Hz, 1 H), 4.62 (d, J = 6.1 Hz, 2 H), 3.95 (s, 3 H), 3.04 (s, 2 H), 2.53 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 161.0, 145.6, 129.5, 124.4, 123.2, 119.5, 110.8, 57.6, 56.6, 49.0 (2 C), 34.5, 29.9, 12.5 (2 C); HRMS-EI calcd for C₁₆H₂₃N₆O₄ [M+H]⁺: 363.1781, found: 363.1783.

N-((1-(2-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(diethylamino)ethanamide (15n)

Yield 78%: yellow oil; R_f 0.77 (EtOAc/hexanes, 1:5); t_R – HPLC: 1.46 min (99.5 %); IR υ_max 3336, 2967, 2931, 2825, 1670, 1519, 1492, 1041 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (br s, 1 H), 7.91 (s, 1 H), 7.73 (dd, J = 1.0, 8.1, 1 H), 7.45-7.53 (app dtd, J = 1.8, 7.8, 16.0 Hz, 2 H), 7.38 (dt, J = 1.8, 7.8 Hz, 1 H), 4.65 (d, J = 6.1 Hz, 2 H), 3.1 (s, 2 H), 2.54 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.47, 144.9, 136.7, 134.1, 131.4, 128.6, 128.3, 124.4, 118.7, 57.5, 49.0 (2 C), 34.5, 12.4 (2 C); HRMS-EI calcd for C₁₅H₂₁BrN₅O [M+H]⁺: 366.0929, found: 366.0934.

2-(Diethylamino)-N-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ethanamide (15o)

Yield 32%: white solid; mp 125-127 °C; R_f 0.11 (EtOAc); t_R – HPLC: 1.47 min (98.8 %); FTIR (KBr pellet) υ_max 2968, 2931, 1682, 1605, 1509, 1473, 1419, 1231, 1128, 1043, 1006 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.84 (br s, 1 H), 7.95 (s, 2 H), 6.92 (s, 1 H), 4.59 (d, J = 6.2 Hz, 2 H), 3.91 (s, 6 H), 3.87 (s, 3 H), 3.05 (s, 2 H), 2.53 (q, J = 7.1 Hz, 4 H), 0.99 (t, J = 7.1 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 154.1, 145.8, 138.4, 133.0 (2 C), 120.8, 98.4 (2 C), 61.23, 57.6, 56.6 (2 C), 48.8 (2 C), 34.5, 12.4 (2 C); HRMS-ESI calcd for C₁₈H₂₈N₅O₄ [M+H]⁺: 378.21358, found: 378.21378.

5-(Azidomethyl)-3-(4-methoxyphenyl)isoxazole (17a)

To a solution of 3-(4- methoxyphenyl)isoxazol-5-yl)methanol (16a) (728 mg, 3.55 mmol, 1.0 equiv) and p-toluenesulfonyl chloride (682 mg, 3.58 mmol, 1.01 equiv) in dry CH₂Cl₂ (9.5 mL) was added Et₃N (0.560 mL, 4.28 mmol, 1.21 equiv) and reaction mixture was stirred at rt for 12 h, until thin-layer chromatography indicated consumption of the alcohol. The reaction was then quenched with brine (15 mL), extracted with CH₂Cl₂ (3 × 15 mL) and the combined organic extracts dried (Na₂SO₄) and concentrated under reduced pressure to provide the corresponding tosylate ester (96%). A mixture of this material (1.23 g, 3.43 mmol, 1.0 equiv) and NaN₃ (0.42 g, 6.51 mmol, 1.9 equiv) in DMF (14 mL) was then stirred at rt for 3 h, quenched with brine (30 mL) and extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with brine (2 × 15 mL), dried (Na₂SO₄) and concentrated under reduced pressure to provide 17a (829 mg, 65%); a colorless oil; R_f 0.31 (EtOAc/hexanes, 1:3); IR (film) υ_max 3129, 2103, 1613, 1431, 1253, 1177, 1028, 837 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 8.8 Hz, 2 H), 6.99 (d, J = 8.8 Hz, 2 H), 6.56 (s, 1 H), 4.50 (s, 2 H), 3.87 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.0, 162.5, 161.4, 128.5 (2 C), 114.6 (2 C), 101.3, 55.6, 45.8; MS-EI calcd for C₁₁H₁₁N₄O₂ [M+H]⁺: 230.2248, found: 231.0882.

1-(3-(4-Methoxyphenyl)isoxazol-5-yl)methaneamine (18a)

To a solution of 5-(Azidomethyl)-3-(4-methoxyphenyl)isoxazole 17a (160 mg, 0.695 mmol, 1.0 equiv) in dry THF (2 mL) at 0 °C was added PPh₃ (192 mg, 0.730 mmol, 1.1 equiv) portionwise over 5 min. After stirring at rt for 12 h reaction mixture was quenched with H₂O (5 mL) and extracted with Et₂O (3 × 15 mL). The combined organic layers ware washed with brine (2 × 10 mL), dried (Na₂SO₄), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography over silica gel (EtOAc) to provide 18a (158 mg, 77%): white solid; mp 184 °C, R_f 0.10 (EtOAc); t_R – HPLC: 20.8 min (THF, 97.9 %); IR (film) υ_max 3240 (br), 3058, 1591, 1437, 1333, 1265, 1158, 909, 814 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 8.8 Hz, 2 H), 6.94 (d, J = 8.8 Hz, 2 H), 6.37 (s, 1 H), 3.97 (s, 2 H), 3.82 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 174.3, 162.1, 161.0, 128.2 (2 C), 121.7, 114.4 (2 C), 98.5, 55.4, 38.5; MS-EI calcd for C₁₁H₁₂N₂O₂ [M+H]⁺: 205.0972, found: 205.0970.

1-(3-(4-Bromophenyl)isoxazol-5-yl)methaneamine (18b)

To a solution of 5-(Azidomethyl)-3-(4-bromophenyl)isoxazole (393 mg, 0.154 mmol, 1.0 equiv) in dry THF (2 mL) at 0 °C was added PPh₃ (125 mg, 0.476 mmol, 1.1 equiv) portionwise over 3 min. After stirring at rt for 12 h reaction mixture was quenched with H₂O (5 mL) and extracted with Et₂O (3 × 15 mL). The combined organic layers ware washed with brine (2 × 10 mL), dried (Na₂SO₄), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography over silica gel (EtOAc) to provide 18b (218 mg, 86%): a colorless liquid; R_f 0.43 (EtOAc/hexanes, 1:1); t_R – HPLC: 20.9 min (THF, 20.8 %); IR (film) υ_max 3236 (br), 3018, 1601, 1402, 1333, 1265, 1158, 886 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J = 8.8 Hz, 2 H), 7.53 (d, J = 8.8 Hz, 2 H), 6.55 (s, 1 H), 4.92 (br s, 2 H), 4.83 (s, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.3, 148.1, 144.3, 129.9 (2 C), 128.2, 126.2 (2C), 101.1, 57.2; MS-EI calcd for C10H₉BrN₂O₂ [M+H]⁺: 253.1800, found: 253.1809.

tert-Butyl(S)-1-((3-(4-methoxyphenyl)isoxazol-5-yl)methylcarbamoyl)-2-phenyl ethylcarbamate (20a)

Yield 84%: white solid; mp 146-148 °C; R_f 0.51 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.44 min (95.6 %); IR (film) υ_max 3296 (br), 2923, 1662, 1612, 1529, 1432, 1366, 1253, 1174, 1029 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.71 (d, J = 8.8 Hz, 2 H), 7.27 (m, 4 H), 7.17 (m, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 6.45 (s, 1 H), 6.32 (s, 1 H), 4.99 (s, 1 H), 4.52 (d, J = 6.0 Hz, 1 H), 3.87 (s, 3 H), 3.10 (m, 2 H), 1.41 (s, 9 H); ¹³C NMR (125 MHz, CDCl₃) δ 171.6, 168.6, 162.3, 161.2, 136.5 (3 C), 129.0 (2 C), 129.1 (2 C), 128.4 (2 C), 127.3, 121.5, 114.5 (2 C), 100.4, 56.1, 55.6, 38.4, 35.4, 28.5 (3 C); HRMS-ESI calcd for C₂₅H₂₉N₃O₅ [M+H]⁺: 474.19997, found: 474.20001.

(S)-2-Amino-N-((3-(4-methoxyphenyl)isoxazol-5-yl)methyl)-3-phenylpropaneamide (20b)

To a tert-butyl (S)-1-((3-(4-methoxyphenyl)isoxazol-5-yl)methylcarbamoyl)-2-phenyl ethylcarbamate (20a) (53 mg, 0.117 mmol) was added mixture of TFA and H₂O (9:1, 1.5 mL). After 10 min, EtOH (3 mL) was added and reaction was concentrated under reduced pressure to provide 20b (36 mg, 88%): white solid; [α]_D −26.1 (c = 0.52, MeOH); mp 166-170 °C; R_f 0.11 (EtOAc); t_R – HPLC: 1.46 min (100 %); FTIR (KBr pellet) υ_max 2920, 2851, 1583, 1652, 1558, 1456, 1134 cm⁻¹; ¹H NMR (400 MHz, CD₃OD) δ 7.71 (d, J = 8.9 Hz, 2 H), 7.26 (m, 5 H), 7.02 (d, J = 8.9 Hz, 2H), 6.49 (s, 1 H), 4.58 (d, J = 16.0 Hz, 1 H), 4.45 (d, J = 16.0 Hz, 1 H), 4.08 (t, J = 7.3 Hz, 1 H), 3.84 (s, 3 H), 3.17 (dd, J = 7.4, 13.8 Hz, 1 H), 3.08 (dd, J = 7.4, 13.8 Hz, H); ¹³C NMR (100 MHz, CDCl₃) δ 170.6, 169.8, 164.5, 163.8, 163.2, 135.6, 130.6 (2 C), 130.2 (2 C), 129.4 (2 C), 129.0, 122.5, 115.6 (2 C), 101.6, 56.0, 38.8, 36.0; HRMS-EI calcd for C₂₀H₂₂N₃O₃ [M+H]⁺: 352.4070, found: 352.4073.

Benzyl (S)-1-((3-(4-methoxyphenyl)isoxazol-5-yl)methylcarbamoyl)-2-methylpropyl carbamate (20c)

Yield: 48% as a colorless oil; R_f 0.26 (EtOAc/hexanes, 1:3); t_R – HPLC: 1.60 min (99.8 %); IR (film) υ_max 3348 (br), 2965, 1722, 1613, 1529, 1432, 1253, 1028, 837 cm⁻¹; ¹ H NMR (500 MHz, CDCl₃) δ 7.73 (d, J = 8.8 Hz, 2 H), 7.36 (m, 3 H), 7.32 (m, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 6.58 (s, 1 H), 5.32 (d, J = 13.7 Hz, 2 H), 5.24 (d J = 13.7 Hz, 2 H), 5.12 (s, 2 H), 4.37 (m, 1 H), 3.86 (s, 3 H), 2.20 (m, 1 H), 1.56 (s, 1 H), 0.97 (d, J = 6.85 Hz, 3 H), 0.88 (d, J = 6.85 Hz, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 171.7, 166.4, 162.4, 161.3, 156.4, 128.8 (2 C), 128.4 (5 C), 121.2, 114.5 (2 C), 102.4, 67.4, 59.2, 57.1, 55.6, 31.4, 19.2, 17.7; HRMS-ESI calcd for C₂₄H₂₈N₃O₅ [M+H]⁺: 438.4962, found: 438.4960.

(S)-Benzyl 2-((3-(4-methoxyphenyl)isoxazol-5-yl)methylcarbamoyl)pyrrolidine-1-carboxylate (20d)

Yield 55%: colorless oil; [α]_D −38.1 (c = 0.40, MeOH); R_f 0.13 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.60 min (95.9 %); IR (film) υ_max 3315 (br), 2953 (br), 1700, 1612, 1529, 1431, 1356, 1253, 1176, 1118, 1028 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 6.9 Hz, 2 H), 7.34 (m, 5 H), 6.94 (d, J = 6.9 Hz, 2 H), 6.44 (s, 1 H), 5.17 (s, 2 H), 4.57 (m, 2 H), 4.40 (m, 1 H), 3.85 (s, 3 H), 3.53 (m, 2 H), 3.46 (m, 1 H), 2.19 (s, 1 H), 1.83-1.86 (m, 4 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 169.4, 162.4, 161.2. 156.7, 136.4, 128.8 (2 C), 128.4 (2 C), 128.2 (2 C), 121.7, 114.5 (2 C), 100.2, 67.8, 60.8, 55.6, 47.4, 41.2, 35.7, 28.3, 25.8; HRMS-EI calcd for C₂₄H₂₆N₃O₅ [M+H]⁺: 436.1867, found: 436.1856.

(S)-2-(Acetylamino)-3-mercapto-N-((3-4(methoxyphenyl)isoxazol-5-yl)methyl)) propanamide (20e)

Yield 37%: white solid; mp 112 °C; R_f 0.51 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.60 min (95.4 %); IR (film) υ_max 2922, 1671, 1612, 1528, 1431, 1253, 1177, 1028 cm⁻¹; ¹ H NMR (500 MHz, CDCl₃) δ 7.91 (m, 1 H), 7.73 (d, J = 8.8 Hz, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 6.45 (s, 1 H), 6.41 (s, 1 H), 4.68 (s, 2 H), 4.02 (s, 2 H), 3.86 (s, 3 H), 3.84 (s, 3 H), 2.07 (d, J = 4.9 Hz, 2 H); ¹³C NMR (125 MHz, CDCl₃) δ 174.3, 170.6, 165.6, 162.3, 161.1, 128.4 (2 C), 121.8, 114.5 (2 C), 100.2, 98.7, 56.2, 55.6, 38.6, 22.7; HRMS-EI calcd for C₂₅H₂₉N₃O₅ [M+H]⁺: 350.1117, found: 350.1108.

tert-Butyl(S)-1-((3-(4-methoxyphenyl)isoxazol-5-yl)methylcarbamoyl)-2-((benzyloxy)carbonyl)ethylcarbamate (20g)

Yield 57%: white solid; [α]_D −5.0 (c = 0.46, MeOH); mp 86 °C; R_f 0.26 (EtOAc/hexanes, 1:3); t_R – HPLC: 1.61 min (97.6 %); IR (film) υ_max 3375 (br), 2975, 1717, 1614, 1498, 1253, 1163, 1028 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.71 (d, J = 8.8 Hz, 2 H), 7.32 (m, 5 H), 6.95 (d, J = 8.8 Hz, 4 H), 6.55 (s, 1 H), 5.55 (d, J = 8.7 Hz, 1 H), 5.21 (d, J = 2.2 Hz, 2 H), 5.10 (s, 2 H), 4.67 (m, 1 H), 3.84 (s, 3 H), 3.09 (dd, J = 4.6, 17.2 Hz, 1 H), 2.90 (dd, J = 4.6, 17.2 Hz, 1 H), 1.43 (s, 9 H); ¹³C NMR (125 MHz, CDCl₃) δ 170.8, 170.6, 166.3, 162.3, 161.2, 155.5, 135.4, 128.7 (5 C), 128.5 (2 C), 114.5 (2 C), 102.3, 80.5, 67.1, 60.5, 57.6, 55.5, 50.1, 36.9, 28.4 (3 C); HRMS-EI calcd for C₂₇H₃₁N₃O₇ [M+H]⁺: 510.5589, found: 510.5598.

Isobutyl((3-(4-methoxyphenyl)isoxazol-5-yl)methyl)carbamate (20h)

To a stirred solution of isobutyl chloroformate (31 μL, 0.183 mmol, 1.01 equiv) in anhydrous CH₂Cl₂ (2 mL) under an atmosphere of N₂ at −40 °C (MeCN/CO₂) and Et₃N (17 μL, 0.182 mmol, 1 equiv) was added 5-aminomethyl-3(4-methoxyphenyl)isoxazole (18b) (159 mg, 0.182 mmol, 1 equiv). After stirring for 10 min the cold bath was removed, reaction mixture was warming up to rt and stirred for 12 h. Reaction was quenched with 1M HCl and the aqueous portion was extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried (Na₂SO₄) filtered and concentrated under reduced pressure. The residue was purified by flash chromatography over silica gel (EtOAc/hexanes, 1:5) to provide 20h (52 mg, 93%): colorless oil; R_f 0.42 (EtOAc/hexanes, 1:5); t_R – HPLC: 1.61 min (97.2 %); IR (film) υ_max 2962, 1751, 1614, 1432, 1253, 1178, 970 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 8.8 Hz, 2 H), 6.96 (d, J = 8.8 Hz, 2 H), 6.62 (s, 1 H), 5.26 (s, 2 H), 3.97 (d, J = 6.6 Hz, 2 H), 3.86 (s, 3 H), 2.17 (s, 2 H), 1.99 (m, 1 H), 1.59 (s, 6 H), 0.95 (d, J = 6.9 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 161.3, 154.8, 129.8, 128.5 (2 C), 121.1, 114.6 (2 C), 102.4, 75.0, 59.7, 55.6, 28.0, 19.1 (2 C); HRMS-EI calcd for C₁₆H₂₁N₂O₄ [M+H]⁺: 305.3489, found: 305.3493.

2-Bromo-N-((3-(4-methoxyphenyl)isoxazol-5-yl)methyl)ethaneamide (20i)

Yield 36%: colorless oil; R_f 0.20 (EtOAc/hexanes, 1:5); t_R – HPLC: 1.62 min (99.2 %); IR (film) υ_max 2961, 1760, 1614, 1431, 1257, 1023, 802 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 8.6 Hz, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 6.61 (s, 1 H), 5.31 (s, 2 H), 4.15 (s, 2 H), 3.86 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 166.9, 165.9, 162.4, 161.3, 128.4 (2 C), 121.1, 114.5 (2 C), 102.8, 57.8, 55.5, 40.7; HRMS-EI calcd for C₁₃H₁₄BrN₂O₃ [M+H]⁺: 326.1659, found: 326.1661.

N-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)ethanamide (20j)

Yield: 88% as a white solid; mp 168-169 °C, R_f 0.41 (EtOAc); t_R – HPLC: 1.61 min (98.5 %); FTIR (KBr pellet) υ_max 3271, 1651, 1558, 1423, 1295, 1224, 1101, 1028, 825 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 8.5 Hz, 2 H), 7.58 (d, J = 8.5 Hz, 2 H), 7.27 (s, 1 H), 6.50 (br s, 1 H), 4.60 (d, J = 6.0 Hz, 2 H), 2.07 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 169.8, 162.0, 132.4(2 C), 128.5(2 C), 127.9, 124.7, 100.6, 34.5, 23.3; MS-EI calcd for C₁₂H₁₀Br₂N₂O₂ [M+H]⁺: 375.0359, found: 375.0364.

N-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)propanamide (20k)

Yield: 86% as a white solid; mp 171-172 °C, R_f 0.19 (EtOAc); t_R – HPLC: 1.60 min (98.9 %); FTIR (KBr pellet) υ_max 3207, 1657, 1524, 1360, 828, 725 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (d, J = 8.5 Hz, 2 H), 7.57 (d, J = 8.5 Hz, 2 H), 6.48 (s, 1 H), 6.17 (br s, 1 H), 4.59 (d, J = 5.9 Hz, 2 H), 2.28 (q, J = 7.1 Hz, 2 H), 1.84 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.0, 169.9, 161.9, 132.4, 128.5, 127.9, 124.6, 100.5, 35.3, 29.6, 9.8; MS-EI calcd for C₁₃H₁₃BrN₂O₂ [M+H]⁺: 310.1665, found: 310.1658.

N-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)-2-methylpropanamide (20l)

Yield 86%: yellow oil; R_f 0.43 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.46 min (99,2 %); IR (film) υ_max 3287, 1648, 1548, 1424, 1222, 826 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 8.5 Hz, 2 H), 7.59 (d, J = 8.5 Hz, 2 H), 6.48 (s, 1 H), 6.48 (br s, 1 H), 4.60 (d, J = 6.0 Hz, 2 H), 2.43 (m, 1 H), 2.19 (d, J = 6.9 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 177.2, 170.0, 161.7, 132.4 (2 C), 128.5 (2 C), 127.9, 124.6, 100.5, 35.7, 35.4, 19.7 (2 C); HRMS-EI calcd for C₁₄H₁₅BrN₂O₂ [M+H]⁺: 323.1851, found: 323.1895.

N-3-(4-Bromophenyl)isoxazol-5-yl)benzamide (20m)

Yield 90%: white solid; mp 166-168 °C; R_f 0.51 (EtOAc/hexanes, 1:1); t_R – HPLC: 1.60 min (95.6 %); FTIR (KBr pellet) υ_max 3357, 3262, 1644, 1532, 1306, 1159, 816 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.81 (m, 3 H), 7.65 (m, 2 H), 7.58 (m, 2 H), 7.50 (d, J = 8.2 Hz, 2 H), 7.46 (m, 2 H), 7.31 (d, J = 8.2 Hz, 2 H), 6.77 (br s, 1 H), 6.57 (s, 1 H), 4.82 (d, J = 6.0, 2 H), 2.44 (s, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 177.2, 170.0, 161.7, 132.4 (5H), 128.8 (2C), 128.5 (2C), 127.9, 124.6, 100.5, 57.7, 35.4; HRMS-EI calcd for C₁₇H₁₄BrN₂O₂ [M+H]⁺: 358.2093, found: 358.2090.

2-Bromo-N-((3-(4-bromophenyl)isoxazol-5-yl)methyl)ethanamide (20n)

Yield 73%: yellow oil; R_f 0.11 (EtOAc/hexanes, 1:5); t_R – HPLC: 1.52 min (100 %); IR (film) υ_max 3296 (br), 1647, 1544, 1420, 1263, 1221, 1027 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.65 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.13 (br s, 1H), 6.53 (s, 1H), 4.67 (d, J = 6.1 Hz, 2H), 4.13 (s, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 168.8, 132.4 (2 C), 128.5 (2 C), 127.8, 100.9, 42.6, 35.7; HRMS-EI calcd for C₁₂H₁₁Br₂N₂O₂ [M+H]⁺: 372.9187, found: 372.9190.

(3Z)-4-Amino-4-(4-methoxyphenyl)but-3-en-2-one (21) and 3-(4-Methoxyphenyl)-5-methylisoxazole (22)

A round-bottom flask was flashed with N₂ and charged with benzyl 2-((3-(4-methoxyphenyl)isoxazol-5-yl)methylamino)-2-oxoethylcarbamate (20f) (21 mg, 0.055 mmol), 5% Pd/C (4 mg, 0.03 mmol) and EtOAc (2.0 mL) then flushed with N₂ and placed under an atmosphere of H₂ (1 atm). After stirring for 3 h at rt, the flask was flashed with N₂ and the reaction mixure filtered through a plug of Celite 521. After thoroughly washing the filter cake with MeOH (25 mL), the combined filtrates were concentrated under reduced pressure and the residue purified by flash chromatography over silica gel (EtOAc/hexanes, 1:1) to afford a mixture of (3Z)-4-amino-4-(4-methoxyphenyl)but-3-en-2-one (21) (2.2 mg, 21 %) and 3-(4-methoxyphemyl)-5-methylisoxazole (22) (4.8 mg, 48 %). Analytical data for 21: white solid; mp 81-83°C (lit⁴⁰ 81-84 °C), R_f 0.66 (EtOAc); t_R – HPLC: 1.48 min (100 %); ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J =6.8 Hz, 2 H), 6.94 (d, J = 6.8 Hz, 2 H), 5.43 (s, 1 H), 3.85 (s, 3 H), 2.15 (s, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 197.4, 161.8, 160.8, 129.4, 127.9 (2 C), 114.5 (2 C), 94.7, 55.6, 30.0. Analytical data for 22: white solid; mp 92 °C (lit⁴¹ 92-93 °C); R_f 0.84 (EtOAc); t_R – HPLC: 1.47 min (97.6 %); ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 8.8 Hz, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 6.24 (s, 1 H), 3.86 (s, 3 H), 2.47 (s, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 169.8, 162.4 161.1, 128.4 (2 C), 122.1, 114.5 (2 C), 99.7, 55.6, 12.6.

N-Allyl-2-(diethylamino)acetamide (24)

Yield 80% colorless oil; R_f 0.34 (EtOAc); IR (film) υ_max 3353, 2969, 2934, 2874, 2821, 1677, 1515, 1455, 1205, 1163, 1088, 990, 916 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.48 (br s, 1 H), 5.80 (m, 1 H), 5.12 (dq, J = 1.5, 17 Hz, 3 H), 5.07 (dq, J = 1.3, 10.4 Hz, 1 H), 3.85 (m, 2 H), 3.00 (s, 2 H), 2.51 (q, J = 7.1 Hz, 4 H), 0.98 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 134.5, 115.9, 57.5, 48.8 (2 C), 41.2, 12.4 (2 C); HRMS-EI calcd for C₉H₁₈N₂O [M+H]⁺: 170.1419, found: 170.1415.

Prop-2-yn-1-yl(diethylamino) acetate (25)

Yield 58%: colorless oil; R_f 0.23 (EtOAc); IR (film) υ_max 3291, 2971, 2844, 1750, 1652, 1456, 1386, 1157, 1008 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 4.70 (d, J = 2.5 Hz, 2 H), 3.37 (s, 3 H), 2.65 (q, J = 7.2 Hz, 4 H), 2.47 (t, J = 2.4 Hz, 1 H), 1.05 (t, J = 7.2 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 170.8, 75.1, 60.6, 54.0, 51.9, 47.9 (2 C), 14.4 (2 C); HRMS-EI calcd for C₉H₁₆NO₂ [M+H]⁺: 170.2288, found: 170.2292.

2-(Diethylamino)-N-prop-2-yn-1-ylethanthioamide (26)

A solution of N’-propargyl-N,N-diethylglycine (13) (222 mg, 1.6 mmol, 1.6 equiv) and Lawesson’s reagent (320 mg, 1.0 mmol, 1 equiv) in 6.5 ml of anhydrous THF was heated at reflux for 12 h. The resulting reaction mixture was cooled down to rt and concentrated under reduced pressure. Crude product was purified by flash chromatography over silica gel (EtOAc/hexanes, 1:1) give 26 (126 mg, 87%) as yellow oil: R_f 0.60 (EtOAc); IR (film) υ_max 3208, 2960, 2836, 1683, 1595, 1499, 1461, 1293, 1251, 1135, 1027, 925, 831, 733 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 9.60 (br s, 1 H), 4.47 (s, 2 H), 3.50 (s, 3 H), 2.56 (q, J = 7.2 Hz, 4 H), 2.31 (t, J = 2.6 Hz, 1 H), 1.01 (t, J = 7.2 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 201.5, 77.9, 72.8, 65.7, 47.8 (2 C), 34.4, 12.6 (2 C); HRMS-EI calcd for C₉H₁₇N₂S [M+H]⁺: 185.3097, found: 185.3096.

2-(Diethylamino)-N-(3-(4-methoxyphenyl)4,5-dihydroisoxazol-5-yl)methyl)ethanamide (27)

Yield 75%: colorless oil; R_f 0.14 (EtOAc); t_R – HPLC: 1.45 min (98.7 %); IR (film) υ_max 2967, 2927, 1672, 1608, 1515, 1354, 1252, 1176, 898, 832 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.84 (br s, 1 H), 7.57 (d, J = 3.2 Hz, 2 H), 7.26 (d, J = 3.2 Hz, 2 H), 4.85 (m, 1 H), 3.84 (s, 3 H), 3.60 (m, 2 H), 3.37 (dd, J = 10.7, 16.8 Hz, 1 H), 3.10 (dd, J = 10.7, 16.8 Hz, 1 H), 3.03 (d, J = 17.1 Hz, 1 H), 2.96 (d, J = 17.1 Hz, 1 H), 2.97 (q, J = 7.2 Hz, 4 H), 0.94 (t, J = 7.2 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃) δ 173.2, 161.3, 156.5, 128.4 (2 C), 122.0, 114.3 (2 C), 79.8, 57.5, 55.6, 49.0 (2 C), 42.0, 37.9, 12.5 (2 C); HRMS-EI calcd for C₁₇H₂₆N₃O₃ [M+H]⁺: 320.1974, found: 320.1976.

(3-(4-Methoxyphenyl)isoxazol-5-yl)methyl(diethylamino) acetate (28)

Yield 79%: colorless oil; R_f 0.44 (EtOAc); t_R – HPLC: 1.46 min (98.2 %); IR (film) υ_max 3370 (br), 2967, 2934, 1752, 1613, 1529, 1431, 1253, 1177, 1028, 837 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 8.6 Hz, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 6.58 (s, 1 H), 5.25 (s, 2 H), 3.86 (s, 3 H), 3.42 (s, 2 H), 2.67 (q, J = 7.2 Hz, 4 H), 1.07 (t, J = 7.2 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 166.9, 162.4, 161.3, 129.8, 128.4 (2 C), 114.5 (2 C), 102.4, 99.9, 55.6, 54.1, 47.9 (2 C), 12.4 (2 C); HRMS-EI calcd for C₁₇H₂₃N₂O₄ [M+H]⁺: 319.1658, found: 319.1657.

2-(Diethylamino)-N-((3-(4-methoxyphenyl)isoxazol-5-yl)ethanethioamide (29)

Yield 54%: yellow oil; R_f 0.67 (EtOAc); t_R – HPLC: 1.45 min (97.1 %); IR (film) υ_max 2918, 2849, 1611, 1528, 1431, 1383, 1254, 1177, 1114, 1028 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 10.05 (br s, 1 H), 7.73 (d, J = 8.8 Hz, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 6.55 (s, 1 H), 5.10 (s, 2 H), 3.86 (s, 3 H), 3.60 (s, 2 H), 2.62 (q, J = 7.1 Hz, 4 H), 1.04 (t, J = 7.1 Hz, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 167.1, 162.5, 161.3, 128.4 (2 C), 121.4, 14.6 (2 C), 101.2, 55.6, 48.8 (2 C), 40.2, 29.9, 12.5 (2 C); HRMS-EI calcd for C₁₇H₂₃N₃O₂S [M+H]⁺: 334.4564, found: 334.4568.

Biology

Cell Lines and Antibodies

293T Human embryonic kidney cells were maintained in Dulbecco’s modified Eagle’s medium (MEM) supplemented with 10% fetal calf serum with penicillin and streptomycin plus 300 g of Geneticin per mL. HeLa cells were maintained in the same medium without Geneticin. A549 Human alveolar adenocarcinoma cells were maintained in Dulbecco’s modified Eagle’s medium (MEM) supplemented with 10% fetal calf serum. The mouse monoclonal antibody 12B5-1-1, which recognizes the GP1 of Ebola virus Zaire (EBOZ) GP, was kindly provided by Mary K. Hart (U.S. Army Medical Research Institute of Infectious Diseases).⁴² The mouse anti-HIV p24 monoclonal antibody was obtained from the National Institutes of Health AIDS Research and Reference Reagent program.⁴³

Preparation of Pseudotyped Viruses

To produce Ebola virus GP pseudotyped HIV virions, 293T producer cells were co-transfected with HIV vector pNL4-3-Luc-R--E-(National Institutes of Health AIDS Research and Reference Reagent program; catalogue number 3418)^37,38 and the DNA of wild-type (wt) Ebola virus Zaire glycoprotein (wt Ebola-GP), which was synthesized by multiple rounds of overlapping PCR based on the EBOZ genome sequence (GenBank accession number L11365). In the pseudotyping experiments, 2 μg DNA of wt Ebola-GP and 2 μg of pNL4-3-Luc-R-E were used to transfect 293T cells (90% confluent) in six-well plates by Lipofectamine 2000 according to the protocol of the supplier (Invitrogen). The supernatants containing the pseudotyped viruses were collected twice (at 24 hours and 48 hours posttransfection), combined, clarified from floating cells and cells debris by low-speed centrifugation, and filtered through a 0.45 μm-pore-size filter (Nalgene). Then, 1 mL of supernatant was used to infect cells, and the rest stored at −80° C for future use.

The plasmid vector expressing the GP of Marburg (Lake Victoria strain) was prepared as previously described.⁴⁴ Marburg virus GP (Lake Victoria strain) and VSV-G pseudotyped HIV virions where produced in a similar manner, i.e. through co-transfection with pNL4-3-Luc-R-E-, according to previous published procedures.⁴⁴

Western Blotting

To evaluate EBOZ GP expression, the 293T producer cells were lysed in 0.2 mL of Triton X-100 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and a protease inhibitors cocktail consisting of 10 g of leupeptin per mL, 5 g of aprotinin per mL, and 2 mM phenylmethylsulfonyl fluoride) at 48 h after cotransfection. The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membrane was first incubated with anti-EBOZ GP1 monoclonal antibody 12B5-1-1(1:5000 dilution) for 1 h and then probed with peroxidase-conjugated goat anti-mouse antiserum (Pierce) for 1 h. The bands were visualized by the chemiluminescence method according to the protocol of the supplier (Pierce).

Detection of GP Incorporation in Pseudotyped Viruses

To evaluate the incorporation of wt GP protein into the pseudotyped viruses, 2 mL of pseudotyped virus was layered onto a 3-mL cushion of 20% (wt/vol) sucrose in phosphate-buffered saline and centrifuged at 55,000 rpm for 30 min in a Beckman SW41 rotor. The pelleted pseudotyped viruses were lysed in 50 μlL of Triton X-100 lysis buffer, and a 25-μL sample was subjected to SDS-PAGE. Expression of the EBOZ GP protein was detected by Western blotting as described above. A mouse anti-HIV p24 monoclonal antibody (1:5,000 dilution) was used as the primary antibody to detect the HIV p24 protein.

Assay of Infectivity of Pseudotyped Viruses

Human 293T, A549, or Hela cells (3×10⁵ cells) were seeded in six-well plates one day prior to infection. These targeted cells were incubated with 1 mL of the pseudotyped viruses for 5 h. The cells were then lysed in 200 μlL of cell culture lysis reagent (Promega) at 48 h post-infection. The luciferase activity was measured using a luciferase assay kit (Promega) and an FB12 luminometer (Berthold detection system) according to the supplier’s protocol. Each experiment was done in triplicate and repeated at least three times.

Compound Blocking Assay

Pseudotype viruses were incubated with individual compounds at different concentration (for initial screening, concentrations or 30-60 μM were employed) briefly at room temperature. This mixture was then added to 293T, A549, or HeLa cells which were seeded one day prior to the assay in poly-L-lysine-treated 24-well plates. After overnight incubation, the supernatant was replaced with fresh media. The cells were lysed in 100 μL of cell culture lysis reagent (Promega) 48 hours post-infection. The luciferase activity was measured using a luciferase assay kit (Promega) and an FB12 luminometer (Berthold detection system), according to the supplier’s protocol.

Figure 6.

Inhibition of Marburg GP-mediated infection. Shown are the effects of compounds 8a, 8j, 15b and 15k (final concentrations, 60 μM) on the infectivity of Marburg GP pseudotyped HIV virions with 293T (■), HeLa (□), and A549 () cell lines. To determine the level of infection by pseudotype viruses, cells were challenged with pseudotypes in the presence and absence of test compounds. After 48 h, luciferase expression was assessed using the method described in the Experimental Section. Infectivity is expressed as a percentage of luciferase activity, relative to the DMSO control. Values are reported as mean of data from three independent experiments, ± standard error.

Abbreviations

EBOV

Ebola virus

EboZ

Ebola Zaire virus

MARV

Marburg virus

VSV

vesicular stomatitis virus

MLV

murine leukemia virus

HIV

human immunodeficiency virus

GP

glycoprotein

IFN-α

interferon alpha

CatB

cathepsin B

CatL

cathepsin L.

env

viral envelope glycoprotein

IC₅₀

half maximal inhibitory concentration

SAH

S-adenosylhomocysteine hydrolase