Discovery of Coronavirus Main Protease Inhibitors with Enhanced Brain Exposure and Potent Oral Efficacy in SARS-CoV-2 and MERS Infection Models

Abstract

The main proteases (M^Pro) of coronaviruses are clinically validated targets for antiviral discovery. Herein, we detail the in vivo optimization of uracil-core M^Pro inhibitors derived from AVI-4516, an in vivo active lead bearing an un-activated propargyl warhead. To expand anti-coronaviral spectrum we introduced diverse C6 substitution to target the S1’ pocket in M^Pro and observed enhanced cellular activity against various nirmatrelvir-resistant mutants. Pharmacokinetic profiling of twelve analogs revealed overall inferior exposure of the C6 aryl analogs. However, PK profiling across three species identified the improved atropisomeric lead (M)-AVI-4773 (5-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-((M)-isoquinolin-4-yl)-6-methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione), which exhibits rapid-onset oral efficacy in both SARS-CoV-2 and middle east respiratory syndrome (MERS) mouse models, highlighting a promising chemotype with the potential to deliver anti-coronaviral development candidates.

Graphical Abstract

INTRODUCTION

A lasting impact of the COVID-19 pandemic has been to emphasize the need for broad-spectrum antiviral agents to complement the development of pathogen-specific vaccines. Among the most promising coronaviral drug targets with broad-spectrum potential are the coronavirus main proteases (M^Pro or 3CLPro), cysteine proteases that are critical for viral replication and are highly conserved across coronavirus families.^1,2 In SARS-CoV-2, the main protease together with the papain-like protease PL^Pro,³ coordinate viral maturation by cleaving and releasing the various non-structural proteins (nsps) from the polyproteins pp1a pp1ab.

Currently, the only FDA-approved M^Pro inhibitor is nirmatrelvir, marketed as Paxlovid^® (Pfizer) and formulated with ritonavir to block CYP450 mediated metabolism and thus extend the in vivo half-life of nirmatrelvir in patients.⁴ Accordingly, the drug can elicit drug-drug interactions in patient populations such as the elderly or immunocompromised, who paradoxically are also the most likely to suffer severe disease from SARS-CoV-2 infection. Moreover, clinical use of nirmatrelvir has spurred the emergence of several nirmatrelvir-resistant strains of SARS-CoV-2 bearing M^Pro mutation(s) that lead to reduced susceptibility.^5–7 There is thus a clear medical need for improved oral agents that overcome these liabilities.

Meanwhile, the MERS-CoV virus, the causative agent of middle east respiratory syndrome, continues to circulate in dromedary camels and causes sporadic zoonotic outbreaks with a high case fatality rate (~35%).⁸ The potential for future spillover of this deadly virus, combined with the fact that nirmatrelvir exhibits markedly reduced activity against MERS-CoV^9,10 highlights a critical gap in coronavirus pandemic preparedness.

Numerous efforts have been undertaken to identify new M^Pro-targeting antivirals.¹¹ Broadly, these efforts have focused either on peptidomimetic chemotypes^12–16 similar to nirmatrelvir, or non-peptidic scaffolds, typically with two or three side-chains projecting from a central cyclic core (Figure 1).^17–21 An exemplar of the latter is ensitrelvir (Shinogi) which is approved in Japan and Singapore.^22,23 Unlike nirmatrelvir, ensitrelvir does not require coadministration of a CYP inhibitor, although its potential for drug-drug interactions has nevertheless been observed in patients.²⁴

Figure 1.

A) The structures of selected clinical and preclinical stage M^Pro inhibitors. The highlighted atoms form specific polar contacts with the indicated active-site M^pro residues (see legend). B) Complex X-ray structure of AVI-4516 bound to SARS-CoV2 M^Pro (PDB: 9MVP) showing important polar contacts as yellow dashes, including the contacts noted in panel A, and also showing the covalent adduct formed with the catalytic Cys145. C) Complex X-ray structure of the C6-chloroaryl analog AVI-4692 bound to M^Pro (PDB: 9MVO) revealing additional contact between the C6 aryl substituent and the S1’ pocket.

In addition to an enhanced antiviral spectrum and safety profile, next-generation anti-coronaviral agents should ideally possess sufficient CNS penetrance to achieve antiviral effects in the brain. It is known that SARS-CoV-2 can persist in the brains of formerly infected patients for months after acute infection.²⁵ Whether this constitutes a viral reservoir involved in rebound infection or post-acute sequelae of SARS-CoV-2 (long COVID) remains controversial. Whatever the case, the currently approved agents possess limited brain exposure, making them poorly suited as tools to study these questions in either the preclinical or clinical setting.

Herein, we describe structure-guided optimization of the covalent, uracil-core lead compound AVI-4516 (Figure 1A). This effort was largely focused on the C6 position, which targets the S1’ subsite of Mpro enzymes. Extensive profiling in biochemical, in vitro ADME panels, cellular assays in viral replicons and live virus assays, and pharmacokinetic (PK) studies in rodents and dog culminated in the identification of (M)-AVI-4773, a single-atropisomer lead compound that confers rapid-onset oral efficacy in SARS-CoV-2 and MERS-CoV infection models.

RESULTS

Recently, we described²⁶ the orally efficacious M^Pro inhibitor lead AVI-4516 (Figure 1A), which was developed from a dihydrouracil hit identified in an ultra-large library docking screen.²⁷ The complex X-ray structure of AVI-4516 revealed key hydrogen bonding contacts with the backbone of E166 and G143, and with the sidechain of His163, key interactions formed by most all potent M^pro inhibitors (Figure 1A). These contacts serve to anchor the uracil core near the catalytic machinery (Cys145 and His41), where proximity to the propargyl sidechain leads to covalent reactivity with Cys145, as evidenced both in the complex X-ray structure (Figure 1B) and confirmed in various biochemical and biophysical analyses.²⁶ The synthetically accessible uracil core provides a convenient vector toward the S1’ pocket, as illustrated with the early C6-chloroaryl analog AVI-4692 (Figure 1C). Most recently, and subsequent to our initial report, a group from Shinogi described the uracil-core drug candidate S-892216,²⁸ (Figure 1A) which employs a nitrile warhead to target Cys145.

The SAR campaign leading to AVI-4516 was initially focused on reversible non-covalent inhibition. However, once the potent and selective reactivity of AVI-4516 was established (and with minimal inhibition of mammalian proteases²⁶) the goal shifted entirely to covalent inhibition. To better understand the requirements for reactivity with Cys145, we evaluated a focused library of AVI-4516 analogs bearing electrophilic and non-electrophilic N1 side chains related to N-propargyl, or bearing other well-established cysteine warheads (Table 1). Thus, cyanomethyl congener AVI-4689 retained reasonable activity (M^Pro IC₅₀ ~160 nM) that was nevertheless ~10-fold weaker than AVI-4516. Methylation of the propargyl group (AVI-4774), extension to homopropargyl (AVI-5764), reduction to N-allyl (AVI-4690) or introduction of an epoxide (AVI-4770) all yielded weak or inactive analogs, presumably due to lack of covalent engagement. The keto acid analog AVI-5854 on the other hand showed reasonable inhibition (IC₅₀ = 92 nM) while the reduced, non-electrophilic hydroxy acid AVI-5853 showed only weak activity.

Table 1.

Biochemical Potency (Expressed as IC₅₀) of AVI-4516 and Closely Related Analogs Bearing Altered N1 Side Chains, Including Examples with Well-Studied Cysteine Electrophiles Such as Nitrile, Epoxide, and Keto-acid.^a

Compound	MPro IC50 (nM)	Compound	MPro IC50 (nM)
AVI-4516	20	AVI-4690	>500
AVI-4689	161	AVI-4770	>500
AVI-4774	>500	AVI-5853	1700
AVI-5764	>500	AVI-5854	92

^aThis SAR suggests strict requirements for cysteine reactivity with the propargyl side chain. M^Pro IC₅₀ assay performed with 40 nM enzyme.

Using mass spectrometry to follow adduct formation we could confirm single-site labelling of recombinant M^pro by keto-acid AVI-5854 and also epoxide AVI-4770 (despite its weak biochemical activity). By contrast, and as expected, M^Pro adducts could not be detected in incubations of non-electrophilic analogs AVI-4690, AVI-4774, or AVI-5853 (Table 1 and Figure S1A). Overall, the SAR survey of N1 sidechains suggested that proximity to Cys145 is necessary but not sufficient to realize covalent inhibition of SARS-CoV-2 M^Pro. To further explore the mechanism of thiol-alkyne reactivity, and the possible intermediacy of an N-allenamide in the reaction pathway (Figure S1B), we prepared AVI-4692 in its deuterated (N–CD₂CCH) form AVI-6602 (Supporting Information, Scheme S1). Incubation of AVI-6602 with M^Pro followed by mass spectrometry analysis (Figure S1B) revealed that both deuterium atoms were retained in the protein adduct, effectively ruling out the intermediacy of an N-allenamide. Overall, the structural and biochemical data are thus consistent with direct nucleophilic attack of Cys145 on the internal carbon of the propargyl function, a reaction mode that was observed previously in peptidic reactivity-based probes targeting the narrow active site of cysteine deubiquitinases.²⁹

Having confirmed potent, irreversible inhibition of M^pro by AVI-4516, subsequent SAR studies were focused entirely on the N-propargyl uracil chemotype with the presumption that these analogs act as covalent, irreversible inhibitors. With the favored P1 isoquinoline and N1 propargyl side chains fixed, focus shifted to the C6 position and the S1’ pocket formed by His41 (the catalytic base) and residues like Thr25 and Met49 that are well conserved across betacoronaviruses (Table 2). We hypothesized that targeting S1’ with C6 sidechains (e.g., as in AVI-4692, Figure 1C) would provide a path toward enhanced antiviral spectrum. We identified a short and efficient synthetic route to such analogs involving, as a key step, the annulation of 4-aminoisoquinoline carbamic esters with the enamine of ethyl 3-aryl-3-oxopropanoates, the latter prepared via Blaise reaction³⁰ of the corresponding aryl nitriles (Scheme 1). Substitution of the C5 position was achieved using the NBS-promoted addition of benzotriazoles into the enamine esters, as described by Liu et al.³¹ followed, finally, by introduction of the propargyl warhead (Scheme 1). One limitation of this route was the necessary use of N-acidic heterocycles (more acidic than the succinimide byproduct produced in the NBS-promoted bromination). Fortunately, the benzotriazole C5 side chain proved nearly optimal with regard to potency and ADME/PK properties and was retained (in fluorinated form) for subsequent SAR studies of the C6 position.

Table 2.

Selected Residues That Form the S1’ Pocket in Several Coronaviruses Main Proteases.

virus	Residue No.
	25	41	49
SARS-CoV-2	Thr	His	Met
SARS-CoV	Thr	His	Met
Bat-SARS-like_Khosta-1	Thr	His	Met
Rhinolophus_affinis_CoV	Thr	His	Met
Pangolin_CoV	Thr	His	Met
MERS_CoV	Met	His	Leu
αCoV_229	Met	His	Thr
βCoV_OC43	Met	His	Met
FIPV	Asn	His	Ser

Scheme 1.

Representative Three-Step Synthesis of Uracil Analogs Described Herein.^a

^aconditions: a) 1H-benzotriazole, NBS, Na₂CO₃, DMF, 80 °C, 2 h, 66%; b) NaH, DMF, 0 °C, 30 min, 120 °C, 2 h, 18%; c) Cs₂CO₃, propargyl bromide, 80 °C, 4 h, 42%. Yields shown are for the synthesis of AVI-4516 in which R¹ = Me and R² = propargyl.

Thus, an initial survey of aliphatic C6 substituents (Me, Et, iPr, iBu, cyclopentyl, cyclohexyl) was performed in the context of 5,6-difluorobenzotriazole at C5 (Chart 1, upper panel, and Table 3). A trend of reduced potency with increasing steric bulk at C6 was mirrored by reduced stability in mouse liver microsomes (MLM) especially in the case of branched C6 substituents (i.e., iPr, cyclopentyl, cyclohexyl). Among other ADME properties, mouse plasma protein binding increased significantly from 81.0% (C6 Me) to 98.6% (C6 cyclohexyl) across the series, while apical to basolateral (A-to-B) permeability in MDCK-MDR cell monolayers improved with larger C6 substituents while efflux ratios remained favorably low across the series (ER <2). Moving to aryl C6 substitution yielded analogs that were considerably more potent than the larger/branched aliphatic analogs (Table 3). Moreover, halogenated C6 aryl analogs in particular retained excellent MLM clearance values (e.g., <11.6 μL/min/mg for AVI-5734), in contrast to the case of the larger C6 aliphatic analogs. On the other hand, mouse plasma protein binding (PPB) was concerningly high for C6 aryl analogs, ranging from 97.2% to 99.7%, whilst aqueous solubility also suffered when compared to analogs AVI-4773 and AVI-6179 bearing small aliphatic side chains at C6 (Chart 1, Table 3). Nevertheless, the totality of the data encouraged further exploration of aromatic and heteroaromatic C6 side chains.

Chart 1.

N-Propargyl analogs bearing the indicated C6 side chains and benzotriazole substitution.

Table 3.

M^Pro IC₅₀ and in vitro ADME Assay Data for Analogs Shown in Chart 1.^a

Compound	MPro IC50 (nM)	MLM CL (μL/min/mg)	MDCK-MDR Papp A to B (× 10−6 cm/s)	Efflux ratio	Ksolb (μM)	Mouse PPB (%)
AVI-4516	20	<11.6	11.3	3.7	350.5	63.0
AVI-4773	17	<11.6	17.8	4.0	430.8	81.0
AVI-6179	76	<11.6	24.5	1.8	339.6	84.4
AVI-6358	62	126.5	35.5	1.5	300.0	93.0
AVI-6259	160	69.3	36.5	1.2	125.1	95.4
AVI-6359	79	476.2	39.7	1.2	137.4	97.2
AVI-6261	170	576.2	34.5	1.7	47.8	98.6
AVI-5734	33	<11.6	15.3	1.9	7.6	99.1
AVI-4692	18	<11.6	18.8	3.3	31.1	97.2
AVI-6329	29	23.1	13.5	1.1	5.3	99.1
AVI-4694	17	14.5	8.0	1.7	2.5	99.7
AVI-6330	36	35.8	20.8	0.8	7.6	98.9
AVI-6322	37	29.8	5.1	7.5	<1	96.7
AVI-6325	43	38.3	26.3	0.6	16.7	98.1
AVI-6011	74	13.6	20.3	1.4	20.0	97.7
AVI-6328	33	22.6	18.4	0.8	8.0	98.4
AVI-6039	32	31.5	7.6	6.4	93.9	94.4
AVI-6195	34	14.9	22.5	1.2	31.3	96.7
AVI-6192	37	<11.6	12.5	3.1	43.2	95.8
AVI-6037	31	15.2	4.2	11.8	214.9	84.9
AVI-6036	45	15.0	4.9	10.1	228.8	82.9

^aIC₅₀ determined 40 nM M^pro enzyme; MLM CL = mouse liver microsome clearance; efflux ratio = P_{appB to A}/P_{appA to B} in MDCK-MDR monolayer assay; Mouse PPB = mouse plasma protein binding; Ksolb = kinetic solubility in pH 7.4 PBS.

With an aim of improving solubility and lowering PPB values of the C6 aryl analogs, a change to C6 pyridyl substitution was explored (Chart 1, lower panel, and Table 3). Gratifyingly, this change had the desired effect on both solubility and PPB, without loss of biochemical potency. Furthermore, permeability and PPB values were well correlated with the extent of halogenation on the C5 and C6 sidechains. Thus, analogs like AVI-6011 and AVI-6328 bearing a total of four halogens on their C5 and C6 sidechains showed moderately improved solubility and PPB values compared to aryl comparators also bearing four halogens (Table 3). Analogs AVI-6039 and AVI-6195 bearing three halogens showed lower PPB values, albeit with an increase in ER for AVI-6039. Analog AVI-6192 (two halogens) yielded the best overall combination of ADME properties while analogs AVI-6037 and AVI-6036 bearing a single ring halogen (at C6) while offering the best solubility and PPB values, showed an ER ~10. In summary, while no perfect solution was identified, pyridine analogs AVI-6011, AVI-6328, AVI-6195, and AVI-6192 offered an improved ADME profile overall, when compared to the original C6 aryl analogs (Table 3). The stage was thus set for their evaluation in antiviral assays, and a test of the hypothesis that better engagement of S1’ would produce broader antiviral effects.

Antiviral activity was first assessed using viral replicons in Vero E6 cells stably expressing ACE2 and TMPRSS2 (Table 4 and Supporting Information Table S1 and Figure S2).³² This included replicons based on the ancestral WA.1 strain, the more recent JN.1.1.1 Omicron variant³³, an ensitrelvir-resistant WA.1 strain bearing the M^Pro M49L mutation³⁴ and finally, an experimental triple “FQF” mutant combining three known nirmatrelvir-resistant mutations (L50F, E166Q and L167F) in a single replicon.³⁵ The experiments with replicons were, in general, performed along with a P-gp inhibitor (CP-100356) as both nirmatrelvir and ensitrelvir are known to be P-gp substrates.^4,23 Compounds were also tested without P-gp inhibitor in the WA.1 replicons, revealing minimal shift in EC₅₀ values (≤3-fold) for the new uracil analogs in the absence of P-gp inhibitor (Table 4 and Table S2). By contrast, nirmatrelvir was nearly 100-fold less potent against WA.1 replicons in the absence of CP-100356 while ensitrelvir showed a ~5-fold loss of potency.

Table 4.

Cellular Antiviral Activity of Analogs in Chart 1 Against the Indicated SARS CoV-2 Replicons in Vero E6-TMPRSS2 cells.^a

compound	EC50 (nM) in VeroE6-TMPRSS2 cells
	WA.1*	WA.1	JN.1.1.1	WA.1 MPro M49L	WA.1 MPro FQF
nirmatrelvir	10700	129	66.5	55.8	2930
ensitrelvir	1170	238	197	6890	17700
AVI-4516	414	197	1480	28.7	9880
AVI-4773	81.2	74	64.6	10.2	1710
AVI-6179	88.5	143	287	4.59	939
AVI-6358	64.9	95.3	37.7	22	2470
AVI-6259	207	201	152	36	3940
AVI-6359	250	192	190	76.2	2690
AVI-6261	17.8	21.6	79.7	14.8	380
AVI-5734	14.7	8.52	36.6	6.68	208
AVI-4692	179	82.3	32.2	46	5030
AVI-6329	34.9	19.8	39.5	4.9	263
AVI-4694	12.4	9.16	50.9	7.2	158
AVI-6330	25.2	10.7	13.9	6.12	204
AVI-6322	143	46.7	23.8	27.4	300
AVI-6011	27.1	19.1	25.3	9.88	495
AVI-6328	8.94	5.69	18.6	1.89	34
AVI-6039	89.4	92.2	67.1	13.7	941
AVI-6195	13.5	13.8	39.5	4.08	147
AVI-6192	137	46.3	59.8	7.25	492
AVI-6037	415	193	358	42.2	3840
AVI-6036	689	233	34	78.6	3080

^aCellular EC₅₀ values. All EC₅₀ determinations performed in the presence of a P-gp inhibitor except in WA.1 strain as indicated (*). Nirmatrelvir and ensitrelvir were used as controls. FQF mutant: L50F, E166Q, L167F. See Supplementary Table S2 for mean ± SD for this data from two independent biological replicates.

Against the WA.1 replicons, the new C6 (hetero)aryl analogs were generally ~10-fold more potent than ensitrelvir or nirmatrelvir (with P-gp inhibitor). For example, the single-digit nM potency of analogs AVI-5734, AVI-4694, and AVI-6328 WA.1 replicon compared favorably to EC₅₀ values of 129 nM and 238 nM for nirmatrelvir and ensitrelvir (Table 4 and Supporting Information, Table S3). Another noteworthy comparison is that of AVI-4692 (EC₅₀ = 82 nM) with AVI-5734 and AVI-4694 (EC₅₀ < 10 nM), which reveals the favorable effect of a second halogen (Cl) on the C6 aryl ring. Against the JN.1.1.1 replicon, we observed similar SAR trends for the uracil analogs, with C6 aryl analogs again showing the most potent EC₅₀ values while larger aliphatic C6 substituents generally afforded weaker activities that were still similar to the approved agents. One surprising observation was the rather weak activity of AVI-4516 (JN.1.1.1 EC₅₀ = 1.5 μM) when compared to its 5,6-difluoro congener AVI-4773 (EC₅₀ = 65 nM). Also of note, AVI-6330 with its bulkier C6 side chain exhibited an excellent EC₅₀ ~ 10 nM against both WA.1 and JN.1.1.1 replicons. Nearly all of the new analogs showed impressive low to single-digit nM activity against the ensitrelvir-resistant WA.1 M^Pro M49L replicon (ensitrelvir EC₅₀ = 6.9 μM). Interestingly, the C6 aliphatic analogs were also potent against the M^Pro M49L WA.1 replicon, with C6 ethyl (AVI-6179) and C6 isopropyl (AVI-6358) analogs showing particularly potent effects (EC₅₀ = 10.2 and 4.6 nM, respectively). This likely reflects better shape complementarity of these C6 aliphatic side chains with the S1’ pocket of the M49L mutant, given that this pocket is formed in part by residue 49.

Against the FQF WA.1 triple mutant, nirmatrelvir and ensitrelvir showed greatly reduced activities in the μM regime, as expected. While the uracil analogs were also less active against this replicon, the majority of C6 hetero/aryl analogs retained EC₅₀ values in the mid-nM regime. A notable exception to this trend was progenitor lead AVI-4516 (EC₅₀ = 9.9 μM). Thus, the C6 SAR campaign described here was notably successful in improving antiviral potency and spectrum over the progenitor AVI-4516. Indeed, the most potent analogs against the challenging FQF mutant were the C6 pyridyl analogs AVI-6328 and AVI-6195, which maintained activity in the mid to low nM range (EC₅₀ = 34 nM and 147 nM, respectively) and were at least 10-fold more potent than the approved agents (Table 4 and Table S3).

Notably, antiviral potency could be roughly correlated with the extent of halogenation of the C5 and C6 side chains, likely reflecting more productive contact with the S2 and S1’ pockets, and possibly also improved cellular permeability. In one notably comparison, the pyrid-2-yl analog AVI-6328 was found to be ~5-fold more potent than its pyrid-3-yl regioisomer AVI-6011 across the replicon panel. In fact, AVI-6328 showed the broadest and most potent spectrum of activity among all uracil analogs evaluated, with low to single-digit nM potencies and a study-best EC₅₀ value of 34 nM against the WA.1 M^Pro FQF mutant (Table 4). Selected analogs were also evaluated in a live-virus cellular assay,²⁶ with AVI-6011 showing picomolar potency (EC₅₀ = 0.018 nM) that compared favorably with that of progenitor C6 aryl analogs AVI-4692 (EC₅₀ = 7.0 nM) and AVI-4694 (EC₅₀ = 71 nM) in this assay (see Supplementary Information, Figure S3).

To better understand the effect of C6 substitution on S1’ engagement vs. cellular uptake, we directly measured intracellular concentrations of representative analogs in the human lung cell line A549 following incubations of 6, 12, and 24 hours (Figure S4). As predicted, C6 aryl analogs AVI-4673, AVI-4692, and AVI-4694 showed much higher accumulation than C6 methyl progenitors AVI-4516 and AVI-4773, a result that was consistent with their superior cellular activity and greater lipophilicity. Thus, we conclude that the impressive replicon activity of C6 hetero/aryl analogs (Table 4) is not purely an effect of engaging the S1’ pocket, but also a consequence of their better cellular uptake compared to the progenitor lead AVI-4516.

Having identified a variety of novel M^Pro inhibitors with good biochemical and cellular activities, and reasonable ADME properties, we moved to rodent PK studies. To evaluate the plasma and CNS exposure of as many leads as possible, whilst minimizing the number of experimental animals used, we employed cassette dosing in SD rat with IP administration at 10 mg/kg/compound in each pool of six compounds (N = 3 rats per study).The twelve compounds selected for two rat studies were chosen based on their ADME and antiviral properties, as well as C6 diversity (Supporting Information, Figure S5A and S5B). Plasma samples were collected at five early timepoints out to 1 hour, and the animals sacrificed for analysis of cerebral spinal fluid (CSF) and brain homogenate (Figure 2A–C).

Figure 2.

Plasma and brain exposure of twelve uracil analogs studied in female SD rat with cassette dosing (6 compounds per group; N = 3).

We detected all twelve analytes in rat plasma across the two studies, indicating technical success of the cassette dosing approach and the bioanalytical methods developed. Plasma exposure values varied widely however, with four compounds exhibiting significantly higher exposure than the remaining eight (Figure 2A). Notably, these four compounds were the ones bearing aliphatic C6 substituents (AVI-4516, AVI-4773, AVI-6179, and AVI-6259). By contrast, all eight C6 aryl or heteroaryl analogs exhibited much lower plasma exposures. Similarly, the four C6 alkyl analogs showed the highest concentrations in CSF and brain homogenate (Figure 2B and C). Four C6 aryl analogs did have measurable concentrations in brain, and analogs AVI-6325 and AVI-6032 showed the highest brain:plasma ratios at 0.28 and 0.25, respectively. However, the plasma C_max for these analogs was only ~10% that of C6 alkyl analogs like AVI-4773 and AVI-6179, which despite lower brain:plasma ratios achieved ≥4-fold higher total brain concentrations than either AVI-6325 or AVI-6032 (Figure 2C). Overall then, the cassette PK experiment revealed a stark difference in vivo exposure by C6 chemotype, with the C6 aryl analogs showing low exposure, despite good in vitro permeability and microsome stability values. This was disappointing, given the superior antiviral effects of the C6 hetero/aryl analogs in the replicon assay (Table 4). Nevertheless, the PK studies did identify analogs AVI-4773 and AVI-6179 as exhibiting a promising combination of antiviral activity, in vitro ADME profile, and in vivo PK exposure profiles, including good exposure in CSF and brain. Accordingly, these two analogs became the focus of further study.

Before proceeding to further in vivo evaluation of AVI-4773 and AVI-6179, the predicted atropisomerism of the compounds was explored with the aim of identifying their biologically active forms. This would potentially allow their evaluation in enantiopure forms and at lower overall doses. Atropisomerism is present in several FDA-approved drugs³⁶ and LaPlante et al.^37,38 have developed a classification scheme based on the torsional rotation barriers ($\Delta E_{rot}$), with implications for clinical development. The congested C–N bonds between the uracil core and isoquinoline and benzotriazole substituents were judged likely to result in atropisomerism within the scaffold. This was suggested by their complex NMR spectra, which implied a mixture of four stereoisomers on NMR timescales leading to diastereomeric resonances. In further support of this hypothesis was the observation that a regioisomer of AVI-6179 bearing a less hindered N2-linked benzotriazole (Supplementary Scheme S2) exhibited a much simplified ¹H NMR spectrum (Supplementary Figure S6).

To determine barriers to rotation experimentally, we employed variable temperature NMR of AVI-4773 with stepwise heating to 80°C, and observed a coalescence of NMR signals, consistent with low-barrier atropoisomerism at one of the two centers (Supporting Information, Figure S7). Using the measured coalescence temperature for three separate sets of isolated NMR resonances, the barrier for rotation (ΔG^‡) could be experimentally determined, yielding similar values (ΔG^‡_{318 K} = 17.2 kcal mol^–1, H_b: ΔG^‡_{343 K} = 17.7 kcal mol^–1, and H_c: ΔG^‡_{333 K} = 17.6 kcal mol^–1; Figure S7). This barrier was presumed to be that of the less hindered uracil(C5)–bezotriazole(N1) bond. To provide further support for this supposition, we employed density functional theory (DFT) calculations at the SMD(water)-wB97XD|def2svp and SMD(DMSO)-wB97XD|def2svp levels of theory to calculate theoretical energy barriers of rotation (${\Delta E}_{rot}\left(\mathit{\text{theoretical}}\right)$) along both the uracil(N3) and uracil(C5) axis for AVI-4773 (Supporting Information, Figure S8). These calculations confirmed the higher energy barrier of the isoquinoline substituent, while providing a calculated rotational barrier for the uracil(C5)–bezotriazole(N1) bond that was in excellent agreement with value from the NMR studies.

Uracil(N3)–isoquinoline:

$${\Delta E}_{rot}\left(\mathit{\text{theoretical}};SMD\left(\mathit{\text{water}}\right)\right)=30.94kcal\bullet {mol}^{-1}$$

Uracil(C5)-benzotriazole:

$${\Delta E}_{rot}\left(\mathit{\text{theoretical}};SMD\left(\mathit{\text{water}}\right)\right)=17.67\mathit{\text{kcal}}\bullet {mol}^{-1}$$

Analogous calculated energy barriers for C5 ethyl analog AVI-6179 are provided as supporting information and show a slightly greater barrier for the uracil(C5)-benzotriazole bond due to the larger C5 substituent (Figures S9). The derivation of rate constants for interconversion based on the calculated energies (Supporting Information, Figure S10, Tables S4, S5, S6) indicates that interconversion half-life of (M) and (P) forms of AVI-4773 is on the order of years.

Consistent with the predicted slow interconversion rates, we found it was possible to separate the enantiomers of AVI-4773 and AVI-6179 on preparative scales using supercritical fluid chromatography (SFC) with a chiral stationary phase, yielding well separated peaks of the same intensity and with identical NMR spectra (Supporting Information, Figure S11 and S12). These were re-crystallized and subjected to X-ray crystallographic analysis to obtain small molecule crystal structures of the forms (M)-AVI-4773, corresponding to the first peak to elute during SFC separation (Figure 3), and (P)-AVI-6179 corresponding to the second peak to elute (Supporting information, Figure S13).

Figure 3.

Molecular structure of the biologically active atropisomeric form (M)-AVI-4773 from the small molecule X-ray structure. Thermal ellipsoids drawn at the 30% probability level. Solvent water molecule omitted for clarity.

Since it was the (M) form of AVI-4516 that we observed bound to M^Pro in its complex structure²⁶ with SARS-CoV-2 M^Pro (PDB:9MVP), we anticipated that (M)-AVI-4773 and (M)-AVI-6179 would be the biologically active forms of the inhibitors (eutomers). Indeed, the measured M^Pro IC₅₀ values for (M)-AVI-4773 and (M)-AVI-6179 were 2.5 nM and 2.1 nM, respectively, at least 150-fold more potent than their corresponding (P) atropisomers and roughly twice as potent as the racemates (Table 5). The potency of the active forms comparable to that of an authentic sample of S-892216 (Table 5). The large differences in biochemical potencies of the enantiomers was consistent with equally large differences in antiviral EC₅₀ values, both by WA.1 replicon assay and in a live virus Incucyte-based³⁹ assay (Table 5 and Supplementary Figures S14 and S15).

Table 5.

Biochemical and Cellular Antiviral Activities of Racemic and SFC-Separated Atropisomers of AVI-4773 and AVI-6179.^a

Compound	MPro IC50 (nM)	WA.1 EC50 (nM)	Incucyte EC50 (nM)
(±)-AVI-4773	4.3	115	5.0
(M)-AVI-4773	2.5	38.8	3.0
(P)-AVI-4773	516	5392	2982
(±)-AVI-6179	5.8	96.0	40
(M)-AVI-6179	2.1	32.9	15
(P)-AVI-6179	>400	3382	2304
S-892216	2.0	n.t.	n.t
nirmatrelvir	4.1	n.t	n.t

^aIC₅₀ were performed at an enzyme concentration of 5 nM. Cellular activity in WA.1 replicons without P-gp inhibitors. Incucyte assay in A549-ACE2h cells treated for 24 hrs with icSARS-CoV-2-mNG virus bearing a mNeonGreen reporter in place of Orf7a and Orf7b coding sequence.

Having identified the active enantiomers, we sought to produce pure (M)-AVI-4773 and (M)-AVI-6179 on gram scales to support further in vivo studies. Notably, we found that heating the undesired, SFC-isolated (P) isomers in DMSO at 100°C for 16 hours returned the racemic mixtures without detectable byproducts (Figure S16). This recycling of (P) form to racemate was followed by further SFC separation to afford additional quantities of the desired active (M) forms.

Next, mouse PK studies of (M)-AVI-4773 and (M)-AVI-6179 were performed using IV and PO doses of 3 mg/kg and 10 mg/kg, respectively. We observed high exposures with notably low clearance (≤ 0.5 L/h/kg) that was approximately ~7% of liver blood flow (Table 6). Overall exposure by AUC and oral bioavailability were higher for (M)-AVI-4773 (AUC = 18,847 h*ng/mL; 79%F) than for (M)-AVI-6179 (AUC = 10,677 h*ng/mL; 52%F) with free drug concentrations at 8 hours ~10-fold higher for (M)-AVI-4773 than for AVI-6179 (Figure 4A and Table 6). Total brain concentrations (Figure 4B, solid bars) were similar for the two compounds, but trending higher for (M)-AVI-4773, while brain/plasma ratios were comparable (Figure 4B, open bars). The significantly higher plasma exposure of (M)-AVI-4773, especially at later timepoints led to its selection for further PK and PD studies.

Table 6.

Selected PK Parameters for M^Pro Inhibitors Following a Single Dose in Mice, Rats, and Dogs.^a

	IV 3 mg/kg CD-1 mice				PO 10 mg/kg CD-1 mice
compound	CL (L/h/Kg)	Vss (L/Kg)	AUClast (h*ng/mL)	MRTinf (h)	Cmax (ng/mL)	T1/2 (h)	AUClast (h*ng/mL)	F (%)
(M)-AVI-4773	0.412	1.01	7085	2.44	3327	6.1	18847	79.8
(M)-AVI-6179	0.458	1.14	6367	2.49	3940	2.0	10677	52.0
	IV 3 mg/kg SD rat				PO 10 mg/kg SD rat
	CL (L/h/Kg)	Vss (L/Kg)	AUClast (h*ng/mL)	MRTinf (h)	Cmax (ng/mL)	T1/2 (h)	AUClast (h*ng/mL)	F (%)
(M)-AVI-4773	0.506	2.02	6012	4.06	559	2.73	3774	25.4
	IV 0.62 mg/kg beagle dog				PO 2 mg/kg beagle dog
	CL (L/h/Kg)	Vss (L/Kg)	AUClast (h*ng/mL)	MRTinf (h)	Cmax (ng/mL)	T1/2 (h)	AUClast (h*ng/mL)	F (%)
(M)-AVI-4773	0.127	0.56	4946	4.5	1867	5.98	18174	78.8

^aPK parameters calculated based on total plasma exposure values employing a formulation of 10% DMSO | 50% PEG400 | 40% (20% hydroxypropyl-β-CD in water, except for the PO arms of rat and dog studies, which used a suspension in 1% (hydroxypropyl)methyl cellulose/1% Tween 80 at 1 mg/mL (rat) or 0.4 mg/mL (dog). Mouse studies involved n=9 male CD-1 mice per arm. Rat studies involved n=3 male SD rat per arm. Dog study involved n=3 male beagle dogs per arm. In all studies n=3 plasma samples were taken per timepoint.

Figure 4.

Pharmacokinetics and efficacy of lead compounds. A) Plasma total and free drug concentration-time profiles of (M)-AVI-4773 and (M)-AVI-6179 after a single oral dose of 10mg/kg in male CD1 mouse (n=3 in each treatment). B) Total brain concentration in solid bars; brain:plasma ratio in outline. C) Plasma concentration-time profiles of (M)-AVI-4773 in rat and dog after PO or IV administration D) efficacy of (M)-AVI-4773 at indicated BID oral doses vs. vehicle or nirmatrelvir control as judged 2 days post-infection. E) body weights of infected and treated animals over the full course of the experiment. F) body weights of uninfected animals treated for 5 days as in B. IFN −/− mice (blk/6) were intranasally infected with 2.5×10⁸ PFU of Adv-hDPP4 and after 5 days, again infected with 1×10⁵ PFU of MERS-CoV EMC2012 and treated orally BID with indicated doses of compound for 5 days (D 0–4). infected N = 5, uninfected N = 3. Data was analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). G. Wild-type (WT) mice were intranasally infected with 10³ PFU of the SARS-CoV-2 Beta variant. Infected animals were treated by oral gavage twice daily (BID) with vehicle, (±)-AVI-4773 at 100 mg/kg, or (M)-AVI-4773 at 50 mg/kg. Lung tissues were collected at 2- and 4-days post-infection (dpi) for analysis (n = 5 per group per time point). H. The graphs show infectious virus titers in the lungs at the indicated time points post infection; each dot represents an individual mouse. Data are presented as mean ± SEM and were analyzed using a two-tailed unpaired Student’s t-test (**P < 0.01).

Next, (M)-AVI-4773 was studied in SD rat and beagle dog (Figure 4C; Table 6). For the oral arms of these studies the compounds were formulated as an oral suspension in 1% (hydroxypropyl)methyl cellulose/1% Tween 80. We found oral bioavailability was lower at 25%F in rat but remained high at 79%F in dog, while clearance remained very low in both species (0.506 L/hr/Kg in rat; 0.127 L/hr/Kg in dog; Table 6). Thus, (M)-AVI-4773 exhibits low clearance across rodent and non-rodent species and modest to excellent oral bioavailability.

Next, we studied (M)-AVI-4773 in a MERS-CoV pathogenesis model employing B6(Cg)-Ifnar1^tm1.2Ees/J mice (interferon knockout mice) transduced with hDPP4-adeonvirus five days prior to inoculation with MERS-CoV.⁴⁰ Using the free plasma exposure values and the cellular MERS EC₅₀ = 0.28 μM (for racemic material) we selected four PO dosing levels from 8 mg/kg BID to 125 mg/kg BID, estimating that trough plasma levels for the 125 and 50 mg/kg doses would exceed the cellular EC₉₀. The positive control was oral nirmatrelvir (without ritonavir) BID at 600 mg/kg. At day −5, mice were treated with 2.5×10⁸ PFU hDPP4 adenovirus to establish expression of the entry factor hDPP4 in lungs of the animals. Mice were infected at day 0 with 1×10⁵ PFU MERS-CoV EMC2012 and test compounds and controls were administered from days 0–5 BID as a suspension in 1% (hydroxypropyl)methyl cellulose/1% Tween 80 by oral gavage, with the first dose administered 1 hour before infection. A separate cohort of uninfected mice were treated with test compounds at the same dose/schedule and viral titers were assessed at 2 dpi in the infected mice and mouse weights monitored for 14 days in infected mice and 5 days in the uninfected cohort.

The antiviral efficacy of (M)-AVI-4773 was clearly evident at the 2 dpi timepoint in both the 50 mg/kg and 125 mg/kg groups, while the 20 mg/kg group showed a reduction in viral titers without reaching significance; no reduction in viral load was evident in the 8 mg/kg group. In the nirmatrelvir control arm viral loads were trending lower, but were not statistically significant (Figure 4D), despite clear protection from weight loss (Figure 4E). Gratifyingly, the 125 mg/kg BID dose of (M)-AVI-4773 produced a robust ~4-log reduction in viral load, to the lowest level detectable in this model (Figure 4D).

We also observed a dose-dependent protection from pathogenesis associated weight loss for (M)-AVI-4773 groups on days 6–7 post-infection (Figure 4E). A dose-dependent reduction in weight well after dosing ended (days 8–12) is of unclear origin but nevertheless was reversed by the end of the study at day 14. In both the infected and uninfected cohorts (Figure 4F), a small decrease in weight was observed from days 2–5 (during dosing) but was not well correlated with dosing levels and therefore is of uncertain significance.

Finally, we compared the efficacy of (M)-AVI-4773 at 50 mg/kg BID to racemic material at 100 mg/kg BID in a non-lethal SARS-CoV-2 infection model. We again used an oral suspension in 1% (hydroxypropyl)methyl cellulose/1% Tween 80 for the oral arm of the study, which was performed with the SARS-CoV-2 Beta variant (B.1.351) in wild-type C57bl6 mice. As expected, 50 mg/kg BID of (M)-AVI-4773 was equally as effective as 100 mg/kg BID dose of racemate, rapidly reducing viral load to nearly undetectable levels after just three doses (2 d.p.i.) and completely suppressing detectable viral load by 4 d.p.i (Figure 4G and H). Previously,²⁶ racemic AVI-4516 was evaluated at both 50 and 100 mg/kg BID in this same model, producing respectively ~1 log and ~3 log reductions in viral titers by plaque assay at 2 d.p.i. Thus, the finding here that a 50 mg/kg BID of (M)-AVI-4773 produces an eight-log reduction (to undetectable levels) by 2 d.p.i. highlights the promising PK/PD profile of this lead, particularly when applied in the biologically active, enantiomerically pure form. No statistically significant body weight loss was observed in AVI-4773 treated animals from either arm of this study.

To assess the potential for further preclinical development of both C6 methyl- and C6 aryl-class lead compounds, we evaluated the off-target profile of (±)-AVI-4773 and (±)-AVI-6011 across a panel of human proteases, receptors, and ion channels (assays performed at Eurofins Panlabs, Inc). AVI-4773 demonstrated excellent selectivity across >30 mammalian proteases, including caspases and cathepsins, with typically ≤ 20% inhibition at 10 μM (Figure 5A). By comparison, AVI-6011 showed comparatively higher, though still modest, inhibition of the off-target proteases, with Cathepsin L2 and Cathepsin S being the most substantially inhibited (IC₅₀ values of 2.9 μM and 1.9 μM, respectively). Against ion channels and receptors (Figure 5B) AVI-4773 showed ≤50% response at 10 μM across the entire panel, while AVI-6011 showed ≥50% response against five off-targets, four with low μM IC₅₀ values and one with sub-μM activity: cannabinoid CB1 receptor (IC₅₀ = 5.2 μM), cannabinoid CB2 receptor (IC₅₀ = 7.5 μM), cholecystokinin CCK1 (IC₅₀ = 10 μM), glucocorticoid receptor (IC₅₀ = 5.1 μM), and Nav1.5 sodium channel (IC₅₀ = 0.23 μM). Finally, AVI-4773 displayed moderate (≤50%) inhibition of CYP isoforms at 10 μM, that was similar to that reported previously²⁶ for AVI-4516 (Figure 5C). Overall, AVI-4773 showed a superior off-target profile compared to AVI-6011, and a notable degree of selectivity for SARS CoV-2 M^Pro (IC₅₀ ~ 5 nM) over all human proteases evaluated (IC₅₀ >> 10 μM).

Figure 5.

Selectivity of (±)-AVI-4773 and (±)-AVI-6011 as judged by inhibition of off-target mammalian proteases (panel A) and receptors and ion channels (panel B) at 10 μM. Inhibition of mammalian CYPs by (±)-AVI-4516 and (±)-AVI-4773 at 10 μM (panel C).

We also studied the CYP induction potential of (±)-AVI-4773 and (±)-AVI-6011 at 1, 10 and 100 μM as well as the potential interaction with eight off-target cardiac channel activities. The full results of these studies are provided in supporting information (Supporting Information, Table S7). Briefly, AVI-4773 showed the potential to induce CYP1A2, CYP2B6, and CYP3A4 at 1 μM and higher concentrations while AVI-6011 showed the potential to induce CYP2B6 at 1μM. The induction of CYP1A2 and CYP2B6 by AVI-4773 was generally <3-fold even at 100 μM, while induction of CYP3A4 at 100 μM was between 10- and 25-fold. Of the eight cardiac channel activities evaluated, AVI-4773 showed a hERG IC₅₀ values of 17.5 μM, but showed IC₅₀ > 100 μM across the other seven channel activities evaluated (Nav1.5 peak, Nav1.5 agonism, Nav1.5 antagonism, Kv4.3, Cav1.2, KCNQ1, Kir2.1). AVI-6011 showed a hERG IC₅₀ = 19.7 μM and an Cav1.2 IC₅₀ = 4.4 μM, with an IC₅₀ > 100 μM across the other six channel activities. In summary, the overall selectivity of AVI-4773 across known off-targets was promising, especially against human proteases, while some potential liabilities with regard to CYP induction were identified and merit consideration in the further optimization of this lead series.

Having noted a discrepancy in the measured hERG activity of (±)-AVI-4773 as reported by different vendors and assay formats (e.g., 0% inhibition @ 10μM in Saftey 44 panel, vs. IC₅₀ = 17 μM in dose-response) we performed additional hERG assays in our own laboratories using a commercial fluorescence polarization assay kit (ThermoFisher). For this study, we employed the active form (M)-AVI-4473 rather than racemate, and in addition to the positive control (E-4031) we also profiled S-892216 as representative of a clinical stage M^Pro inhibitor from the uracil chemotype. We found that (M)-AVI-4773 exhibited minimal hERG inhibition even at the highest concentration tested, whereas S-892216 exhibited modest hERG activity in the mid μM regime (Figure 6). Accordingly, we judge that the uracil scaffold and the lead (M)-AVI-4773 in particular presents a low potential for hERG inhibition.

Figure 6.

Dose response of hERG inhibition for (M)-AVI-4773 and S-892216 and positive control.

Finally, we sought to assess the potential for mutagenicity of the uracil analogs described herein, with a particular focus on the isoquinoline sidechain at P1. While this moiety can be regarded as a privileged P1 side chain in SARS-CoV-2 M^pro inhibitors, and has been employed in M^pro inhibitors from several other groups,^19,21,41,42 the parent isoquinoline-4-amine has been reported previously to be an AMES-positive mutagen.⁴³ To explore this possibility, we assessed both AVI-4773 and isoquinoline-4-amine itself in a mini-AMES assay (performed at Frontage Labs, Hayward, CA). In this study, we found that neither isoquinoline-4-amine nor AVI-4773 were AMES positive, while all three positive controls produced the expected positive response (Supporting Information, Table S8). Notwithstanding this result, we acknowledge that it may still be advisable to consider alternate P1 moieties in the ongoing search for a development candidate from this series.

CONCLUSIONS

In summary, here we explored diverse warhead chemotypes and C5/C6 SAR in the context of the potent, in vivo active lead compound AVI-4516 described in our initial report. We discovered the favorable effect of larger C6/P1’ substituents on antiviral spectrum and potency, and the ability of halogenated pyridyl C6 sidechains to modulate plasma protein binding and solubility while retaining other ADME properties in a favorable regime. On the other hand, C6 (hetero)aryl side chains were found to be associated with reduced plasma exposure in rat, despite excellent in vitro ADME profiles, thus identifying a possible in vitro-in vivo disconnect in this C6 sub-type. We also described the off-target profiles of exemplar C6 methyl and C6 pyridyl analogs, finding both chemotypes to exhibit considerable selectivity over mammalian proteases and other off targets, with C6 methyl exemplar AVI-4773 showing the best overall off-target profile. We confirmed stable atropisomerism in P1 isoquinoline analogs using NMR and DFT methods, assigned the eutomer rigorously by crystallography, and prepared enantiomerically pure forms on gram scales, recycling the undesired form by thermal racemization. We evaluated the PK profile of (M)-AVI-4773 in rodent and dog, revealing good to high oral bioavailability and very low-clearance, with CNS exposure superior to currently available antiviral agents, and possibly sufficient to treat infections of the brain. In mouse models of MERS-CoV and SARS-CoV-2 infection, oral administration of (M)-AVI-4773 produced a rapid onset antiviral effect that was superior to currently available agents, particularly in the case of MERS infections. These findings encourage the further optimization of uracil leads towards a development candidate that could find utility in treating infections caused by currently circulating as well as emerging coronavirus pathogens with pandemic potential.

MATERIALS AND METHODS

General Methods (Synthesis).

unless otherwise noted all chemical reagents and solvents used are commercially available. Air and/or moisture sensitive reactions were carried out under an argon atmosphere in oven-dried glassware using anhydrous solvents from commercial suppliers. Air and/or moisture sensitive reagents were transferred via syringe or cannula and were introduced into reaction vessels through rubber septa. Solvent removal was accomplished with a rotary evaporator at ca. 10–50 Torr. NMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer. Chemical shifts are reported in δ units (ppm). NMR spectra were referenced relative to residual NMR solvent peaks. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants (Hz) and integration. Chromatography was carried out using Isolera Four and CombiFlash NextGen 300 flash chromatography systems with SiliaSep silica gel and C18 cartridges from Silicycle. Reverse phase chromatography was carried out on Waters 2535 Separation module with Waters 2998 Photodiode Array Detector. Separations were carried out on XBridge Preparative C18, 19 × 50 mm column at ambient temperature using a mobile phase of water-acetonitrile containing a constant 0.1% formic acid. LC/MS data were acquired on a Waters Acquity UPLC QDa mass spectrometer equipped with Quaternary Solvent Manager, Photodiode Array Detector and Evaporative Light Scattering Detector. Separations were carried out with Acquity UPLC^® BEH C18 1.7 μm, 2.1 × 50 mm column at 25 °C, using a mobile phase of water-acetonitrile containing a constant 0.1 % formic acid. The synthesis of AVI-4673, AVI-4692, AVI-4694, AVI-4516, and AVI-4773 in their racemic forms were described.²⁶ All final analogs subjected to in vitro, cellular, or in vivo assay were judged ≥95% pure by UPLC and ¹H NMR analyses.

Synthesis of carbamic acid-4-isoquinolinyl-ethyl ester.

Pyridine (1.7 mL, 20.8 mmol, 3 eq) was added to a suspension of 4-aminoisoquinoline (1 g, 6.94 mmol) in DCM (25 mL) at 0°C, followed by a dropwise addition of ethyl chloroformate (0.995 mL, 10.41 mmol, 1.5 eq) dissolved in 5 mL of DCM. Then the mixture was warmed to room temperature and stirred for 1 h and quenched with 1N HCl (20 mL). The aqueous phase was extracted with DCM (3×20 mL). The organic phase was dried over Na₂SO₄ and concentrated in vacuo. The crude material (1 g, 6.02 mmol, 87%) was used in the next step without further purification. General: C₁₂H₁₂N₂O₂; MW = 216.24. ¹H-NMR (400 MHz, CDCl₃): δ (ppm): 9.10 (s, 1H); 8.91 (s, 1H); 8.04 (d, J = 8.2 Hz, 1H); 7.95 (d, J = 8.4 Hz, 1H); 7.79 (t, J = 6.9 Hz, 1H); 7.67 (t, J = 7.6 Hz, 1H); 7.11 (s, 1H); 4.30 (q, J = 7.1 Hz, 2H); 1.34 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 217.1 [M+H]⁺ (calcd for C₁₂H₁₃N₂O₂: 217.09).

General Procedure A.

To a solution of the aryl nitrile (1 mmol) in 1,2-dichloroethane (10 mL), were added ZnCl₂ (1.2 eq), potassium ethyl malonate (2.3 eq) and DIPEA (0.3 eq). The mixture was stirred at 100°C for 16 h, then cooled to room temperature and washed with saturated NH₄Cl aqueous solution. The aqueous phase was extracted with CH₂Cl₂ and the organic extracts were dried over Na₂SO₄ and concentrated in vacuo. Unless otherwise stated, the crude was used in the next step without further purification.

General Procedure B.

N-Bromosuccinimide or N-bromophthalimide (1.2 eq) were added to a solution of enaminone a (1 mmol) in DMF (2 mL) or toluene (2 mL) and the mixture was stirred for 10 min at room temperature. Then, the corresponding N-heterocycle (1.2 eq), and Na₂CO₃ (1.2 eq) or DBU (1.2 eq) were added, and the mixture was heated to 80°C and stirred for 2 h. The reaction was quenched with saturated Na₂S₂O₃ aqueous solution and the aqueous phase was extracted with EtOAc. The organic extracts were dried over Na₂SO₄ and concentrated in vacuo. The crude was purified by flash chromatography on silica gel (EtOAc in hexane 0% to 60%). In the case of reaction with a benzotriazole, the product was isolated along with its regioisomer Bt-iso as a minor by-product (3:1 mixture, Scheme S2), which was removed in the next step.

General procedure C.

The enaminone intermediate (0.2 mmol) was dissolved in DMF (1 mL) and added dropwise to a suspension of NaH (60% in mineral oil; 2.5 eq) in DMF (0.5 mL) at 0°C. The mixture was stirred for 30 min at 0°C and then added to a solution of carbamic acid-4-isoquinolinyl-ethyl ester (1.5 eq) in DMF (1 mL) at 0°C. The mixture was heated to 120°C and stirred for 2 h; then cooled to room temperature and directly purified by preparative HPLC. Note: given reports of EXPLOSION RISK on heating NaH in DMF an alternative procedure using NaOtBu was adopted and is recommended: the enaminone (0.2 mmol) was dissolved in DMF (1 mL) and added dropwise to a suspension of NaOtBu (2 eq) in DMF (1 mL) at 0°C. The mixture was stirred for 30 min at 0°C and then added to a solution of carbamic acid-4-isoquinolinyl-ethyl ester (1.7 eq) in DMF (1 mL) at 0°C. The mixture was heated to 120°C and stirred for 2 h; then cooled to room temperature and directly purified by preparative HPLC.

General Procedure D.

The unsubstituted uracil intermediate (0.3 mmol) was dissolved in DMF (3 mL), after which Cs₂CO₃ (1.7 eq) and propargyl bromide (1.5 eq) were added at room temperature. The mixture was stirred at 80°C for 4 h, and directly purified by preparative HPLC (40% to 90% CH₃CN in H₂O + 0.1% formic acid.

Genearl Procedure E.

When the Blaise reaction was not suitable (alkyl enaminones) and the starting enaminone was not commercially available, the enaminones were synthesized through the following procedure: the appropriate β-ketoester and ammonium formate (5 eq) were taken in a round bottom flask and ethanol was added. The mixture was heated at reflux for 24h and allowed to reach room temperature. The solvent was evaporated and extracted with ethyl acetate (3x). The combined organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated on a rotary evaporator. The crude product was purified by CombiFlash column chromatography, eluting with 10 to 60% EtOAc in hexane to afford the desired enaminone.

Synthesis of AVI-4689.

General Procedure D was followed using 2-bromoacetonitrile and uracil intermediate 5-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(isoquinolin-4-yl)-6-methylpyrimidine-2,4(1H,3H)-dione, prepared as described.²⁶ General: C₂₂H₁₅N₇O₂; MW = 409.41. ¹H NMR (400 MHz, Acetone-d₆) δ 9.42 (s, 1H), 8.67 (d, J = 12.7 Hz, 1H), 8.31 – 8.22 (m, 1H), 8.19 – 7.99 (m, 2H), 7.85 (q, J = 7.8 Hz, 1H), 7.77 (t, J = 7.8 Hz, 2H), 7.63 – 7.51 (m, 1H), 7.50 – 7.40 (m, 1H), 5.52 – 5.20 (m, 2H), 2.42 (s, 3H). ¹³C-NMR (100 MHz, DMSO) δ 159.0, 154.8, 154.5, 154.0, 150.9, 145.3, 143.5, 134.9, 133.0, 132.2, 129.2, 129.0, 128.6, 127.6, 125.0, 122.2, 120.0, 116.1, 111.5, 110.8, 34.6, 16.3. LCMS (ESI): m/z = 410.13 [M+H]⁺ (calcd for C₂₂H₁₅N₇O₂: 410.41).

Synthesis of AVI-4774.

General Procedure D was followed using 1-bromobut-2-yne and uracil intermediate 5-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(isoquinolin-4-yl)-6-methylpyrimidine-2,4(1H,3H)-dione (30 mg, 0.081 mmol). Yield: 10 mg, 0.024 mmol, 29%. General: C₂₄H₁₈N₆O₂; MW = 422.45. ¹H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.67 (d, J = 23.0 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.3 Hz, 2H), 7.99 – 7.83 (m, 2H), 7.82 – 7.72 (m, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 4.89 (dd, J = 49.9, 18.3 Hz, 2H), 2.28 (d, J = 8.0 Hz, 3H), 1.89 (s, 3H). ¹³C-NMR (100 MHz, DMSO) δ 159.0, 155.5, 153.9, 151.0, 145.3, 143.6, 134.9, 133.0, 132.2, 128.9, 128.5, 127.8, 125.0, 122.3, 121.9, 119.9, 111.7, 110.1, 81.8, 73.9, 36.4, 31.2, 15.9, 3.7. LCMS (ESI): m/z = 423.33 [M+H]⁺ (calcd for C₂₂H₁₅N₇O₂: 423.45).

Synthesis of AVI-5764.

General Procedure D was followed using 4-bromobut-1-yne and uracil intermediate 5-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(isoquinolin-4-yl)-6-methylpyrimidine-2,4(1H,3H)-dione. General: C₂₄H₁₈N₆O₂; MW = 422.45. ¹H-NMR ¹H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.62 (d, J = 7.9 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.94 – 7.85 (m, 1H), 7.79 (q, J = 7.1, 6.6 Hz, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 9.0 Hz, 1H), 4.20 (q, J = 7.4 Hz, 2H), 3.11 (s, 1H), 2.76 (t, J = 6.6 Hz, 2H), 2.23 (d, J = 4.2 Hz, 3H). ¹³C-NMR (100 MHz, DMSO) δ 159.1, 156.1, 155.8, 153.9, 146.5, 145.2, 143.6, 134.9, 133.0, 132.2, 129.2, 129.0, 128.5, 127.8, 125.0, 122.0, 120.0, 111.4, 111.3, 109.7, 74.3, 44.8, 17.9, 16.2. LCMS (ESI): m/z = 423.13 [M+H]⁺ (calcd for C₂₄H₁₈N₆O₂: 423.45).

Synthesis of AVI-4690.

General Procedure D was followed using allyl bromide and uracil intermediate 5-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(isoquinolin-4-yl)-6-methylpyrimidine-2,4(1H,3H)-dione. General: C₂₃H₁₈N₆O₂; MW = 410.44. ¹H NMR (400 MHz, Acetone-d₆) δ 9.40 (s, 1H), 8.65 (d, J = 26.9 Hz, 1H), 8.33 – 8.17 (m, 1H), 8.16 – 7.96 (m, 2H), 7.84 (t, J = 7.6 Hz, 1H), 7.75 (dd, J = 14.4, 7.7 Hz, 1H), 7.58 (d, J = 10.5 Hz, 1H), 7.49 – 7.36 (m, 1H), 6.28 – 6.00 (m, 1H), 5.43 (dd, J = 63.9, 13.9 Hz, 2H), 4.87 (d, J = 4.9 Hz, 2H), 2.27 (s, 33H). ¹³C-NMR (100 MHz, DMSO) δ 159.1, 155.9, 155.7, 153.8, 151.1, 145.3, 143.6, 134.9, 132.9, 132.2, 129.2, 128.9, 128.5, 128.5, 127.9, 124.9, 122.1, 119.9, 117.7, 111.5, 109.9, 48.3, 15.7. LCMS (ESI): m/z = 411.18 [M+H]⁺ (calcd for C₂₃H₁₈N₆O₂: 411.44).

Synthesis of AVI-4770.

General Procedure D was followed using 2-(bromomethyl)oxirane and uracil intermediate 5-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(isoquinolin-4-yl)-6-methylpyrimidine-2,4(1H,3H)-dione. General: C₂₃H₁₈N₆O₃; MW = 426.14. ¹H-NMR (400 MHz, DMSO-d6): δ (ppm): 9.44 (s, 1H), 8.66 (d, J = 9.5 Hz, 1H), 8.28 (d, J = 9.4 Hz, 1H), 8.02 (dd, J = 7.0, 3.5, 1H), 7.97 – 7.70 (m, 4H), 7.60 – 7.44 (m, 2H), 4.62 – 4.35 (m, 1H), 4.21 – 3.84 (m, 1H), 3.41 (ddt, J = 6.7, 4.9, 2.8 Hz, 1H), 2.98 – 2.67 (m, 2H), 2.13 (s, 3H). ¹³C-NMR (101 MHz, DMSO-d6): δ (ppm): 158.9, 155.7, 155.6, 153.9, 151.2, 144.9, 143.6, 135.4, 132.9, 132.9, 132.2, 128.6, 128.5, 128.1, 127.7, 127.7, 121.8, 118.8, 116.5, 50.0, 45.6, 45.2, 16.5. LCMS (ESI): m/z = 427.29 [M+H]⁺ (calcd for C₂₃H₁₈N₆O₃: 427.14).

Synthesis of AVI-6179.

Step 1: Synthesis of ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)pent-2-enoate: General Procedure B was followed using ethyl (2Z)-3-amino-2-pentenoate (500 mg, 3.49 mmol) and 5,6-difluoro-1H-benzotriazole (1.2 eq, 4.19 mmol). Yield: 857 mg, 2.89 mmol, 83%. General: C₁₃H₁₄F₂N₄O₂; MW = 296.11. LCMS (ESI): m/z = 297.09 [M+H]⁺ (calcd for C₁₃H₁₄F₂N₄O₂: 297.11). Step 2: General Procedure C was followed from ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)pent-2-enoate (400 mg, 1.35 mmol). Purification by preparative HPLC (10% to 100% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil product (271 mg, 0.645 mmol, 48%) as a brown solid. General: C₂₁H₁₄F₂N₆O₂; MW = 420.38. ¹H-NMR (400 MHz, Acetone-d₆) δ 9.39 (d, J = 0.9 Hz, 1H), 8.63 (s, 1H), 8.24 (dt, J = 8.1, 1.0 Hz, 1H), 8.08 (dd, J = 9.7, 7.1 Hz, 2H), 7.98 (s, 1H), 7.92 – 7.79 (m, 2H), 7.75 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 2.07 (p, J = 2.2 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). LCMS (ESI): m/z = 421.44 [M+H]⁺ (calcd for C₂₁H₁₄F₂N₆O₂: 420.38). Step 3: General Procedure D was followed from the uracil intermediate (8 mg, 0.020 mmol). Yield: 1.5 mg, 3.3 μmol, 20%. General: C₂₄H₁₆F₂N₆O₂; MW = 458.43. ¹H-NMR (400 MHz, Acetone-d₆) δ 9.40 (d, J = 0.9 Hz, 1H), 8.64 (d, J = 10.2 Hz, 1H), 8.31 – 8.19 (m, 1H), 8.17 – 8.05 (m, 1H), 8.02 – 7.66 (m, 4H), 5.27 – 4.85 (m, 2H), 3.11 (q, J = 2.6 Hz, 1H), 2.09 (dd, J = 6.1, 1.7 Hz, 2H), 1.38 (t, J = 7.5 Hz, 3H). ¹³C-NMR (101 MHz, DMSO-d₆): δ (ppm): 160.0, 159.7, 159.1, 153.7, 151.0, 143.1, 143.0, 133.2, 133.1, 132.4, 129.1, 128.7, 122.6, 122.5, 122.0, 119.0, 112.3, 109.6, 109.5, 78.9, 76.6, 36.0, 22.7, 13.6. LCMS (ESI): m/z = 459.35 [M+H]⁺ (calcd for C₂₄H₁₆F₂N₆O₂: 459.43).

Synthesis of AVI-6358.

Step 1: Synthesis of ethyl (Z)-3-amino-4-methylpent-2-enoate: General Procedure E was used using ethyl 4-methyl-3-oxopentanoate (1.5 g, 9 mmol), ammonium formate (5 eq, 45 mmol) and ethanol (15 mL). General: C₈H₁₅NO₂; MW = 157.21. LCMS (ESI): m/z = 158.15 [M+H]⁺ (calcd for C₈H₁₅NO₂: 158.21). Step 2: Synthesis of ethyl (E)-3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-5-methylpent-2-enoate: General Procedure B was followed using ethyl (2Z)-3-amino-2-pentenoate (596 mg, 3.49 mmol) and 5,6-difluoro-1H-benzotriazole (1.2 eq, 4.19 mmol). Yield: 760 mg, 2.44 mmol, 70%. General: C₁₄H₁₆F₂N₄O₂; MW = 310.30. LCMS (ESI): m/z = 311.15 [M+H]⁺ (calcd for C₁₄H₁₆F₂N₄O₂: 311.30). Step 3: General procedure C was used from ethyl (E)-3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-5-methylpent-2-enoate (273 mg, 0.88 mmol). Purification by preparative HPLC (10% to 100% CH₃CN in H₂O + 0.1% formic acid) afforded AVI-6260 (82 mg, 0.19 mmol, 21%) as a yellow solid. General: C₂₂H₁₆F₂N₆O₂; MW = 434.41. ¹H-NMR (400 MHz, Acetone-d₆) δ 9.50 (s, 1H), 8.69 (d, J = 22.1 Hz, 1H), 8.32 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 16.8 Hz, 1H), 8.08 – 7.94 (m, 1H), 7.88 (q, J = 7.4 Hz, 1H), 7.79 – 7.65 (m, 1H), 2.71 (p, J = 7.0 Hz, 1H), 1.52 – 1.39 (m, 3H), 1.39 – 1.23 (m, 3H). LCMS (ESI): m/z = 435.29 [M+H]⁺ (calcd for C₂₂H₁₆F₂N₆O₂: 435.41). Step 4: General Procedure D was followed using the uracil intermediate (30 mg, 0.0692 mmol). Yield: 8.6 mg, 0.018 mmol, 26%. General: C₂₅H₁₈F₂N₆O₂; MW = 472.46. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.42 (d, J = 5.4 Hz, 1H), 8.63 (d, J = 27.7 Hz, 1H), 8.39 – 8.29 (m, 1H), 8.25 (t, J = 7.5 Hz, 2H), 7.90 (ddd, J = 32.2, 15.4, 8.1 Hz, 2H), 7.82 – 7.68 (m, 1H), 5.14 – 4.68 (m, 2H), 3.05 (q, J = 7.2 Hz, 1H), 1.41 (dd, J = 14.2, 7.0 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 0.71 (d, J = 24.4 Hz, 1H). ¹³C-NMR (100 MHz, DMSO) δ 163.8, 153.9, 151.2, 151.1, 143.5, 143.4, 140.3, 138.6, 132.1, 129.1, 128.6, 127.8, 122.4, 122.1, 122.1, 109.9, 109.7, 107.2, 76.6, 46.0, 36.3, 30.5, 21.6, 19.6, 9.0. LCMS (ESI): m/z = 473.26 [M+H]⁺ (calcd for C₂₅H₁₈F₂N₆O₂: 473.46).

Synthesis of AVI-6259.

Step 1: Synthesis of ethyl (Z)-3-amino-5-methylhex-2-enoate: General Procedure E was followed, using ethyl 4-isobutyl-3-oxopentanoate (0.97 g, 5.4 mmol), ammonium formate (5 eq, 45 mmol) and ethanol (10 mL). General: C₉H₁₇NO₂; MW = 171.24. LCMS (ESI): m/z = 172.05 [M+H]⁺ (calcd for C₉H₁₇NO₂: 172.24). Step 2: Synthesis of ethyl (E)-3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-5-methylhex-2-enoate: General Procedure B was followed using ethyl (Z)-3-amino-2-pentenoate (596 mg, 3.49 mmol) and 5,6-difluoro-1H-benzotriazole (1.2 eq, 4.19 mmol). Yield: 867 mg, 2.67 mmol, 77%. General: C₁₅H₁₈F₂N₄O₂; MW = 324.33. LCMS (ESI): m/z = 324.95 [M+H]⁺ (calcd for C₁₅H₁₈F₂N₄O₂: 325.33). Step 3: General Procedure C was followed using ethyl (E)-3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-5-methylhex-2-enoate (400 mg, 1.23 mmol). Purification by preparative HPLC (10% to 100% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (271 mg, 0.645 mmol, 48%) as a yellow solid. General: C₂₃H₁₈F₂N₆O₂; MW = 448.43. ¹H-NMR (400 MHz, DMSO-d₆) δ 12.26 (s, 1H), 9.47 (s, 1H), 8.68 (d, J = 6.5 Hz, 1H), 8.39 – 8.26 (m, 2H), 8.17 (t, J = 8.8 Hz, 1H), 7.92 (dt, J = 8.6, 4.9 Hz, 2H), 7.80 (t, J = 7.4 Hz, 1H), 2.39 – 1.89 (m, 3H), 0.86 (d, J = 10.0 Hz, 6H). LCMS (ESI): m/z = 448.45 [M+H]⁺ (calcd for C₂₃H₁₈F₂N₆O₂: 448.43). Step 4: General Procedure D was followed using the uracil intermediate. Yield: (9.7 mg, 0.020 mmol, 21%). General: C₂₆H₂₀F₂N₆O₂; MW = 486.8. ¹H-NMR (400 MHz, Acetone-d₆) δ 9.40 (s, 1H), 8.65 (d, J = 17.3 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H), 8.09 (ddd, J = 9.5, 5.8, 2.3 Hz, 2H), 7.97 – 7.87 (m, 1H), 7.86 – 7.79 (m, 1H), 7.79 – 7.72 (m, 1H), 5.10 – 4.95 (m, 2H), 3.09 (q, J = 1.8, 1.2 Hz, 1H), 2.74 (m, 2H), 2.34 – 2.21 (m, 1H), 1.02 (dd, J = 6.6, 3.1 Hz, 3H), 0.79 (dd, J = 23.4, 6.6 Hz, 3H). ¹³C-NMR (100 MHz, DMSO-d₆) δ 158.9, 157.3, 156.9, 153.9, 150.9, 143.6, 143.5, 132.8, 132.2, 132.1, 129.2, 128.5, 127.7, 122.5, 121.9, 110.9, 110.6, 107.2, 107.0, 79.0, 76.3, 48.2, 37.3, 26.9, 23.0, 22.4. LCMS (ESI): m/z = 487.22 [M+H]⁺ (calcd for C₂₆H₂₀F₂N₆O₂: 486.80).

Synthesis of AVI-6359.

Step 1: Synthesis of ethyl (Z)-3-amino-3-cyclopentylacrylate: General Procedure E was followed using ethyl 3-cyclopentyl-3-oxopropanoate (1 g, 5.16 mmol), ammonium formate (5 eq, 26 mmol) and ethanol (10 mL). General: C₁₀H₁₇NO₂; MW = 183.25. LCMS (ESI): m/z = 184.05 [M+H]⁺ (calcd for C₁₀H₁₈NO₂: 184.25). Step 2: Synthesis of ethyl (E)-3-amino-3-cyclopentyl-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)acrylate (S42): General Procedure B (Amination of Enaminone) was followed using ethyl (Z)-3-amino-cyclopentylacrylate S41 (893 mg, 4.87 mmol) and 5,6-difluoro-1H-benzotriazole (1.1 eq, 5.36 mmol). Yield: 900 mg, 2.67 mmol, 55%. General: C₁₆H₁₈F₂N₄O₂; MW = 336.34. LCMS (ESI): m/z = 336.95 [M+H]⁺ (calcd for C₁₆H₁₉F₂N₄O₂: 337.34). Step 3: General Procedure C was followed using ethyl (E)-3-amino-3-cyclopentyl-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)acrylate (900 mg, 2.68 mmol). Purification by preparative HPLC (10% to 100% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (464 mg, 1.01 mmol, 38%) as a white solid. General: C₂₄H₁₈F₂N₆O₂; MW = 460.44. ¹H-NMR (400 MHz, CD₃CN) δ 9.36 (s, 1H), 8.57 (d, J = 21.6 Hz, 1H), 8.21 (d, J = 8.09 Hz, 1H), 8.10 (s, 1H), 8.02–7.95 (m, 1H), 7.88–7.79 (m,, 1H), 764–7.60 (m, 1H), 1.48 (t, J = 6.92 Hz, 1H), 1.35–1.22 (m, J = 10.0 Hz, 8H). LCMS (ESI): m/z = 461.45 [M+H]⁺ (calcd for C₂₄H₁₉F₂N₆O₂: 461.44). Step 4: General Procedure D was followed using the uracil intermediate. Yield: (11 mg, 0.022 mmol, 34%). General: C₂₇H₂₀F₂N₆O₂; MW = 498.49. ¹H-NMR (400 MHz, CD₃CN) δ 9.38 (s, 1H), 8.53 (d, J = 17.3 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.03–7.98 (m, 2H), 7.92 – 7.83 (m, 2H), 7.80 – 7.74 (m, 1H), 7.66 – 7.61 (m, 1H), 5.06 – 4.74 (m, 2H), 3.34 (q, J = 1.8, 1.2 Hz, 1H), 2.83 (m, 1H), 1.69–1.17 (m, 8H). LCMS (ESI): m/z = 499.22 [M+H]⁺ (calcd for C₂₇H₂₁F₂N₆O₂: 499.49)

Synthesis of AVI-6261.

Step 1: Synthesis of ethyl (Z)-3-amino-3-cyclohexylacrylate: General Procedure E was followed using ethyl 3-cyclohexyl-3-oxopropanoate (500 mg, 2.4 mmol), ammonium formate (5 eq., 12 mmol) and ethanol (10 mL). General: C₁₁H₁₉NO₂; MW = 197.28. LCMS (ESI): m/z = 198.29 [M+H]⁺ (calcd for C₁₁H₁₉NO₂: 198.28). Step 2: synthesis of ethyl (E)-3-amino-3-cyclohexyl-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)acrylate: General Procedure B was followed using ethyl (Z)-3-amino-3-cyclohexylacrylate (300 mg, 1.52 mmol) and 5,6-difluoro-1H-benzotriazole (1.1 eq, 1.67 mmol). Yield: 360 mg, 1.03 mmol, 68%. General: C₁₇H₂₀F₂N₄O₂; MW = 350.37. LCMS (ESI): m/z = 351.3 [M+H]⁺ (calcd for C₁₇H₂₀F₂N₄O₂: 351.37). Step 3: General Procedure C was followed using ethyl (E)-3-amino-3-cyclohexyl-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)acrylate (150 mg, 0.43 mmol). Purification by preparative HPLC (10% to 100% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (40.6 mg, 0.0856 mmol, 20%) as a yellow solid. General: C₂₅H₂₀F₂N₆O₂; MW = 474.47. ¹H-NMR (400 MHz, CD₃CN) δ 9.38 (s, 1H), 8.56 (d, J = 22.6 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.06 – 7.82 (m, 2H), 7.78 (dd, J = 9.1, 4.5 Hz, 2H), 7.60 (dd, J = 9.2, 6.8 Hz, 1H), 2.30 – 2.18 (m, 1H), 1.85 – 1.56 (m, 8H), 1.19 (d, J = 13.3 Hz, 2H). LCMS (ESI): m/z = 475.31 [M+H]⁺ (calcd for C₂₅H₂₀F₂N₆O₂: 475.47). Step 4: General Procedure D was followed using the uracil intermediate(63 mg, 0.085 mmol). Yield: 8.6 mg, 0.017 mmol, 20%. General: C₂₈H₂₂F₂N₆O₂; MW = 512.52. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.42 (d, J = 5.7 Hz, 1H), 8.62 (d, J = 23.1 Hz, 1H), 8.33 (dd, J = 9.8, 7.2 Hz, 1H), 8.28 – 8.19 (m, 3H), 7.98 – 7.83 (m, 2H), 7.81 – 7.74 (m, 1H), 5.19 – 4.74 (m, 2H), 3.62 (d, J = 5.4 Hz, 1H), 2.22 (s, 1H), 1.72 (d, J = 14.7 Hz, 4H), 1.54 (d, J = 13.3 Hz, 4H), 1.38 – 1.04 (m, 2H). ¹³C-NMR (100 MHz, DMSO) δ 168.4, 163.7, 153.9, 151.2, 151.1, 143.4, 143.4, 132.8, 132.1, 132.1, 129.1, 128.5, 128.1, 127.8, 122.4, 122.4, 122.1, 114.2, 110.2, 107.0, 79.1, 76.7, 76.6, 42.0, 41.9, 26.4, 25.5, 23.1. LCMS (ESI): m/z = 513.13 [M+H]⁺ (calcd for C₂₈H₂₂F₂N₆O₂: 513.52)

Synthesis of AVI-5734.

Step 1: Synthesis of ethyl 3-amino-3-(3,4-dichlorophenyl)-2-propenoate: The general procedure A was followed, using 3,4-dichlorobenzonitrile (1 g, 5.81 mmol). Yield: 1.42 g, 5.50 mmol, 95%. General: C₁₁H₁₁Cl₂NO₂; MW = 260.11. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.63 (d, J = 2.2 Hz, 1H); 7.48 (d, J = 8.3 Hz, 1H); 7.37 (dd, J = 8.4, 2.1 Hz, 1H); 4.92 (s, 1H); 4.16 (q, J = 7.2 Hz, 2H); 1.29 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 226.1 [M+H]⁺. LCMS (ESI): m/z = 260.1 [M+H]⁺ (calcd for C₁₁H₁₂Cl₂NO₂: 260.02). Step 2: Synthesis of ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(3,4-dichlorophenyl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(3,4-dichlorophenyl)-2-propenoate (100 mg, 0.386 mmol), N-bromosuccinimide, 1H-benzotriazole and Na₂CO₃ in DMF. Yield: 87 mg, 0.231 mmol, 60%. General: C₁₇H₁₄Cl₂N₄O₂; MW = 377.23. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.91 (brs, 1H); 7.96 (d, J = 8.4 Hz, 1H); 7.45 (m, 1H); 7.37–7.28 (m, 3H); 7.13 (d, J = 8.3 Hz, 1H); 6.98 (dd, J = 8.3, 2.0 Hz, 1H); 5.26 (brs, 1H); 4.13–4.05 (m, 2H); 1.02 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 377.1 [M+H]⁺ (calcd for C₁₇H₁₅Cl₂N₄O₂: 377.05). Step 3: General Procedure C was followed using ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(3,4-dichlorophenyl)acrylate (66 mg, 0.176 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (30.7 mg, 0.0612 mmol, 35%) as a white solid. General: C₂₅H₁₄Cl₂N₆O₂; MW = 501.33. ¹H-NMR (400 MHz, DMSO-d₆): δ (ppm): 12.61 (brs, 1H); 9.45 (s, 1H); 8.65 (s, 1H); 8.28 (d, J = 8.2 Hz, 1H); 8.25–8.06 (m, 1H); 8.03 (d, J = 8.3 Hz, 1H); 7.97–7.83 (m, 2H); 7.79 (t, J = 7.5 Hz, 1H); 7.76 (d, J = 1.8 Hz, 1H); 7.64–7.52 (m, 2H); 7.39 (t, J = 7.7 Hz, 1H); 7.26 (brs, 1H). LCMS (ESI): m/z = 501.2 [M+H]⁺ (calcd for C₂₅H₁₅Cl₂N₆O₂: 501.06). Step 4: General Procedure D was followed using the uracil intermediate (12 mg, 0.0239 mmol). Yield: 5.2 mg, 9.64 μmol, 40%. General: C₂₈H₁₆Cl₂N₆O₂; MW = 539.38. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.62 (d, J = 18.5 Hz, 1H); 8.23 (d, J = 8.3 Hz, 1H); 8.17–7.98 (m, 1H); 7.97–7.87 (m, 2H); 7.84–7.49 (m, 5H); 7.42–7.29 (m, 2H); 4.68–4.27 (m, 2H); 2.70 (brs, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.3, 154.9, 151.6, 145.9, 143.9, 135.7, 135.6, 133.7, 132.9, 132.8, 132.0, 131.9, 131.8, 130.1, 129.9, 129.6, 129.4, 129.3, 129.2, 128.4, 125.4, 122.1, 122.0, 120.5, 111.2, 78.5, 74.8, 38.6. LCMS (ESI): m/z = 539.0 [M+H]⁺ (calcd for C₂₈H₁₇Cl₂N₆O₂: 539.07).

Synthesis of AVI-6329.

Step 1: Synthesis of ethyl 3-amino-3-(7-chloro-1-benzofuran-5-yl)-2-propenoate: The general procedure A was followed, using 7-chloro-5-benzofurancarbonitrile (300 mg, 1.69 mmol). Yield: 337 mg, 1.27 mmol, 75%. General: C₁₃H₁₂ClNO₃; MW = 265.69. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.77–7.67 (m, 1H); 7.74 (d, J = 2.2 Hz, 1H); 7.53 (s, 1H); 6.85 (d, J = 2.1 Hz, 1H); 4.96 (s, 1H); 4.19 (q, J = 7.2 Hz, 2H); 1.30 (t, J = 7.2 Hz, 3H). LCMS (ESI): m/z = 266.1 [M+H]⁺ (calcd for C₁₃H₁₃ClNO₃: 266.05). Step 2: Synthesis of ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(7-chloro-1-benzofuran-5-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(7-chloro-1-benzofuran-5-yl)-2-propenoate (100 mg, 0.376 mmol), N-bromosuccinimide, 1H-benzotriazole and Na₂CO₃ in DMF. Yield: 81 mg, 0.211 mmol, 56%. General: C₁₉H₁₅ClN₄O₃; MW = 382.80. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.99 (brs, 1H); 7.88 (d, J = 8.3 Hz, 1H); 7.55 (m, 1H); 7.42–7.33 (m, 3H); 7.17 (d, J = 1.5 Hz, 1H); 6.60 (d, J = 2.2 Hz, 1H); 5.40 (brs, 1H); 4.13–4.04 (m, 2H); 1.02 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 383.0 [M+H]⁺ (calcd for C₁₉H₁₆ClN₄O₃: 383.08). Step 3: General Procedure C was followed using ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(7-chloro-1-benzofuran-5-yl)acrylate (70 mg, 0.194 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (13.4 mg, 0.0264 mmol, 22%) as a slightly yellow solid. General: C₂₇H₁₅ClN₆O₃; MW = 506.91. ¹H-NMR (400 MHz, DMSO-d₆): δ (ppm): 8.52 (s, 1H); 7.41 (s, 1H); 7.37 (d, J = 8.1 Hz, 1H); 7.34–7.13 (m, 1H); 7.21 (d, J = 2.1 Hz, 1H); 7.06 (d, J = 8.4 Hz, 1H); 7.04–6.91 (m, 2H); 6.88 (t, J = 7.7 Hz, 1H); 6.79 (d, J = 12.9 Hz, 1H); 6.63 (t, J = 7.6 Hz, 1H); 6.55 (m, 1H); 6.44 (t, J = 7.7 Hz, 1H); 6.16 (s, 1H). ¹³C-NMR (101 MHz, DMSO-d₆): δ (ppm): 160.4, 153.4, 150.8, 150.5, 148.5, 145.7, 144.5, 143.2, 141.0, 134.9, 132.8, 131.7, 128.9, 128.8, 128.6, 128.4, 128.2, 128.1, 127.3, 124.4, 124.0, 120.7, 120.6, 119.3, 115.5, 110.8, 108.0. LCMS (ESI): m/z = 507.0 [M+H]⁺ (calcd for C₂₇H₁₆ClN₆O₃: 507.09). Step 4: General Procedure D was followed using the uracil intermediate (10 mg, 0.0197 mmol). Yield: 5.4 mg, 9.91 μmol, 50%. General: C₃₀H₁₇ClN₆O₃; MW = 544.95. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.64 (dd, J = 20.2, 3.8 Hz, 1H); 8.22 (d, J = 8.1 Hz, 1H); 8.16–8.03 (m, 1H); 7.96–7.83 (m, 3H); 7.82–7.73 (m, 3H); 7.64 (m, 1H); 7.54 (m, 1H); 7.33 (m, 1H); 6.99–6.80 (m, 1H); 4.64–4.30 (m, 2H); 2.67 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.5, 156.4, 154.9, 152.1, 151.7, 149.1, 145.8, 143.9, 135.6, 133.7, 132.9, 132.8, 130.1, 129.5, 129.4, 129.3, 129.2, 128.5, 125.3, 124.1, 122.5, 122.1, 120.7, 120.3, 111.2, 108.6, 108.5, 78.7, 74.5, 38.7. LCMS (ESI): m/z = 545.1 [M+H]⁺ (calcd for C₃₀H₁₈ClN₆O₃: 545.11).

Synthesis of AVI-6330.

Step 1: Synthesis of ethyl 3-amino-3-(3-chlorophenyl-4-methoxy)-2-propenoate: The general procedure A was followed, using 3-chloro-4-methoxybenzonitrile (500 mg, 2.98 mmol). Due to incomplete conversion, the crude was purified on silica gel chromatography (0% to 50% EtOAc in hexane). Yield: 212 mg, 0.829 mmol, 28%. General: C₁₂H₁₄ClNO₃; MW = 255.70. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.58 (s, 1H); 7.43 (m, 1H); 6.94 (d, J = 8.6 Hz, 1H); 4.91 (s, 1H); 4.17 (q, J = 7.1 Hz, 2H); 3.93 (s, 3H); 1.29 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 256.1 [M+H]⁺ (calcd for C₁₂H₁₅ClNO₃: 256.07). Step 2: Synthesis of ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(3-chlorophenyl-4-methoxy) acrylate: General Procedure B was followed using ethyl 3-amino-3-(3-chlorophenyl-4methoxy)-2-propenoate (187 mg, 0.731 mmol), N-bromosuccinimide, 5,6-difluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 208 mg, 0.509 mmol, 70%. General: C₁₈H₁₅ClF₂N₄O₃; MW = 408.79. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.95 (brs, 1H); 7.68 (m, 1H); 7.50 (t, J = 8.4 Hz, 1H); 7.09 (m, 1H); 7.02 (dd, J = 8.5, 2.2 Hz, 1H); 6.61 (d, J = 8.6 Hz, 1H); 5.32 (brs, 1H); 4.14–4.06 (m, 2H); 3.76 (s, 3H); 1.04 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 409.1 [M+H]⁺ (calcd for C₁₈H₁₆ClF₂N₄O₃: 409.08). Step 3: General Procedure C was followed using ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(3-chlorophenyl-4-methoxy) acrylate (165 mg, 0.404 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (60 mg, 0.113 mmol, 28%) as a white solid. General: C₂₆H₁₅ClF₂N₆O₃; MW = 532.89. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.38 (s, 1H); 8.58 (s, 1H); 8.20 (d, J = 8.2 Hz, 1H); 8.04 (m, 1H); 7.90 (dd, J = 9.6, 7.0 Hz, 2H); 7.77 (t, J = 8.0 Hz, 1H); 7.56 (dd, J = 9.2, 6.7 Hz, 1H); 7.47 (d, J = 8.2 Hz, 1H); 7.25 (dd, J = 8.6, 2.1 Hz, 1H); 6.94 (d, J = 8.7 Hz, 1H); 3.83 (s, 3H). LCMS (ESI): m/z = 533.1 [M+H]⁺ (calcd for C₂₆H₁₆ClF₂N₆O₃: 533.09). Step 4: General Procedure D was followed using the uracil intermediate (39 mg, 0.0732 mmol). Yield: 26.4 mg, 0.0462 mmol, 63%. General: C₂₉H₁₇ClF₂N₆O₃; MW = 570.94. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.40 (s, 1H); 8.61 (d, J = 20.8 Hz, 1H); 8.22 (d, J = 8.1 Hz, 1H); 8.15–7.98 (m, 1H); 7.91 (m, 1H); 7.85–7.75 (m, 2H); 7.63 (m, 1H); 7.58–7.32 (m, 2H); 7.06–6.90 (m, 1H); 4.68–4.25 (m, 2H); 3.81 (d, J = 11.2 Hz, 1H); 2.71 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.3, 157.7, 156.5, 154.9, 151.6, 143.9, 143.8, 141.0, 140.9, 133.6, 132.9, 132.8, 131.2, 130.1, 129.7, 129.3, 129.2, 128.5, 128.4, 123.0, 122.0, 121.7, 113.5, 107.4, 98.9, 78.7, 74.6, 57.1, 38.7. LCMS (ESI): m/z = 571.1 [M+H]⁺ (calcd for C₂₉H₁₈ClF₂N₆O₃: 571.10).

Synthesis of AVI-6322.

Step 1: Synthesis of ethyl 3-amino-3-(3,4-dimethoxyphenyl)-2-propenoate. The general procedure A was followed, using 3,4-dimethoxybenzonitrile (500 mg, 3.06 mmol). Due to incomplete conversion, the crude was purified on silica gel chromatography (0% to 50% EtOAc in hexane). Yield: 225 mg, 0.895 mmol, 29%. General: C₁₃H₁₇NO₄; MW = 251.28. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.14 (dd, J = 8.3, 2.1 Hz, 1H); 7.04 (d, J = 2.0 Hz, 1H); 6.88 (d, J = 8.4 Hz, 1H); 4.94 (s, 1H); 4.17 (q, J = 7.1 Hz, 2H); 3.91 (s, 6H); 1.30 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 252.1 [M+H]⁺ (calcd for C₁₃H₁₈NO₄: 252.12). Step 2: Synthesis of ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(3,4-dimethoxyphenyl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(3,4-dimethoxyphenyl)-2-propenoate (200 mg, 0.796 mmol), N-bromosuccinimide, 5,6-difluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 242 mg, 0.598 mmol, 75%. General: C₁₉H₁₈F₂N₄O₄; MW = 404.37. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.99 (brs, 1H); 7.68 (dd, J = 9.2, 7.0 Hz, 1H); 7.06 (dd, J = 8.6, 6.5 Hz, 1H); 6.88 (dd, J = 8.4, 2.1 Hz, 1H); 6.65 (d, J = 8.4 Hz, 1H); 6.56 (d, J = 2.0 Hz, 1H); 5.32 (brs, 1H); 4.14–4.07 (m, 2H); 3.75 (s, 3H); 3.55 (s, 3H); 1.06 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 405.1 [M+H]⁺ (calcd for C₁₉H₁₉F₂N₄O₄: 405.13). Step 3: General Procedure C was followed using ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(3,4-dimethoxyphenyl) acrylate (100 mg, 0.247 mmol) and carbamic acid-4-isoquinolinyl-ethyl ester. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (26.7 mg, 0.0505 mmol, 20%) as a white solid. General: C₂₇H₁₈F₂N₆O₄; MW = 528.48. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.37 (s, 1H); 8.58 (d, J = 9.5 Hz, 1H); 8.20 (d, J = 8.2 Hz, 1H); 8.04 (m, 1H); 7.89 (dd, J = 9.6, 7.0 Hz, 2H); 7.76 (t, J = 7.5 Hz, 1H); 7.56 (dd, J = 9.2, 6.9 Hz, 1H); 6.99 (d, J = 8.4 Hz, 1H); 6.83 (d, J = 8.4 Hz, 1H); 6.76 (s, 1H); 3.76 (s, 3H); 3.54 (s, 3H). LCMS (ESI): m/z = 529.1 [M+H]⁺ (calcd for C₂₇H₁₉F₂N₆O₄: 529.14). Step 4: General Procedure D was followed using the urail intermediate (16 mg, 0.0303 mmol). Yield: 10.5 mg, 0.0185 mmol, 61%. General: C₃₀H₂₀F₂N₆O₄; MW = 566.52. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.40 (s, 1H); 8.61 (d, J = 15.8 Hz, 1H); 8.22 (d, J = 8.3 Hz, 1H); 8.14–7.99 (m, 1H); 7.96–7.86 (m, 1H); 7.84–7.75 (m, 2H); 7.72–7.58 (m, 1H); 7.12–6.95 (m, 2H); 6.89–6.76 (m, 1H); 4.68–4.26 (m, 2H); 3.75 (m, 3H); 3.68 (m, 3H); 2.70 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.5, 157.9, 154.9, 151.9, 151.7, 149.8, 143.9, 143.8, 133.7, 132.9, 130.1, 129.3, 129.3, 129.2, 128.5, 122.6, 122.0, 121.1, 112.7, 112.2, 111.4, 107.1, 98.9, 79.1, 74.5, 56.5, 56.3, 38.9. LCMS (ESI): m/z = 567.2 [M+H]⁺ (calcd for C₃₀H₂₁F₂N₆O₄: 567.15).

Synthesis of AVI-6325.

Step 1: Synthesis of ethyl 3-amino-2-(4-cyano-1H-1,2,3-triazol1-yl)-3-(3,4-dichlorophenyl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(3,4-dichlorophenyl)-2-propenoate (100 mg, 0.386 mmol), N-bromosuccinimide, 1H-triazole-5-carbonitrile and DBU in toluene. Yield: 36 mg, 0.102 mmol, 27%. General: C₁₄H₁₁Cl₂N₅O₂; MW = 352.18. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.70 (brs, 1H); 7.88 (s, 1H); 7.39 (d, J = 2.1 Hz, 1H); 7.34 (d, J = 8.3 Hz, 1H); 7.05 (dd, J = 8.4, 2.1 Hz, 1H); 5.18 (brs, 1H); 4.16 (q, J = 7.1 Hz, 2H); 1.16 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 351.0 [M+H]⁺ (calcd for C₁₄H₁₂Cl₂N₅O₂: 352.03). Step 2: General Procedure C was followed using ethyl 3-amino-2-(4-cyano-1H-1,2,3-triazol-1-yl)-3-(3,4-dichlorophenyl) acrylate (36 mg, 0.102 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (18.7 mg, 0.0393 mmol, 38%) as a white solid, one peak observed by LCMS. General: C₂₂H₁₁Cl₂N₇O₂; MW = 476.28. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.39 (s, 1H); 8.53 (s, 1H); 8.26 (s, 1H); 8.21 (d, J = 8.3 Hz, 1H); 7.97 (dd, J = 8.3, 0.8 Hz, 1H); 7.91–7.83 (m, 1H); 7.80–7.73 (m, 1H); 7.65 (d, J = 2.2 Hz, 1H); 7.58 (d, J = 8.2 Hz, 1H); 7.25 (dd, J = 8.3, 2.1 Hz, 1H). LCMS (ESI): m/z = 476.0 [M+H]⁺ (calcd for C₂₂H₁₂Cl₂N₇O₂: 476.04). Step 3: General Procedure D was followed using the uracil intermediate (14.5 mg, 0.0304 mmol). Yield: 4.3 mg, 8.36 μmol, 28%. General: C₂₅H₁₃Cl₂N₇O₂; MW = 514.33. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.56 (s, 1H); 8.22 (d, J = 8.2 Hz, 1H); 8.14 (s, 1H); 7.99 (dd, J = 8.4, 3.8 Hz, 1H); 7.89 (t, J = 7.9 Hz, 1H); 7.78 (t, J = 7.9 Hz, 1H); 7.70 (dd, J = 15.9, 2.0 Hz, 1H); 7.62 (d, J = 8.3 Hz, 1H); 7.43 (m, 1H); 4.56–4.31 (m, 2H); 2.68 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 159.8, 155.0, 151.3, 143.9, 141.1, 136.2, 133.6, 132.9, 132.2, 132.1, 131.2, 130.1, 129.3, 129.1, 129.0, 128.6, 128.1, 124.1, 121.9, 117.0, 111.8, 78.3, 75.0, 38.6. LCMS (ESI): m/z = 514.1 [M+H]⁺ (calcd for C₂₅H₁₄Cl₂N₇O₂: 514.05).

Synthesis of AVI-6011.

Step 1: Synthesis of ethyl 3-amino-3-(5,6-dichloropyridin-3-yl)-2-propenoate: The general procedure A was followed, using 5,6-dichloro-3-pyridinecarbonitrile (500 mg, 2.89 mmol). Yield: 747 mg, 2.86 mmol, 99%. General: C₁₀H₁₀Cl₂N₂O₂; MW = 261.10. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.48 (d, J = 2.2 Hz, 1H); 7.93 (d, J = 2.2 Hz, 1H); 4.94 (s, 1H); 4.19 (q, J = 7.1 Hz, 2H); 1.30 (t, J = 7.2 Hz, 3H). LCMS (ESI): m/z = 261.0 [M+H]⁺ (calcd for C₁₀H₁₁Cl₂N₂O₂: 261.01). Step2: Sythesis of ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(5,6-dichloropyridin-3-yl)-2-propenoate (150 mg, 0.574 mmol), N-bromosuccinimide, 5,6-difluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 147 mg, 0.355 mmol, 62%. General: C₁₆H₁₄ClN₅O₂; MW = 414.19. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.93 (brs, 1H); 8.10 (d, J = 2.2 Hz, 1H); 7.74 (m, 1H); 7.70 (m, 1H); 7.15 (dd, J = 8.4, 6.5 Hz, 1H); 5.37 (brs, 1H); 4.17–4.10 (m, 2H); 1.05 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 413.0 [M+H]⁺ (calcd for C₁₆H₁₅ClN₅O₂: 414.03). Step 3: General Procedure C was followed using ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate (100 mg, 0.241 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (33.7 mg, 0.0626 mmol, 26%) as a white solid. General: C₂₄H₁₁Cl₂F₂N₇O₂; MW = 538.30. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.38 (s, 1H); 8.58 (s, 1H); 8.27 (d, J = 2.2 Hz, 1H); 8.21 (d, J = 8.4 Hz, 1H); 8.03 (m, 2H); 7.90 (dd, J = 9.5, 7.0 Hz, 2H); 7.81–7.74 (m, 1H); 7.57 (dd, J = 9.1, 6.7 Hz, 1H). LCMS (ESI): m/z = 538.0 [M+H]⁺ (calcd for C₂₄H₁₂Cl₂F₂N₇O₂: 538.03) Step 4: General Procedure D was followed using the uracil intermedaite (10 mg, 0.0186 mmol). Yield: 3.9 mg, 6.77 μmol, 36%. General: C₂₇H₁₃Cl₂F₂N₇O₂; MW = 576.34. ¹H-NMR (400 MHz, DMSO-d₆): δ (ppm): 9.51 (s, 1H); 8.69 (d, J = 19.7 Hz, 1H); 8.60–8.45 (m, 1H); 8.43–8.27 (m, 2H); 8.25–8.07 (m, 3H); 8.04–7.90 (m, 1H); 7.83 (t, J = 7.7 Hz, 1H); 4.81–4.37 (m, 2H); 3.53 (m, 1H). ¹³C-NMR (101 MHz, DMSO-d₆): δ (ppm): 158.7, 153.7, 153.6, 150.2, 150.1, 150.0, 142.4, 142.3, 139.7, 139.6, 132.7, 132.4, 130.8, 128.8, 128.6, 128.5, 127.4, 127.3, 121.8, 121.5, 114.3, 111.3, 106.9, 106.8, 78.2, 76.5, 37.8. LCMS (ESI): m/z = 576.1 [M+H]⁺ (calcd for C₂₇H₁₄Cl₂F₂N₇O₂: 576.05).

Synthesis of AVI-6328.

Step 1: Synthesis of ethyl 3-amino-3-(5,6-dichloropyridin-2-yl)-2-propenoate: The general procedure A was followed, using 5,6-dichloropicolinonitrile (500 mg, 2.89 mmol). Yield: 747 mg, 2.86 mmol, 99%. General: C₁₀H₁₀Cl₂N₂O₂; MW = 261.1. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.81 (d, J = 8.3 Hz, 1H); 7.63 (d, J = 8.3 Hz, 1H); 5.30 (s, 1H); 4.20 (q, J = 7.1 Hz, 2H); 1.31 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 261.0 [M+H]⁺ (calcd for C₁₀H₁₀Cl₂N₂O₂: 261.01). Step 2: Synthesis of ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-2-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(5,6-dichloropyridin-2-yl)-2-propenoate (200 mg, 0.766 mmol), N-bromosuccinimide, 5,6-difluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 166 mg, 0.401 mmol, 52%. General: C₁₆H₁₁Cl₂F₂N₅O₂; MW = 414.19. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 9.03 (brs, 1H); 7.79 (dd, J = 9.0, 6.9 Hz, 1H); 7.41 (d, J = 8.3 Hz, 1H); 7.15 (dd, J = 8.5, 6.5 Hz, 1H); 6.46 (d, J = 8.3 Hz, 1H); 6.35 (brs, 1H); 4.16–4.07 (m, 2H); 1.05 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 414.0 [M+H]⁺ (calcd for C₁₆H₁₂Cl₂F₂N₅O₂: 414.03). Step 3: Synthesis of AVI-6326: General Procedure C was followed using ethyl 3-amino-2-(5,6-difluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-2-yl) acrylate (130 mg, 0.314 mmol) and carbamic acid-4-isoquinolinyl-ethyl ester. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded AVI-6326 (57 mg, 0.106 mmol, 34%) as a white solid. General: C₂₄H₁₁Cl₂F₂N₇O₂; MW = 538.30. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.38 (s, 1H); 8.59 (s, 1H); 8.20 (d, J = 8.2 Hz, 1H); 8.04 (m, 1H); 7.97 (dd, J = 9.6, 7.0 Hz, 1H); 7.87 (t, J = 7.5 Hz, 1H); 7.79 (d, J = 8.2 Hz, 1H); 7.75 (d, J = 7.5 Hz, 1H); 6.61 (dd, J = 9.2, 6.7 Hz, 1H); 6.92 (d, J = 8.3 Hz, 1H). LCMS (ESI): m/z = 538.0 [M+H]⁺ (calcd for C₂₄H₁₂Cl₂F₂N₇O₂: 538.03). Step 4: General Procedure D was followed using the uracil intermediate (34 mg, 0.0632 mmol). Yield: 13.3 mg, 0.0231 mmol, 37%. General: C₂₇H₁₃Cl₂F₂N₇O₂; MW = 576.34. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.40 (s, 1H); 8.61 (s, 1H); 8.22 (d, J = 8.0 Hz, 1H); 8.09 (m, 1H); 7.93–7.81 (m, 3H); 7.78 (t, J = 7.4 Hz, 1H); 7.76 (t, J = 7.4 Hz, 1H); 7.50 (m, 1H); 4.72–4.55 (m, 2H); 2.62 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.2, 155.0, 151.5, 149.9, 146.0, 143.8, 141.2, 141.1, 141.1, 133.6, 133.5, 132.9, 132.1, 130.1, 129.3, 129.2, 128.3, 126.0, 122.2, 112.4, 107.5, 107.3, 99.5, 99.2, 78.0, 74.8, 38.0. LCMS (ESI): m/z = 576.1 [M+H]⁺ (calcd for C₂₇H₁₄Cl₂F₂N₇O₂: 576.05).

Synthesis of AVI-6039.

Step 1: Synthesis of ethyl 3-amino-2-(5,6-difluoro1H-benzo[d][1,2,3]triazol-1-yl)-3-(2-chloropyridin-4-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(2-chloropyridin-4-yl)-2-propenoate (200 mg, 0.882 mmol), N-bromosuccinimide, 5,6-difluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 202 mg, 0.532 mmol, 60%. General: C₁₆H₁₃ClF₂N₆O₂; MW = 378.77. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.88 (brs, 1H); 8.17 (d, J = 5.1 Hz, 1H); 7.72 (dd, J = 9.1, 7.0 Hz, 1H); 7.21 (d, J = 0.9 Hz, 1H); 7.12 (dd, J = 8.5, 6.5 Hz, 1H); 6.99 (dd, J = 5.1, 1.4 Hz, 1H); 5.38 (brs, 1H); 4.17–4.09 (m, 2H); 1.06 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 380.1 [M+H]⁺ (calcd for C₁₆H₁₄ClF₂N₆O₂: 380.06). Ste[2: General Procedure C was followed using ethyl 3-amino-2-(5,6-difluoro1H-benzo[d][1,2,3]triazol-1-yl)-3-(2-chloropyridin-4-yl) acrylate (76 mg, 0.200 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (41.4 mg, 0.0822 mmol, 41%) as a white solid. General: C₂₄H₁₂ClF₂N₇O₂; MW = 503.85. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.38 (s, 1H); 8.60 (s, 1H); 8.34 (d, J = 5.1 Hz, 1H); 8.21 (d, J = 8.2 Hz, 1H); 8.06 (m, 1H); 7.90 (dd, J = 9.5, 6.9 Hz, 2H); 7.77 (t, J = 8.0 Hz, 1H); 7.60 (dd, J = 9.2, 6.7 Hz, 1H); 7.51 (m, 1H); 7.26 (dd, J = 5.1, 1.4 Hz, 1H). LCMS (ESI): m/z = 504.1 [M+H]⁺ (calcd for C₂₄H₁₃ClF₂N₇O₂: 504.07). Step 3: General Procedure D was followed using the uracil intermediate (25 mg, 0.0496 mmol). Yield: 8.4 mg, 0.0155 mmol, 31%. General: C₂₇H₁₄ClF₂N₇O₂; MW = 541.90. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.62 (s, 1H); 8.49–8.26 (m, 1H); 8.23 (d, J = 8.3 Hz, 1H); 8.15–7.99 (m, 1H); 7.92 (m, 1H); 7.83 (dd, J = 9.6, 7.1 Hz, 1H); 7.79 (m, 1H); 7.70–7.56 (m, 2H); 7.52–7.31 (m, 1H); 4.72–4.23 (m, 2H); 2.74 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.0, 155.1, 153.6, 152.2, 151.6, 151.3, 143.8, 141.2, 141.1, 139.8, 133.6, 133.0, 132.9, 131.9, 131.8, 130.1, 129.4, 129.3, 129.2, 128.2, 121.9, 107.6, 107.4, 98.9, 78.2, 75.2, 38.7. LCMS (ESI): m/z = 542.1 [M+H]⁺ (calcd for C₂₇H₁₅ClF₂N₇O₂: 542.09).

Synthesis of AVI-6195.

Step 1: Synthesis of ethyl 3-amino-2-(5/6-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(5,6-dichloropyridin-3-yl)-2-propenoate (261 mg, 1.00 mmol), N-bromosuccinimide, 5-fluoro-1H-benzotriazole and Na₂CO₃ in DMF. Yield: 161 mg, 0.406 mmol, 41% (mixture of regioisomers, 5 and 6-fluorobenzotriazole). A small amount of crude was further purified to separate the regioisomers for analytical purposes. General: C₁₆H₁₂Cl₂FN₅O₂; MW = 396.20. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.92 (brs, 1H); 8.09 (dd, J = 6.7, 2.2 Hz, 1H); 7.94 (m, 1H); 7.69 (dd, J = 7.4, 2.1 Hz, 1H); 7.47 (dd, J = 8.0, 2.0 Hz, 1H); 7.22 (m, 1H); 5.47 (brs, 1H); 4.17–4.07 (m, 2H); 1.03 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 396.1 [M+H]⁺ (calcd for C₁₆H₁₃Cl₂FN₅O₂: 396.04). Step 2: General Procedure C was followed using ethyl 3-amino-2-(5/6-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate (115 mg, 0.290 mmol) and carbamic acid-4-isoquinolinyl-ethyl ester. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (40.5 mg, 0.0778 mmol, 27%, mixture of regioisomers) as a white solid. General: C₂₄H₁₂Cl₂FN₇O₂; MW = 520.31. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.39 (s, 1H); 8.59 (s, 1H); 8.29 (dd, J = 7.7, 2.1 Hz, 1H); 8.21 (d, J = 8.3 Hz, 1H); 8.03 (m, 2H); 7.90 (t, J = 8.0 Hz, 1H); 7.78 (t, J = 7.5 Hz, 1H); 7.69 (m, 1H); 7.39 (m, 1H); 7.22 (td, J = 9.2, 2.3 Hz, 1H). LCMS (ESI): m/z = 520.1 [M+H]⁺ (calcd for C₂₄H₁₃Cl₂FN₇O₂: 520.04). Step 3: General Procedure D was followed using the uracil intermediate (29.6 mg, 0.0569 mmol). Yield: 11.4 mg, 0.0204 mmol, 36% (mixture of regioisomers). General: C₂₇H₁₄Cl₂FN₇O₂; MW = 558.35. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.68–8.31 (m, 2H); 8.23 (d, J = 8.2 Hz, 1H); 8.12–7.86 (m, 3H); 7.82–7.61 (m, 2H); 7.48–7.14 (m, 2H); 4.73–4.32 (m, 2H); 2.75 (m, 1H). LCMS (ESI): m/z = 558.1 [M+H]⁺. ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 159.9, 155.0, 152.0, 151.4, 143.8, 143.7, 143.0, 133.6, 133.0, 131.3, 130.1, 129.4, 129.3, 128.2, 122.7, 122.6, 122.0, 121.8, 115.4, 115.1, 105.4, 105.2, 97.2, 96.9, 78.3, 75.3, 38.7. LCMS (ESI): m/z = 558.1 [M+H]⁺ (calcd for C₂₇H₁₅Cl₂FN₇O₂: 558.06).

Synthesis of AVI-6192.

Step 1: Synthesis of ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(5,6-dichloropyridin-3-yl)-2-propenoate (210 mg, 0.804 mmol), N-bromosuccinimide, 1H-benzotriazole and Na₂CO₃ in DMF. Yield: 200 mg, 0.529 mmol, 66%. General: C₁₆H₁₃Cl₂N₅O₂; MW = 378.21. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.90 (brs, 1H); 8.09 (d, J = 2.1 Hz, 1H); 7.97 (d, J = 8.3 Hz, 1H); 7.67 (d, J = 2.2 Hz, 1H); 7.46 (d, J = 7.3 Hz, 1H); 7.34 (m, 2H); 5.31 (brs, 1H); 4.15–4.06 (m, 2H); 1.02 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 378.0 [M+H]⁺ (calcd for C₁₆H₁₄Cl₂N₅O₂: 378.04). Step 2: General Procedure C was followed using ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(5,6-dichloropyridin-3-yl) acrylate (83 mg, 0.219 mmol) and carbamic acid-4-isoquinolinyl-ethyl ester. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (41 mg, 0.0816 mmol, 37%) as a white solid. General: C₂₄H₁₃Cl₂N₇O₂; MW = 502.31. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.39 (s, 1H); 8.60 (s, 1H); 8.28 (d, J = 2.1 Hz, 1H); 8.21 (d, J = 8.1 Hz, 1H); 8.09–7.99 (m, 3H); 7.90 (t, J = 7.8 Hz, 1H); 7.78 (t, J = 7.5 Hz, 1H); 7.67 (m, 1H); 7.58 (m, 1H); 7.42 (m, 1H). LCMS (ESI): m/z = 502.1 [M+H]⁺ (calcd for C₂₄H₁₄Cl₂N₇O₂: 502.05). Step 3: General Procedure D was followed using the uracil intermediate (25 mg, 0.0498 mmol). Yield: 7.2 mg, 0.0133 mmol, 27%. General: C₂₇H₁₅Cl₂N₇O₂; MW = 540.36. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.44 (s, 1H); 8.71–8.34 (m, 2H); 8.26 (d, J = 8.2 Hz, 1H); 8.25–8.11 (m, 1H); 8.08–7.89 (m, 2H); 7.98 (d, J = 8.4 Hz, 1H); 7.82 (t, J = 7.4 Hz, 1H); 7.75 (m, 1H); 7.62 (t, J = 7.8 Hz, 1H); 7.43 (t, J = 7.6 Hz, 1H); 4.75–4.36 (m, 2H); 2.78 (m, 1H). LCMS (ESI): m/z = 540.1 [M+H]⁺. ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.1, 155.0, 152.0, 151.9, 151.5, 146.0, 143.8, 135.5, 133.6, 133.5, 133.0, 132.9, 130.1, 129.9, 129.4, 129.3, 129.3, 128.3, 125.6, 122.0, 121.9, 120.7, 113.4, 111.1, 78.4, 75.3, 38.7. LCMS (ESI): m/z = 540.07 [M+H]⁺ (calcd for C₂₇H₁₆Cl₂N₇O₂: 540.07).

Synthesis of AVI-6037.

Step 1: Synthesis of ethyl 3-amino-3-(2-chloropyridin-4-yl)-2-propenoate: The general procedure A was followed, using 2-chloro-4-cyanopyridine (1 g, 7.22 mmol). Yield: 1.62 g, 7.15 mmol, 99%. General: C₁₀H₁₁N₂O₂; MW = 226.66. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.46 (d, J = 5.2 Hz, 1H); 7.47 (d, J = 1.1 Hz, 1H); 7.35 (dd, J = 5.2, 1.6 Hz, 1H); 5.01 (s, 1H); 4.19 (q, J = 7.1 Hz, 2H); 1.30 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 227.1 [M+H]⁺ (calcd for C₁₀H₁₂N₂O₂: 227.05). Step 2: Synthesis of ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(2-chloropyridin-4-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(2-chloropyridin-4-yl)-2-propenoate (200 mg, 0.882 mmol), N-bromosuccinimide, 1H-benzotriazole and Na₂CO₃ in DMF. Yield: 165 mg, 0.480 mmol, 54%. General: C₁₆H₁₄ClN₅O₂; MW = 343.77. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.86 (brs, 1H); 8.09 (d, J = 5.1 Hz, 1H); 7.95 (d, J = 8.1 Hz, 1H); 7.45 (t, J = 7.4 Hz, 1H); 7.36–7.29 (m, 2H); 7.20 (s, 1H); 6.97 (dd, J = 5.1, 1.3 Hz, 1H); 5.39 (brs, 1H); 4.16–4.05 (m, 2H); 1.03 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 344.1 [M+H]⁺ (calcd for C₁₆H₁₅ClN₅O₂: 344.08). Step 3: General Procedure C was followed using ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(2-chloropyridin-4-yl)acrylate (36 mg, 0.105 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (20.3 mg, 0.0434 mmol, 41%) as a white solid. General: C₂₄H₁₄ClN₇O₂; MW = 467.87. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.38 (s, 1H); 8.59 (s, 1H); 8.31 (d, J = 5.0 Hz, 1H); 8.21 (d, J = 8.1 Hz, 1H); 8.05 (m, 1H); 8.01 (d, J = 8.4 Hz, 1H); 7.90 (t, J = 7.4 Hz, 1H); 7.78 (t, J = 7.6 Hz, 1H); 7.67 (dt, J = 8.4, 1.0 Hz, 1H); 7.58 (t, J = 7.4 Hz, 1H); 7.46 (m, 1H); 7.42 (m, 1H); 7.24 (dd, J = 5.1, 1.4 Hz, 1H). LCMS (ESI): m/z = 468.1 [M+H]⁺ (calcd for C₂₄H₁₅ClN₇O₂: 468.09). Step 4: General Procedure D was followed using the uracil intermediate (16 mg, 0.0342 mmol). Yield: 5.8 mg, 0.0115 mmol, 34%. General: C₂₇H₁₆ClN₇O₂; MW = 505.92. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.62 (d, J = 11.4 Hz, 1H); 8.46–8.19 (m, 1H); 8.23 (d, J = 8.2 Hz, 1H); 8.16–7.99 (m, 1H); 7.94 (d, J = 8.4 Hz, 1H); 7.91 (m, 1H); 7.79 (dd, J = 7.7, 7.3 Hz, 1H); 7.75–7.54 (m, 1H); 7.72 (d, J = 8.3 Hz, 1H); 7.59 (t, J = 7.8 Hz, 1H); 7.46–7.29 (m, 1H); 7.39 (m, 1H); 4.66–4.30 (m, 2H); 2.73 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.2, 155.0, 151.5, 151.45, 151.41, 145.9, 143.8, 140.1, 135.6, 135.6, 135.5, 133.6, 133.0, 132.9, 130.1, 129.8, 129.33, 129.32, 129.28, 128.3, 125.5, 121.9, 120.6, 111.2, 78.3, 75.1, 38.6. LCMS (ESI): m/z = 506.2 [M+H]⁺ (calcd for C₂₇H₁₇ClN₇O₂: 506.11).

Synthesis of AVI-6036.

Step 1: Synthesis of ethyl 3-amino-3-(5-chloropyridin-3-yl)-2-propenoate: The general procedure A was followed, using 2-chloro-4-cyanopyridine (505 mg, 3.64 mmol). Yield: 777 mg, 3.43 mmol, 94%. General: C₁₀H₁₁N₂O₂; MW = 226.66. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.69 (d, J = 2.0 Hz, 1H); 8.64 (d, J = 2.3 Hz, 1H); 7.84 (t, J = 2.2 Hz, 1H); 4.96 (s, 1H); 4.19 (q, J = 7.1 Hz, 2H); 1.30 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 227.1 [M+H]⁺ (calcd for C₁₀H₁₂N₂O₂: 227.05). Step 2: Synthesis of ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(5-chloropyridin-3-yl) acrylate: General Procedure B was followed using ethyl 3-amino-3-(5-chloropyridin-3-yl)-2-propenoate (200 mg, 0.882 mmol), N-bromosuccinimide, 1H-benzotriazole and Na₂CO₃ in DMF. Yield: 103 mg, 0.299 mmol, 34%. General: C₁₆H₁₄ClN₅O₂; MW = 343.77. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.90 (brs, 1H); 8.33 (d, J = 2.3 Hz, 1H); 8.30 (d, J = 1.9 Hz, 1H); 7.93 (dt, J = 8.4, 0.9 Hz, 1H); 7.56 (t, J = 2.1 Hz, 1H); 7.44 (m, 1H); 7.35 (dt, J = 8.3, 0.9 Hz, 1H); 7.32–7.27 (m, 1H); 5.50 (brs, 1H); 4.16–4.05 (m, 2H); 1.02 (t, J = 7.1 Hz, 3H). LCMS (ESI): m/z = 344.1 [M+H]⁺ (calcd for C₁₆H₁₅ClN₅O₂: 344.08). Step 3: General Procedure C was followed using ethyl 3-amino-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(5-chloropyridin-3-yl)acrylate (74 mg, 0.215 mmol) and S1. Purification by preparative HPLC (30% to 70% CH₃CN in H₂O + 0.1% formic acid) afforded the uracil intermediate (30.5 mg, 0.0652 mmol, 30%) as a white solid. General: C₂₄H₁₄ClN₇O₂; MW = 467.87. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.39 (s, 1H); 8.61 (s, 1H); 8.57 (d, J = 2.4 Hz, 1H); 8.45 (d, J = 1.9 Hz, 1H); 8.21 (d, J = 8.3 Hz, 1H); 8.01 (m, 1H); 8.00 (dt, J = 8.4, 0.9 Hz, 1H); 7.91 (m, 1H); 7.88 (t, J = 2.1 Hz, 1H); 7.78 (t, J = 7.8 Hz, 1H); 7.68 (dt, J = 8.5, 1.1 Hz, 1H); 7.58 (m, 1H); 7.42 (m, 1H). LCMS (ESI): m/z = 468.1 [M+H]⁺ (calcd for C₂₄H₁₅ClN₇O₂: 468.09). Step 4: General Procedure D was followed using the uracil intermediate (25 mg, 0.0534 mmol). Yield: 10.1 mg, 0.0120 mmol, 37%. General: C₂₇H₁₆ClN₇O₂; MW = 505.92. ¹H-NMR (400 MHz, CD₃CN): δ (ppm): 9.41 (s, 1H); 8.80–8.47 (m, 3H); 8.22 (d, J = 8.2 Hz, 1H); 8.14–7.83 (m, 4H); 7.81–7.69 (m, 2H); 7.61–7.54 (m, 1H); 7.41–7.35 (m, 1H); 4.70–4.34 (m, 2H); 2.72 (m, 1H). ¹³C-NMR (101 MHz, CD₃CN): δ (ppm): 160.2, 155.0, 152.9, 151.7, 151.6, 151.5, 145.9, 143.9, 135.5, 133.6, 133.6, 133.0, 132.9, 130.1, 129.8, 129.4, 129.3, 129.3, 128.4, 125.5, 122.1, 121.9, 120.6, 111.1, 78.4, 75.1, 38.6. LCMS (ESI): m/z = 506.1 [M+H]⁺ (calcd for C₂₇H₁₇ClN₇O₂: 506.11).

Biochemical inhibition of MPro constructs.

In the major process of SAR, we used GST-MPro-His6 construct to obtain wild-type SARS-CoV-2 M^Pro protease. In our assay, we determined the potency of M^Pro inhibitors with M^Pro at 40nM. For testing of (M)-AVI-4773 and (M)-AVI-6179, we adopted the His6-SUMO-M^Pro-coil construct described in a recent study.¹⁴ The fusion construct (M^Pro-Coil) promotes dimerization of M^Pro and activity at lower concentrations of enzyme, providing better signal than M^Pro produced from the GST-M^Pro-His6 construct. Herein, we determined the potency of M^Pro inhibitors with M^Pro-Coil at 5 nM.

In vitro inhibition of MPro followed previously described enzymatic assays by our group.^27,44 Briefly, 50 mM Tris, 150 mM NaCl, 1mM EDTA, 0.05% Tween-20 & 1 mM TCEP pH 7.4 (Buffer C) is used, and TCEP is added fresh for each assay with pH readjusted to 7.4 upon addition. M^Pro is incubated in the buffer with TCEP for 10 min to ensure full activation of M^Pro. The compounds are then incubated with M^Pro at room temperature for 1 hour on Corning3820 384 well black plates. After incubation with compounds, the M^Pro substrate (dR)(dR)(MCA)KATVQAIAS(DNP)K that we synthesized²⁷ is used to initiate the reaction at a final concentration of 10 μM, with 1% DMSO. An increase in fluorescence with an excitation of 328 nm and an emission of 393 nm over time is monitored for the first 60 min of the reaction with a BioTek Neo2 plate reader. The substrate cleavage rate (v) was extracted as the initial linear fluorescence increased and normalized against the substrate cleavage rate without an inhibitor to obtain the relative activity. From this, IC₅₀ was obtained using a four-parameter logistic function in GraphPad Prism 10.2.0.

Pharmacokinetic studies in mice.

Mouse pharmacokinetic studies of AVI-4773 and AVI-6179 at 3 mg/kg (IV) and 10 mg/kg (PO) doses (Table 6 and Figure 4A) were performed in male CD1 mice (n = 9 per group) with a formulation of 10% DMSO/50% PEG400/40% of a 20% HP-β-CD solution in water. Animals were restrained manually at designated timepoints and ca. 110 mL of blood was taken into K₂EDTA tubes via facial vein. The blood samples were collected on ice and centrifuged (2000 g, 5 min) to obtain plasma samples within 15 minutes post sampling. Three blood samples were collected from each mouse; three samples were collected at each time point. Plasma samples (20 μL) were diluted into 200 μL acetonitrile with internal standard (glipizide, 60 ng/mL) were analyzed by LC-MS/MS (triple quad 6500+) and quantified by comparison with a standard curve (1.0–3000 ng/mL) generated from authentic analyte. Data was processed by Phoenix WinNonlin (version 8.3); samples below limit of quantitation were excluded in the PK parameters and mean concentration calculation.

Rat cassette pharmacokinetic study.

The rat PK study with cassette dosing (Figure 2) was performed in female Sprague Dawley (SD) rats, aged 6–8 weeks, weighing 180~200 g. Six rats were separated into two groups with each group being dosed via intraperitoneal (IP) injection with six different MPro inhibitors. Specifically, the three rats in group 1 were dosed with AVI-6011, AVI-6195, AVI-6192, AVI-6325, AVI-6259, and AVI-6032 at 10 mg/kg; the three rats in group 2 were dosed with AVI-4516, AVI-4773, AVI-6179, AVI-6328, AVI-6360, and AVI-6330 at 10 mg/kg of each compound. The animals were restrained manually and 150 μL samples of blood were taken into K₂EDTA tubes via jugular vein at 0 (pre-dose), 0.083, 0.25, 0.5, and 1 hour timepoints. Blood samples were put on ice and centrifuged to obtain plasma samples (2000 g, 5 min under 4°C) within 15 minutes post sampling. The animals were anesthetized with CO₂ at 1 hour post drug administration. A perfusion with pre-cold saline was conducted via cardiac puncture before brain collection. After perfusion, a mid-line incision was made in the animal’s scalp and skin retracted. The skull overlying the brain was removed and whole brain collected, rinsed with cold saline, dried on filtrate paper, weighted, and snap frozen by placing into dry-ice. The CSF was collected by direct needle puncture into the cisterna magna. Samples were stored in dry ice temporarily and transferred into −70°C freezer for long term preservation. The drug concentrations were determined by a LC-MS/MS (triple quad 6500+) and quantified by comparison with a standard curve (1.0–3000 ng/mL) generated from authentic analyte. Data was processed by Phoenix WinNonlin (version 8.3); samples below limit of quantitation were excluded in the PK parameters and mean concentration calculation.

Pharmacokinetic studies of (M)-AVI-4773 in rat and dog.

Male SD rats were administered 3 mg/kg (IV) or 10 mg/kg (PO) while beagle dogs were administered 0.615 mg/kg (IV) or 2 mg/kg (PO) of test compound formulated in 10% DMSO/50% PEG400/40% of a 20% HP-β-CD solution for the IV arms and for PO arms as a suspension in 1% (hydroxypropyl)methyl cellulose/1% Tween 80. Animals were restrained manually at designated timepoints and ca. 500 μL samples of blood collected via cephalic vein into K₂EDTA tubes. The blood samples were collected on ice and centrifuged (3000 g, 5 min) to obtain plasma samples within 15 minutes post sampling. Plasma samples (20 μL) were diluted into 200 μL acetonitrile with internal standard (diclofenac, 60 ng/mL) were analyzed by LC-MS/MS (triple quad 6500+) and quantified by comparison with a standard curve (1.0–3000 ng/mL) generated from authentic analyte. Data was processed by Phoenix WinNonlin (version 8.3); samples below limit of quantitation were excluded in the PK parameters and mean concentration calculation.

ADME Assays.

In vitro ADME assays were performed at Quintara Discovery (Hayward, CA) using experimental procedures as further detailed in the Supporting Information File. The hERG assay dose response data in Figure 7 was performed in our laboratories using the Predictor^™ hERG Fluorescence Polarization Assay kit (ThermoFisher) following the recommended procedures.

SARS CoV-2 replicon assay.

SARS-CoV-2 single-round infectious particles were generated as previously described with some modifications.³² BHK-21 cells were seeded in 15-cm dish (3×10⁶) and were transfected the next day with 40 μg pBAC SARS-CoV-2 Spike replicon plasmid (WA1, WA1 M49L, WA1 nsp5 L50F/E166Q/L167F, or JN.1.1.1), 20 μg Spike Delta variant plasmid, 45 and 5 ug Nucleocapsid R203M plasmid⁴⁶ using Xtremegene 9 DNA transfection reagent (Sigma Aldrich). The media was changed 4–6 hours later, and the cells were incubated at 37 °C and 5% CO₂. At 70 hours post transfection, 20K VAT cells in 50 μL culture medium were mixed with 50 μL compound at 4x final concentration and 50 μL of 4x final concentration P-gp inhibitor (CP-100356) and plated in 96-well tissue culture plates. At 72 hours post transfection, the supernatant was 0.45 μm filtered and 50 μL was added to each well of compound treated VAT cells and the cells were incubated for 6–8 hours at 37 °C and 5% CO2. The cells were washed once with culture medium and 100 μL of compound containing culture medium was added with and without 1 μM P-gp inhibitor (CP-100356). The cells were incubated for 24 hours and 50 μL of supernatant was transferred to white 96-well plate. 50 μL of Promega nanoGlo reagent was added and luminescence was recorded in a Tecan plate reader. Experiments were conducted in at least two independent biological replicates.

SARS-CoV-2 live virus antiviral activity assay.

Compound antiviral activity was determined using the IncucyteⓇ live cell analysis system. A549-ACE2h cells were seeded and incubated as for the cytotoxicity assay. The next day, cells were pre-treated with compounds for 2 h prior to removal of compounds and infection with the mNeon expressing virus icSARS-CoV-2-mNG (MOI 0.1). Cells were infected with 50 μl viral inoculum for 2 h before removal and addition of fresh compounds and controls. Fresh compounds and controls were diluted in DMEM complete (10% FBS, 1% L-Glutamine, 1X P/S, 1X NEAA) supplemented with Incucyte^® Cytotox Dye (4632, Sartorius) to control for cell death. After addition of fresh compounds, infected cells were placed in an Incucyte S3 (Sartorius) and infection/cell death measured for 48 h in 1 h intervals using a 10x objective and capturing 3 images/well per time point under cell maintenance conditions (37°C, 5% CO2). Infection was quantified as Total Green Object Integrated Intensity (GCU × μm2/Image) with an acquisition time of 300 ms and cell death as Red Object Integrated Intensity (GCU × μm2/Image) for 400 ms. Image analysis for measurements were done with the following parameters: Phase, AI confluence segmentation. Green, Top-hat segmentation with a 50 μM Radius, GCU threshold of 0.5, and Edge Split On. Red was similar to Green with a 100 μM radius and a threshold of 1 RCU. A 2 % spectral unmixing of the red channel into the green was predefined to prevent signal spillover. Post in-built software analysis, raw data was exported and antiviral efficacy determined as the percentage of infection normalized to the vehicle control. A positive control (Nirmatrelvir, HY-138687, MedChemExpress) at efficacious concentrations and uninfected cells were used as an intra-assay positive and negative control. Unless otherwise stated, experiments were performed in triplicate with 3 technical replicates. EC50 values were calculated using GraphPad Prism 10 (La Jolla, CA, USA) using a dose-response inhibition equation with non-linear fit regression model.

DFT Methods.

All calculations were performed using Gaussian16 (Revision C.01),47 and input file generation and output file parsing was performed with GaussView.⁴⁸ An initial geometry optimization for compounds AVI-4773 and AVI-6179 was calculated using the w-B97XD|def2SVP level of theory. Subsequently, an independent dihedral scan coordinate for each chemical bond of interest (the uracil(N3)–isoquinoline(C) bond and the uracil-benzotriazole C–N bond) was defined using the Redundant Coordinates tool. A scan coordinate of a 360° rotation was divided into 72 total steps, with a step size of 5° increments. A relaxed potential energy surface (PES) was obtained through sequential geometry optimizations at each step along the reaction coordinate, with the starting geometry resulting from the specified degree of rotation [Opt=(modredunant)]. PES scans were performed with an implicit solvent SMD model49 to evaluate potential solvent effects of water (ε=78.3553) and dimethyl sulfoxide (ε=46.826). Furthermore, we considered two additional DFT functional and basis set combinations: B3LYP|6–31G(d,p) and w-B97XD|def2TZVP. Our choice of theory (w-B97XD|def2SVP) was determined to provide the best tradeoff between accuracy and computational expense (Tables S4–S5). All calculations were performed at the default temperature (298K) and pressure (1 atm). Additionally, all calculations were performed utilizing 30 CPUs, made available through UCSF’s Wynton HPC Center.

SARS-CoV-2 Mouse Infection Model.

The SARS-CoV-2 infection model was conducted in an animal biosafety level 3 (ABSL3) facility at the Gladstone Institutes following guidelines in the “NIH Guide for the Care and Use of Laboratory Animals”. Approval for the evaluation of antiviral efficacy of M^pro inhibitors was obtained from Institutional Animal Care and Use Committees (IACUC, protocol AN203103–00E) at the Gladstone Institutes and University of California, San Francisco. The antiviral efficacy of (±)-AVI-4773 and (M)-AVI-4773 were evaluated in wild-type (WT) mice infected with the SARS-CoV-2 Beta variant using an oral suspension in 1% (hydroxypropyl)methyl cellulose and 1% Tween 80 in water. Thirty female WT mice (6–8 weeks old) were intranasally infected with 10³ PFU of the SARS-CoV-2 Beta variant. Mice were administered (±)-AVI-4773 at 100 mg/kg or (M)-AVI-4773 at 50 mg/kg by oral gavage. Treatment was initiated 4 hours post-infection and continued a twice-daily (BID) schedule starting from day 1 post-infection. Half of the animals were euthanized on day 2 post-infection, and the remaining mice were euthanized on day 4, following continued dosing through day 3. Lung tissues were collected, and viral titers were quantified to assess viral replication.

MERS-CoV Mouse Infection Model.

All in vivo studies were conducted in an animal biosafety level 3 (ABSL-3) facility at the Icahn School of Medicine at Mount Sinai. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC ID IPROTO202200000054) and conducted in compliance with institutional and federal guidelines. Female B6(Cg)-Ifnar1tm1.2Ees/J mice (6–8 weeks old; Jackson Laboratory) were anesthetized with isoflurane and transduced intranasally with 2.5 × 10⁸ PFU of recombinant adenovirus expressing human DPP4 (AdV-hDPP4) in 50 μL of PBS to facilitate susceptibility to MERS-CoV. Five days post-transduction, mice were infected intranasally with 1 × 10⁵ PFU of MERS-CoV EMC2012 in 50 μL of PBS.

Test compounds were administered orally BID (twice daily) at doses of 8, 20, 50, or 125 mg/kg in a formulation of 1% (hydroxypropyl)methyl cellulose and 1% Tween 80 in water, with the first dose delivered 1 hour prior to viral challenge (Day 0). Oral gavage was continued for 5 consecutive days. A positive control group received oral nirmatrelvir at 600 mg/kg BID (without ritonavir) on the same schedule. Uninfected cohorts were included to assess compound tolerability.

At 2 days post-infection (2 dpi), mice were euthanized by CO₂ asphyxiation and lungs were harvested for virological assessment. The lower right lung lobe was homogenized in PBS using glass beads and clarified by centrifugation. Viral titers were quantified by plaque assay on Vero E6 cells and expressed as PFU per lung. Additional cohorts were monitored daily for body weight through 14 dpi (infected animals) or 5 dpi (uninfected controls). All weight and titer data were analyzed using two-way ANOVA with appropriate post-hoc corrections.

Supplementary Material

The Supporting Information is available free of charge on the ACS Publications website.

These include supplementary synthetic schemes (Schemes S1 and S2), supplementary figure illustrating native MS analyses (Figure S1), dose-response curves (Figure S2 and S3), quantification of cellular uptake (Figure S4), compounds selected for rat PK study (Figure S5), 1H NMR spectra of AVI-6179 regioisomer (Figure S6), VT-NMR data and spectra (Figure S7), DFT data and figures (Figures S8 and S9), derivation of rate law for atropoisomeric interconversion (Figure S10), chromatograms for SFC separations (Figures S11 and S12), small molecule X-ray structures (Figure S13), dose-response curves (Figure S14 and S15), and SFC analysis of (P)-AVI-6179 (Figure S16), tables of antiviral activities (Tables S1–S3), calculated rate constants for AVI-4773 (Table S4), results of DFT calculations (Table S5 and S6), CYP induction results (Table S7), table of AMES assay data (Table S8), supporting materials and methods section, and crystallographic data and refinement statistics (Table S9 and S10). (PDF)

Smiles strings and associated data for final analogs (CSV)

ABBREVIATIONS

ADME

absorption, distribution, metabolism, and excretion

BID

twice daily

CoV

coronavirus

CYP

cytochrome P

DDI

drug-drug interactions

DCM

dichloromethane

DFT

density functional theory

DMF

dimethylformamide

hDPP4

human dipeptidyl peptidase-4

EC₅₀

half-maximum effective concentration

ESI

electrospray ionization

F%

oral bioavailability

FDA

Food and Drug Administration

hERG

human Ether-a-go-go-Related Gene

HPLC

high-performance liquid chromatography

HLM

human liver microsome

IC₅₀

half-maximum inhibitory concentration

IP

intraperitoneal

IV

intravenous

J

coupling constant

MERS

middle-east respiratory syndrome

MLM

mouse liver microsome

Mpro

main protease

NBS

N-bromosuccinimide

PBS

phosphate buffered saline

PD

pharmacodynamics

PDB

Protein Data Bank

PFU

plaque-forming units

PK

pharmacokinetics

PO

orally administered

P-gp

P-glycoprotein

PPB

plasma protein binding

PSA

polar surface area

QD

once-daily

SARS

sudden acute respiratory syndrome

SFC

supercritical fluid chromatography