Discovery of a 4′‐Azido Fleximer Nucleoside Analog With Broad‐Spectrum Antiviral Activity Against DENV, EBOV, and Other RNA Viruses

Abstract

The ongoing emergence and reemergence of RNA viruses from multiple viral families highlights the urgent need for broad‐spectrum antiviral therapeutics. To address this issue, a series of 4′‐azido fleximer nucleoside analogs were synthesized and evaluated for broad‐spectrum antiviral activity against pathogens of epidemic/pandemic concern. One compound, KAD‐039, featuring a 2′, 3′‐diisobutyrate‐5′‐ProTide prodrug, exhibited potent antiviral activity across flaviviruses, filoviruses, and coronaviruses, with EC₅₀ values ranging from 1 to 10 µM and minimal cytotoxicity (CC₅₀ > 50 µM). Pharmacokinetic evaluation of KAD‐039 demonstrated high plasma stability in dog, monkey, and human plasma, with half‐lives ranging from 19.9 to 28 h but exhibited poor stability in human liver microsomes (t _1/2 = 6.1 min). To help elucidate the mechanism of action of KAD‐039 against these viral families, computational molecular docking studies were performed using the expected triphosphate active form within the cap/GTP‐binding pockets of several viral methyltransferases and showed that KAD‐039TP docked into all of the viral MTases with similar or higher predicted affinity compared to the natural substrate. In addition, KAD‐039TP displayed reduced predicted affinity for the human N7 methyltransferase, suggesting low host toxicity. These results highlight 4′‐azido fleximer analogs as promising broad‐spectrum antivirals, potentially functioning through viral methyltransferase inhibition, and support further investigation.

A series of 4′‐azido fleximer nucleoside analogs were designed, synthesized, and evaluated for their antiviral activity against RNA viruses of epidemic/pandemic concern. One compound, KAD‐039, featuring a 2′‐3′‐diisobutyrate‐5′‐ProTide prodrug strategy was found to inhibit viruses from across multiple viral families, including flaviviruses, filoviruses, and coronaviruses, with EC₅₀ values below 10 µM.

1. Introduction

Emerging and reemerging viral pathogens with epidemic or pandemic potential represent a serious and growing global public health threat. Factors such as increasing international travel and trade, urbanization, and climate change have increased human exposure to zoonotic viruses from multiple viral families, including flaviviruses, filoviruses, and coronaviruses. These viruses are capable of causing serious outbreaks with high morbidity and mortality, placing a significant burden on healthcare systems and exposing gaps in current medical countermeasures [1, 2].

Orthoflaviviruses remain one of the arboviruses of greatest public health concern, with more than 4 billion people currently living in regions at risk for dengue virus (DENV) and other flaviviral infections [3, 4, 5, 6]. In 2025, dengue infections reached exceptionally high levels with over 5 million confirmed infections and nearly 2000 deaths worldwide [7]. Approved vaccines do exist for some of these viruses; however, their utility is severely limited due to restricted eligibility and safety considerations [8, 9]. Another mosquito‐borne flavivirus, West Nile virus (WNV), represents a substantial public health threat, where in 2024 nearly 2000 cases and approximately 300 deaths were reported in the United States [10, 11]. Other clinically relevant orthoflaviviruses transmitted by mosquitoes include yellow fever virus, which causes severe hemorrhagic disease with an estimated 200,000 cases and 30,000 deaths per year globally [12, 13], and Zika virus (ZIKV), which causes severe outbreaks with teratogenic effects [14].

In addition to mosquito‐borne flaviviruses, tick‐borne flaviviruses also represent a significant public health threat. Tick‐borne encephalitis virus (TBEV) is endemic across both Europe and Asia, causing an estimated 12,000 clinical cases worldwide each year. Many of these infections result in chronic neurological complications or death [15]. Other tick‐borne flaviviruses, including Omsk hemorrhagic fever virus, Kyasanur Forest disease virus (KFDV), and Powassan virus (POWV), cause sporadic infections that range in severity from mild illness to severe disease with hemorrhagic and/or neurological manifestations [16, 17, 18].

Filoviruses, such as Ebola virus (EBOV), are one of the most lethal viruses with significant epidemic potential [19]. These zoonotic viruses are primarily transmitted through direct contact with infected bodily fluids and have caused repeated outbreaks that spread rapidly with extremely high fatality rates ranging from 25% to as high as 90% in some cases [19, 20]. Large outbreaks, such as the West Africa epidemic from 2014 to 2016, highlight the severity and the continued risk of reemergence [21, 22].

Coronaviruses are another group of clinically important RNA viruses with clear pandemic potential. Severe Acute Respiratory Syndrome Coronavirus (SARS‐CoV) emerged in 2002, causing severe respiratory disease with a fatality rate of approximately 10% [23]. Later in 2012, Middle East Respiratory Syndrome Coronavirus (MERS‐CoV) was first identified and continues to cause sporadic outbreaks with an even higher mortality rate of 30%–40% [24]. Most notably, the emergence of SARS‐CoV‐2 in late 2019 led to the COVID‐19 pandemic, which resulted in unprecedented global health, societal, and economic consequences [25, 26, 27]. Collectively, these events highlight coronaviruses as persistent reemerging threats with a strong capacity to cause global pandemics.

Despite the recognition of emerging and reemerging viral threats from across multiple viral families and the availability of vaccines for several of these viral pathogens, approved antiviral therapies remain severely limited or unavailable for many of them, highlighting the urgent need for improved antiviral preparedness and the development of broad‐spectrum antiviral therapeutics [28, 29, 30].

Nucleoside analogs have long served as antiviral therapeutics due to their ability to mimic endogenous nucleosides and interfere with viral replication through several different mechanisms [31, 32]. Importantly, modifications to the 4′‐position of the sugar moiety have demonstrated a critical role in modulating antiviral activity and selectivity [33, 34]. Introduction of substituents at the 4′‐position plays an important structural role by enforcing the C3′‐endo sugar pucker which is the conformation that is preferentially bound by viral enzymes due to its resemblance to natural ribonucleotides [31, 35]. One particularly successful class of 4′‐modified analogs are those bearing the 4′‐azido group. There are currently three 4′‐azido nucleoside analogs that have advanced into clinical trials including balapiravir, azvudine, and 4′‐azidothymidine (Figure 1) [36]. Balapiravir, the tri‐isobutyrate ester prodrug of 4′‐azidocytidine, was initially developed for the treatment of hepatitis C infection and was later repurposed for use against DENV [37, 38]. Although balapiravir progressed into early‐stage clinical trials in dengue‐infected patients, its development was discontinued due to lack of clinical benefit [39, 40, 41]. Azvudine, 2′‐fluoro‐4′‐azidocytidine, while not approved by U.S. Food and Drug Administration (FDA), received regulatory approval in China for the treatment of HIV‐1 infection in 2021 and later obtained emergency approval for the treatment of COVID‐19 making it the most clinically advanced 4′‐azido NA to date [36, 42]. In contrast, 4′‐azidothymidine demonstrated potent anti‐HIV activity in preclinical and early clinical studies but was discontinued in 1995 due to an unfavorable therapeutic index [36, 43].

FIGURE 1.

Structures of clinically relevant 4′‐azido nucleoside analogs.

Fleximer nucleoside analogs, developed by the Seley‐Radtke group, feature a modified purine ring scaffold in which the imidazole and pyrimidine rings are separated by a single carbon–carbon bond between the C‐4 position of the imidazole and C‐5 of the pyrimidine, forming proximal fleximers [44, 45, 46]. Their unique structure preserves the crucial hydrogen‐bonding interactions required for molecular recognition while also allowing for new interactions within the enzyme binding pocket [47, 48]. Furthermore, the inherent flexibility of the fleximer design enables these compounds to maintain their antiviral activity in the presence of point mutations [49]. Collectively, the fleximer strategy has been shown to enhance antiviral potency relative to rigid nucleoside analogs and to confer novel antiviral activity across multiple viral families [50, 51].

Building upon the clinical relevance of 4′‐azido nucleoside analous and our previous report of 4′‐fluoro fleximer analogs with broad‐spectrum antiviral activity [52], we herein describe the design, synthesis, and biological evaluation of a new series of 4′‐azido fleximer analogs (Figure 2) (Table 1). To overcome well‐known limitations of nucleoside analogs, including poor cellular uptake and inefficient activation, several different prodrug strategies were employed. First, tri‐isobutyrate ester prodrugs, as seen on balapiravir, were prepared to mask the highly polar hydroxyl groups and enhance cellular penetration [38, 53]. Second, McGuigan ProTide derivatives, as used in FDA‐approved antivirals including remdesivir (RDV), sofosbuvir (SOF), and tenofovir alafenamide (TAF), were synthesized to bypass rate‐limiting monophosphorylation while also increasing cellular uptake [54, 55, 56, 57]. Finally, a combined prodrug strategy was explored, inspired by GS‐7682, a 2′, 3′‐diisobutyrate‐5′‐ProTide mixed prodrug with broad‐spectrum activity against respiratory viruses [58, 59]. These fleximer compounds were ultimately evaluated across a diverse panel of pathogens of public health concern, spanning multiple viral families, including flaviviruses, filoviruses, and coronaviruses, with the goal of developing broadly active nucleoside therapeutics capable of addressing both current and future viral threats.

FIGURE 2.

Synthesized 4′‐azido fleximer analogs.

TABLE 1.

Synthesized 4′‐azido fleximer analogs (Figure 2).

Compound	X	Y	R 1	R 2
KAD‐020	OMe	OMe	H	H
KAD‐021	H	NH2	H	H
KAD‐022	OMe	OMe	C(O)CH(CH3)2	C(O)CH(CH3)2
KAD‐038	OMe	NH2	McG	H
KAD‐039	OMe	NH2	McG	C(O)CH(CH3)2
KAD‐041	OMe	NH2	H	H
KAD‐042	OMe	NH2	C(O)CH(CH3)2	C(O)CH(CH3)2
KAD‐043	H	OMe	H	H
KAD‐044	H	OMe	C(O)CH(CH3)2	C(O)CH(CH3)2
KAD‐045	OMe	OMe	McG	H
KAD‐046	OMe	OMe	McG	C(O)CH(CH3)2
KAD‐047	H	NH2	McG	H
KAD‐048	H	NH2	McG	C(O)CH(CH3)2
KAD‐049	NH2	OMe	McG	H
KAD‐050	NH2	OMe	McG	C(O)CH(CH3)2
KAD‐051	NH2	OMe	H	H
KAD‐052	NH2	OMe	C(O)CH(CH3)2	C(O)CH(CH3)2
KAD‐056	OMe	H	McG	H
KAD‐057	OMe	H	McG	C(O)CH(CH3)2
KAD‐058	OMe	H	H	H
KAD‐059	OMe	H	C(O)CH(CH3)2	C(O)CH(CH3)2
KAD‐060	H	NH2	H	H
KAD‐061	H	OMe	McG	H
KAD‐062	H	OMe	McG	C(O)CH(CH3)2
KAD‐065	NH2	H	McG	H
KAD‐06 6	NH2	H	McG	C(O)CH(CH3)2

2. Results and Discussion

2.1. Chemical Synthesis

The synthetic route began by synthesizing olefin intermediate 1 in six steps starting from commercially available 4,5‐diiodo‐1H‐imidazole and beta‐D‐ribofuranose 1,2,3,5‐tetraacetate following our previously reported route [52] (Scheme 1). Stereoselective introduction of the 4′‐azido group onto the α‐phase of the furanose ring was achieved by generating iodine azide in situ from iodine monochloride and sodium azide. Following dropwise addition of olefin intermediate 1, intermediate 2 was realized in excellent yield (84%). In order to restore the 5′‐hydroxyl group, the 5′‐iodine of intermediate 2 was displaced by acetate using potassium acetate and 18‐crown‐6 to give 3 which was immediately deprotected using potassium carbonate in methanol to afford the 5′‐hydroxyl intermediate 4 in an acceptable yield of 35% across the two steps (Scheme 1). The stereochemistry of the new C4′ chiral center was confirmed using 2D Nuclear Overhauser Effect Spectroscopy (NOESY) that showed interactions between H5′ and H5″ with both H2 and H5 of the imidazole ring.

SCHEME 1.

Synthesis of 4′‐azido sugar scaffold. Reaction conditions: (a) NaN₃, ICl, DMF, rt, 2 h; (b) KOAc, 18‐Crown‐6, DMF, 120 °C, 72 h; and (c) K₂CO₃, MeOH, rt, 16 h.

Once the 4‐iodoimidazole intermediate 4 was in hand, it could be coupled to several different pyrimidine moieties using palladium‐catalyzed cross‐coupling reactions. The first set of reactions was Suzuki couplings with commercially available pyrimidine‐5‐boronic acids. Synthesis of 5a, 5b, and 5c began by coupling 4 with 2,4‐dimethoxypyrimidine‐5‐boronic acid for 5a (78%), 2‐aminopyrimidine‐5‐boronic acid for 5b (71%), and 2‐methoxypyrimidine‐5‐boronic acid for 5c (76%) in the presence of potassium carbonate and PdCl₂(PPh₃)₂ to afford the coupled intermediates in good yield (Scheme 2). For additional pyrimidine moieties where the corresponding pyrimidine‐5‐boronic acid or pinacol ester was not commercially available, a two‐step, one‐pot Miyaura borylation followed by Suzuki coupling was used. Synthesis of 5d, 5e, and 5f was achieved by reacting 2‐amino‐5‐bromo‐4‐methoxypyrimidine for 5d, 5‐bromo‐2‐methoxypyrimidin‐4‐amine for 5e, and 5‐bromo‐4‐methoxypyrimidine for 5f with bis(pinacolato)diboron, potassium acetate, and PdCl₂(PPh₃)₂. Once the Miyaura borylation was complete, 4, potassium carbonate, water, and additional PdCl₂(PPh₃)₂ were added to afford coupled intermediates 5d (44%), 5e (42%), and 5f (49%) in acceptable yields across the two steps. Attempts to perform the two‐step‐one‐pot reaction with 5‐bromopyrimidin‐4‐amine to achieve 5g were fruitless, so an alternative route was taken. Ammonolysis of 4‐methoxy intermediate 5f with ammonia in methanol was successful to afford 5g in an acceptable yield of 29% (Scheme 2).

SCHEME 2.

Synthesis of coupled intermediates. Reaction conditions: (a) for 5a: 2,4‐dimethoxypyrimidine‐5‐boronic acid; for 5b: 2‐aminopyrimidine‐5‐boronic acid; for 5c: 2‐methoxypyrimidine‐5‐boronic acid; K₂CO₃, PdCl₂(PPh₃)₂, 1,4‐dioxanes:H₂O (4:1), µWave, 80 °C, 2 h; (b) (i) for 5d 2‐amino‐5‐bromo‐4‐methoxypyrimidine; for 5e 5‐bromo‐2‐methoxypyrimidin‐4‐amine; for 5f 5‐bromo‐4‐methoxypyrimidine; bis(pinacolato)diboron, KOAc, PdCl₂(PPh₃)₂, 1,4‐dioxanes, µWave, 100 °C, 3 h; (ii) 4, K₂CO₃, PdCl₂(PPh₃)₂, H₂O, µWave, 80 °C, 2 h; and (c) 5f, NH₃, MeOH, 160 °C, 48 h.

Synthesis of parent nucleoside analogs KAD‐020, KAD‐021, KAD‐041, KAD‐043, KAD‐051, and KAD‐058 was achieved through removal of the isopropylidene protecting group using trifluoroacetic acid (Scheme 3). Significant nucleobase cleavage was observed during this step, resulting in the poor yields. Several attempts to purify KAD‐021 were unsuccessful; therefore, this analog was not tested for its antiviral activity but was used without further purification in the next step.

SCHEME 3.

Synthesis of parent and triisobutyrate analogs. Reaction conditions: (a) TFA, DCM, H₂O, rt, 4–6 h. (b) Isobutyric anhydride, DMAP (cat.), pyridine, rt, 4 h.

Once the parent analogs were synthesized, the focus turned to synthesizing the triisobutyrate ester prodrugs. Treatment of parent nucleosides KAD‐020, KAD‐021, KAD‐041, KAD‐043, KAD‐051, and KAD‐058 with isobutyric anhydride and a catalytic amount of DMAP afforded KAD‐022, KAD‐042, KAD‐044, KAD‐052, KAD‐059, and KAD‐060 in excellent yields (Scheme 3).

Synthesis of the ProTide analogs began by treating isopropylidene‐protected intermediates 5a–g with tert‐butylmagnesium chloride, followed by addition of commercially available (S)‐(perfluorophenoxy)(phenoxy)‐phosphoryl)‐L‐alaninate to afford the isopropylidene‐protected ProTide intermediates 6a–g in good yields, which were subsequently deprotected using trifluoroacetic acid to give ProTides KAD‐038, KAD‐045, KAD‐047, KAD‐049, KAD‐056, KAD‐061, and KAD‐065 in acceptable yields (Scheme 4). Finally, the 2′‐3′‐diisobutyrate‐5′‐ProTide mixed prodrugs were synthesized by treating the ProTides with isobutyric anhydride and a catalytic amount of DMAP to afford the 2′‐3′‐diisobutyrate‐5′‐ProTide mixed prodrugs KAD‐039, KAD‐046, KAD‐048, KAD‐050, KAD‐057, KAD‐062, and KAD‐066 in excellent yields (Scheme 4).

SCHEME 4.

Synthesis of ProTides and 2′‐3′‐diisobutyrate‐5′‐ProTide mixed prodrugs. Reaction conditions: (a) t‐BuMgCl, N‐[(S)‐(2,3,4,5,6‐Pentafluorophenoxy)phenoxyphosphinyl]‐L‐alanine 1‐methylethyl ester, DMF, µWave, 30 min, 60 °C; (b) TFA, DCM, H₂O rt, 16 h; and (c) isobutyric anhydride, DMAP (cat.), pyridine, rt, 2 h.

2.2. Antiviral Testing

Initial antiviral testing was performed by our NIH Antiviral Drug Discovery (AViDD) collaborators against pathogens of public health concern, including serotypes 2 and 4 of DENV as well as EBOV. The EC₅₀'s of all tested compounds are summarized in Table 2.

TABLE 2.

Initial antiviral activity of tested compounds against DENV and EBOV.

Compound	DENV‐2	DENV‐4	EBOV
Strain	New Guinea C	H241	ΔVP30
Cell line	Huh‐7	Huh‐7	Huh‐7 VP30
KAD‐020	>100	>100	>100
KAD‐022	34.1	>100	>100
KAD‐038	>100	>100	>100
KAD‐039	6.6	12.1	9.6
KAD‐041	>100	>100	>100
KAD‐042	12.7	25.4	48.0
KAD‐043	>100	>100	>100
KAD‐044	>100	>100	96.7
KAD‐045	>100	>100	>100
KAD‐046	4.8	18.7	55.0
KAD‐047	>100	>100	>100
KAD‐048	>100	>100	29.2
KAD‐049	>100	>100	>100
KAD‐050	2.1	6.3	33.4
KAD‐051	>100	>100	>100
KAD‐052	18.3	21.0	92.7
KAD‐056	>100	>100	>100
KAD‐057	43.0	>50	68.5
KAD‐058	>100	>100	>100
KAD‐059	>100	>100	47.6
KAD‐060	>100	>100	>100
KAD‐061	>100	>100	>100
KAD‐062	>100	>100	>100
KAD‐065	>50	>50	>100
KAD‐066	21.7	33.5	14.3

Note: Reported values correspond to EC₅₀ and are given in µM.

As shown in Table 2, the parent analogs without any prodrugs exhibited no antiviral activity against DENV or EBOV at concentrations up to 100 µM, likely due to their highly polar nature, which limits cellular uptake. In contrast, several tri‐isobutyrate ester prodrugs displayed moderate activity, most notably KAD‐042 and KAD‐052, which showed EC₅₀ values in the 10–25 µM range against DENV. KAD‐042 also exhibited weak antiviral activity against EBOV with an EC₅₀ of 48 µM, whereas KAD‐052 showed no activity. The increased activity of these tri‐isobutyrate analogs is likely attributable to the increased lipophilicity and cell penetration.

Interestingly, analogs containing only the ProTide prodrug did not exhibit antiviral activity against either virus. However, nearly all of the 2′‐3′‐diisobutyrate‐5′‐ProTide analogs demonstrated activity against both DENV and EBOV. Similar to the tri‐isobutyrate analogs, the mixed prodrug analogs are hypothesized to benefit from the increased lipophilicity and membrane permeability from the ester groups. However, unlike the tri‐isobutyrate analogs, these mixed prodrug analogs also incorporate the ProTide moiety, which is designed to enhance intracellular activation. Therefore, the improved antiviral activity of these 2′‐3′‐diisobutyrate‐5′‐ProTide analogs likely results from complementary effects from the ester and ProTide moieties. Of these analogs, KAD‐050 was the most potent, showing single‐digit micromolar activity against DENV‐2 and DENV‐4 (EC₅₀ = 2.1 and 6.3 µM, respectively) and moderate activity against EBOV (EC₅₀ = 33.4 µM). KAD‐046, which differs from KAD‐050 only by the substitution of a 4‐amino group with a methoxy group on the pyrimidine ring, displayed comparable activity against DENV‐2 (EC₅₀ = 4.8 µM) but reduced activity against EBOV (EC₅₀ = 48 µM). Although not the most potent analog, KAD‐039, an isomer of KAD‐050 with the 2‐methoxy and 4‐amino substituents on the pyrimidine ring switched, exhibited the best broad‐spectrum profile with EC₅₀ values of 6.6 µM against DENV‐2, 12.1 µM against DENV‐4, and 9.6 µM against EBOV. The improved activity of these mixed prodrugs is likely due to a combination of the ProTide and ester modifications, which together enhance activation and intracellular delivery.

Given the potent and broad‐spectrum activity of KAD‐039, KAD‐046, and KAD‐050, these analogs were evaluated against a wider variety of orthoflaviviruses. These include mosquito‐transmitted viruses such as WNV, ZIKV, and Japanese encephalitis virus (JEV), as well as those transmitted by ticks, including TBEV, POWV, KFDV, Langat virus (LGTV), and louping ill virus (LIV). The results for KAD‐039 are summarized in Table 3. Notably, KAD‐046 and KAD‐050 did not show antiviral activity below 50 µM and are therefore not included in the table. As shown in Table 3, KAD‐039 displayed potent, low‐micromolar antiviral activity against all of the tested viruses, with EC₅₀ values as low as approximately 1–2 µM against POWV and LGTV. These results suggest that the 4′‐azido substitution constitutes a relevant structural feature associated with effective inhibition of orthoflavivirus replication [60].

TABLE 3.

Additional antiviral testing for KAD‐039 against several orthoflaviviruses and SARS‐CoV‐2.

Virus	Strain	KAD‐039
		Huh‐7	A549
WNV	Eg‐101	4.31 ± 0.99	6.54 ± 0.65
ZIKV	Paraiba_01	4.18 ± 2.84	10.74 ± 2.84
JEV	P3	2.13 ± 0.20	9.10 ± 1.62
TBEV	Hypr	11.27 ± 5.04	9.49 ± 2.10
POWV	LB	1.77 ± 0.58	8.24 ± 0.59
KFDV	W‐377	2.69 ± 1.20	6.14 ± 0.43
LGTV	TP21	1.04 ± 0.12	4.61 ± 1.57
LIV	LI/31	7.66 ± 0.42	21.04 ± 0.42
SARS‐CoV‐2	WA‐1	n.d.	9.75

Note: Reported values correspond to EC₅₀ and are given in µM. EC₅₀ values were expressed as a 50% reduction in viral titers and calculated from the inflection points of sigmoidal dose–response curves, which were obtained by a nonlinear fit of log‐transformed inhibitor concentrations versus normalized response using GraphPad Prism 7.04.

The compound showed no obvious differences in activity among orthoflaviviruses, regardless of whether they are transmitted by mosquitoes or ticks, which may reflect their close phylogenetic relationships and the high sequence similarity of the target enzymes. On the other hand, KAD‐039 exhibited a strict cell‐dependent antiviral effect where EC₅₀ values in A549 cells are 1.5–4.6‐fold higher compared with those in Huh‐7 cells (Table 3). This is likely due to the higher metabolic competence of Huh‐7 cells, which is required for the cleavage of masked ProTide groups and the efficient conversion of the prodrug analogs into their active forms. A similar phenomenon has been repeatedly observed for other ProTides when tested in hepatocyte‐derived cell lines, emphasizing the importance of selecting clinically and metabolically relevant cell lines for screening nucleos(t)ide prodrugs for antiviral activity [61, 62].

Furthermore, KAD‐039 also exhibited low‐micromolar antiviral activity against SARS‐CoV‐2, a representative of the Coronaviridae family (Table 3), highlighting its broad‐spectrum antiviral potential across multiple families of RNA viruses. Importantly, none of the tested analogs showed signs of cytotoxicity (CC₅₀ > 50 µM) in both the Huh‐7 and A549 cell lines, showing a favorable in vitro safety profile for these compounds.

2.3. Pharmacokinetic Evaluation of KAD‐039

To evaluate the pharmacokinetic properties of KAD‐039, plasma and in vitro metabolic studies were conducted (Table 4). As seen in Table 4, KAD‐039 showed species‐dependent plasma stability. Rapid degradation was seen in mouse plasma (t _1/2 = 0.7 min), likely due to the increased esterase activity in mice. In contrast, KAD‐039 demonstrated good stability in the other higher‐order species, with half‐lives of 28, 21, and 19.9 h in beagle dog, cynomolgus monkey, and human plasma, respectively. In addition, KAD‐039 showed poor stability in liver microsomes, with rapid metabolism in human liver microsomes (t _1/2 = 6.1 min) and even faster degradation in mouse microsomes (t _1/2 = 0.9 min). Notably, degradation was observed under both NADPH‐dependent and non‐NADPH‐dependent conditions. These findings suggest that KAD‐039 may undergo significant first‐pass metabolism, particularly following oral administration, and highlight the need for further optimization or alternative administration strategies to overcome poor metabolic stability.

TABLE 4.

Plasma stability and in vitro metabolic stabilities of KAD‐039.

Compound	Plasma stability				Microsomal stabilitya
	Mouse t 1/2 (min), n = 3	Dog t 1/2 (h), n = 3	Monkey t 1/2 (h), n = 3	Human t 1/2 (h), n = 3	Mouse t 1/2 (min), n = 3	Human t 1/2 (min), n = 3
KAD‐039	0.7 ± 0.0	28 ± 1	21 ± 1	19.9 ± 0.8	0.9 ± 0.0b	6.1 ± 0.0b

Note: Data is presented as mean ± SD.

^a CYP enzyme cofactor: NADPH.

^b Control in the absence of NADPH showed remaining percentage less than 5% at the end of incubation of 60 min (susceptible to non‐NADPH dependent regulation).

2.4. Computational Modeling

Finally, to investigate the mechanism of action of KAD‐039 against multiple viral families, computational modeling and molecular docking studies were performed using the expected triphosphate active form, KAD‐039TP (Figure 3F). These docking studies focused on the viral methyltransferases (MTases) based on previous reports from our group demonstrating that several fleximer nucleoside triphosphates bearing the same 2‐amino‐4‐methoxypyrimidine nucleobase as KAD‐039 exert their antiviral effects through inhibition of the DENV NS5 MTase [51], rather than through RNA‐dependent RNA polymerase (RdRp) inhibition, which is the expected mechanism of action of most nucleoside analogs.

FIGURE 3.

Docking of KAD‐039TP to selected viral and human MTase enzymatic cap/GTP binding sites. In all structures, the substrate (light gray) is superimposed with KAD‐039TP (dark green), with nitrogen (blue), oxygen (red), and phosphorus (orange) atoms colored for clarity. Hydrogen bonds are depicted as pink dashed lines. Residue numbers correspond to those annotated in the PDB structure. (A) The DENV NS5 MTase domain is shown in teal. (B) SARS‐CoV‐2 nsp14 is shown in cyan. (C) WNV NS5 is shown in purple. (D) The EBOV L‐protein MTase domain is shown in beige. (E) The human N7 MTase domain is shown in light blue. (F) Structure of KAD‐039TP.

We used reported structures of the DENV, SARS‐CoV‐2 nsp14, WNV, and human N7 MTase domains, as well as a homology model of the MTase domain of the EBOV L‐protein. Docking of KAD‐039TP was restricted to the cap/GTP‐binding site of these enzymes. Binding pose scores for the substrate or KAD‐039TP are listed in Table 5. These scores are intended to be compared directly within the same enzyme to rank the relative affinity of the docked compounds. They are not intended to compare the binding affinities of docked molecules across different enzymes [63]. KAD‐039TP docked to the enzymatic GTP/cap‐binding site with similar or higher affinity than the native substrate in the case of DENV NS5 (ΔΔG = 0.09), WNV NS5 (ΔΔG = −1.92), SARS‐CoV‐2 nsp14 (ΔΔG = 0.31), and the EBOV L‐protein MTase domain (ΔΔG = −1.90). Conversely, docking of KAD‐039TP to the human N7 MTase domain was less favorable than that of the native substrate (ΔΔG = 1.31).

TABLE 5.

Relative docking scores for substrate or KAD‐039 for viral and human MTase domains.

Enzyme		DENV NS5	SARS‐CoV‐2 nsp14	WNV NS5	EBOV L‐protein	Human N7 MTase
Binding score (kcal/mol)	Substrate	−8.63	−10.11	−6.60	−5.34	−7.23
	KAD‐039TP	−8.54	−9.80	−8.52	−7.24	−5.92

Within KAD‐039TP, the imidazole ring maintains stacking interactions with the DENV NS5 residue F25, as observed for the purine base in the GTP substrate analog, AT‐9010. Residue K14 forms an additional hydrogen bond with the 2′‐OH of the KAD‐039TP ribose ring, while K14 and E157 form additional electrostatic interactions with the 4′‐azido moiety, which is not present in the AT‐9010 structure. Hydrogen bonding between residues K29, S150, R211, and S213 and the phosphate groups is maintained in both structures (Figure 3A).

In the SARS‐CoV‐2 nsp14 GTP/cap‐binding site (Figure 3B), the imidazole ring of KAD‐039TP participates in a stacking interaction with F506, and hydrogen bonds form between the 2‐amino group of the pyrimidine ring and T428, as well as the ribose 2′‐OH and W385. Residue K423 forms electrostatic interactions with the terminal nitrogen of the 4′‐azido group and with oxygens in the α‐ and γ‐phosphate groups. R310 participates in hydrogen bonding with the β‐phosphate group. In the substrate‐bound structure, the GTP base stacks with F426, and the heterocyclic amine in the pyrimidine ring forms a hydrogen bond with T428. The side chain of residue N306 acts as an H‐bond acceptor for the GTP 3′‐OH, while the N306 backbone acts as an H‐bond donor to the β‐phosphate group. Residue K423 forms electrostatic interactions with all three phosphate groups in the bound GTP substrate, and R310 participates in hydrogen bonding with the β‐phosphate group. The primary difference between the KAD‐039TP and GTP docked poses appears to be the coordination of the phosphate groups, with the K423/4′‐azido electrostatic interaction displacing the β‐phosphate group, leading to greater solvent exposure of the phosphate groups as a whole relative to the coordination observed in the GTP‐bound structure. This may explain the slightly decreased binding score for KAD‐039TP compared to the substrate.

Docking of WNV NS5 (Figure 3C) shows pi–pi stacking interactions with the imidazole and pyrimidine rings in KAD‐039TP, consistent with those observed in the purine base of the bound GTP. In the docked structure of KAD‐039TP, the 2‐amino moiety of the pyrimidine maintains a hydrogen bond with residue M19 and establishes two additional hydrogen bonds with residue S215, one with the 3′‐OH and another with the 4′‐azido moiety of the ribose ring. The phosphate groups are coordinated by residues R28 and R213 in both cases, and by K29 or S150 in the case of KAD‐039TP or GTP, respectively.

In the EBOV L‐protein GTP/cap‐binding site (Figure 3D), the pyrimidine rings of GTP and KAD‐039TP stack with residue F214. KAD‐039TP also forms an additional hydrogen bond between its pyrimidine ring and the S216 side chain, whereas the GTP pyrimidine ring hydrogen bonds with S248. Both compounds’ phosphate groups coordinate with the active‐site magnesium ion, and the 4′‐azido group in KAD‐039TP forms an additional electrostatic interaction with the ion. Furthermore, the phosphate groups of both molecules hydrogen bond with R55, and the S247 side chain forms an additional hydrogen bond with the α‐phosphate of GTP.

The human N7 MTase domain forms hydrogen bonds with the GTP base via residues N176 and Y467. The 2‐amino group of the pyrimidine in KAD‐039TP is sufficiently displaced relative to the GTP base to establish a hydrogen bond with Y289. Furthermore, the ribose 2′‐OH and 3′‐OH groups form hydrogen bonds with N177 and K180, respectively, in both the GTP and KAD‐039TP docking structures. In both cases, residues R173, K214, and R239 coordinate the phosphate groups.

The similarity of docking poses of KAD‐039TP across the respective GTP/cap‐binding sites (Figure 3) and similar or better binding scores compared to the native substrate (Table 5) support the measured antiviral activity against DENV NS5 (Table 2), WNV NS5 (Table 3), SARS‐CoV‐2 (Table 3), and EBOV L‐protein (Table 2). Although KAD‐039TP could be docked into the human N7 MTase domain, the fleximer nucleobase is considerably displaced relative to that of the GTP substrate (Figure 3), which, in part, accounts for the lower binding affinity of KAD‐039TP compared to GTP (Table 5). Additional biochemical mechanism of action studies are currently underway to confirm the mechanism of action of KAD‐039 against these viral families.

3. Conclusion

A series of novel 4′‐azido fleximer nucleoside analogs were designed, synthesized, and evaluated for their broad‐spectrum antiviral potential against pathogens of epidemic/pandemic concern. Several analogs featuring the 2′, 3′‐diisobutyrate‐5′‐ProTide prodrug strategy exhibited promising antiviral activity against some or all of these viruses. In particular, KAD‐046 and KAD‐050 exhibited potent antiviral activity against DENV‐2 with EC₅₀ values of 4.8 and 2.1 µM but did not possess significant activity against any of the other viruses. The most promising broad‐spectrum analog, KAD‐039, displayed impressive antiviral activity, with low single‐digit micromolar EC₅₀ values against all of the viruses tested. Pharmacokinetic evaluation of KAD‐039 showed high plasma stability in higher‐order species, but poor stability in human liver microsomes, suggesting significant first‐pass hepatic metabolism is possible. In order to gain a deeper understanding of KAD‐039's mechanism of action against flaviviruses, filoviruses, as well as coronaviruses, computational molecular docking studies were performed with the anticipated active metabolite, KAD‐039TP. These docking studies were performed in the conserved cap/GTP binding pockets of the viral methyltransferases based on our group's prior studies that found structurally similar compounds to be inhibitors of the DENV NS5 MTase. The results of these studies found that KAD‐039TP docked into the enzymatic GTP/cap‐binding site with similar or higher predicted affinity compared to the native substrate for all of the viral methyltransferases but had a lower affinity compared to the native GTP in the case of the human N7 methyltransferase. Additional biochemical mechanism of action studies are currently being conducted to determine the exact mechanism of action of KAD‐039 against these different viruses.

4. Experimental Section

All reactions were performed using oven‐dried glassware under argon atmosphere, unless otherwise noted. A CEM Discover 2.0 microwave synthesizer was used for all microwave reactions. All reagents and solvents were purchased from Sigma–Aldrich, Fisher Scientific, or Combi‐Blocks. Analytical thin‐layer chromatography was done on Sigma Aldrich silica gel 60 Å glass‐backed plates with 254 nm fluorescent indicator. Flash column chromatography was performed on a Teledyne Isco Combi‐Flash NEXTGEN 300 system using RediSep Rf silica and RediSep Rf GOLD C18aq High Performance cartridges, monitoring at 254, 280, and/or 235 nm. All ¹H, ¹³C, ³¹P and NOESY spectra were recorded on a JEOL Eclipse ECX 400 MHz NMR or JEOL YH 400 MHz NMR. ¹H chemical shifts are reported in parts per million (δ ppm) referenced to internal tetramethylsilane (TMS) standard at 0.0 ppm. ¹³C chemical shifts are reported in parts per million (δ ppm) with solvent resonance as the internal standard. ³¹P chemical shifts were indirectly referenced via the unified chemical shift scale relative to the ¹H signal calibrated to TMS. Splitting patterns are designed as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), hept (heptuplet), and m (multiplet). Coupling constant (J) are listed in hertz (Hz). Mass spectrometry (MS) and High‐resolution mass spectrometry (HRMS) were performed on a LTQ Orbitrap hybrid mass spectrometer with an electrospray ionization probe by direct infusion. The purity of the final compounds was at least 95% by elemental analysis performed by Atlantic Microlabs.

4.1. Compound 2: 1‐((3aR,4R,6S,6aS)‐6‐azido‐6‐(iodomethyl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)‐4‐iodo‐1H‐imidazole

A stirred solution of sodium azide (9.41 g, 144.9 mmol, 5 eq.) in anhydrous DMF (50 mL) was added iodine monochloride (3.8 mL, 72.4 mmol, 2.5 eq.) and stirred at room temperature for 20 min. 1 (10 g, 29.0 mmol, 1 eq.), which was synthesized by our previously reported route [52], was suspended in anhydrous DMF (10 mL) and added dropwise across 10 min and the reaction was stirred at room temperature for an additional 2 h. Upon completion, the reaction was quenched with saturated aqueous NaHCO₃ (10 mL) and then slow addition of 10% w/v aqueous Na₂S₂O₃ (10 mL). The reaction mixture was then extracted in EtOAc 3x. The combined organic layers were dried with MgSO₄, gravity filtered and purified by flash chromatography on silica gel (0%–50% EtOAc/Hexanes) to afford 2 (12.6 g, 84.1%) as an off‐white solid. TLC: Rf  = 0.62 (50% EtOAc/Hexanes). ¹ H NMR (400 MHz, DMSO‐D ₆) δ 7.92 (d, J = 1.5 Hz, 1H), 7.63 (d, J = 1.5 Hz, 1H), 6.29 (s, 1H), 5.53 (dd, J = 5.6, 1.2 Hz, 1H), 4.78 (d, J = 5.6 Hz, 1H), 3.63 (d, J = 11.1 Hz, 1H), 3.56 (d, J = 11.3 Hz, 1H), 1.50 (s, 3H), 1.34 (s, 3H). ¹³ C NMR (101 MHz, DMSO‐D ₆) δ 140.23, 124.31, 113.96, 101.25, 91.03, 84.90, 84.37, 84.22, 26.36, 25.11, 5.73. MS (ESI): m/z 517.92 [M+H]⁺.

4.2. Compound 4: ((3aS,4R,6R,6aR)‐4‐azido‐6‐(4‐iodo‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a solution of 2 (12.61 g, 24.4 mmol, 1 eq.) in anhydrous DMF (200 mL) was added potassium acetate (23.9 g, 243.7 mmol, 10 eq.) and 18‐Crown‐6 (32.2 g, 122.0 mmol, 5 eq.) and refluxed at 120 °C for 72 h. The reaction was then cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was washed with saturated aqueous sodium chloride and extracted in EtOAc 3x. The combined organic layers were dried with MgSO₄ and gravity filtered to afford crude 3 that was then dissolved in MeOH (100 mL) and added potassium carbonate (6.7 g, 48.8 mmol, 2 eq.). The reaction was stirred at room temperature for 16 h before the salts were filtered off and the solvent was removed under reduced pressure. The resulting residue was purified by flash chromatography on silica gel (0%–75% EtOAc/Hexanes) to afford 4 (3.47 g, 35.0% across two steps) as a flaky white solid. TLC: R f  = 0.38 (50% EtOAc/Hexanes). ¹ H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 1.4 Hz, 1H), 7.26 (d, J = 1.3 Hz, 1H), 5.92 (d, J = 1.4 Hz, 1H), 5.13 (dt, J = 5.8, 1.3 Hz, 1H), 4.66 (dd, J = 5.7, 1.1 Hz, 1H), 4.09–3.96 (m, 2H), 2.72 (t, J = 6.8 Hz, 1H), 1.56 (s, 3H), 1.36 (s, 3H). ¹³ C NMR (101 MHz, CDCl₃) δ 138.37, 122.98, 114.99, 102.52, 92.85, 85.05, 84.87, 83.87, 63.93, 26.38, 24.82. MS (ESI): m/z 408.02 [M+H]⁺.

4.3. Compound 5a: ((3aS,4R,6R,6aR)‐4‐azido‐6‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a solution of 4 (0.500 g, 1.23 mmol, 1 eq.) in 1,4‐dioxane (12 mL) and H₂O (3 mL) was added 2,4‐dimethoxypyrimidine‐5‐boronic acid (0.407 g, 2.21 mmol, 1.8 eq.), K₂CO₃ (0.509 g, 3.68 mmol, 3.0 eq.), and PdCl₂(PPh₃)₂ (0.086 g, 0.12 mmol, 0.1 eq.) in a microwave vial. The reaction was placed in a microwave reactor and heated to 80 °C for 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in DCM, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–10% MeOH/ DCM) to afford 5a (0.404 g, 78.4%) as a white solid. TLC: R f  = 0.56 (8% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.99 (s, 1H), 7.73 (d, J = 1.4 Hz, 1H), 7.59 (d, J = 1.4 Hz, 1H), 6.00 (d, J = 1.4 Hz, 1H), 5.22 (dd, J = 5.7, 1.4 Hz, 1H), 4.69 (d, J = 5.7 Hz, 1H), 4.10 (s, 3H), 4.04 (d, J = 3.4 Hz, 2H), 4.02 (s, 3H), 3.14 (s, 1H), 1.57 (s, 3H), 1.36 (s, 3H). ¹³ C NMR (101 MHz, CDCl₃) δ 167.09, 163.89, 155.90, 136.48, 135.34, 116.41, 114.87, 109.19, 102.41, 93.29, 85.10, 84.96, 64.00, 55.00, 54.28, 26.44, 24.92. MS (ESI): m/z 420.17 [M+H]⁺.

4.4. Compound 5b: ((3aS, 4R,6R, 6aR)‐6‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a stirred solution of 4 (0.500 g, 1.23 mmol, 1 eq.) in 1,4‐dioxane (12 mL) and H₂O (3 mL) was added 2‐aminopyrimidine‐5‐boronic acid (0.307 g, 2.21 mmol, 1.8 eq.), K₂CO₃ (0.509 g, 3.68 mmol, 3.0 eq.), and PdCl₂(PPh₃)₂ (0.086 g, 0.12 mmol, 0.1 eq.) in a microwave vial. The reaction was placed in a microwave reactor and heated to 80 °C for 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in EtOAc, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–10% MeOH/ DCM) to afford 5b (0.321 g, 69.9%) as a white solid. TLC: R f  = 0.27 (10% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.59 (s, 2H), 7.97 (d, J = 1.3 Hz, 1H), 7.76 (d, J = 1.3 Hz, 1H), 6.65 (s, 2H), 6.24 (s, 1H), 5.53 − 5.46 (m, 2H), 4.72 (dd, J = 5.8, 1.1 Hz, 1H), 3.83 − 3.69 (m, 2H), 1.49 (s, 3H), 1.34 (s, 3H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 163.11, 154.83, 145.88, 138.56, 137.75, 117.57, 113.63, 113.10, 102.43, 91.49, 84.76, 84.13, 64.03, 26.47, 25.09. MS (ESI): m/z 375.16 [M+H]⁺.

4.5. Compound 5c: ((3aS, 4R,6R, 6aR)‐4‐azido‐6‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a solution of 4 (0.500 g, 1.23 mmol, 1 eq.) in 1,4‐dioxane (12 mL) and H₂O (3 mL) was added 2‐methoxypyrimidine‐5‐boronic acid (0.340 g, 2.21 mmol, 1.8 eq.), K₂CO₃ (0.509 g, 3.68 mmol, 3.0 eq.), and PdCl₂(PPh₃)₂ (0.086 g, 0.12 mmol, 0.1 eq.) in a microwave vial. The reaction was placed in a microwave reactor and heated to 80 °C for 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in DCM, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–8% MeOH/ DCM) to afford 5c (0.364 g, 76.2%) as a white solid. TLC: R f  = 0.56 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.89 (s, 2H), 7.78 (d, J = 1.4 Hz, 1H), 7.44 (d, J = 1.4 Hz, 1H), 6.02 (d, J = 1.3 Hz, 1H), 5.25 (dd, J = 5.7, 1.4 Hz, 1H), 4.71 (d, J = 5.7 Hz, 1H), 4.05 (s, 5H), 3.24 (s, 1H), 1.59 (s, 3H), 1.38 (s, 3H). ¹³ C NMR (101 MHz, CDCl₃) δ 164.81, 156.00, 137.81, 137.54, 121.67, 114.86, 112.77, 102.45, 93.07, 84.89, 84.79, 63.84, 26.29, 24.74. MS (ESI): m/z 390.15 [M+H]⁺.

4.6. Compound 5d: ((3aS, 4R,6R, 6aR)‐6‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a microwave vial was added 2‐amino‐5‐bromo‐4‐methoxypyrimidine (0.380 g, 1.86 mmol, 1 eq.), bis(pinacolato)diboron (0.567 g, 2.23 mmol, 1.2 eq.), potassium acetate (0.548 g, 5.58 mmol, 3 eq.), and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) in 1,4‐dioxane (12 mL). The reaction was placed in a microwave reactor and heated to 100 °C for 3 h. Then, 4 (0.250 g, 0.61 mmol, 0.33 eq.), K₂CO₃ (0.514 g, 3.72 mmol, 2 eq.) and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) were added along with H₂O (3 mL) and stirred at 80 °C for an additional 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in EtOAc, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–10% MeOH/ DCM) to afford 5d (0.110 g, 44.4%) as a white solid. TLC: R f  = 0.23 (10% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D ₆) δ 8.62 (s, 1H), 7.95 (d, J = 1.4 Hz, 1H), 7.54 (d, J = 1.4 Hz, 1H), 6.63 (s, 2H), 6.26 (d, J = 1.2 Hz, 1H), 5.53 (t, J = 5.9 Hz, 1H), 5.48 (dd, J = 5.6, 1.3 Hz, 1H), 4.71 (d, J = 5.7 Hz, 1H), 3.95 (s, 3H), 3.85 − 3.71 (m, 2H), 1.49 (s, 3H), 1.34 (s, 3H). ¹³ C NMR (101 MHz, DMSO‐D ₆) δ 165.40, 161.81, 155.02, 136.87, 134.72, 115.43, 113.19, 103.95, 101.96, 91.39, 84.28, 83.77, 63.47, 53.24, 26.08, 24.73. MS (ESI): m/z 405.17 [M+H]⁺.

4.7. Compound 5e: ((3aS, 4R,6R, 6aR)‐6‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a microwave vial was added 5‐bromo‐2‐methoxypyrimidin‐4‐amine (0.380 g, 1.86 mmol, 1 eq.), bis(pinacolato)diboron (0.567 g, 2.23 mmol, 1.2 eq.), potassium acetate (0.548 g, 5.58 mmol, 3 eq.), and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) in 1,4‐dioxane (12 mL). The reaction was placed in a microwave reactor and heated to 100 °C for 3 h. Then, 4 (0.250 g, 0.61 mmol, 0.33 eq.), K₂CO₃ (0.514 g, 3.72 mmol, 2 eq.) and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) were added along with H₂O (3 mL) and stirred at 80 °C for an additional 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in DCM, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–7% MeOH/ DCM) to afford 5e (0.103 g, 41.6%) as a white solid. TLC: R f  = 0.50 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H), 7.73 (d, J = 1.3 Hz, 1H), 7.37 (d, J = 1.4 Hz, 1H), 5.99 (d, J = 1.4 Hz, 1H), 5.23 (dd, J = 5.8, 1.4 Hz, 1H), 4.70 (d, J = 5.7 Hz, 1H), 4.06 (d, J = 2.6 Hz, 2H), 3.93 (s, 3H), 3.78 (s, 1H), 1.58 (s, 3H), 1.37 (s, 3H). ¹³ C NMR (101 MHz, CDCl₃) δ 164.31, 162.11, 153.41, 139.16, 135.81, 114.80, 111.77, 104.55, 102.55, 93.13, 84.93, 84.76, 63.62, 54.40, 26.31, 24.77. MS (ESI): m/z 405.17 [M+H]⁺.

4.8. Compound 5f: ((3aS, 4R,6R, 6aR)‐4‐azido‐6‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

To a microwave vial were added 5‐bromo‐4‐methoxypyrimidine (0.352 g, 1.86 mmol, 1 eq.), bis(pinacolato)diboron (0.567 g, 2.23 mmol, 1.2 eq.), potassium acetate (0.548 g, 5.58 mmol, 3 eq.), and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) in 1,4‐dioxane (12 mL). The reaction was placed in a microwave reactor and heated to 100 °C for 3 h. Then, 4 (0.250 g, 0.61 mmol, 0.33 eq.), K₂CO₃ (0.514 g, 3.72 mmol, 2 eq.) and PdCl₂(PPh₃)₂ (0.065 g, 0.093 mmol, 0.05 eq.) were added along with H₂O (3 mL) and stirred at 80 °C for an additional 2 h. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was suspended in DCM, dried with Mg₂SO₄ and filtered over a celite pad. The filtrate was then concentrated under reduced pressure and purified by flash chromatography on silica gel (0%–7% MeOH/ DCM) to afford 5f (0.118 g, 49.3%) as a flaky white solid. TLC: R f  = 0.42 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.26 (s, 1H), 8.70 (s, 1H), 7.77 (d, J = 1.4 Hz, 1H), 7.74 (d, J = 1.4 Hz, 1H), 6.03 (d, J = 1.3 Hz, 1H), 5.25 (dd, J = 5.8, 1.4 Hz, 1H), 4.72 (d, J = 5.8 Hz, 1H), 4.14 (s, 3H), 4.05 (s, 2H), 2.42 (s, 1H), 1.60 (s, 3H), 1.39 (s, 3H). ¹³ C NMR (101 MHz, CDCl₃) δ 164.81, 155.76, 153.80, 136.58, 134.83, 118.21, 114.91, 114.86, 102.27, 93.18, 85.02, 84.90, 63.95, 54.06, 26.32, 24.79. MS (ESI): m/z 390.16 [M+H]⁺.

4.9. Compound 5g: ((3aS, 4R,6R, 6aR)‐6‐(4‐(4‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methanol

A solution of 5f (0.359 g, 0.92 mmol, 1 eq.) in anhydrous MeOH (10 mL) was added to a steel Parr bomb and cooled to −78 °C. Once the solution reached −78 °C, NH₃ (g) was bubbled in until the volume of the solution doubled. The reaction vessel was sealed and allowed to warm to room temperature before being heated to 160 °C for 48 h. Upon completion, the reaction was cooled to 0 °C, degassed, and the solvent was evaporated to give a brown residue that was purified by flash chromatography on silica gel (0%–10% MeOH/ DCM) to afford 5g (0.101 g, 29.2%) as an off‐white solid. TLC: R f  = 0.31 (10% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.51 (s, 1H), 8.29 (s, 1H), 8.14 (d, J = 1.3 Hz, 1H), 8.02 (d, J = 1.4 Hz, 1H), 7.62 (s, 2H), 6.31 (d, J = 1.0 Hz, 1H), 5.61 − 5.48 (m, 2H), 4.74 (d, J = 5.6 Hz, 1H), 3.76 (h, J = 6.4 Hz, 2H), 1.49 (s, 3H), 1.34 (s, 3H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 159.47, 156.31, 151.08, 137.18, 136.96, 114.95, 113.14, 109.35, 102.06, 91.06, 84.18, 83.54, 63.43, 25.92, 24.59. MS (ESI): m/z 375.15 [M+H]⁺.

4.10. General Procedure A: Isopropylidene Deprotection to Afford Parent Nucleosides

The starting material (100–150 mg) was suspended in DCM (1.5 mL) and H₂O (50 µL). TFA (1.5 mL) was added, and the reaction was stirred at room temperature for 4–6 h. The solvent was removed under reduced pressure and coevaporated with toluene 3X to remove any excess TFA. The resulting residue was purified by reverse phase flash chromatography (0%–40% MeCN/H₂O) and lyophilized to afford the title compound.

4.10.1. KAD‐020: (2R, 3S,4R, 5R)‐2‐azido‐5‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐(hydroxymethyl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as a white solid (0.036 g, 36.4%) from 5a (0.111 g, 0.26 mmol) following general procedure A. TLC: Rf = 0.51 (25% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.87 (s, 1H), 8.04 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.3 Hz, 1H), 5.87 − 5.79 (m, 2H), 5.74 (d, J = 6.9 Hz, 1H), 5.36 (t, J = 5.9 Hz, 1H), 4.66 (td, J = 7.2, 4.1 Hz, 1H), 4.03 (s, 3H), 3.92 (s, 3H), 3.85 − 3.71 (m, 3H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 166.40, 163.21, 154.76, 137.69, 133.67, 115.76, 109.22, 107.74, 100.48, 89.76, 74.84, 73.86, 62.81, 54.57, 54.08. HRMS‐ESI (m/z): calcd for C₁₄H₁₇N₇O₆ [M+H]⁺ 380.13131, found 380.13155. Elemental analysis: Anal. calcd for C₁₄H₁₇N₇O₆ + 0.20 CH₃OH: C, 44.22; H, 4.65; N, 25.42. Found: C, 44.10; H, 4.89; N, 25.66.

4.10.2. KAD‐021: (2R, 3S,4R, 5R)‐5‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐(hydroxymethyl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as an off‐white solid (0.030 g, 23.4%) from 5b (0.145 g, 0.39 mmol) following general procedure A. Attempts to purify the compound by column chromatography were unsuccessful; therefore, the crude product was used directly in the next step. TLC: Rf = 0.30 (25% MeOH/DCM). HRMS‐ESI (m/z): calcd for C₁₂H₁₄N₈O₄ [M+H]⁺ 335.12108, found 335.12143.

4.10.3. KAD‐043: (2R, 3S,4R, 5R)‐2‐azido‐2‐(hydroxymethyl)‐5‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as a white solid (0.035 g, 26.8%) from 5c (0.144 g, 0.37 mmol) following general procedure A. TLC: Rf = 0.68 (25% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.98 (s, 2H), 8.06 (d, J = 1.3 Hz, 1H), 8.03 (d, J = 1.3 Hz, 1H), 5.83 − 5.78 (m, 2H), 5.73 (d, J = 7.0 Hz, 1H), 5.31 (t, J = 5.9 Hz, 1H), 4.75 (td, J = 7.2, 4.2 Hz, 1H), 3.94 (s, 3H), 3.85 − 3.71 (m, 3H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 164.03, 155.36, 138.82, 136.14, 122.27, 113.69, 100.35, 99.66, 89.60, 74.41, 73.78, 62.65, 54.59. HRMS‐ESI (m/z): calcd for C₁₃H₁₅N₇O₅ [M+H]⁺ 350.12074, found 350.12115. Elemental analysis: Anal. calcd for C₁₃H₁₅N₇O₅ + 0.05 CH₃OH + 0.05 H₂O: C, 44.55; H, 4.38; N, 27.87. Found: C, 44.43; H, 4.56; N, 27.98.

4.10.4. KAD‐041: (2R, 3S,4R, 5R)‐5‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐(hydroxymethyl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as a white solid (0.036 g, 28.8%) from 5d (0.137 g, 0.34 mmol) following general procedure A. TLC: Rf = 0.32 (25% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.61 (d, J = 1.7 Hz, 1H), 7.92 (d, J = 1.5 Hz, 1H), 7.46 (d, J = 1.5 Hz, 1H), 6.58 (s, 2H), 5.82 − 5.74 (m, 2H), 5.68 (d, J = 7.0 Hz, 1H), 5.31 (t, J = 5.9 Hz, 1H), 4.62 (td, J = 7.2, 4.1 Hz, 1H), 3.93 (s, 3H), 3.85 − 3.72 (m, 3H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 165.89, 162.35, 155.55, 137.71, 135.65, 114.24, 104.48, 100.90, 90.19, 75.26, 74.33, 63.29, 53.66. HRMS‐ESI (m/z): calcd for C₁₃H₁₆N₈O₅ [M+H]⁺ 365.13164, found 365.13301. Elemental analysis: Anal. calcd for C₁₃H₁₆N₈O₅ + 0.30 CH₃OH + 1.40 H₂O: C, 40.02; H, 5.05; N, 28.07. Found: C, 39.63; H, 4.61; N, 27.68.

4.10.5. KAD‐051: (2R, 3S,4R, 5R)‐5‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐(hydroxymethyl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as a white solid (0.038 g, 31.3%) from 5e (0.133 g, 0.33 mmol) following general procedure A. Attempts to purify the compound by column chromatography were unsuccessful; therefore, the crude product was used directly in the next step. TLC: Rf = 0.66 (25% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 8.41 (s, 1H), 8.06 (d, J = 1.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.55 (s, 2H), 5.89 (d, J = 1.0 Hz, 1H), 5.85 (t, J = 7.0 Hz, 1H), 5.79 (d, J = 7.0 Hz, 1H), 5.19 − 5.12 (m, 1H), 4.75 − 4.65 (m, 1H), 4.15 − 4.09 (m, 1H), 3.80 (s, 3H), 3.79 − 3.70 (m, 1H), 3.57 − 3.48 (m, 1H) [13]. C NMR (101 MHz, DMSO‐D₆) δ 163.70, 161.67, 153.40, 137.76, 137.06, 112.34, 104.35, 100.39, 89.79, 74.37, 73.86, 62.71, 53.69. HRMS‐ESI (m/z): calcd for C₁₃H₁₆N₈O₅ [M+H]⁺ 365.13164, found 335.13332. Elemental analysis: Anal. calcd for C₁₃H₁₆N₈O₅ + 0.60 H₂O: C, 41.62; H, 4.62; N, 29.87. Found: C, 31.41; H, 4.71; N, 30.06.

4.10.6. KAD‐058: (2R, 3S,4R, 5R)‐2‐azido‐2‐(hydroxymethyl)‐5‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diol

The title compound was obtained as a white solid (0.039 g, 33.4%) from 5f (0.129 g, 0.33 mmol) following general procedure A. TLC: Rf = 0.64 (25% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D₆) δ 9.10 (s, 1H), 8.71 (s, 1H), 8.08 (d, J = 1.5 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H), 5.97 − 5.89 (m, 2H), 5.79 (d, J = 7.0 Hz, 1H), 5.15 (d, J = 6.5 Hz, 1H), 4.70 − 4.59 (m, 1H), 4.15 − 4.11 (m, 1H), 4.10 (s, 3H), 3.86 − 3.70 (m, 1H), 3.62–3.50 (m, 1H). ¹³ C NMR (101 MHz, DMSO‐D₆) δ 164.19, 155.34, 152.74, 137.97, 133.05, 117.91, 114.92, 100.59, 89.84, 74.90, 73.85, 62.69, 53.96. HRMS‐ESI (m/z): calcd for C₁₃H₁₅N₇O₅ [M+H]⁺ 350.12074, found 350.12290. Elemental analysis: Anal. calcd for C₁₃H₁₅N₇O₅ + 0.40 H₂O: C, 43.80; H, 4.47; N, 27.50. Found: C, 43.50; H, 4.60; N, 27.88.

4.11. General Procedure B: Synthesis of Triisobutyrate Ester Prodrugs

To a stirred solution of the parent nucleoside starting material (50–100 mg) in anhydrous pyridine (3 mL) was added isobutyric anhydride (4 eq.) and a catalytic amount of DMAP. The reaction was stirred at room temperature for 4 h. Upon completion, the solvent was removed under reduced pressure. The resulting residue was washed with 1 M aqueous HCl and extracted in DCM 3x. The combined organic layers were dried with MgSO₄, gravity filtered, and purified by flash chromatography on silica gel (0%–8% MeOH/DCM) and lyophilized to afford the title compound.

4.11.1. KAD‐022: (2R, 3S,4R, 5R)‐2‐azido‐5‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐((isobutyryloxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a powdery white solid (0.102 g, 92.2%) from KAD‐020 (0.071 g, 0.19 mmol) following general procedure B. TLC: Rf = 0.67 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.01 (s, 1H), 7.71 (d, J = 1.4 Hz, 1H), 7.61 (d, J = 1.5 Hz, 1H), 5.97 (d, J = 7.3 Hz, 1H), 5.89 (dd, J = 7.3, 4.3 Hz, 1H), 5.43 (d, J = 4.3 Hz, 1H), 4.56 (d, J = 11.9 Hz, 1H), 4.37 (d, J = 12.0 Hz, 1H), 4.11 (s, 3H), 4.03 (s, 3H), 2.73–2.43 (m, 3H), 1.26–1.08 (m, 18H). ¹³ C NMR (101 MHz, CDCl₃) δ 176.04, 175.00, 174.61, 167.13, 163.99, 155.90, 136.60, 136.15, 115.11, 109.13, 98.13, 88.30, 74.00, 73.00, 62.33, 55.00, 54.25, 34.02, 33.95, 33.72, 19.04, 18.95, 18.91, 18.80, 18.73. HRMS‐ESI (m/z): calcd for C₂₆H₃₅N₇O₉ [M+H]⁺ 590.25690, found 590.25908. Elemental analysis: Anal. calcd for C₂₆H₃₅N₇O₉ + 0.20 CH₃OH + 0.10 H₂O: C, 52.64; H, 6.07; N, 16.40. Found: C, 52.58; H, 5.96; N, 16.30.

4.11.2. KAD‐060: (2R, 3S,4R, 5R)‐5‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((isobutyryloxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.086 g, 87.6%) from KAD‐021 (0.060 g, 0.18 mmol) following general procedure B. TLC: Rf = 0.52 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.70 (s, 2H), 7.72 (d, J = 1.3 Hz, 1H), 7.35 (d, J = 1.4 Hz, 1H), 5.98–5.86 (m, 2H), 5.44 (d, J = 4.2 Hz, 1H), 5.22 (s, 2H), 4.54 (d, J = 12.0 Hz, 1H), 4.39 (d, J = 11.9 Hz, 1H), 2.75–2.41 (m, 3H), 1.27–1.06 (m, 18H). ¹³ C NMR (101 MHz, CDCl₃) δ 176.01, 175.02, 174.60, 162.28, 155.43, 139.51, 137.52, 118.46, 110.61, 98.16, 88.24, 73.79, 72.96, 62.29, 34.01, 33.93, 33.72, 19.05, 18.94, 18.89, 18.79, 18.73. HRMS‐ESI (m/z): calcd for C₂₄H₃₂N₈O₇ [M+H]⁺ 545.24667, found 545.24682. Elemental analysis: Anal. calcd for C₂₄H₃₂N₈O₇ + 0.10 CH₃OH + 0.05 H₂O: C, 52.76; H, 5.97; N, 20.42. Found: C, 52.74; H, 6.01; N, 20.45.

4.11.3. KAD‐044: (2R, 3S,4R, 5R)‐2‐azido‐2‐((isobutyryloxy)methyl)‐5‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.094 g, 89.9%) from KAD‐043 (0.065 g, 0.19 mmol) following general procedure B. TLC: Rf = 0.61 (10% MeOH/DCM). ¹H NMR (400 MHz, CDCl₃) δ 8.89 (s, 2H), 7.75 (d, J = 1.4 Hz, 1H), 7.43 (d, J = 1.4 Hz, 1H), 5.96 (d, J = 7.2 Hz, 1H), 5.91 (dd, J = 7.2, 4.2 Hz, 1H), 5.45 (d, J = 4.2 Hz, 1H), 4.55 (d, J = 11.9 Hz, 1H), 4.40 (d, J = 12.1 Hz, 1H), 4.04 (s, 3H), 2.74–2.54 (m, 2H), 2.49 (p, J = 7.0 Hz, 1H), 1.29–1.07 (m, 18H). ¹³C NMR (101 MHz, CDCl₃) δ 176.01, 175.03, 174.60, 165.05, 156.18, 138.68, 137.73, 121.70, 111.69, 98.27, 88.31, 73.86, 72.97, 62.26, 55.16, 34.01, 33.94, 33.73, 19.06, 18.94, 18.90, 18.79, 18.74. HRMS‐ESI (m/z): calcd for C₂₅H₃₃N₇O₈ [M+H]⁺ 560.24634, found 560.24670. Elemental analysis: Anal. calcd for C₂₅H₃₃N₇O₈ + 0.50 H₂O: C, 52.81; H, 6.03; N, 17.24. Found: C, 52.72; H, 6.22; N, 17.42.

4.11.4. KAD‐042: (2R, 3S,4R, 5R)‐5‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((isobutyryloxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.098 g, 91.5%) from KAD‐041 (0.068 g, 0.19 mmol) following general procedure B. TLC: Rf = 0.53 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.81 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 5.95 (d, J = 7.4 Hz, 1H), 5.91–5.83 (m, 1H), 5.41 (d, J = 4.3 Hz, 1H), 5.31 (s, 2H), 4.54 (d, J = 11.9 Hz, 1H), 4.34 (d, J = 11.9 Hz, 1H), 4.00 (s, 3H), 2.70–2.38 (m, 3H), 1.28–1.04 (m, 18H). ¹³ C NMR (101 MHz, CDCl₃) δ 175.99, 174.96, 174.57, 166.38, 161.47, 155.19, 136.85, 136.33, 114.00, 105.70, 97.98, 88.19, 73.89, 72.93, 62.29, 53.76, 33.95, 33.88, 33.65, 18.98, 18.89, 18.85, 18.83, 18.74, 18.68. HRMS‐ESI (m/z): calcd for C₂₅H₃₄N₈O₈ [M+H]⁺ 575.25724, found 575.25691. Elemental analysis: Anal. calcd for C₂₅H₃₄N₈O₈ + 0.25 CH₃OH: C, 52.06; H, 6.06; N, 19.23. Found: C, 52.12; H, 6.12; N, 19.23.

4.11.5. KAD‐052: (2R, 3S,4R, 5R)‐5‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((isobutyryloxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a powdery white solid (0.104 g, 86.6%) from KAD‐051 (0.076 g, 0.21 mmol) following general procedure B. TLC: Rf = 0.70 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.74 (d, J = 1.4 Hz, 1H), 7.36 (d, J = 1.4 Hz, 1H), 5.95 (d, J = 7.2 Hz, 1H), 5.89 (dd, J = 7.2, 4.3 Hz, 1H), 5.44 (d, J = 4.4 Hz, 1H), 4.54 (d, J = 11.9 Hz, 1H), 4.39 (d, J = 12.0 Hz, 1H), 3.93 (s, 3H), 2.75–2.42 (m, 3H), 1.28–1.06 (m, 18H). ¹³ C NMR (101 MHz, CDCl₃) δ 176.00, 174.97, 174.58, 164.58, 162.20, 153.93, 139.85, 135.61, 111.03, 104.50, 98.30, 88.40, 73.95, 72.99, 62.26, 54.50, 34.00, 33.93, 33.72, 19.04, 18.93, 18.89, 18.88, 18.78, 18.73. HRMS‐ESI (m/z): calcd for C₂₅H₃₄N₈O₈ [M+H]⁺ 575.25724, found 575.25923. Elemental analysis: Anal. calcd for C₂₅H₃₄N₈O₈ + 0.40 CH₃OH: C, 51.94; H, 6.11; N, 19.08. Found: C, 52.01; H, 6.17; N, 19.02.

4.11.6. KAD‐059: (2R, 3S,4R, 5R)‐2‐azido‐2‐((isobutyryloxy)methyl)‐5‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.112 g, 89.3%) from KAD‐058 (0.078 g, 0.22 mmol) following general procedure B. TLC: Rf = 0.58 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.24 (s, 1H), 8.70 (s, 1H), 7.76 (s, 2H), 5.98 (d, J = 7.3 Hz, 1H), 5.90 (dd, J = 7.2, 4.3 Hz, 1H), 5.44 (d, J = 4.3 Hz, 1H), 4.57 (d, J = 12.0 Hz, 1H), 4.37 (d, J = 12.0 Hz, 1H), 4.14 (d, J = 2.2 Hz, 3H), 2.74–2.41 (m, 3H), 1.28–1.04 (m, 18H). ¹³ C NMR (101 MHz, CDCl₃) δ 176.03, 175.00, 174.61, 164.99, 155.98, 153.81, 136.93, 135.55, 117.11, 114.89, 98.23, 88.35, 74.07, 73.00, 62.29, 54.18, 34.01, 33.94, 33.72, 19.04, 18.94, 18.91, 18.88, 18.80, 18.72. HRMS‐ESI (m/z): calcd for C₂₅H₃₃N₇O₈ [M+H]⁺ 560.24634, found 560.24975. Elemental analysis: Anal. calcd for C₂₅H₃₃N₇O₈ + 0.70 H₂O: C, 52.48; H, 6.06; N, 17.14. Found: C, 52.18; H, 6.21; N, 17.48.

4.12. General Procedure C: Synthesis of Isopropylidene‐Protected ProTides

To a microwave vial was added a solution of the isopropylidene‐protected nucleoside starting material (100–125 mg, 1 eq.) in anhydrous DMF (3 mL). To this solution was added tert‐butylmagnesium chloride (1.5 eq., 1 M solution in THF), and the reaction was stirred at room temperature for 10 min. Isopropyl((S)‐(perfluorophenoxy)(phenoxy)phosphoryl)‐L‐alaninate (2 eq.) was then added, and the microwave vial was capped and placed into the microwave reactor and stirred at 60 °C for 30 min. Upon completion, the reaction was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting crude material was purified by flash chromatography on silica gel (0%–8% MeOH/DCM) to afford the title compound.

4.12.1. Compound 6a: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐4‐azido‐6‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.152 g, 90.0%) from 5a (0.103 g, 0.25 mmol) following general procedure C. TLC: Rf = 0.65 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.99 (s, 1H), 7.70 (d, J = 1.4 Hz, 1H), 7.55 (d, J = 1.4 Hz, 1H), 7.34–7.28 (m, 2H), 7.24–7.20 (m, 2H), 7.19–7.10 (m, 1H), 5.99 (d, J = 1.4 Hz, 1H), 5.23 (dd, J = 5.7, 1.5 Hz, 1H), 5.01 (hept, J = 6.2 Hz, 1H), 4.60 (d, J = 5.7 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 4.11 (s, 3H), 4.07–3.96 (m, 4H), 3.77 (dd, J = 11.5, 9.3 Hz, 1H), 1.55 (s, 3H), 1.41–1.31 (m, 6H), 1.28–1.17 (m, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 172.98 (d, J _CP = 8.1 Hz), 167.08, 163.93, 155.96, 150.72 (d, J _CP = 7.1 Hz), 136.47, 135.51, 129.79, 125.17, 120.33 (d, J _CP = 5.0 Hz), 116.32, 115.07, 109.21, 100.68 (d, J _CP = 10.1 Hz), 93.47, 84.85 (d, J _CP = 4.0 Hz), 69.48, 67.01 (d, J _CP = 4.0 Hz), 55.00, 54.26, 50.37, 50.35, 26.49, 25.02, 21.77, 21.74, 21.29, 21.25. ³¹ P NMR (162 MHz, CDCl₃) δ 3.91. MS (ESI): m/z 689.25 [M+H]⁺.

4.12.2. Compound 6b: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐6‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.172 g, 86.2%) from 5b (0.116 g, 0.31 mmol) following general procedure C. TLC: Rf = 0.37 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.67 (s, 2H), 7.71 (d, J = 1.3 Hz, 1H), 7.35–7.27 (m, 3H), 7.26–7.19 (m, 2H), 7.15 (t, J = 7.3 Hz, 1H), 5.97 (d, J = 1.3 Hz, 1H), 5.29–5.20 (m, 3H), 5.01 (hept, J = 6.3 Hz, 1H), 4.60 (d, J = 5.7 Hz, 1H), 4.44 (d, J = 5.6 Hz, 2H), 4.08–3.88 (m, 2H), 1.54 (s, 3H), 1.41–1.32 (m, 6H), 1.23 (dd, J = 6.2, 1.3 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.03 (d, J _CP = 8.0 Hz), 162.17, 155.36, 150.72 (d, J _CP = 8.0 Hz), 138.89, 137.40, 129.79, 125.17, 120.32 (d, J _CP = 5.0 Hz), 118.47, 115.09, 111.72, 100.72 (d, J _CP = 10.1 Hz), 93.26, 84.76 (d, J _CP = 8.0 Hz), 69.49, 67.06 (d, J _CP = 4.0 Hz), 50.35, 26.42, 24.94, 21.78, 21.75, 21.28, 21.24. ³¹ P NMR (162 MHz, CDCl₃) δ 2.81. MS (ESI): m/z 644.24 [M+H]⁺.

4.12.3. Compound 6c: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐4‐azido‐6‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.153 g, 88.8%) from 5c (0.102 g, 0.26 mmol) following general procedure C. TLC: Rf = 0.61 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.87 (s, 2H), 7.73 (d, J = 1.3 Hz, 1H), 7.38 (d, J = 1.4 Hz, 1H), 7.35–7.28 (m, 2H), 7.25–7.20 (m, 2H), 7.19–7.11 (m, 1H), 5.99 (d, J = 1.4 Hz, 1H), 5.24 (dd, J = 5.7, 1.4 Hz, 1H), 5.01 (hept, J = 6.2 Hz, 1H), 4.60 (d, J = 5.6 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 4.04 (s, 4H), 3.76 (dd, J = 11.4, 9.3 Hz, 1H), 1.55 (s, 3H), 1.41–1.31 (m, 6H), 1.23 (dd, J = 6.3, 1.2 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 172.97 (d, J _CP = 8.0 Hz), 165.00, 156.10, 150.72 (d, J _CP = 7.0 Hz), 138.12, 137.61, 129.80, 125.20, 121.80, 120.29 (d, J _CP = 10.1 Hz), 115.19, 112.75, 100.80 (d, J _CP = 9.1 Hz), 93.33, 84.77 (d, J _CP = 3.0 Hz), 69.51, 67.03 (d, J _CP = 5.0 Hz), 55.15, 50.38, 26.44, 24.94, 21.78, 21.75, 21.29, 21.24. ³¹ P NMR (162 MHz, CDCl₃) δ 2.59. MS (ESI): m/z 659.24 [M+H]⁺.

4.12.4. Compound 6d: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐6‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.158 g, 88.4%) from 5d (0.107 g, 0.26 mmol) following general procedure C. TLC: Rf = 0.39 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.83 (s, 1H), 7.66 (d, J = 1.2 Hz, 1H), 7.47 (d, J = 1.2 Hz, 1H), 7.34–7.28 (m, 2H), 7.23 (dt, J = 7.6, 1.2 Hz, 2H), 7.18–7.11 (m, 1H), 5.97 (d, J = 1.6 Hz, 1H), 5.22 (dt, J = 5.7, 1.2 Hz, 1H), 5.08–4.94 (m, 3H), 4.59 (dd, J = 5.7, 1.1 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 4.09–3.94 (m, 4H), 3.90–3.82 (m, 1H), 1.55 (s, 3H), 1.41–1.33 (m, 6H), 1.23 (dt, J = 6.3, 1.2 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.01 (d, J _CP = 8.0 Hz), 166.36, 161.43, 155.79, 150.74 (d, J _CP = 7.0 Hz), 136.35, 136.19, 129.78, 125.15, 120.35 (d, J _CP = 5.0 Hz), 115.30, 115.00, 105.94, 100.60 (d, J _CP = 10.1 Hz), 93.46, 84.86, 69.47, 67.06 (d, J _CP = 4.0 Hz), 53.73, 50.37, 26.49, 25.02, 21.77, 21.75, 21.30, 21.26. ³¹ P NMR (162 MHz, CDCl₃) δ 3.91. MS (ESI): m/z 674.25 [M+H]⁺.

4.12.5. Compound 6e: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐6‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.142 g, 83.7%) from 5e (0.102 g, 0.25 mmol) following general procedure C. TLC: Rf = 0.58 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.70 (d, J = 1.4 Hz, 1H), 7.36–7.27 (m, 3H), 7.22 (dt, J = 8.5, 1.3 Hz, 2H), 7.19–7.12 (m, 1H), 5.98 (d, J = 1.4 Hz, 1H), 5.23 (dd, J = 5.7, 1.4 Hz, 1H), 5.01 (hept, J = 6.2 Hz, 1H), 4.59 (d, J = 5.6 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 4.08–3.94 (m, 1H), 3.93 (s, 3H), 3.84–3.73 (m, 1H), 1.55 (s, 3H), 1.42–1.31 (m, 6H), 1.23 (dd, J = 6.3, 1.6 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 172.98 (d, J _CP = 9.1 Hz), 164.62, 162.23, 153.80, 150.73 (d, J _CP = 6.0 Hz), 139.54, 135.88, 129.80, 125.19, 120.32 (d, J _CP = 4.0 Hz), 115.17, 111.74, 104.59, 100.80 (d, J _CP = 10.1 Hz), 93.41, 84.79, 69.50, 67.02 (d, J _CP = 5.0 Hz), 54.47, 50.39, 50.37, 26.45, 24.96, 21.78, 21.75, 21.27, 21.22. ³¹ P NMR (162 MHz, CDCl₃) δ 2.63. MS (ESI): m/z 674.25 [M+H]⁺.

4.12.6. Compound 6f: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐4‐azido‐6‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.165 g, 87.9%) from 5f (0.111 g, 0.29 mmol) following general procedure C. TLC: Rf = 0.46 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 8.70 (s, 1H), 7.74 (d, J = 1.4 Hz, 1H), 7.71 (d, J = 1.4 Hz, 1H), 7.37–7.27 (m, 2H), 7.25–7.21 (m, 2H), 7.19–7.12 (m, 1H), 6.01 (d, J = 1.5 Hz, 1H), 5.24 (dd, J = 5.7, 1.5 Hz, 1H), 5.02 (hept, J = 6.2 Hz, 1H), 4.61 (d, J = 5.6 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 4.14 (s, 3H), 4.09–3.95 (m, 1H), 3.74 (dd, J = 11.4, 9.3 Hz, 1H), 1.56 (s, 3H), 1.42–1.30 (m, 6H), 1.23 (d, J = 6.3 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 172.99 (d, J _CP = 8.0 Hz), 164.94, 155.93, 153.99, 150. 73 (d, J _CP = 5.0 Hz), 136.75, 135.01, 129.82, 125.20, 120.35 (d, J _CP = 5.0 Hz), 118.30, 115.16, 115.01, 100.79 (d, J _CP = 10.1 Hz), 93.53, 84.91, 84.83, 69.52, 67.00 (d, J _CP = 5.0 Hz), 54.20, 50.39, 26.52, 25.05, 21.79, 21.77, 21.32, 21.28. ³¹ P NMR (162 MHz, CDCl₃) δ 2.63. MS (ESI): m/z 659.24 [M+H]⁺.

4.12.7. Compound 6g: isopropyl ((S)‐(((3aS, 4R,6R, 6aR)‐6‐(4‐(4‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐4‐azido‐2,2‐dimethyltetrahydrofuro[3,4‐d[1,3]dioxol‐4‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.146 g, 85.2%) from 5g (0.100 g, 0.27 mmol) following general procedure C. TLC: Rf = 0.68 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.51–8.33 (m, 2H), 7.73 (d, J = 1.4 Hz, 1H), 7.45 (d, J = 1.5 Hz, 1H), 7.35–7.27 (m, 2H), 7.25–7.19 (m, 2H), 7.18–7.10 (m, 1H), 6.00 (d, J = 1.4 Hz, 1H), 5.24 (dd, J = 5.7, 1.4 Hz, 1H), 5.01 (hept, J = 6.1 Hz, 1H), 4.60 (d, J = 5.6 Hz, 1H), 4.47–4.41 (m, 2H), 4.06–3.93 (m, 2H), 1.55 (s, 3H), 1.39–1.33 (m, 6H), 1.25–1.20 (m, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.01 (d, J _CP = 8.0 Hz), 160.04, 157.04, 151.50, 150.70 (d, J _CP = 7.0 Hz), 139.08, 136.10, 129.79, 125.18, 120.30 (d, J _CP = 5.0 Hz), 115.20, 113.17, 100.85 (d, J _CP = 10.1 Hz), 93.35, 84.75, 69.48, 67.00 (d, J _CP = 5.0 Hz), 50.35, 26.41, 24.91, 21.77, 21.73, 21.25, 21.20. ³¹ P NMR (162 MHz, CDCl₃) δ 3.64. MS (ESI): m/z 644.23 [M+H]⁺.

4.13. General Procedure D: Isopropylidene Deprotection to Afford ProTides

The isopropylidene‐protecting ProTide starting material (100–200 mg) was suspended in DCM (1.5 mL) and H₂O (50 µL). TFA (1.5 mL) was added, and the reaction was stirred at room temperature for 16 h. The solvent was removed under reduced pressure and coevaporated with toluene to remove excess TFA. The resulting residue was purified by reverse‐phase flash chromatography (0%–50% MeCN/H₂O) and lyophilized to afford the title compound.

4.13.1. KAD‐045: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐2‐azido‐5‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐3,4‐dihydroxytetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.093 g, 65.6%) from 6a (0.151 g, 0.22 mmol) following general procedure D. TLC: Rf = 0.39 (10% MeOH/DCM). ¹ H NMR (400 MHz, CD₃OD) δ 8.83 (s, 1H), 7.98 (d, J = 1.4 Hz, 1H), 7.72 (d, J = 1.3 Hz, 1H), 7.35–7.28 (m, 2H), 7.27–7.23 (m, 2H), 7.19–7.12 (m, 1H), 5.91 (d, J = 7.3 Hz, 1H), 4.99 (hept, J = 6.3 Hz, 1H), 4.77 (dd, J = 7.3, 4.3 Hz, 1H), 4.52–4.45 (m, 2H), 4.13 (s, 3H), 4.02 (s, 3H), 3.99–3.89 (m, 2H), 1.35 (d, J = 7.1 Hz, 3H), 1.23 (dd, J = 6.3, 4.4 Hz, 6H). ¹³ C NMR (101 MHz, CD₃OD) δ 174.42 (d, J _CP = 6.0 Hz), 168.58, 165.20, 156.16, 152.15 (d, J _CP = 7.0 Hz), 138.60, 135.12, 130.71, 126.16, 121.36 (d, J _CP = 4.0 Hz), 117.26, 110.07, 99.76 (d, J _CP = 10.1 Hz), 92.17, 76.39, 75.85, 70.22, 68.17 (d, J _CP = 4.0 Hz), 55.46, 54.84, 51.62, 21.93, 21.89, 20.51, 20.45. ³¹ P NMR (162 MHz, CD₃OD) δ 4.08. HRMS‐ESI (m/z): calcd for C₂₆H₃₃N₈O₁₀P [M+H]⁺ 649.21300, found 649.21366. Elemental analysis: Anal. calcd for C₂₆H₃₃N₈O₁₀P + 0.75 H₂O: C, 47.17; H, 5.25; N, 16.92. Found: C, 47.32; H, 5.44; N, 16.88.

4.13.2. KAD‐047: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐5‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐3,4‐dihydroxytetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.098 g, 61.3%) from 6b (0.170 g, 0.26 mmol) following general procedure D. TLC: Rf = 0.20 (10% MeOH/DCM). ¹ H NMR (400 MHz, DMSO‐D ₆) δ 8.64 (s, 2H), 8.01 (s, 1H), 7.85 (s, 1H), 7.38–7.29 (m, 2H), 7.27–7.11 (m, 3H), 6.64 (s, 2H), 6.16–6.01 (m, 2H), 5.84 (dd, J = 14.7, 7.1 Hz, 2H), 4.93–4.83 (m, 1H), 4.82–4.75 (m, 1H), 4.30 (d, J = 5.4 Hz, 2H), 3.87–3.72 (m, 2H), 1.23 (d, J = 7.2 Hz, 3H), 1.16 (d, J = 6.3 Hz, 6H). ¹³ C NMR (101 MHz, DMSO‐D ₆) δ 172.55 (d, J _CP = 5.0 Hz), 162.57, 154.38, 150.59 (d, J _CP = 6.0 Hz), 138.48, 137.74, 129.58, 124.61, 120.07 (d, J _CP = 4.0 Hz), 117.14, 111.50, 97.69 (d, J _CP = 9.1 Hz), 89.61, 74.12, 73.76, 68.05, 66.87 (d, J _CP = 4.0 Hz), 49.73, 21.37, 19.71, 19.65. ³¹ P NMR (162 MHz, DMSO‐D ₆) δ 2.81. HRMS‐ESI (m/z): calcd for C₂₄H₃₀N₉O₈P [M+H]⁺ 604.20277, found 604.20272. Elemental analysis: Anal. calcd for C₂₄H₃₀N₉O₈P + 0.55 H₂O: C, 46.99; H, 5.11; N, 20.55. Found: C, 47.09; H, 5.31; N, 20.72.

4.13.3. KAD‐061: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐2‐azido‐3,4‐dihydroxy‐5‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.099 g, 69.4%) from 6c (0.152 g, 0.23 mmol) following general procedure D. TLC: Rf = 0.46 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.83 (s, 2H), 7.74 (d, J = 1.3 Hz, 1H), 7.38–7.29 (m, 3H), 7.25–7.14 (m, 3H), 6.22 (s, 1H), 5.75 (d, J = 7.0 Hz, 1H), 5.04 (hept, J = 6.2 Hz, 1H), 4.72–4.61 (m, 2H), 4.36 (dd, J = 12.5, 9.7 Hz, 1H), 4.23 (dd, J = 11.6, 10.3 Hz, 1H), 4.13 (d, J = 4.4 Hz, 1H), 4.05–3.91 (m, 4H), 1.39–1.33 (m, 3H), 1.25 (dd, J = 10.9, 6.3 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.11 (d, J _CP = 7.0 Hz), 164.81, 156.02, 150.35 (d, J _CP = 7.0 Hz), 137.59, 130.06, 125.63, 121.92, 120.05 (d, J _CP = 5.0 Hz), 112.09, 99.49 (d, J _CP = 6.0 Hz), 91.73, 75.65, 73.56, 69.89, 64.58 (d, J _CP = 6.0 Hz), 55.16, 50.42, 21.76, 21.73, 20.87, 20.81. ³¹ P NMR (162 MHz, CDCl₃) δ 4.27. HRMS‐ESI (m/z): calcd for C₂₅H₃₁N₈O₉P [M+H]⁺ 619.20244, found 619.20330. Elemental analysis: Anal. calcd for C₂₅H₃₁N₈O₉P + 0.50 H₂O: C, 47.85; H, 5.14; N, 17.86. Found: C, 47.54; H, 5.30; N, 18.22.

4.13.4. KAD‐038: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐5‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐3,4‐dihydroxytetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.097 g, 66.2%) from 6d (0.156 g, 0.23 mmol) following general procedure D. TLC: Rf = 0.74 (25% MeOH/DCM). ¹ H NMR (400 MHz, CD₃OD) δ 8.57 (s, 1H), 7.91 (d, J = 3.9 Hz, 1H), 7.57 (d, J = 4.4 Hz, 1H), 7.36–7.21 (m, 4H), 7.20–7.10 (m, 1H), 5.89 (d, J = 7.2 Hz, 1H), 5.04–4.93 (m, 1H), 4.81–4.72 (m, 1H), 4.55 – 4.43 (m, 2H), 4.01 (s, 3H), 3.97–3.89 (m, 2H), 1.35 (t, J = 5.8 Hz, 3H), 1.22 (dd, J = 6.2, 3.6 Hz, 6H). ¹³ C NMR (101 MHz, CD₃OD) δ 174.42 (d, J _CP = 6.0 Hz), 167.76, 163.23, 155.22, 152.10 (d, J _CP = 6.0 Hz), 138.19, 136.44, 130.71, 126.15, 121.36 (d, J _CP = 5.0 Hz), 115.85, 105.94, 99.67 (d, J _CP = 9.1 Hz), 92.09, 76.31, 75.80, 70.20, 68.13 (d, J _CP = 6.0 Hz), 54.12, 51.58, 21.93, 21.89, 20.54, 20.47. ³¹ P NMR (162 MHz, CD₃OD) δ 4.16. HRMS‐ESI (m/z): calcd for C₂₅H₃₂N₉O₉P [M+H]⁺ 634.21334, found 634.21644. Elemental analysis: Anal. calcd for C₂₅H₃₂N₉O₉P + 1.25 H₂O: C, 45.77; H, 5.30; N, 19.21. Found: C, 45.70; H, 5.17; N, 19.28.

4.13.5. KAD‐049: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐5‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐Azido‐3,4‐dihydroxytetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.076 g, 57.4%) from 6e (0.141 g, 0.21 mmol) following general procedure D. TLC: Rf = 0.57 (10% MeOH/DCM). ¹ H NMR (400 MHz, CD₃OD) δ 8.22 (s, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 7.36–7.27 (m, 2H), 7.27–7.22 (m, 2H), 7.19–7.12 (m, 1H), 5.90 (d, J = 7.4 Hz, 1H), 4.98 (dt, J = 12.5, 6.3 Hz, 1H), 4.85 (dd, J = 7.4, 4.3 Hz, 1H), 4.52–4.42 (m, 2H), 3.98–3.92 (m, 2H), 3.91 (s, 3H), 1.35 (d, J = 0.9 Hz, 3H), 1.23 (dd, J = 6.3, 4.4 Hz, 6H). ¹³ C NMR (101 MHz, CD₃OD) δ 174.43 (d, J _CP = 5.0 Hz), 165.40, 163.90, 154.33, 152.13 (d, J _CP = 9.1 Hz), 139.27, 138.23, 131.46, 126.17, 121.35 (d, J _CP = 5.0 Hz), 113.80, 105.98, 99.61 (d, J _CP = 10.1 Hz), 92.01, 76.08, 75.82, 70.21, 68.19 (d, J _CP = 4.0 Hz), 54.83, 51.59, 21.94, 21.91, 20.51, 20.45. ³¹ P NMR (162 MHz, CD₃OD) δ 4.06. HRMS‐ESI (m/z): calcd for C₂₅H₃₂N₉O₉P [M+H]⁺ 634.21334, found 634.21299. Elemental analysis: Anal. calcd for C₂₅H₃₂N₉O₉P + 1.30 H₂O: C, 45.71; H, 5.31; N, 19.19. Found: C, 45.54; H, 5.29; N, 19.33.

4.13.6. KAD‐056: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐2‐azido‐3,4‐dihydroxy‐5‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.107 g, 70.2%) from 6f (0.161 g, 0.25 mmol) following general procedure D. TLC: Rf = 0.44 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 8.64 (s, 1H), 7.72 (d, J = 1.3 Hz, 1H), 7.63 (d, J = 1.3 Hz, 1H), 7.37–7.29 (m, 2H), 7.24–7.12 (m, 3H), 6.13 (s, 1H), 5.79 (d, J = 6.9 Hz, 1H), 5.05 (h, J = 6.3 Hz, 1H), 4.96 (s, 1H), 4.72–4.60 (m, 2H), 4.40 (dd, J = 12.3, 9.7 Hz, 1H), 4.25 (t, J = 10.8 Hz, 1H), 4.13 (d, J = 4.3 Hz, 1H), 4.06 (s, 3H), 4.03–3.92 (m, 1H), 1.37 (d, J = 7.0 Hz, 3H), 1.25 (dd, J = 10.1, 6.3 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.12 (d, J _CP = 7.0 Hz), 164.90, 155.57, 153.24, 150.43 (d, J _CP = 7.0 Hz), 136.94, 134.37, 130.02, 125.54, 120.11 (d, J _CP = 5.0 Hz), 115.12, 99.40 (d, J _CP = 7.0 Hz), 91.69, 75.63, 73.73 (d, J _CP = 6.0 Hz), 69.79, 64.69, 54.14, 50.45, 21.76, 21.74, 20.93, 20.87. ³¹ P NMR (162 MHz, CDCl₃) δ 4.26. HRMS‐ESI (m/z): calcd for C₂₅H₃₁N₈O₉P [M+H]⁺ 619.20244, found 619.20528. Elemental analysis: Anal. calcd for C₂₅H₃₁N₈O₉P + 1.20 H₂O: C, 46.91; H, 5.26; N, 17.50. Found: C, 46.53; H, 5.18; N, 17.88.

4.13.7. KAD‐065: isopropyl ((S)‐(((2R, 3S,4R, 5R)‐5‐(4‐(4‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐3,4‐dihydroxytetrahydrofuran‐2‐yl)methoxy)(phenoxy)phosphoryl)‐L‐alaninate

The title compound was obtained as a white solid (0.084 g, 63.4%) from 6g (0.141 g, 0.22 mmol) following general procedure D. TLC: Rf = 0.31 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.37–8.29 (m, 2H), 7.72 (d, J = 1.3 Hz, 1H), 7.44 (d, J = 1.3 Hz, 1H), 7.37–7.29 (m, 2H), 7.25–7.13 (m, 3H), 5.79 (d, J = 7.4 Hz, 1H), 5.09–4.98 (m, 1H), 4.78–4.60 (m, 2H), 4.51–4.35 (m, 1H), 4.31–4.20 (m, 1H), 4.15 (s, 1H), 4.03–3.93 (m, 1H), 1.37 (d, J = 7.1 Hz, 3H), 1.25 (dd, J = 9.4, 6.6 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 173.08 (d, J _CP = 7.0 Hz), 160.00, 156.27, 150.39 (d, J _CP = 7.0 Hz), 138.33, 135.89, 130.05, 125.61, 120.09 (d, J _CP = 5.0 Hz), 112.89, 110.13, 99.53 (d, J _CP = 6.0 Hz), 91.67, 75.74, 73.71, 70.24, 64.66, 50.46, 21.77, 21.74, 20.91, 20.87. ³¹ P NMR (162 MHz, CDCl₃) δ 4.49. HRMS‐ESI (m/z): calcd for C₂₄H₃₀N₉O₈P [M+H]⁺ 604.20277, found 604.20305. Elemental analysis: Anal. calcd for C₂₄H₃₀N₉O₈P + 0.90 H₂O: C, 46.51; H, 5.17; N, 20.34. Found: C, 46.77; H, 5.22; N, 20.11.

4.14. General Procedure E: Synthesis of 2′‐3′‐Diisobutyrate‐5′‐ProTide Mixed Prodrugs

To a stirred solution of the ProTide starting material (50–100 mg) in anhydrous pyridine (3 mL) were added isobutyric anhydride (3 eq.) and a catalytic amount of DMAP. The reaction was stirred at room temperature for 2 h. Upon completion, the solvent was removed under reduced pressure. The resulting was purified by flash chromatography on silica gel (0%–8% MeOH/DCM) and lyophilized to afford the title compound.

4.14.1. KAD‐046: (2R, 3S,4R, 5R)‐2‐azido‐5‐(4‐(2,4‐dimethoxypyrimidin‐5‐yl)‐1H‐Imidazol‐1‐yl)‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a powdery white solid (0.076 g, 91.9 %) from KAD‐045 (0.068 g, 0.10 mmol) following general procedure E. TLC: Rf = 0.62 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.01 (s, 1H), 7.70 (d, J = 1.4 Hz, 1H), 7.58 (d, J = 1.4 Hz, 1H), 7.32–7.26 (m, 2H), 7.22–7.17 (m, 2H), 7.17–7.09 (m, 1H), 5.94 (d, J = 7.3 Hz, 1H), 5.91–5.86 (m, 1H), 5.40 (d, J = 4.2 Hz, 1H), 5.02 (hept, J = 6.3 Hz, 1H), 4.39 (d, J = 6.2 Hz, 2H), 4.11 (s, 3H), 4.03 (s, 3H), 4.01–3.90 (m, 1H), 3.77–3.67 (m, 1H), 2.70 (hept, J = 7.0 Hz, 1H), 2.47 (hept, J = 7.0 Hz, 1H), 1.36 (d, J = 7.0 Hz, 3H), 1.27–1.18 (m, 12H), 1.10 (dd, J = 7.0, 4.4 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.96, 174.69, 172.80 (d, J _CP = 8.0 Hz), 167.13, 163.99, 155.88, 150.56 (d, J _CP = 7.0 Hz), 136.59, 136.05, 130.48, 125.31, 120.14 (d, J _CP = 5.0 Hz), 115.13, 109.12, 97.83 (d, J _CP = 9.1 Hz), 88.32, 73.88, 73.02, 69.63, 65.64 (d, J _CP = 5.0 Hz), 55.02, 54.25, 50.39, 33.95, 33.71, 21.75, 21.13, 21.09, 19.15, 18.85, 18.82, 18.70. ³¹ P NMR (162 MHz, CDCl₃) δ 2.72. HRMS‐ESI (m/z): calcd for C₃₄H₄₅N₈O₁₂P [M+H]⁺ 789.29673, found 789.29777. Elemental analysis: Anal. calcd for C₃₄H₄₅N₈O₁₂P + 0.20 H₂O: C, 51.54; H, 5.78; N, 14.14. Found: C, 51.55; H, 5.79; N, 14.14.

4.14.2. KAD‐048: (2R, 3S,4R, 5R)‐5‐(4‐(2‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.076 g, 86.4%) from KAD‐047 (0.071 g, 0.12 mmol) following general procedure E. TLC: Rf = 0.54 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.68 (s, 2H), 7.69 (d, J = 1.4 Hz, 1H), 7.34–7.24 (m, 3H), 7.22–7.17 (m, 2H), 7.16–7.10 (m, 1H), 5.94–5.85 (m, 2H), 5.40 (d, J = 3.7 Hz, 1H), 5.30 (s, 2H), 5.02 (hept, J = 6.2 Hz, 1H), 4.45–4.34 (m, 2H), 4.03–3.89 (m, 2H), 2.69 (hept, J = 6.9 Hz, 1H), 2.47 (hept, J = 7.0 Hz, 1H), 1.35 (d, J = 6.3 Hz, 3H), 1.29–1.16 (m, 12H), 1.09 (dd, J = 6.9, 4.8 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.98, 174.69, 172.88 (d, J _CP = 7.0 Hz), 162.26, 155.39, 150.56 (d, J _CP = 7.0 Hz), 139.41, 137.48, 129.85, 125.28, 120.12 (d, J _CP = 5.0 Hz), 118.39, 110.59, 97.88 (d, J _CP = 9.1 Hz), 88.24, 73.66, 72.97, 69.59, 65.52 (d, J _CP = 5.0 Hz), 50.39, 33.93, 33.70, 21.75, 21.74, 21.11, 21.06, 19.14, 18.82, 18.80, 18.69. ³¹ P NMR (162 MHz, CDCl₃) δ 2.81. HRMS‐ESI (m/z): calcd for C₃₂H₄₂N₉O₁₀P [M+H]⁺ 744.28650, found 744.28832. Elemental analysis: Anal. calcd for C₃₂H₄₂N₉O₁₀P + 0.30 H₂O: C, 51.31; H, 5.73; N, 16.83. Found: C, 51.44; H, 5.87; N, 16.77.

4.14.3. KAD‐062: (2R, 3S,4R, 5R)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)‐5‐(4‐(2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.074 g, 87.3%) from KAD‐061 (0.069 g, 0.11 mmol) following general procedure E. TLC: Rf = 0.64 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.89 (s, 2H), 7.73 (d, J = 1.3 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 7.32–7.27 (m, 2H), 7.23–7.17 (m, 2H), 7.17–7.10 (m, 1H), 5.96–5.86 (m, 2H), 5.41 (d, J = 4.1 Hz, 1H), 5.02 (hept, J = 6.3 Hz, 1H), 4.46–4.34 (m, 2H), 4.04 (s, 3H), 4.02–3.88 (m, 1H), 3.70 (dd, J = 11.5, 9.3 Hz, 1H), 2.70 (hept, J = 7.0 Hz, 1H), 2.48 (hept, J = 7.0 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 1.29–1.18 (m, 12H), 1.10 (dd, J = 7.0, 4.7 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.99, 174.69, 172.79 (d, J _CP = 8.0 Hz), 165.05, 156.16, 150.56 (d, J _CP = 7.0 Hz), 138.59, 137.68, 129.87, 125.32, 121.70, 120.13 (d, J _CP = 5.0 Hz), 111.69, 97.98 (d, J _CP = 9.1 Hz), 88.32, 73.75, 72.98, 69.64, 65.51 (d, J _CP = 5.0 Hz), 55.16, 50.41, 33.95, 33.71, 21.77, 21.75, 21.13, 21.08, 19.15, 18.84, 18.81, 18.69. ³¹ P NMR (162 MHz, CDCl₃) δ 2.67. HRMS‐ESI (m/z): calcd for C₃₃H₄₃N₈O₁₁P [M+H]⁺ 759.28617, found 759.28586. Elemental analysis: Anal. calcd for C₃₃H₄₃N₈O₁₁P + 0.90 H₂O: C, 51.15; H, 5.83; N, 14.46. Found: C, 50.98; H, 6.01; N, 14.66.

4.14.4. KAD‐039: (2R, 3S,4R, 5R)‐5‐(4‐(2‐amino‐4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a powdery white solid (0.069 g, 84.8%) from KAD‐038 (0.067 g, 0.11 mmol) following general procedure E. TLC: Rf = 0.53 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H), 7.65 (d, J = 1.3 Hz, 1H), 7.48 (d, J = 1.4 Hz, 1H), 7.31–7.27 (m, 2H), 7.22–7.16 (m, 2H), 7.15–7.08 (m, 1H), 5.93 (d, J = 7.4 Hz, 1H), 5.90–5.85 (m, 1H), 5.39 (d, J = 4.2 Hz, 1H), 5.11 (s, 2H), 5.02 (hept, J = 6.3 Hz, 1H), 4.38 (d, J = 6.2 Hz, 2H), 4.00 (s, 3H), 3.97–3.85 (m, 2H), 2.68 (hept, J = 7.1 Hz, 1H), 2.46 (hept, J = 7.0 Hz, 1H), 1.35 (d, J = 6.4 Hz, 3H), 1.29–1.14 (m, 12H), 1.09 (dd, J = 7.0, 4.6 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.93, 174.68, 172.83 (d, J _CP = 8.0 Hz), 166.38, 161.49, 155.59, 150.57 (d, J _CP = 7.0 Hz), 136.88, 136.27, 129.84, 125.25, 120.13 (d, J _CP = 5.0 Hz), 114.06, 105.82, 97.72 (d, J _CP = 9.1 Hz), 88.24, 73.81, 73.01, 69.56, 65.63 (d, J _CP = 5.0 Hz), 53.68, 50.39, 33.92, 33.68, 21.73, 21.10, 21.05, 19.12, 18.83, 18.80, 18.73. ³¹ P NMR (162 MHz, CDCl₃) δ 3.15. HRMS‐ESI (m/z): calcd for C₃₃H₄₄N₉O₁₁P [M+H]⁺ 774.29707, found 774.30040. Elemental analysis: Anal. calcd for C₃₃H₄₄N₉O₁₁P + 0.30 H₂O: C, 50.87; H, 5.77; N, 16.18. Found: C, 51.05; H, 5.81; N, 16.00.

4.14.5. KAD‐050: (2R, 3S,4R, 5R)‐5‐(4‐(4‐amino‐2‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a powdery white solid (0.082 g, 90.2%) from KAD‐049 (0.074 g, 0.12 mmol) following general procedure E. TLC: Rf = 0.72 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.71 (d, J = 1.4 Hz, 1H), 7.33 (d, J = 1.4 Hz, 1H), 7.32–7.26 (m, 2H), 7.22–7.18 (m, 2H), 7.17–7.11 (m, 1H), 5.95–5.85 (m, 2H), 5.41 (d, J = 4.2 Hz, 1H), 5.03 (hept, J = 6.3 Hz, 1H), 4.44–4.35 (m, 2H), 4.01–3.87 (m, 4H), 3.74 (dd, J = 11.5, 9.3 Hz, 1H), 2.70 (hept, J = 7.0 Hz, 1H), 2.48 (hept, J = 7.0 Hz, 1H), 1.36 (d, J = 7.1 Hz, 3H), 1.28–1.17 (m, 12H), 1.10 (dd, J = 7.0, 4.1 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.95, 174.68, 172.81 (d, J _CP = 7.0 Hz), 164.57, 162.21, 153.90, 150.55 (d, J _CP = 6.0 Hz), 139.80, 135.59, 129.87, 125.32, 120.13 (d, J _CP = 5.0 Hz), 110.99, 104.54, 98.01 (d, J _CP = 9.1 Hz), 88.40, 73.83, 73.01, 69.64, 65.54 (d, J _CP = 5.0 Hz), 54.50, 50.39, 33.94, 33.71, 21.77, 21.75, 21.10, 21.06, 19.14, 18.84, 18.81, 18.70. ³¹ P NMR (162 MHz, CDCl₃) δ 2.70. HRMS‐ESI (m/z): calcd for C₃₃H₄₄N₉O₁₁P [M+H]⁺ 774.29707, found 774.29876. Elemental analysis: Anal. calcd for C₃₃H₄₄N₉O₁₁P + 0.50 H₂O: C, 50.64; H, 5.79; N, 16.10. Found: C, 50.98; H, 5.88; N, 15.91.

4.14.6. KAD‐057: (2R, 3S,4R, 5R)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)‐5‐(4‐(4‐methoxypyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.086 g, 91.1%) from KAD‐056 (0.077 g, 0.12 mmol) following general procedure E. TLC: Rf = 0.56 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 9.26 (s, 1H), 8.70 (s, 1H), 7.76–7.70 (m, 2H), 7.29 (dd, J = 8.6, 7.2 Hz, 2H), 7.23–7.16 (m, 2H), 7.16–7.10 (m, 1H), 5.95 (d, J = 7.3 Hz, 1H), 5.89 (dd, J = 7.2, 4.3 Hz, 1H), 5.41 (d, J = 4.3 Hz, 1H), 5.03 (hept, J = 6.3 Hz, 1H), 4.40 (d, J = 6.3 Hz, 2H), 4.13 (s, 3H), 4.03–3.88 (m, 1H), 3.71 (dd, J = 11.5, 9.3 Hz, 1H), 2.70 (hept, J = 7.0 Hz, 1H), 2.48 (hept, J = 7.0 Hz, 1H), 1.36 (d, J = 7.1 Hz, 3H), 1.29–1.17 (m, 12H), 1.10 (dd, J = 7.0, 4.6 Hz, 6H). ¹³ C NMR (101 MHz, CDCl₃) δ 174.96, 174.69, 172.80 (d, J _CP = 8.0 Hz), 164.97, 156.01, 153.89, 150.57 (d, J _CP = 7.0 Hz), 136.80, 135.55, 129.87, 125.31, 120.14 (d, J _CP = 5.0 Hz), 117.12, 97.93 (d, J _CP = 9.1 Hz), 88.36, 73.96, 73.03, 69.63, 65.60 (d, J _CP = 6.0 Hz), 54.16, 50.42, 33.95, 33.71, 21.75, 21.14, 21.10, 19.15, 18.86, 18.83, 18.69. ³¹ P NMR (162 MHz, CDCl₃) δ 2.73. HRMS‐ESI (m/z): calcd for C₃₃H₄₃N₈O₁₁P [M+H]⁺ 759.28617, found 759.28910. Elemental analysis: Anal. calcd for C₃₃H₄₃N₈O₁₁P + 0.55 H₂O: C, 51.57; H, 5.78; N, 14.58. Found: C, 51.45; H, 5.66; N, 14.54.

4.14.7. KAD‐066: (2R, 3S,4R, 5R)‐5‐(4‐(4‐aminopyrimidin‐5‐yl)‐1H‐imidazol‐1‐yl)‐2‐azido‐2‐((((S)‐(((S)‐1‐isopropoxy‐1‐oxopropan‐2‐yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran‐3,4‐diyl bis(2‐methylpropanoate)

The title compound was obtained as a white solid (0.086 g, 86.3%) from KAD‐065 (0.081 g, 0.13 mmol) following general procedure E. TLC: Rf = 0.55 (10% MeOH/DCM). ¹ H NMR (400 MHz, CDCl₃) δ 8.46 (s, 2H), 7.75 (d, J = 1.3 Hz, 1H), 7.46 (d, J = 1.4 Hz, 1H), 7.34–7.27 (m, 2H), 7.24–7.17 (m, 2H), 7.17–7.11 (m, 1H), 5.97–5.86 (m, 2H), 5.42 (d, J = 4.2 Hz, 1H), 5.03 (hept, J = 6.2 Hz, 1H), 4.47–4.35 (m, 2H), 4.02–3.88 (m, 1H), 3.76 (dd, J = 11.5, 9.4 Hz, 1H), 2.71 (hept, J = 7.0 Hz, 1H), 2.49 (hept, J = 7.0 Hz, 1H), 1.36 (d, J = 7.0 Hz, 3H), 1.32–1.16 (m, 12H), 1.11 (dd, J = 7.0, 4.3 Hz, 6H). ³ C NMR (101 MHz, CDCl₃) δ 174.97, 174.69, 172.82 (d, J _CP = 8.0 Hz), 160.02, 157.08, 151.71, 150.56 (d, J _CP = 7.0 Hz), 139.40, 135.79, 129.89, 125.35, 120.14 (d, J _CP = 5.0 Hz), 112.48, 109.78, 98.11 (d, J _CP = 9.1 Hz), 88.47, 73.89, 73.03, 69.67, 65.53 (d, J _CP = 5.0 Hz), 50.42, 33.95, 33.72, 21.78, 21.76, 21.13, 21.09, 19.16, 18.85, 18.82, 18.71. ³¹ P NMR (162 MHz, CDCl₃) δ 2.72. HRMS‐ESI (m/z): calcd for C₃₂H₄₂N₉O₁₀P [M+H]⁺ 744.28650, found 744.28628. Elemental analysis: Anal. calcd for C₃₂H₄₂N₉O₁₀P + 0.50 H₂O: C, 51.06; H, 5.76; N, 16.75. Found: C, 51.12; H, 5.83; N, 16.79.

5. Antiviral Methods

5.1. Orthoflavivirus‐Based Antiviral Assays and Cytotoxicity Assay

Huh7 cells were infected with DENV2 or DENV4 reporter viruses containing a nanoLuc expression cassette. Infections were performed at a multiplicity of infection (MOI) of 0.05 focus‐forming units per cell. Dose response curves were performed in the presence of 7 × 3‐fold dilutions of compound starting at 100 μM, in triplicate. At 48 h postinfection, luciferase expression was quantified using the NanoGlo Luciferase Assay System (Promega #N1130) according to manufacturer's instructions. EC₅₀ values were determined by fitting normalized data points to a nonlinear regression model using GraphPad Prism software.

5.2. Viruses and Cell Lines

For our antiviral studies, following orthoflaviviruses were used: TBEV, (strain Hypr), LGTV, (strain TP21), POWV, (strain LB), KFDV, (strain W‐377), LIV, (strain LI/31), JEV (strain P3), and WNV, (strain Eg‐101). These viruses was provided by the Collection of Arboviruses, Institute of Parasitology, Biology Center of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic (http://www.arboviruscollection.cz/index.php?lang=en). Furthermore, ZIKV, (the Brasilian strain Paraiba_01) was kindly provided by Prof. Paolo M. de A. Zanotto, University of São Paulo, Brasil. Experiments using authentic TBEV, LGTV, LIV, and ZIKV were performed in our BSL2 facility and experiments with POWV, KFDV, WNV, and JEV were performed in our BLS3 facility.

A549 cells (ATCC CRM‐CCL‐185, human lung epithelial cells originally isolated from the alveolar basal epithelial tissue of a male patient with nonsmall cell lung cancer) and human hepatocarcinoma cells (Huh‐7) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% newborn calf serum plus 100 U/mL penicillin, 100 µg/mL streptomycin, and 1% glutamine (Sigma–Aldrich, Prague, Czech Republic), and cultured at 37 °C under 5% CO₂.

5.3. Cytotoxicity Assay

To assess the potential toxicity of the compounds under investigation, A549 or Huh‐7 cells were seeded into 96‐well plates at a density of 2 × 10⁴ cells per well. Following a 24 h incubation period to allow for the formation of a confluent cell monolayer, the existing medium was removed and replaced with fresh medium containing the tested compounds in the concentration range from 0 to 100 μM. The cells were then cultured at 37 °C for 48 h. The degree of cytotoxicity was determined by evaluating cell viability using a Cell Counting Kit‐8 (Dojindo Molecular Technologies, Munich, Germany) in accordance with the manufacturer's guidelines and used to calculate the CC₅₀ values (concentrations achieving 50% cytotoxic efficacy).

5.4. Viral Titer Reduction Assay

A549 or Huh‐7 cells were seeded in 96‐well plates at a density of 2 × 10⁴ cells per well and allowed to incubate for 24 h. Then, the cell monolayers were infected with the respective virus at a MOI of 0.1. Following infection, the cells were treated with varying concentrations up to 50 μM (in A549 cells) or 25 μM (in Huh‐7 cells) of the compounds and further incubated at 37 °C for 48 h postinfection (p.i.). Viral titers were estimated using plaque assays. The resulting virus titer values were utilized to generate comprehensive dose–response curves, and, upon conversion to percent inhibition, concentrations achieving 50% antiviral efficacy (EC₅₀) were determined.

5.5. Plaque Assay

Plaque assays were conducted utilizing A549 cells following a modified procedure outlined elsewhere [64]. In brief, virus dilutions were prepared at 10‐fold increments in 24‐well tissue culture plates, with cells added at a density ranging from 0.6 to 1.5 × 10⁵ cells per well. Following a 4 h incubation period at 37 °C, the suspension was overlaid with a 1.5% (w/v) solution of carboxymethyl cellulose in DMEM medium. After incubating for 5 days at 37 °C, the plates were rinsed with phosphate‐buffered saline, and the cell monolayers were stained using naphthalene black. Plaques were counted, and the viral titer was quantified as plaque‐forming units (PFU) per milliliter (i.e., PFU/mL).

5.6. Antiviral Assay for EBOV

A biologically contained EBOV (EBOVΔVP30) was used with Huh 7 cells that stably express EBOV VP30 to assay antiviral activity of compounds [65]. Cells were propagated in growth medium (DMEM base, antibiotics, L‐glutamine, and fetal bovine serum; 10% final concentration) and seeded in 96‐well plates (white, clear bottom) at a density of 3 × 10⁴ cells per well. The following day, test compounds were added after removing the existing medium. The test compounds were diluted 2‐fold in a 10‐point dilution series starting at a concentration of 200 µM. After a pretreatment of 1 h, the same volume of EBOVΔVP30 (MOI of 0.01) encoding for Renilla luciferase was added to the cells, resulting in a final starsing concentration of 100 µM for each test compound. 3 days after infection, luciferase expression was measured on a Tecan M1000 plate reader after the luciferase reagent, ViviRen live cell substrate (Promega), was added. Cell viability was measured using Real Time‐Glo MT Cell Viability reagent (Promega) with a separate set of compound0‐treated cells without EBOVΔVP30 infection. Viral‐driven luciferase and cell viability values were normalized to DMSO‐treated and backgrounds, and EC₅₀ or CC₅₀ values, respectively, were calculated in GraphPad Prism by fitting normalized data to a nonlinear regression model.

5.6.1. Mouse and Human Plasma Stability Assay

The plasma stability assay was performed in triplicate by incubating each selected compound (1 µmol/L final concentration) in normal mouse (CD‐1) or human plasma (Innovative Research, Novi, MI, USA) diluted to 80% with 0.1 mol/L potassium phosphate buffer (pH 7.4) at 37 °C. At 0, 1, 3, 6, and 24 h, a 50 µL aliquot of the plasma mixture was taken and quenched with 200 µL of acetonitrile containing 0.1% formic acid. In case of CD‐1 mouse plasma samples were quenched at 0, 5 and 15 min. The samples were then vortexed and centrifuged at 14,000 rpm (Thermo Scientific Sorvall ST 8R, NI, Germany) for 10 min. The supernatants were collected and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to determine the in vitro plasma half‐life (t _1/2).

5.6.2. Dog and Monkey Plasma Stability Assay

The plasma stability assay was performed in triplicate by incubating each selected compound (1 µmol/L final concentration) in Cynomolgus monkey (Cat no. NHP01PLK2‐0000781) or Beagle dog (Cat no. CAN00PLK2‐9979‐D) plasma (BioIVT, Hicksville, New York, USA) diluted to 80% with 0.1 mol/L potassium phosphate buffer (pH 7.4) at 37 °C. At 0, 1, 3, 6, and 24 h, a 50 µL aliquot of the plasma mixture was taken and quenched with 200 µL of acetonitrile containing 0.1% formic acid. The samples were then vortexed and centrifuged at 15,000 rpm (Thermo Scientific Sorvall ST 8R, NI, Germany) for 8 min. The supernatants were collected and analyzed by LC‐MS/MS to determine the in vitro plasma half‐life (t _1/2).

5.6.3. Microsomal Stability Assay

The in vitro microsomal stability assay was conducted in triplicate in commercially available mouse and human liver microsomes (Sekisui XenoTech, Kansas City, KS, USA), which were supplemented with nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. Briefly, a compound (1 µmol/L final concentration) was spiked into the reaction mixture containing liver microsomal protein (0.5 mg/mL final concentration) and MgCl₂ (1 mmol/L final concentration) in 0.1 mol/L potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 1 mmol/L NADPH, followed by incubation at 37 °C. A negative control was performed in parallel without NADPH to reveal any chemical instability or non‐NADPH dependent enzymatic degradation for each compound. A reaction with positive control verapamil was also performed as in‐house quality control to confirm the proper functionality of the incubation systems. At various time points, a 50 µL of reaction aliquot was taken and quenched with 200 µL of acetonitrile containing 0.1% formic acid. The samples were then vortexed and centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatants were collected and analyzed by LC‐MS/MS to determine the in vitro metabolic half‐life (t _1/2).

5.6.4. LC/MS/MS Analysis

The LC‐MS/MS system consists of an AB Sciex QTrap 5500 mass spectrometer and an Agilent 1260 Infinity HPLC. The chromatographic separation of analytes was achieved using a Phenomenex Kintex C18 column (50 × 2.1 mm, 2.6 µm). The two eluents were: (A) 0.1% formic acid in water and (B) 0.1 % formic acid in acetonitrile. The mobile phase was delivered at a flow rate of 0.5 mL/min using the following gradients of A and B: 0–1 min, 10%–90% B (v/v); 1–2.8 min, 90% B (v/v); 2.8–3 min, 90%–10% B (v/v); 3–6 min, 10% B (v/v). MS/MS detection of the analytes was conducted using ESI ion source in positive mode. The MRM transitions used for KAD‐039 were m/z 774.4 to m/z 192.1.

5.6.5. Computational Modeling

Using reported structures for the DENV (PDB: 8BCR) [66], SARS‐CoV‐2 (PDB: 7QIF) [67], WNV (PDB: 2OY0) [68], and human N7 (PDB: 5E9W) [69] MTase domains, and a homology model based on the MTase domain of the EBOV L‐protein (residues 1803–2001, UniProtKB‐Q8JPX5 (L_EBORR). RNA‐directed RNA polymerase L (Reston ebolavirus) [70]. Briefly, the EBOV L‐protein MTase domain sequence was used to generate a predicted structural model using Boltz [71, 72, 73] implemented in UCSF ChimeraX 1.10 [74, 75], including GTP and Mg²⁺ bound at the cap/GTP active site.

GTP coordinates for the human N7 MTase domain (PDB: 5E9W) were obtained as previously described [51]. Structural superimposition of 5E9W with the GTP‐bound Ecm1 cap‐MTase crystal structure (PDB: 1RI1) [76] was performed in UCSF ChimeraX 1.10 with the MatchMaker tool [77]. This analysis revealed a highly conserved GTP‐binding pocket architecture and residue composition between the two proteins. Using the superimposed structures, GTP coordinates were transferred to the human N7 MTase domain before structure preparation, as detailed below.

All structures were prepared for docking using the Protein Preparation workflow within the Schrödinger suite (Schrödinger Release 2025‐2: Protein Preparation Workflow; Epik, Schrödinger, LLC, New York, NY, 2024; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2025) [78].

Docking of KAD‐039TP at the cap/GTP active site was performed using the Glide module in Schrödinger (Schrödinger release 2025‐2: Glide, Schrödinger, LLC, New York, NY, 2025) [79]. Docking was confined to a 20 × 20 × 20 Å box centered on the cap/GTP active site. Docking poses were scored with the OPLS4 force field [80] within Schrödinger and directly compared with the native cap/GTP substrate (or the GTP analog, AT‐9010, for the DENV structure). Docking poses were manually inspected and rendered using UCSF ChimeraX 1.10.

Funding

This work was supported by the National Institutes of Health (GM158458, U19 AI1271292, 5U01GM152511); Ministry of Education, Youth and Sports of the Czech Republic (LUAUS25011); UMBC Technology Catalyst Fund.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.