1-Hydroxy-1,8-napthyridinone (DHN) analogs potently inhibit monkeypox virus resolvase (Mpr)

Abstract

The virally encoded Holliday junction resolvase is required for poxvirus genome replication and viral maturation. Previously, a 1-hydroxy-1,8-napthyridinone (DHN) analog (3) was discovered as an inhibitor hit of mpox resolvase (Mpr). Herein we have conducted Mpr-based comprehensive structure-activity relationship (SAR) of compound 3 via the synthesis of 70 analogs of four distinct subtypes. The SAR identified a phenyl ring and a biphenyl moiety as the optimal substituent for C-3 and C-6/C-5, respectively, and that C-5 analogs are generally better than their C-6 regio-isomers. Against vaccinia virus (VACV), tested new analogs demonstrated antiviral activity in the low μM to nM range. In the end, the best compound 5–1 conferred drastically improved inhibitory profiles against Mpr (IC₅₀ = 36 nM, 10-fold improvement) and VACV (EC₅₀ = 3.2 nM, 400-fold improvement) over compound 3, with significantly lower binding free energy as predicted from free energy perturbation, and highly favorable ADME properties.

Graphical Abstract

Introduction

Monkeypox virus (MPXV) is an orthopoxvirus in the same genus as variola virus, the causative agent of smallpox. Although historically endemic to Central Africa (Clade I) and Western Africa (Clade II) with occasional small-scale zoonotic outbreaks^{1, 2}, MPXV has evolved to substantially gain human-to-human transmission², culminating in the global Mpox outbreaks in 2022 (Clade IIb)³ and 2024 (Clade Ib)⁴, resulting in two WHO declarations of mpox as Public Health Emergency of International Concern (PHEIC). As of September 2025, the recent outbreaks have reported nearly 170,000 confirmed Mpox cases from 140 countries, a scale far exceeding previous outbreaks⁵. MPXV infections cause significant morbidity and mortality, with different clades/subclades having different biological characteristics, including transmission, morbidity and mortality^6–8. For example, Clade Ib has been associated with >1200 deaths in Africa over the last several years, with children being disproportionately affected.^{9, 10}. Additionally, Mpox could manifest as persistent scarring in affected individuals, which may lead to social stigmatization¹¹. Therefore, there are clear benefits in treating Mpox with efficacious antiviral drugs. Notably, two drugs have been developed under the animal rule and approved by FDA for stockpiling against smallpox (Figure 1): tecovirimat 1¹² which targets the viral extracellular envelope protein VP37 to inhibit virus release^{13, 14}; and brincidofovir 2¹⁵, a cidofovir prodrug targeting viral DNA polymerase¹⁶. Tecovirimat has been used as stopgap measures to manage MPXV in outbreak regions¹⁷, yet in human clinical trials it failed to reduce the time to lesion resolution against Clade IIb¹⁸ and Clade Ib¹⁹ MPXV. In addition, tecovirimat exhibited low barrier to viral resistance²⁰. For brincidofovir, clinical efficacy in humans remains unclear and cidofovir is associated with significant side effects^{21, 22}. Developing a chemically novel antiviral, preferably with a distinct mechanism of action, will provide a major countermeasure to Mpox and other emerging and re-emerging orthopoxviruses.

Figure 1.

FDA-approved drugs for stockpiling against smallpox

Poxvirus replicates its genome as DNA concatemers with cruciform junctions between monomers resembling the four-stranded Holliday junctions involved in homologous recombination²³. These junctions are cleaved by a virally encoded Holliday junction resolvase to generate monomeric genomic viral DNA^{24, 25}. Importantly, genetically repressing the vaccinia virus (VACV) resolvase, A22, resulted in strong antiviral phenotypes, including a reduction in viral titer; a decrease in late-viral DNA; and the arrest of viral morphogenesis at a premature stage²⁶. Mechanistically, these phenotypes are associated with the accumulation of viral concatemeric DNA. These observations strongly suggest that poxvirus resolvase can be a viable therapeutic target, and that pharmacologically inhibiting the resolvase activity by small molecules may produce similar antiviral phenotypes.

Poxvirus resolvase belongs to the RNase H-like (RNHL) superfamily of enzymes, which have similar acidic residues at the active site and require the coordination to a divalent metal, mostly magnesium, for catalysis²⁷. Since metalloenzymes are key to viral replication, metal-binding chemotypes have shown effectiveness as antiviral drugs^{28, 29}. Our research group has long been invested in the development of small molecule metal-binding chemotypes to inhibit RNHL viral enzymes^{30, 31}. Previously, Culyba et al. developed a bulged double-stranded DNA substrate used to measure the activity and inhibition of fowlpox virus (an avipoxvirus) resolvase (Fpr) in a biochemical fluorescence polarization (FP) assay³². The subsequent screening of a Merck metal-binding compound library identified 1-hydroxy-1,8-napthyridinone (DHN) analog 3 as a sub-μM inhibitor of Fpr³² (Figure 2). Although the FP assay was not applied to any orthopoxvirus resolvases due to their poor solubility, the seminal work supports the general approach of resolvase inhibition using metal chelating compounds. Recently, we developed a robust Förster resonance energy transfer (FRET) assay capable of reporting the activity and inhibition of both Mpox resolvase (Mpr) and the VACV resolvase A22³³. Utilizing the bulged double-stranded DNA substrate identified by Culyba et al., we appended a fluorophore and a quencher on the complementary DNA strands. This quenched substrate exhibits low fluorescence emission until Mg²⁺-dependent cleavage by resolvase generates an unquenched product with increased emission intensity. High-throughput screening of our in-house metal-chelating library using our optimized FRET assay allowed us to identify several metal-binding chemotypes as potential inhibitors of orthopoxvirus resolvases. One such chemotype is DHN as compound 3 inhibited both Mpr and A22 at sub-μM concentrations (Figure 2)³³. However, there were limited SAR insights into the DHN chemotype for Fpr activity. Herein, this current study details our medicinal chemistry approach which is centered around the synthesis of DHN subtypes 4–7 with modifications at the C3, C4, C5, C6 and N-1 positions of the DHN ring (Figure 2), to provide a comprehensive SAR understanding and thereby arriving at more potent and selective metal-binding inhibitors against Mpr and MPVX.

Figure 2.

DHN as a poxvirus inhibitor type and subtypes 4-7 for SAR exploration.

Results and Discussion

Chemical Synthesis

Although the generic synthesis of the DHN chemotype has been reported^{34, 35}, our synthetic chemistry efforts focused on generating a few different subtypes (4–7, Figure 2) to expand the structure-activity relationship (SAR). For the synthesis of subtypes 4a–b (Scheme 1), the benzyloxy-amino 9 was synthesized from the direct coupling of commercially available 5-bromo-2-chloronicotinate 8 with o-benzylhydroxylamine. Intermediate 8 was coupled with the appropriate acid chloride through an n-acylation reaction in the presence of a base to form the amido intermediate 10. The amido intermediate subsequently underwent a Dieckmann condensation, resulting in the formation of the DHN ring for intermediate 11. Suzuki coupling of intermediate 11 with the appropriate boronic acid with a palladium catalyst and under microwave irradiation resulted in intermediate 12. Subsequent deprotection of the benzyl groups under acidic conditions with moderate heating resulted in the formation of subtypes 4a–b. Interestingly, during the synthesis of subtype 4, an N-1 deoxygenated subtype 7 was also obtained (Scheme 1) which contributes significantly to the SAR. It was observed that extending the reaction time during the Suzuki coupling under conventional heating and at elevated temperatures led to the formation of subtypes 7 (7–1 – 7–5) from 11b. However, in certain cases, the over-reduction of the benzyl group with palladium on carbon resulted in the formation of subtypes 7 (7–7 – 7–8) from 12a (Scheme 1).

Scheme 1.

Synthesis of analogs for subtypes 4a˗b and 7

Reagents and conditions: a) O-benzylhydroxylamine, 110 °C, 12 h, 68 %; b) R²acetyl chloride, TEA, DCM, 12h, 57 %; c) LiHMDS/THF, −78 °C, 1 h, 79 % or d) KOtBu, o-xylene, 145 °C, 4 h, 25 %; e) R¹B(OH)₂, Pd(dppf)Cl₂, K₂CO₃, DMF/H₂O (4:1 v/v), 20 min. 110 °C MW; f) 48% HBr in H₂O/HOAc, 16 – 56 % over two steps or g) 10 % Pd/C, MeOH/EtOAc, 1:2 v/v, 32 – 87 %

For the synthesis of subtype 4c, ethyl malonyl chloride was coupled to 9 through a N-acylation reaction with pyridine as the base to yield 18. Dieckmann condensation of 18 with sodium ethoxide under reflux with ethanol solvent afforded the ethyl ester 19. Decarboxylation of the ethyl ester 19 yielded the enone 20. The benzyl group in 20 was deprotected under acidic conditions with moderate heating to afford subtype 4c (Scheme 2).

Scheme 2.

Synthesis of analogs for subtype 4c

Reagents and conditions: a) ethyl malonyl chloride, pyridine, DCM, rt, 12 h, 81 %; b) NaOEt, EtOH, 80 °C, 1 h, 81 %; c) 5 N aq, NaOH/EtOH (1:1 v/v), reflux, 94 %; d) R¹B(OH)₂, Pd(dppf)Cl₂, K₂CO₃, DMF/H₂O (4:1 v/v), 20 min. 110 °C MW; e) 48% HBr in H₂O/HOAc, 18 – 41 % over two steps.

The synthesis of C5 substituted DHN subtype 5 proceeded in a similar manner to the C6 substituted DHN subtypes 4 and 6, except that the C5 substituent was first installed by Suzuki coupling of the boronic acid/ester to the commercially available methyl 2-fluoro-4-iodonicotinate 13 to yield 14. Subsequently, direct coupling with the o-benzylhydroxylamine produced 15 which was subjected to the n-acylation reaction with the acid chloride to afford 16. Dieckmann condensation on 16 furnished the DHN core 17. Deprotection of the benzyl group yielded subtype 5 (Scheme 3).

Scheme 3.

Representative synthesis of analogs for subtype 5

Reagents and conditions: a) R¹B(OH)₂, Pd(PPh₃), K₂CO₃, EtOH/Tol/H₂O (8:1:1 v/v/v), 110 °C, 12 h, 45 %; b) O-benzylhydroxylamine, DMSO, 110 °C, 12 h, 53 – 55 %; c) (i) Phenylacetyl chloride, TEA, DCM, 12 h, 58 % (ii) ethyl malonyl chloride, pyridine, DCM, rt, 12 h, 75 %; d) (i) LiHMDS/THF, −78 °C, 1 h, 22 % (ii) NaOEt, EtOH, 80 °C, 1 h, 77 %; e) 48% HBr in H₂O/HOAc, 80 °C, 1 h, 51 – 58 %

The synthesis of subtype 6 commenced with the preparation of phenylacetamide 23, achieved via the n-acylation of O-benzylhydroxylamine 22 with phenyl acetyl chloride 21 under basic conditions. Building block 25 for the key amination and aldol condensation step was obtained by Suzuki coupling of boronic acid with commercially available nicotinaldehyde 24. Gratifyingly, Buchwald-Hartwig coupling of 23 and 25 under palladium catalyzed condition in tandem with intramolecular aldol condensation completed furnished the napthyridinone ring with relatively good yields. Deprotection under acidic conditions³⁶ completed the synthesis of subtype 6 (Scheme 4).

Scheme 4.

Synthesis of analogs of subtype 6

Reagents and conditions: Reagents and conditions: a) O-benzylhydroxylamine, phenyl acetyl chloride, Na₂CO₃, rt, toluene/H₂O (1:1 v/v), 12 h, 44 %; b) R¹B(OH)₂, Pd(PPh₃), K₂CO₃, EtOH/Tol/H₂O (8:1:1 v/v/v), 110 °C, 12 h, 45 %; c) Pd₂(dba)₃, Cs₂CO₃, Xphos, Tol., 110 °C; d) 48% HBr in H₂O/HOAc, 80 °C, 36 – 44 % over two steps

Inhibition of Mpr in a FRET assay

All final compounds were first screened at 50 μM against Mpr in a FRET assay recently developed by us³³. Compounds producing ≥50% inhibition without aggregation or florescence quenching were selected for dose response testing in the same assay. We recently reported that compound 3, previously reported by Culyba et al as an inhibitor of Fpr (IC₅₀ = 0.3 μM), potently inhibited Mpr in the FRET assay (IC₅₀ = 0.34 μM). For the current SAR, compound 3 served as the control and benchmark. Subtype 4 consists of three distinct series with C3 position substituted by a phenyl moiety (4a, R² = Ph, Table 1), an ethyl group (4b, R² = Et, Table 1), or unsubstituted (4c, R² = H, Table 1), respectively. Analogs within each series were designed and synthesized to explore the SAR at the C6 position. Overall, direct comparisons (4a-1–4a-11 vs 4b-1–4b-11 vs 4c-1–4c-11) show that phenyl ring at C-3 is preferred over Et and H. Remarkably, 4a analogs 4a-1–4a-11 all conferred potent inhibition against Mpr (IC₅₀ = 0.12–1.6 μM, Table 1), moderately better than 4b analogs 4b-1–4b-11 (IC₅₀ = 0.12–5.3 μM, Table 1). The Mpr inhibitory profile of the 4c subtype appeared intractable. A few 4c analogs such as 4c-1 (IC₅₀ = 0.12 μM), 4c-7 (IC₅₀ = 0.47 μM), 4c-4 (IC₅₀ = 0.24 μM) and 4c-6 (IC₅₀ = 1.0 μM) exhibited increased Mpr inhibition when directly compared to their corresponding 4a and 4b congeners. However, 4c-5 (IC₅₀ = 1.8 μM), 4c-10 (IC₅₀ = 2.5 μM), 4c-9 (IC₅₀ = 1.6 μM) and 4c-19 (IC₅₀ = 8.5 μM) showed reduced inhibition. Others, including 4c-2, 4c-3, 4c-13, 4c-8 and 4c-11 demonstrated direct quenching of fluorophore used in the FRET assay.

Table 1.

Mpr inhibition by analogs of DHN subtypes 4a-c

Compd	R1	R2	IC50a (μM)
3 (4a-1)			0.34 ± 0.06
4a-2			0.25 ± 0.01
4a-3			0.12 ± 0.01
4a-4			0.97 ± 0.03
4a-5			0.9 ± 0.2
4a-6			1.1 ± 0.1
4a-7			0.6 ± 0.1
4a-8			0.37 ± 0.03
4a-9			1.4 ± 0.1
4a-10			1.6 ± 0.2
4a-11			1.3 ± 0.3
4a-12			FQb
4a-13			1.4 ± 0.2
4a-14	Br		7.6 ± 0.1
4a-15			1.5 ± 0.1
4a-16			1.9 ± 0.4
4a-17			0.6 ± 0.1
4a-18			FQb
4a-19			1.1 ± 0.1
4a-20			1.9 ± 0.2
4a-21			1.5 ± 0.1
4a-22			FQb
4a-23			2.7 ± 0.4
4a-24			FQb
4a-25			12 ± 1
4a-26			15 ± 1
4a-27			4.0 ± 0.2
4a-28			FQb
4a-29			NIc
4a-30			8 ± 1
4a-31	H		17 ± 1
4b-1		Et	0.80 ± 0.03
4b-2		Et	0.36 ± 0.02
4b-3		Et	0.32 ± 0.06
4b-4		Et	1.6 ± 0.3
4b-5		Et	5.3 ± 0.2
4b-6		Et	2.1 ± 0.9
4b-7		Et	2.5 ± 0.1
4b-8		Et	0.7 ± 0.1
4b-9		Et	1.5 ± 0.1
4b-10		Et	5 ± 1
4b-11		Et	3.9 ± 0.1
4c-1		H	0.12 ± 0.01
4c-2		H	FQb
4c-3		H	FQb
4c-4		H	0.24 ± 0.03
4c-5		H	1.8 ± 0.01
4c-6		H	1.0 ± 0.1
4c-7		H	0.47 ± 0.05
4c-8		H	FQb
4c-9		H	1.6 ± 0.1
4c-10		H	2.5 ± 0.5
4c-11		H	FQb
4c-12		H	15 ± 2
4c-13		H	FQb
4c-14	Br	H	9 ± 1

^aIC₅₀: concentration of a compound that produces half-maximal inhibition. Reported as mean ± standard deviation from two independent experiments, each in duplicate.

^bFQ: fluorescence quenching. Not selected for dose-response testing.

^cNI: <50% inhibition at 50 μM. Not selected for dose-response testing.

To comprehensively probe the C-6 SAR, a set of 30 hit 3 analogs of subtype 4a were synthesized (Table 1), many of which demonstrated activity in the sub-micromolar range. Analog 4a-5 (IC₅₀ = 0.9 μM) with a neutral phenyl moiety, 4a-8 (IC₅₀ = 0.37 μM) and 4a-17 (IC₅₀ = 0.6 μM) with the electron donating thioanisole substituents at the meta and para positions respectively all exhibited potency against Mpr in the sub-μM range. The meta-anisole group 4a-8 (IC₅₀ = 0.37 μM) had a comparable level of potency to 3 (IC₅₀ = 0.34 μM) but was slightly more potent than the corresponding 4a-17 para-anisole group (IC₅₀ = 0.6 μM). However, 4a-5 (IC₅₀ = 0.9 μM) and 4a-17 (IC₅₀ = 0.6 μM) were slightly less potent than 3 (IC₅₀ = 0.34 μM).

As for electron withdrawing substituents for subtype 4a, a few fluorinated analogs such as 4a-22 (difluoro phenyl) and 4a-24 (trifluoro phenyl), and non-fluorinated analogs like 4a-12 (3-benzamide) and 4a-28 (acetyl thiophene) displayed fluorescence quenching in the biochemical assay. For active compounds, 4a-7 (IC₅₀ = 0.6 μM) displayed inhibition against Mpr in the sub micromolar range, albeit slightly less potent than 3 (IC₅₀ = 0.34 μM).

The heteroaryl substitution at C6 generally led to reduced Mpr inhibition (Table 1). For analogs with an electron-deficient C-6 heteroaryl, 4a-25 (pyridine, IC₅₀ = 12 μM) and 4a-26 (pyrimidine, IC₅₀ = 15 μM) exhibited modest potency, far less potent than 4a-5 (IC₅₀ = 0.9 μM). Similarly, 4a-29 did not inhibit Mpr while 4a-4 (IC₅₀ = 0.97 μM) produced sub-μM potency. For the electron-rich 5-membered heteroaryls, the thiophene analog 4a-11 (IC₅₀ = 1.3 μM) and furan analog 4a-27 (IC₅₀ = 4.0 μM) also conferred decreased Mpr inhibition as compared to 4a-5, albeit significantly better than 4a-25 and 4a-26. For biaryl analogs of subtype 4a, both 4a-3 (IC₅₀ = 0.12 μM) and 4a-2 (IC₅₀ = 0.25μM) showed greater inhibition than 3 (IC₅₀ = 0.34 μM).

Finally, replacing the C-6 aryl moiety with a cyclopropyl group (4a-30, IC₅₀ = 8 μM) or H (4a-31, IC₅₀ = 17 μM) led to substantially decreased potency in Mpr inhibition (Table 1), suggesting that a group of proper size is required for C-6.

The effect of moving the R¹ substituent from the 6-position to the 5-position was explored via a few analogs of subtype 5 (Table 2). In general, the reposition produced substantially increased potency inhibiting Mpr in the FRET assay. Particularly, 5–1 (IC₅₀ = 0.036 μM) and 5–3 (IC₅₀ = 0.015 μM) both showed a 10-fold increase in potency over their C-6 regioisomers. Subtype 5 is the focus of our future Mpr-based SAR efforts.

Table 2.

Mpr Inhibition by analogs of DHN subtypes 5.

Compd	R1	R2	IC50a (μM)
5-1			0.036 ± 0.001
5-2			FQb
5-3		H	0.015 ± 0.001
5-4		H	0.12 ± 0.01

^aIC₅₀: concentration of a compound that produces half-maximal inhibition. Reported as mean ± standard deviation from two independent experiments, each in duplicate.

^bFQ: fluorescence quenching. Not selected for dose-response testing.

Removing the hydroxyl group at the C-4 position resulted in subtype 6 (Table 3). Notably, neither 6–1 nor 6–2 exhibited significant Mpr inhibition (IC₅₀ = >50 μM). This suggests that the hydroxyl group at the C-4 position is vital for Mpr inhibition.

Table 3.

Mpr Inhibition by analogs of DHN subtype 6.

Compd	R1	IC50a (μM)
6-1		NIb
6-2		NIb

^aIC₅₀: concentration of a compound that produces half-maximal inhibition.

^bNI: <50% inhibition at 50 μM. Not selected for dose-response testing.

Subtype 7 lacks the N-1 OH group, the middle prong of the chelating triad which is central to inhibiting the RNHL viral nucleases. Not surprisingly, none of the subtype 7 analogs inhibited Mpr at 50 μM (Table 4), confirming that the chelating triad is absolutely required for inhibiting Mpr.

Table 4.

Mpr Inhibition by analogs of DHN subtype 7.

Compd	R1	R2	IC50a (μM)
7-1		Et	FQb
7-2		Et	NIc
7-3		Et	NIc
7-4		Et	NIc
7-5		Et	NIc
7-6	Br	Et	NIc
7-7			NIc
7-8			NIc

^aIC₅₀: concentration of a compound that produces half-maximal inhibition.

^bFQ: fluorescence quenching. Not selected for dose-response testing.

^cNI: <50% inhibition at 50 μM. Not selected for dose-response testing.

Overall, our Mpr-based SAR so far has identified a few subtype 4 analogs with increased potency over hit 3 against Mpr in the biochemical FRET assay. More importantly, we have shown that the regioisomeric subtype 5 appeared to confer energetically more favorable target engagement (see Table 6), and hence significantly better inhibitory profiles in vitro.

Table 6.

Predicted relative binding free energy using free energy perturbation calculation

Entry	Predicted ΔΔG (kcal/mol)
3, no constraint	5.5 ± 1.7
5-1, no constraint	2.9 ± 2.4
3, metal constraint	0.0 ± 0.4
5-1, metal constraint	−3.3 ± 2.4

Antiviral activity in a VACV-GLuc reporter assay

From the Mpr biochemical assay, all analogs with an IC₅₀ value of ≤10 μM were selected for testing in our primary antiviral assay against a VACV-Gluc reporter virus in HFF cells^{37, 38}. Top ranked compounds with highest Gluc reduction and no significant reduction in cell viability at 10 μM were further evaluated for potency and cytotoxicity in a dose-response fashion where an EC₅₀ value and a CC₅₀ value were produced. Brincidofovir (2) was used as the control. From these assays, a total of eight new analogs exhibited antiviral potency against VACV in the low μM to nM range without discernible cytotoxicity (Table 5). Of these, five analogs, 4b-2 (EC₅₀ = 0.39 μM), 4a-3 (EC₅₀ = 1.3 μM), 4b-3 (EC₅₀ = 1.6 μM), 4b-13 (EC₅₀ = 1.6 μM), and 4a-5 (EC₅₀ = 2.4 μM), showed comparable antiviral potency to hit 3 (EC₅₀ = 1.3 μM). Strikingly, analog 5–1 (EC₅₀ = 0.0032 μM), the C-5 regioisomer of hit 3, conferred single-digit nM antiviral potency 400-fold better than hit 3 and 2-fold better than the control drug brincidofovir (Table 5). A major antiviral-based SAR observation was that all three analogs (3, 4b-2, 5–1) showing nM potency chemically feature a biphenyl moiety, suggesting that the additional phenyl ring contributes largely to target engagement. It is noteworthy that all these analogs passed the aggregation test in our biochemical assay.

Table 5.

Dose response antiviral activity of selected compounds in a VACV-Gluc reporter assay

Compd	VACV EC50a (μM)	HFF CC50b (μM)	SIc	Mpr IC50 (μM)
3 (4a-1)	1.3 ± 0.3	>250	>1.9 × 102	0.34 ± 0.06
4a-3	1.3 ± 0.4	>250	>1.9 × 102	0.12 ± 0.01
4a-5	2.4 ± 0.3	>250	>1.0 × 102	0.9 ± 0.2
4a-10	9.7 ± 0.3	>250	>26	1.6 ± 0.3
4a-13	1.6 ± 0.1	>250	>1.5 × 102	1.4 ± 0.2
4b-2	0.39 ± 0.01	>250	>6.4 × 102	0.36 ± 0.02
4b-3	1.6 ± 0.1	>250	>1.5 × 102	0.32 ± 0.06
5-1	0.0032 ± 0.0007	150	4.7 × 104	0.036 ± 0.001
5-3	6 ± 1	>250	>4 × 10	0.015 ± 0.001
Brincidofovir	0.007 ± 0.004	>250	>3 × 104	--

^aEC₅₀: concentration of a compound that produces half-maximal effect. Reported as mean ± standard deviation from biological replicates (n ≥3).

^bCC₅₀: concentration of a compound that reduces the number of viable cells by 50%.

^cSI: selectivity index, defined by CC₅₀ / EC₅₀.

Molecular modeling

Compounds 3 and 5–1 are regioisomers with the biphenyl group (R¹) at C-6 and C-5 position, respectively. Interestingly, the small difference in chemical structure drastically altered both the Mpr inhibition in the biochemical assay (the C-5 isomer 5–1 is 10-fold more potent) and the antiviral potency against the VACV-Gluc reporter virus (the C-5 isomer 5–1 is 400-fold more potent). To understand this pharmacological difference and provide atomistic insights, we have performed two different types of molecular modeling: molecular docking and relative binding free energy calculation.

The docked poses of 3 and 5–1 are shown in Figure 3. The most intriguing aspect of the two types of docked poses (with and without metal constraint) is the different orientations of the chelating poses. Depending on whether the metal constraint is used or not, both compounds can take either chelating docked pose. Specifically, we note that when using the same docking parameter, the common chelating core between 3 and 5–1 adopts very similar docked poses (Figure 3, A–D). While the docked poses without metal constraint produce lower docking scores for both 3 and 5–1, the differences are less than 1 kcal/mol. Furthermore, 5–1 shows lower docking scores than 3 for both orientations, but the differences are less than 1 kcal/mol as well.

Figure 3.

Docked poses of 3 and 5–1 with and without metal constraints. The interacting residues for each ligand orientation are shown in yellow licorice representation. The Mg ions are shown in orange spheres. (A) 3 without constraint shown in green (docking score = −9.0 kcal/mol). (B) 5–1 without constraint shown in teal (docking score = −9.5 kcal/mol). (C) 3 with metal constraint shown in magenta (docking score = −8.7 kcal /mol). (D) 5–1 with metal constraint shown in purple (docking score = −8.9 kcal /mol).

To further distinguish between the two binding pose orientations, and more importantly, between isomers 3 and 5–1, we have performed relative binding free energy calculations using the docked poses shown in Figure 1 as the initial structures, with 3 with metal constraint as the reference point. The predicted relative binding free energy for the four entries are shown in Table 6. The predicted relative binding free energy in conjunction with the protein-ligand complex trajectory from free energy calculations provides deeper insights into the orientation of the two compounds as well as the difference in their potency.

As shown in Table 6, metal constraint docked poses consistently show lower binding free energy, approximately 6 kcal/mol for both compounds, than no constraint docked poses. Both orientations share common core protein-ligand interactions: metal chelation, salt bridge or hydrogen bond with LYS 124. Other interacting residues include LYS 149, ARG 123, TYR 120, GLN 83, and ASN 35, observed during the 5 ns protein-ligand complex molecular dynamics simulation within free energy calculation. Detailed ligand interaction diagrams are shown in Figures S1, S2, S3, and S4. The metal constraint orientation is likely favored over the no-constraint orientation because it puts the biphenyl group (for both 3 and 5–1) between α4 and the loop between α2 and β4, which allows stronger and consistent protein-ligand interactions compared to the no constraint orientation in which the biphenyl group is placed between the loop between α4 and α5 and the loop between β1 and β2, two loop regions with greater flexibility³⁹. The atomistic basis for greater affinity for 5–1 over 3 also likely comes down to the more favored ligand orientation within the binding site. The biphenyl group at the C-5 position gives a better angle for protein-ligand interaction. This effect of biphenyl orientation can be observed in the interaction count plot (Figures S5 and S6) in which 5–1 maintains greater number of protein-ligand interactions during the 5 ns trajectory compared to 3.

ADME profiling

To gauge the drug-like properties of the DHN chemotype, we have selected five strongly antiviral analogs representative of subtypes 4a, 4b and 5 for absorption, distribution, metabolism, and excretion (ADME) profiling in four major in vitro assays: aqueous solubility, human plasma stability, human microsomal stability, and PAMPA permeability (Table 7). Notably, all tested analogs showed good human plasma stability (remaining >90% at 24h) and high predictive passive transcellular transport (PAMPA permeability). Apart from 4b-2, all analogs, especially the regiosiomeric pair 5–1 and 3, exhibited acceptable solubility. In addition, 5–1 and 3 both demonstrated excellent human microsomal stability, suggesting the high resistance to oxidative metabolism. Taken together, these data clearly characterize 5–1 and 3 as promising drug-like compounds with favorable pharmacokinetics (PK) predicted.

Table 7.

Solubility, permeability and in vitro metabolic stabilities of selected compounds

Compd	Aqueous Solubilitya (μmol/L), n=3	Human Plasma Stability t1/2 (h), n = 3	Human Microsomal Stabilityb t1/2 (min), n = 3	PAMPA Permeabilityc Pe (10−6 cm/s), n =4
3 (4a-1)	130 ± 8	> 24d	>120e	6 ± 2
4a-3	86 ± 5	> 24d	42.9 ± 0.7	8.6 ± 0.8
4b-2	1.4 ± 0.1	> 24d	>120e	3.8 ± 0.1
4b-3	47 ± 4	> 24d	35.2 ± 0.8	5.2 ± 0.6
5-1	130 ± 4	> 24d	>120e	9 ± 1
Verapamil	--	--	6.9 ± 0.1	--

Data is presented as mean ± SD.

^aAqueous solubility was determined in Dulbecco’s Phosphate-Buffered Saline (DPBS, pH 7.2).

^bCYP enzyme cofactor: NADPH.

^cPe: The apparent permeability coefficient. High Permeability: >1.5 × 10⁻⁶ cm/s; Low Permeability: < 1.5× 10⁻⁶ cm/s.

^dRemaining percentage was observed greater than 90% at the end of incubation (24h).

^eRemaining percentage was observed greater than 75% at the end of incubation (60min).

Conclusion

DHN analog 3 has been shown to inhibit both Fpr and Mpr with significant antiviral activity. To explore the SAR and optimize hit 3, we have designed and synthesized a total of 70 DHN analogs belonging to four distinct subtypes. The SAR revealed that a phenyl group is preferred at the C-3 group over a small alkyl group or H; a two phenyl moiety is favored at the C-6 position; both the C-4 and N-1 hydroxyl groups are essential for Mpr inhibition; and the C-5 substituted subtype conferred significantly better inhibitory profiles than the C-6 isomers. Overall, the SAR identified a new analog 5–1 with 10-fold improved inhibition against Mpr, and more importantly, 400-fold improved antiviral potency against VACV. Binding free energy for compound 5–1 is predicted to be much lower than that for compound 3 from the free energy perturbation calculation. In ADME profiling both 5–1 and 3 produced highly favorable drug-like properties in all four assays conducted, predicting good PK. Together, these results indicate that 5–1 can be a strong antiviral lead. Further studies on expanded antiviral profiling against other orthopoxviruses, mice PK and toxicology, in vivo efficacy, are currently underway. In addition to identifying more potent analogs, the current SAR also supports subtype 5 as the focus of future ad hoc medicinal chemistry in developing the DHN resolvase inhibitor type for potent antivirals against MPXV and other orthopoxviruses.

EXPERIMENTAL SECTION

Chemistry

Experimental

General procedures.

All commercial chemicals were used as supplied unless otherwise indicated. Dry solvents (THF, Et₂O, CH₂Cl₂ and DMF) were dispensed under argon from an anhydrous solvent system with two packed columns of neutral alumina or molecular sieves. Anhydrous ethanol was purchased from Sigma-Aldrich. Flash chromatography was performed on a Teledyne Combiflash RF-200 with RediSep silica (silica) with indicated mobile phase. All moisture sensitive reactions were performed under an inert atmosphere of ultra-pure argon with oven-dried glassware. A Bruker 400 MHz NMR was used to obtain the NMR spectra for compound characterization with MestreNova software. HRMS data were acquired using an Agilent 6230 TOF LC/MS spectrometer equipped with ESI and APCI ion modules. Glass backed plates for thin-layer chromatography (TLC) analysis were used (TLC silica gel 60 F₂₅₄) with fluorescent indicator (254 nm). Compounds on the TLC plates were visualized with a UV lamp (254 nm) and subsequently stained with ceric ammonium molybdate (CAM) stain (235 ml of H₂O, 15 ml of H₂SO₄, 12 g of ammonium molybdate, 0.5 g of ceric ammonium molybdate). HPLC conditions: flow rate, 1.0 mL/min; solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile; gradient (B, %): 0–6 min (5–100), 6–8 min (100), 8–9 min (100–5). Purity was determined by total absorbance at 254 nm. Determined purity was >95% for all final compounds.

General procedure for the Suzuki coupling

In a 10 ml microwave vial containing the 6-bromo DHN intermediate (2.07 mmol, 1 eq) was added the boronic acid (3.49 mmol, 1.68 eq), K₂CO₃ (5.197 mmol) and Pd(dppf)Cl₂ (0.17 mmol, 8 mol %). To the constituents was added DMF and H₂O (5 ml total, 4:1 v/v). The microwave vial was sealed, and argon bubbled through the solution. The reaction was heated using the microwave reactor for 20 minutes at 110 °C. The reaction was cooled to room temperature, quenched with the addition of 1 N HCl to bring the pH of solution to 4, and extracted with ethyl acetate (3 × 20 ml). The combined organics were dried over Na₂SO₄ and filtered over a short pad of celite and concentrated to dryness under reduced pressure. The crude obtained was purified by flash chromatography (gradient 15 % ethyl acetate → 60 % ethyl acetate in hexanes) to yield the titled product.

General procedure for the debenzylation for subtypes 4, 5 and 6 compounds

The final compounds for DHN subtypes 4, 5 and 6 were obtained through the following deprotection procedure as described below:

To a 50 ml round bottom flask containing benzyloxy intermediates was added sequentially glacial acetic acid (3 ml) and 48 % HBr in acetic acid (1 ml). The solution was stirred at 80 °C for 2 h. After complete deprotection of the benzyloxy group, the solution was cooled to room temperature and co-evaporated with MeOH under reduced pressure to form a crude reddish-brown residue. The crude residue was purified by flash chromatography (100 % ethyl acetate then 10 % MeOH in CH₂Cl₂, 12 g RediSep Rf gold column) to obtain the final product. In other instances, water or ethyl acetate was added to the reaction solution after cooling to precipitate the product. The solids obtained were filtered, washed sequentially with water, diethyl ether and dried under vacuum.

Characterizations for subtype 4 analogs

6-([1,1’-biphenyl]-3-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (3 or (4a-1))

This compound was synthesized according to the procedure described above. Yield over two steps (55 mg, 18 %) as a cream solid; HPLC purity 97 %; ¹H NMR(400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.64 (s, 1H), 9.18 – 9.02 (d, J = 2.2 Hz, 1H), 8.85 – 8.63 (d, J = 2.3 Hz, 1H), 8.15 – 8.00 (t, J = 1.8 Hz, 1H), 7.85 – 7.78 (m, 3H), 7.76 – 7.71 (dt, J = 7.9, 1.3 Hz, 1H), 7.68 – 7.62 (t, J = 7.7 Hz, 1H), 7.55 – 7.49 (dd, J = 8.4, 7.0 Hz, 2H), 7.48 – 7.39 (m, 5H), 7.39 – 7.32 (m, 1H); ¹³C NMR (101 MHz, DMSO) δ 160.1, 154.8, 149.6, 147.2, 141.7, 140.4, 137.7, 133.4, 131.5, 130.7, 130.3, 130.3, 129.5, 128.3, 128.2, 127.8, 127.5, 126.8, 126.3, 125.6, 114.2, 111.1; HRMS (m/z) calculated for C₂₆H₁₉N₂O₃⁺ [M + H]⁺, 407.1390; found, 407.1321 (Δ 16 ppm).

6-([1,1’-biphenyl]-4-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-2)

This compound was synthesized according to the procedure described above. Yield over two steps (23 mg, 24 %) as a light brown solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 9.09 (s, 1H), 8.77 (s, 1H), 7.97 – 7.91 (d, J = 7.9 Hz, 2H), 7.90 – 7.83 (d, J = 7.6 Hz, 2H), 7.80 – 7.73 (d, J = 7.5 Hz, 2H), 7.56 – 7.30 (m, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 160.1, 154.8, 149.3, 147.1, 140.1, 139.9, 135.9, 133.4, 131.5, 130.3, 130.0, 130.0, 129.5, 129.2, 128.8, 128.3, 128.2, 128.0, 127.8, 127.6, 127.1, 114.2, 111.2; HRMS (m/z) calculated for C₂₆H₁₉N₂O₃⁺ [M+H]⁺, 407.1389; found 407.1390 (Δ 0.2 ppm).

1,4-dihydroxy-6-(4-phenoxyphenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-3)

This compound was synthesized according to the procedure described above. Yield over two steps (45 mg, 45 %) as grey solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.79 (s, 1H), 10.60 (s, 1H), 9.03 – 8.98 (d, J = 2.2 Hz, 1H), 8.70 – 8.64 (d, J = 2.3 Hz, 1H), 7.89 – 7.79 (d, J = 8.6 Hz, 2H), 7.54 – 7.32 (m, 8H), 7.26 – 7.00 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 157.3, 156.9, 154.7, 149.2, 146.9, 133.3, 132.1, 131.5, 130.64, 130.2, 129.8, 128.9, 128.4, 127.8, 124.3, 119.7, 119.4, 114.2, 111.1; HRMS (m/z) calculated for C₂₆H₁₉N₂O₄⁺ [M + H]⁺, 423.1338; found 423.1339 (Δ 0.2 ppm).

1,4-dihydroxy-6-(6-methoxynaphthalen-2-yl)-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-4)

This compound was synthesized according to the procedure described above. Yield over two steps (37 mg, 38 %) as a grey solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.63 (s, 1H), 9.16 – 9.11 (d, J = 2.2 Hz, 1H), 8.81 – 8.76 (d, J = 2.3 Hz, 1H), 8.31 (s, 1H), 8.05 – 7.87 (m, 3H), 7.52 – 7.29 (m, 6H), 7.26 – 7.21 (dd, J = 8.9, 2.5 Hz, 1H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 158.1, 154.9, 149.5, 147.0, 134.3, 133.4, 131.9, 131.5, 130.4, 130.2, 129.3, 128.4, 128.2, 127.8, 125.7, 125.6, 119.8, 114.1, 111.2, 106.3, 55.7; HRMS (m/z) calculated for C₂₅H₁₉N₂O₄⁺ [M+H]⁺, 411.1336; found 411.1339 (Δ 0.7 ppm).

1,4-dihydroxy-3,6-diphenyl-1,8-naphthyridin-2(1H)-one (4a-5)

This compound was synthesized according to the procedure described above. Yield over two steps (24 mg, 30 %) as a brown solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.78 (s, 1H), 9.01 (apparent s, 1H), 8.69 (apparent s, 1H), 7.85 – 7.79 (d, J = 7.5 Hz, 2H), 7.59 – 7.52 (d, J = 7.5 Hz, 2H), 7.48 – 7.40 (m, 5H), 7.39 – 7.33 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 149.3, 147.1, 137.0, 133.5, 131.5, 130.5, 130.3, 129.7, 128.4, 128.3, 127.7, 127.2, 111.3; HRMS (m/z) calculated for C₂₀H₁₅N₂O₃⁺ [M + H]⁺, 331.1077; found 331.1069 (Δ 2.4 ppm).

6-(2-fluorophenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-6)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 31 %) as a cream solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.82 (s, 1H), 10.61 (s, 1H), 8.87 – 8.85 (t, J = 1.9 Hz, 1H), 8.59 – 8.57 (dd, J = 2.3, 1.2 Hz, 1H), 7.79 – 7.62 (td, J = 7.8, 1.7 Hz, 1H), 7.59 – 7.22 (m, 8H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.9, 160.1, 158.5, 154.7, 150.8 (d, J_CF = 3.0 Hz), 150.8, 147.2, 133.3, 132.7 (d, J_CF = 3.0 Hz), 131.5, 131.3 (d, J_CF = 3.0 Hz), 130.8 (d, J_CF = 8.1 Hz), 128.4, 127.8, 125.8 (d, J_CF = 3.0 Hz), 125.4, 124.8 (d, J_CF = 14.1 Hz), 116.7 (d, J_CF = 62.6 Hz), 114.2, 111.1; HRMS (m/z) calculated for C₂₀H₁₅N₂O₃⁺ [M+H]⁺, 349.0983; found 349.0973 (Δ 2.9 ppm).

1,4-dihydroxy-3-phenyl-6-(4-(trifluoromethyl) phenyl)-1,8-naphthyridin-2(1H)-one (4a-7)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 21 %) as a grey solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.69 (s, 1H), 9.12 – 9.07 (d, J = 2.3 Hz, 1H), 8.78 – 8.74 (d, J = 2.3 Hz, 1H), 8.10 – 8.04 (d, J = 8.1 Hz, 2H), 7.93 – 7.87 (d, J = 8.2 Hz, 2H), 7.48 – 7.38 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 154.7, 149.6, 147.6, 141.0, 133.2, 131.5, 131.1, 128.7 (q, J_CF = 15.2 Hz), 128.4, 127.9, 127.9, 126.5 (q, J_CF = 2.97 Hz), 124.8 (q, J_CF = 270 Hz), 114.3, 111.2; HRMS (m/z) calculated for C₂₁H₁₄F₃N₂O₃⁺ [M + H]⁺, 399.0951; found 399.0952 (Δ 0.3 ppm).

1,4-dihydroxy-6-(4-(methylthio)phenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-8)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 17 %) as a cream solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.79 (s, 1H), 10.62 (s, 1H), 9.03 – 8.98 (d, J = 2.3 Hz, 1H), 8.72 – 8.65 (d, J = 2.3 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.49 – 7.32 (m, 7H), 2.57 – 2.52 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 207.0, 160.0, 154.8, 149.1, 147.0, 138.8, 133.4, 133.3, 131.5, 130.0, 129.7, 128.3, 127.8, 127.5, 127.0, 114.1, 111.2, 15.1; HRMS (m/z) calculated for C₂₁H₁₇N₂O₃S⁺ [M+H]⁺ 377.0954; found 377.0934 (Δ 5.3 ppm).

6-(2,5-dimethoxyphenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-9)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 16 %) as a grey solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 10.52 (s, 1H), 8.83 – 8.79 (d, J = 2.2 Hz, 1H), 8.56 – 8.49 (d, J = 2.2 Hz, 1H), 7.50 – 7.32 (m, 6H), 7.14 – 7.10 (d, J = 9.0 Hz, 1H), 7.07 – 7.04 (d, J = 3.1 Hz, 1H), 7.02 – 6.97 (dd, J = 8.9, 3.1 Hz, 1H), 3.80 – 3.78 (s, 3H), 3.77 – 3.76 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 154.7, 154.0, 151.5, 150.9, 146.7, 133.3, 133.0, 131.5, 128.5, 128.4, 128.1, 127.8, 127.0, 116.6, 114.6, 114.0, 113.7, 110.6, 56.7, 56.0; HRMS (m/z) calculated for C₂₂H₁₉N₂O₅⁺ [M+H]⁺, 391.1288; found 391.1287 (Δ 0.2 ppm);

6-(3-fluoro-4-methoxyphenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-10)

This compound was synthesized according to the procedure described above. Yield over two steps (23 mg, 26 %) as grey solid; HPLC purity 97 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 9.03 – 8.98 (d, J = 2.3 Hz, 1H), 8.67 – 8.65 (d, J = 2.3 Hz, 1H), 7.77 – 7.71 (dd, J = 12.8, 2.3 Hz, 1H), 7.64 – 7.58 (ddd, J = 8.6, 2.2, 1.0 Hz, 1H), 7.49 – 7.28 (m, 7H), 3.93 – 3.89 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 154.8, 153.6, 151.2, 149.1, 147.6 (d, J_CF = 8 Hz), 146.9, 133.4, 131.5, 130.1, 129.8 (d, J_CF = 7 Hz), 128.9 (d, J_CF = 2 Hz), 128.3, 127.8, 123.4 (d, J_CF = 3 Hz), 115.0 (d, J_CF = 2 Hz), 114.6 (d, J_CF = 22.2 Hz), 114.1, 111.1, 56.6; HRMS (m/z) calculated for C₂₁H₁₆FN₂O₄⁺ [M+H]⁺, 379.1085; found 379.1089 (Δ 1.0 ppm);

1,4-dihydroxy-3-phenyl-6-(thiophen-3-yl)-1,8-naphthyridin-2(1H)-one (4a-11)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 31 %) as grey solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.57 (s, 1H), 9.14 – 9.02 (d, J = 2.2 Hz, 1H), 8.74 – 8.59 (d, J = 2.3 Hz, 1H), 8.12 – 8.01 (apparent s, 1H), 7.77 – 7.74 (m, 1H), 7.71 – 7.69 (m, 1H), 7.49 – 7.31 (m, 5H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9, 154.7, 149.1, 146.7, 138.1, 133.3, 131.5, 129.6, 128.4, 128.3, 127.8, 126.4, 125.9, 122.1, 114.1, 111.1; HRMS (m/z) calculated for C₁₈H₁₁N₂O₃S⁻ [M−H]⁻, 335.0496; found 335.0526 (Δ 8.9 ppm).

3-(5,8-dihydroxy-7-oxo-6-phenyl-7,8-dihydro-1,8-naphthyridin-3-yl) benzamide (4a-12)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 22 %) as a cream solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.70 (s, 1H), 9.10 – 9.04 (d, J = 2.3 Hz, 1H), 8.79 – 8.75 (d, J = 2.3 Hz, 1H), 8.31 (apparent s, 1H), 8.17 (s, 1H), 8.01 – 7.97 (dt, J = 7.8, 1.4 Hz, 1H), 7.96 – 7.91 (dt, J = 7.8, 1.4 Hz, 1H), 7.66 – 7.60 (t, J = 7.7 Hz, 1H), 7.51 (s, 1H), 7.49 – 7.41 (m, 4H), 7.40 – 7.34 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 207.0, 168.1, 160.1, 149.5, 147.3, 137.0, 135.7, 133.4, 131.5, 130.7, 129.8, 129.7, 128.3, 127.8, 127.5, 126.1, 114.0, 111.3; HRMS (m/z) calculated for C₂₁H₁₄N₃O₄⁻ [M − H]⁻, 372.0990; found, 372.1005 (Δ 4.0 ppm).

6-(3-fluoro-4-methylphenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-13)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 29 %) as a cream solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (s, 1H), 8.96 (s, 1H), 8.64 (s, 1H), 7.75 (dd, J = 7.3, 2.2 Hz, 1H), 7.70 – 7.62 (m, 1H), 7.49 – 7.26 (m, 6H), 2.34 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.4, 160.0, 154.7, 149.3, 147.0, 133.2 (d, J_CF = 17.2 Hz), 131.5, 130.4 (t, J_CF = 4 Hz), 129.5, 128.4, 127.8, 126.5 (d, J_CF = 8 Hz), 125.6 (d, J_CF = 18.2 Hz), 116.2 (d, J_CF = 20 Hz), 114.2, 111.1, 14.8; HRMS (m/z) calculated for C₂₁H₁₆FN₂O₃⁺ [M+H]⁺, 363.1139; found 363.1134 (Δ 1.4 ppm).

6-bromo-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-14)

This compound was synthesized according to the procedure described above. Yield (23 mg, 63 %) as a cream solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.71 – 10.67 (s, 0H), 8.81 – 8.74 (d, J = 2.3 Hz, 1H), 8.59 – 8.54 (d, J = 2.3 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.41 – 7.32 (m, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 153.7, 151.4, 146.6, 134.7, 132.9, 131.4, 128.4, 128.0, 114.9, 112.9, 112.6,

1,4-dihydroxy-6-(4-hydroxyphenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-15)

This compound was synthesized according to the procedure described above. Yield over two steps (24 %, 20 mg) as a cream solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 10.59 (s, 1H), 9.74 (s, 1H), 8.99 (d, J = 2.3 Hz, 1H), 8.64 (s, 1H), 7.72 – 7.65 (m, 2H), 7.54 – 7.43 (m, 4H), 7.00 – 6.94 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9, 158.1, 154.7, 148.8, 146.5, 136.4, 133.4, 131.6, 131.5, 130.5, 129.4, 128.3, 128.2, 127.8, 116.5, 116.5, 114.8, 114.1, 111.1; HRMS (m/z) calculated for C₂₀H₁₅N₂O₄⁺ [M+H]⁺, 347.1003; found 347.1026 (Δ 6.6 ppm).

1,4-dihydroxy-3-phenyl-6-(p-tolyl)-1,8-naphthyridin-2(1H)-one (4a-16)

This compound was synthesized according to the procedure described above. Yield over two steps (41 mg, 50 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR (400 MHz, DMSO) δ 10.50 (s, 1H), 8.90 (s, 1H), 8.58 (s, 1H), 7.62 (d, J = 7.1 Hz, 2H), 7.41 – 7.22 (m, 7H), 2.29 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 154.8, 149.1, 146.9, 137.9, 134.0, 133.4, 131.5, 130.3, 130.1, 128.3, 127.8, 127.0, 114.2, 111.1, 21.2; HRMS (m/z) calculated for C₂₁H₁₇N₂O₃⁺ [M+H]⁺ 345.1234; found 345.1234 (Δ 0 ppm)

1,4-dihydroxy-6-(3-(methylthio)phenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-17)

This compound was synthesized according to the procedure described above. Yield over two steps (45 mg, 50 %) as a cream solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.82 (s, 1H), 10.62 (s, 1H), 9.03 (s, 1H), 8.70 (s, 1H), 7.64 (s, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.53 – 7.26 (m, 14H), 2.60 – 2.56 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 139.9, 131.5, 130.6, 130.2, 129.4, 128.3, 127.8, 125.8, 124.4, 123.8, 15.2; HRMS (m/z) calculated for C₂₁H₁₇N₂O₃S⁺ [M+H]⁺ 377.0954; found 377.0950 (Δ 1.0 ppm).

6-(3,4-dimethoxyphenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-18)

Yield over two steps (15 mg, 16 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.78 (s, 1H), 9.00 (d, J = 2.2 Hz, 1H), 8.64 (d, J = 2.3 Hz, 1H), 7.40 (m, 7H), 7.11 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H), 3.82 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 154.8, 149.8, 149.4, 149.2, 146.7, 133.4, 131.5, 130.4, 129.9, 129.6, 128.3, 127.8, 119.5, 114.0, 112.9, 111.0, 110.8, 56.2, 56.1; HRMS (m/z) calculated for C₂₂H₁₇N₂O₅⁻ [M−H]⁻, 389.1143; found 389.1164 (Δ 5.3 ppm).

6-(benzo[d][1,3]dioxol-5-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-19)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 19 %) as a cream solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 10.56 (s, 1H), 8.98 – 8.92 (d, J = 2.3 Hz, 1H), 8.64 – 8.53 (d, J = 2.3 Hz, 1H), 7.49 – 7.26 (m, 7H), 7.11 – 7.05 (d, J = 8.1 Hz, 1H), 6.12 – 6.08 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 154.7, 149.2, 148.7, 147.8, 146.9, 133.4, 131.5, 131.1, 130.2, 130.1, 128.3, 127.8, 120.9, 114.1, 111.0, 109.5, 107.6, 101.8; HRMS (m/z) calculated for C₂₁H₁₅N₂O₅⁺ [M+H]⁺, 375.0969; found 375.0975 (Δ 1.5 ppm).

4-(5,8-dihydroxy-7-oxo-6-phenyl-7,8-dihydro-1,8-naphthyridin-3-yl) benzonitrile (4a-20)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 23 %) as a cream solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.86 (s, 1H), 10.69 (s, 1H), 9.16 – 9.00 (d, J = 2.3 Hz, 1H), 8.84 – 8.70 (d, J = 2.4 Hz, 1H), 8.13 – 7.81 (m, 4H), 7.49 – 7.22 (m, 5H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 154.9, 149.7, 147.7, 141.6, 133.6, 133.3, 131.5, 131.2, 128.4, 127.9, 127.8, 119.3, 114.2, 111.3, 110.9; HRMS (m/z) calculated for C₂₁H₁₄N₃O₃⁺ [M + H]⁺, 356.1030; found 356.0997 (Δ 9.0 ppm).

4-(5,8-dihydroxy-7-oxo-6-phenyl-7,8-dihydro-1,8-naphthyridin-3-yl)benzenesulfonamide (4a-21)

This compound was synthesized according to the procedure described above. Yield over two steps (54 mg, 56 %) as a grey solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.85 (s, 1H), 10.67 (s, 1H), 9.11 – 9.06 (d, J = 2.2 Hz, 1H), 8.78 – 8.73 (d, J = 2.3 Hz, 1H), 8.07 – 8.00 (d, J = 8.2 Hz, 2H), 8.00 – 7.93 (d, J = 8.1 Hz, 2H), 7.50 – 7.33 (m, 8H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 154.7, 149.6, 147.5, 143.8, 140.2, 133.2, 131.5, 130.9, 128.8, 128.4, 127.9, 127.6, 127.0, 114.3, 111.2; HRMS (m/z) calculated for C₂₀H₁₆N₃O₅S⁺ [M+H]⁺, 410.0805; found 410.0781 (Δ 5.8 ppm)

6-(2,4-difluorophenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-22)

This compound was synthesized according to the procedure described above. Yield over two steps (40 mg, 46 %) as a light brown solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.83 (s, 1H), 10.61 (s, 1H), 8.85 (s, 1H), 8.56 (s, 1H), 7.77 (q, J = 8.3 Hz, 1H), 7.53 – 7.25 (m, 10H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 154.6, 150.7 (d, J_CF = 2.0 Hz), 147.2 (d, J_CF = 2.0 Hz), 133.2, 132.8 (d, J_CF = 2.0 Hz), 132.7 (dd, J_CF = 6.1, 2.0 Hz), 132.6, 131.5, 128.4, 127.9, 124.6, 114.3, 111.0, 105.3 (d, J_CF = 26.3 Hz); HRMS (m/z) calculated for C₂₀H₁₃F₂N₂O₃⁺ [M+H]⁺, 367.0889; found 367.0889 (Δ 0 ppm).

6-(2,4-bis(trifluoromethyl)phenyl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-23)

This compound was synthesized according to the procedure described above. Yield over two steps (45 mg, 41 %) as a light brown solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 10.62 (s, 1H), 8.67 (d, J = 2.2 Hz, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.20 (d, J = 6.9 Hz, 2H), 7.86 (d, J = 8.2 Hz, 1H), 7.50 – 7.33 (m, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2, 154.5, 150.2, 147.7, 141.4, 134.8, 133.9 (d, J_CF = 8.1 Hz), 131.5, 129.6 (q, J_CF = 2 Hz), 129.2, 128.9, 128.5, 128.1 (d, J_CF = 8 Hz), 128.0, 125.2 (d, J_CF = 11.1 Hz), 123.8 (q, J_CF = 2 Hz), 122.5 (d, J_CF = 15 Hz) 114.6, 110.5; HRMS (m/z) calculated for C₂₂H₁₃F₆N₂O₃⁺ [M+H]⁺, 467.0824; found 467.0825 (Δ 0.2 ppm).

1,4-dihydroxy-3-phenyl-6-(2,4,6-trifluorophenyl)-1,8-naphthyridin-2(1H)-one (4a-24)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 28 %) as a light yellow solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.85 (s, 1H), 10.62 (s, 1H), 8.76 (s, 1H), 8.50 (s, 1H), 7.52 – 7.28 (m, 7H); ¹³C NMR (101 MHz, DMSO-d₆) δ 161.6 (apparent q, J_CF = 2 Hz), 160.2, 159.0 (dd, J_CF =42, 10 Hz), 154.4, 151.8, 147.5, 134.3, 133.1, 131.5, 128.5, 128.0, 118.4, 114.4, 111.4 (q, J_CF = 2 Hz), 111.1 , 101.7 (apparent t, J_CF = 29 Hz); HRMS (m/z) calculated for C₂₀H₁₀F₃N₂O₃⁻ [M−H]⁻, 383.0649; found 383.0654 (Δ 1.3 ppm).

1,4-dihydroxy-3-phenyl-6-(pyridin-3-yl)-1,8-naphthyridin-2(1H)-one (4a-25)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 19 %) as a cream solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆ ) δ 12.10 (s, 1H), 10.61 (s, 1H), 9.26 (s, 1H), 9.01 (d, J = 2.3 Hz, 1H), 8.88 – 8.78 (dd, J = 8.6, 3.7 Hz, 2H), 8.74 – 8.62 (dd, J = 8.1, 2.1 Hz, 1H), 7.97 – 7.87 (dd, J = 8.1, 5.1 Hz, 1H), 7.50 – 7.32 (m, 5H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.8, 157.0, 149.7, 149.6, 144.8, 143.9, 139.6, 134.9, 133.2, 131.5, 131.1, 128.3, 127.7, 126.3, 125.4, 114.3, 111.4; HRMS (m/z) calculated for C₁₉H₁₄N₃O₂^•2 [M-H₂O+2H]⁺ 316.1075; found 316.1078 (Δ 0.9 ppm)

1,4-dihydroxy-3-phenyl-6-(pyrimidin-5-yl)-1,8-naphthyridin-2(1H)-one (4a-26)

This compound was synthesized according to the procedure described above. Yield over two steps (41 mg, 52 %) as a pale yellow solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H), 9.38 – 9.19 (d, J = 17.4 Hz, 3H), 9.22 – 9.00 (m, 1H), 8.87 – 8.77 (s, 1H), 7.51 – 7.28 (m, 5H); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.0, 155.3, 131.5, 131.3, 129.4, 128.4, 127.9, 126.3; HRMS (m/z) calculated for C₁₈H₁₃N₄O₃⁺ [M+H]⁺, 333.0982; found 333.0984 (Δ 0.6 ppm).

6-(furan-2-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-27)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 20 %) as a light brown solid.¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.63 (s, 1H), 9.07 – 9.01 (d, J = 2.2 Hz, 1H), 8.67 – 8.62 (d, J = 2.2 Hz, 1H), 7.87 – 7.80 (dd, J = 1.8, 0.7 Hz, 1H), 7.48 – 7.38 (m, 4H), 7.38 – 7.33 (td, J = 6.5, 2.4 Hz, 1H), 7.14 – 7.11 (dd, J = 3.4, 0.8 Hz, 1H), 6.70 – 6.64 (dd, J = 3.4, 1.8 Hz, 1H), 5.78 – 5.74 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.5, 160.0, 151.0, 147.0, 146.7, 144.0, 133.5, 131.5, 127.7, 127.1, 121.5, 112.8, 111.5, 106.9

6-(5-acetylthiophen-2-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-28)

This compound was synthesized according to the procedure described above. Yield over two steps (35 mg, 39 %) as a light yellow solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 10.72 (s, 1H), 9.17 – 9.02 (d, J = 2.3 Hz, 1H), 8.76 – 8.63 (d, J = 2.3 Hz, 1H), 8.08 – 7.95 (d, J = 3.9 Hz, 1H), 7.87 – 7.76 (d, J = 3.9 Hz, 1H), 7.49 – 7.29 (m, 5H), 2,58 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 191.1, 172.5, 160.0, 154.5, 148.6, 147.9, 147.7, 143.7, 135.7, 133.1, 131.5, 129.8, 128.4, 127.9, 126.1, 123.7, 114.5, 111.2, 26.9, 21.5; HRMS (m/z) calculated for C₂₀H₁₅N₂O₄S⁺ [M+H]⁺, 379.0747; found 379.0746 (Δ 0.3 ppm).

1,4-dihydroxy-3-phenyl-6-(quinolin-3-yl)-1,8-naphthyridin-2(1H)-one (4a-29)

This compound was synthesized according to the procedure described above. Yield over two steps (22 mg, 23 %) as a grey solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 12.05 (s, 1H), 10.62 (s, 1H), 9.42 – 9.28 (d, J = 2.4 Hz, 1H), 9.16 – 9.03 (d, J = 2.3 Hz, 1H), 8.85 – 8.73 (m, 2H), 8.15 – 8.04 (ddd, J = 8.5, 2.6, 1.3 Hz, 2H), 7.85 – 7.76 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 7.76 – 7.60 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.48 – 7.24 (m, 5H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.7, 157.0, 149.8, 149.6, 149.1, 147.3, 133.3, 133.3, 131.6, 130.6, 130.3, 130.2, 129.2, 128.9, 128.3, 128.1, 127.7, 127.6, 127.2, 114.3, 111.5; HRMS (m/z) calculated for C₂₃H₁₆N₃O₂^•2+ [M-H₂O+2H]⁺, 366.1232; found 366.1219 (Δ 3.5 ppm).

6-cyclopropyl-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-30)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 21 %) as a cream solid; HPLC purity 95 %; ¹H NMR (400 MHz, CD₃OD-d₄) δ 8.41 – 8.39 (d, J = 2.3 Hz, 1H), 8.02 – 7.99 (d, J = 2.3 Hz, 1H), 7.42 – 7.25 (m, 6H), 2.05 – 1.96 (tt, J = 8.4, 5.1 Hz, 1H), 1.04 – 0.97 (m, 2H), 0.74 – 0.69 (m, 2H); ¹³C NMR (101 MHz, CD₃OD-d₄) δ 160.7, 155.3, 149.2, 145.2, 134.8, 131.8, 130.8, 129.2, 128.3, 127.8, 113.0, 111.4, 11.9, 8.1; HRMS (m/z) calculated for C₁₇H₁₅N₂O₃⁺ [M + H]⁺, 295.1077; found 295.1062 (Δ 5.0 ppm).

1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (4a-31)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 25 %) as an off-white solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.67 (s, 1H), 8.71 – 8.65 (dd, J = 4.7, 1.7 Hz, 1H), 8.45 – 8.39 (dd, J = 7.9, 1.7 Hz, 1H), 7.49 – 7.29 (m, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 154.7, 151.2, 147.8, 133.3, 133.1, 131.5, 128.4, 127.8, 118.4, 113.8, 111.3; HRMS (m/z) calculated for C₁₄H₁₁N₂O₃⁺ [M+H]⁺ 255.0764; found 255.0765 (Δ 0.4 ppm)

6-([1,1’-biphenyl]-3-yl)-3-ethyl-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4b-1)

This compound was synthesized according to the procedure described above. Yield over two steps (18 mg, 26 %) as an off-white solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 1H), 9.05 (d, J = 2.2 Hz, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.84 – 7.59 (m, 6H), 7.52 (dd, J = 8.3, 6.9 Hz, 2H), 7.45 – 7.40 (m, 1H), 2.68 (q, J = 7.3 Hz, 3H), 1.08 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.6, 154.0, 148.7, 146.6, 141.7, 140.5, 137.8, 130.3, 130.2, 130.1, 129.5, 129.4, 128.2, 127.5, 127.5, 126.8, 126.3, 125.6, 115.1, 111.0, 17.7, 13.5; HRMS (m/z) calculated for C₂₂H₁₉N₂O₃⁺ [M+H]⁺, 359.1390; found 359.1392 (Δ 0.6 ppm).

6-([1,1’-biphenyl]-4-yl)-3-ethyl-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4b-2)

This compound was synthesized according to the procedure described above. Yield over two steps (22 mg, 31 %) as an off-white solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR (400 MHz, ) δ 10.59 (s, 1H), 9.01 (d, J = 2.3 Hz, 1H), 8.66 (d, J = 2.3 Hz, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.76 (m, 2H), 7.51 (dd, J = 8.4, 6.9 Hz, 2H), 7.45 – 7.36 (m, 1H), 2.68 (q, J = 7.3 Hz, 2H), 1.08 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 13C NMR (101 MHz, DMSO) δ 160.6, 153.9, 148.4, 146.6, 140.1, 139.9, 136.0, 129.7, 129.7, 129.5, 128.2, 127.9, 127.6, 127.1, 115.1, 111.1, 17.7, 13.5; HRMS (m/z) calculated for C₂₂H₁₉N₂O₃⁺ [M+H]⁺, 359.1390; found 359.1386 (Δ 1.1 ppm).

3-ethyl-1,4-dihydroxy-6-(4-phenoxyphenyl)-1,8-naphthyridin-2(1H)-one (4b-3)

This compound was synthesized according to the procedure described above. Yield over two steps (21 mg, 31 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H), 8.92 (s, 1H), 8.57 (d, J = 2.0 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.44 (dd, J = 8.5, 7.3 Hz, 3H), 7.23 – 7.06 (m, 6H), 2.64 (q, J = 7.3 Hz, 2H), 1.11 – 1.00 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.5, 157.2, 156.9, 153.9, 148.4, 146.4, 132.2, 130.6, 129.5, 128.9, 128.8, 124.2, 119.6, 119.4, 115.1, 111.1, 17.7, 13.5; HRMS (m/z) calculated for C₂₂H₁₇N₂O₄⁻ [M−H]⁻, 373.1194; found 373.1204 (Δ 2.7 ppm).

3-ethyl-1,4-dihydroxy-6-(6-methoxynaphthalen-2-yl)-1,8-naphthyridin-2(1H)-one (4b-4)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 22 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 9.06 (dd, J = 8.9, 2.3 Hz, 1H), 8.71 (dd, J = 8.2, 2.2 Hz, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.02 – 7.82 (m, 3H), 7.39 (d, J = 2.5 Hz, 1H), 7.16 (m, 1H), 3.91 (s, 2H), 2.65 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 172.5, 160.5, 158.1, 153.9, 148.5, 134.2, 132.0, 130.2, 129.8, 129.3, 128.2, 125.7, 119.7, 115.0, 111.2, 106.3, 55.7, 49.1, 21.5, 17.8, 13.6; HRMS (m/z) calculated for C₂₁H₁₇N₂O₄⁻ [M−H]⁻, 361.1194; found 361.1213 (Δ 5.2 ppm).

3-ethyl-1,4-dihydroxy-6-phenyl-1,8-naphthyridin-2(1H)-one (4b-5)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 42 %) as an off-white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (s, 1H), 8.91 (d, J = 2.2 Hz, 1H), 8.61 (d, J = 2.3 Hz, 1H), 7.80 (apparent d, J = 8.2 Hz, 2H), 7.54 (dd, J = 8.4, 7.0 Hz, 2H), 7.44 (m, 1H), 2.67 (q, J = 7.3 Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.5, 153.9, 148.3, 146.4, 137.0, 130.2, 130.0, 129.7, 128.4, 127.2, 115.1, 111.2, 17.7, 13.5; HRMS (m/z) calculated for C₁₆H₁₅N₂O₃⁺ [M+H]⁺, 283.1077; found 283.1078 (Δ 0.35 ppm).

3-ethyl-6-(2-fluorophenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4b-6)

This compound was synthesized according to the procedure described above. Yield (22 mg, 36 %) as an off-white solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR (400 MHz, ) δ 10.58 (s, 1H), 8.80 (s, 1H), 8.50 (t, J = 1.8 Hz, 1H), 7.69 (m, 1H), 7.49 (m, 1H), 7.39 (q, J = 8.1 Hz, 2H), 2.67 (q, J = 7.3 Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.9, 160.6, 158.5, 153.8, 150.0 (d, J_CF = 2.0 Hz), 146.6, 132.1 (d, J_CF = 3.2 Hz), 131.3 (d, J_CF = 3.0 Hz), 130.7 (d, J_CF = 8.1 Hz), 125.8 (d, J_CF = 4.2 Hz), 125.2, 125.1 (d, J_CF = 13.1 Hz), 116.8 (d, J_CF = 22.1 Hz), 115.1, 110.9, 17.7, 13.5; HRMS (m/z) calculated for C₁₆H₁₄FN₂O₃⁺ [M+H]⁺, 301.0983; found 301.0986 (Δ 1.0 ppm).

3-ethyl-1,4-dihydroxy-6-(4-(trifluoromethyl)phenyl)-1,8-naphthyridin-2(1H)-one (4b-7)

This compound was synthesized according to the procedure described above. Yield over two steps (18 mg, 27 %) as an off-white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 9.02 (s, 1H), 8.67 (s, 1H), 8.07 (dd, J = 24.0, 7.9 Hz, 3H), 7.89 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 1H), 2.68 (q, J = 7.3 Hz, 2H), 1.08 (t, J = 7.3 Hz, 4H); ¹³C NMR (101 MHz, DMSO-d₆) 172.5, 160.8, 153.9 (q, J_CF = 2.0 Hz), 148.8, 147.1 (q, J_CF = 2.0 Hz), 135.1, 130.4, 128.8, 128.5, 127.9, 126.5 (q, J_CF = 2.0 Hz), 124.4 (q, J_CF = 4.1 Hz), 21.5, 13.5; HRMS (m/z) calculated for C₁₇H₁₂F₃N₂O₃⁻ [M−H]⁻, 349.0806; found 349.0816 (Δ 2.8 ppm).

3-ethyl-1,4-dihydroxy-6-(4-(methylthio)phenyl)-1,8-naphthyridin-2(1H)-one (4b-8)

This compound was synthesized according to the procedure described above. Yield over two steps (21 mg, 34 %) as an off-white solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.55 (s, 1H), 8.93 (s, 1H), 8.57 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 2.66 (q, J = 7.3 Hz, 2H), 2.54 (s, 3H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.5, 153.9, 148.3, 146.4, 138.7, 133.5, 129.6, 129.4, 127.5, 127.0, 115.0, 111.1, 17.7, 15.1, 13.5; HRMS (m/z) calculated for C₁₇H₁₅N₂O₃S⁻ [M−H]⁻, 327.0809; found 327.0821 (Δ 3.8 ppm).

6-(2,5-dimethoxyphenyl)-3-ethyl-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4b-9)

This compound was synthesized according to the procedure described above. Yield over two steps (22 mg, 34 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 8.69 (dd, J = 15.5, 2.2 Hz, 1H), 8.44 (dd, J = 14.9, 2.2 Hz, 1H), 7.10 (d, J = 9.0 Hz, 1H), 7.06 – 6.95 (m, 2H), 6.85 – 6.79 (m, 0H), 3.78 (s, 3H), 3.76 (s, 3H), 2.67 (q, J = 7.2 Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, DMSO-d₆) δ 160.3, 160.3, 154.0, 153.9, 152.0, 150.8, 149.6, 147.9, 144.7, 134.4, 128.0, 126.2, 116.6, 115.2, 114.9, 113.7, 111.5, 56.7, 56.1, 17.8, 13.5; HRMS (m/z) calculated for C₁₈H₁₉N₂O₅⁺ [M+H]⁺,343.1287; found 343.1288 (Δ 0.3 ppm).

3-ethyl-6-(3-fluoro-4-methoxyphenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4b-10)

This compound was synthesized according to the procedure described above. Yield over two steps (24 mg, 39 %) as an off-white solid; HPLC purity 95 %; ¹H NMR (400 MHz, DMSO-d₆) δ 10.59 (s, 1H), 8.98 (d, J = 2.3 Hz, 1H), 8.62 (d, J = 2.3 Hz, 1H), 7.75 (dd, J = 12.8, 2.3 Hz, 1H), 7.63 (ddd, J = 8.6, 2.3, 1.1 Hz, 1H), 7.37 (t, J = 8.8 Hz, 1H), 3.96 (s, 3H), 2.70 (q, J = 7.3 Hz, 2H), 1.12 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.5, 153.9, 153.6, 148.1, 147.4 (d, J_CF = 11.1 Hz), 146.3, 129.9 (d, J_CF = 7.1 Hz), 129.6, 128.9 (d, J_CF = 2.0 Hz), 123.4 (d, J_CF = 3.0 Hz), 115.1, 115.0 (d, J_CF = 2.1 Hz), 114.7, 114.5, 111.1, 56.6, 17.7, 13.5; HRMS (m/z) calculated for C₁₇H₁₆FN₂O₄⁺ [M+H]⁻, 331.1089; found 331.1084 (Δ 1.5 ppm).

3-ethyl-1,4-dihydroxy-6-(thiophen-3-yl)-1,8-naphthyridin-2(1H)-one (4b-11)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 28 %) as an off-white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 – 8.93 (d, J = 1.9 Hz, 1H), 8.65 – 8.59 (m, 1H), 8.03 – 7.99 (s, 1H), 7.76 – 7.59 (m, 2H), 2.70 – 2.61 (q, J = 7.2 Hz, 2H), 1.10 – 1.01 (t, J = 7.3 Hz, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.6, 155.1, 148.0, 146.3, 138.3, 129.2, 128.1, 126.4, 125.6, 121.7, 114.4, 111.6, 17.8, 13.6; HRMS (m/z) calculated for C₁₄H₁₁N₂O₃S⁻ [M − H]⁻, 287.0496; found, 287.0510 (Δ 4.8 ppm)

6-([1,1’-biphenyl]-3-yl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-1)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 41 %) as an off-white solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.77 (s, 1H), 9.10 (d, J = 2.3 Hz, 1H), 8.52 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 1.8 Hz, 1H), 7.84 – 7.69 (m, 4H), 7.67 – 7.58 (t, J = 7.6 Hz, 1H), 7.56 – 7.38 (dt, J = 38.8, 7.4 Hz, 4H), 6.00 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) 160.3, 159.5, 150.2, 148.4, 141.7, 140.4, 137.6, 130.3, 130.3, 130.2, 129.4, 128.2, 127.5, 126.8, 126.4, 125.7, 111.0, 99.5; HRMS (m/z) calculated for C₂₀H₁₅N₂O₃⁺ [M+H]⁺, 331.1077; found 331.1077 (Δ 0 ppm).

6-([1,1’-biphenyl]-4-yl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-2)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 21 %) as an off-white solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.79 (s, 1H), 9.06 (s, 1H), 8.48 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 6.00 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 172.9, 160.6, 159.7, 149.8, 148.1, 140.2, 139.8, 135.6, 129.9, 129.6, 128.2, 128.0, 127.7, 127.1, 111.2, 99.3; HRMS (m/z) calculated for C₂₀H₁₅N₂O₃⁺ [M+H]⁺, 331.1077; found 323.0626 (Δ 2.7 ppm).

1,4-dihydroxy-6-(4-phenoxyphenyl)-1,8-naphthyridin-2(1H)-one (4c-3)

This compound was synthesized according to the procedure described above. Yield over two steps (35 mg, 35 %) as an off-white solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.76 (s, 1H), 8.98 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 7.82 (m, 3H), 7.49 – 7.39 (ddd, J = 9.8, 5.9, 2.3 Hz, 3H), 7.02 (m, 7H), 5.96 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2, 159.4, 157.3, 156.8, 149.6, 148.1, 131.9, 130.6, 129.7, 129.6, 128.9, 124.3, 119.5, 119.4, 111.0, 99.5; HRMS (m/z) calculated for C₂₀H₁₅N₂O₄⁺ [M+H]⁺, 347.1024; found 347.1026 (Δ 0.3 ppm).

1,4-dihydroxy-6-(6-methoxynaphthalen-2-yl)-1,8-naphthyridin-2(1H)-one (4c-4)

This compound was synthesized according to the procedure described above. Yield over two steps (35 mg, 36 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 9.18 – 9.10 (d, J = 2.4 Hz, 1H), 8.61 – 8.54 (d, J = 2.4 Hz, 1H), 8.47 – 8.42 (d, J = 1.9 Hz, 1H), 8.28 – 8.06 (m, 3H), 7.66 – 7.58 (d, J = 9.1 Hz, 1H), 6.03 (s, 1H), 4.03 (s, 5H); 13C NMR (101 MHz, DMSO) δ 160.3, 159.5, 154.5, 150.0, 148.3, 132.4, 132.2, 130.4, 130.2, 130.0, 129.5, 127.3, 126.7, 126.3, 115.4, 111.1, 107.2, 99.6, 57.4; HRMS (m/z) calculated for C₁₉H₁₃N₂O₄⁻ [M−H]⁻, 333.0881; found 333.0886 (Δ 1.5 ppm).

1,4-dihydroxy-6-phenyl-1,8-naphthyridin-2(1H)-one (4c-5)

This compound was synthesized according to the procedure described above. Yield over two steps (18 mg, 24 %) as an off-white solid; HPLC purity 97 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s, 1H), 10.68 (s, 1H), 9.00 (s, 1H), 8.42 (s, 1H), 7.79 (d, J = 6.5 Hz, 2H), 7.53 (s, 2H), 7.44 (s, 1H), 5.99 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.3, 159.4, 149.9, 148.3, 136.8, 130.2, 129.9, 129.7, 128.4, 127.2, 111.0, 99.5; HRMS (m/z) calculated for C₁₄H₁₁N₂O₃⁺ [M+H]⁺, 255.0764; found 255.0765 (Δ 0.4 ppm).

6-(2-fluorophenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-6)

This compound was synthesized according to the procedure described above. Yield over two steps (22 mg, 30 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (s, 1H), 8.86 (s, 1H), 8.37 (s, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.55 – 7.45 (m, 1H), 7.44 – 7.33 (m, 2H), 5.99 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.94, 160.31, 159.33, 158.50, 151.2 (d, J_CF = 1.0 Hz), 148.3, 132.3 (d, J_CF = 2 Hz), 131.2 (d, J_CF = 2 Hz), 130.8 (2, J_CF = 8.2 Hz), 125.8 (d, J_CF = 3.1 Hz), 125.2, 124.8 (d, J_CF = 13.2 Hz), 116.8 (d, J_CF = 22.2 Hz), 110.8, 99.6; HRMS (m/z) calculated for C₁₄H₁₀FN₂O₃⁺ [M+H]⁺, 273.0670; found 273.0671 (Δ 0.3 ppm).

1,4-dihydroxy-6-(4-(trifluoromethyl)phenyl)-1,8-naphthyridin-2(1H)-one (4c-7)

This compound was synthesized according to the procedure described above. Yield over two steps (30 mg, 32 %) as an off-white solid; HPLC purity 97 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 10.74 (s, 1H), 9.07 (d, J = 2.3 Hz, 1H), 8.51 (d, J = 2.3 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H), 6.00 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.3, 159.4, 150.2, 148.8, 140.9, 130.5, 128.7 (q, J_CF = 2 Hz), 128.0, 126.5 (d, J_CF = 2.0 Hz), 111.0, 99.6; HRMS (m/z) calculated for C₁₅H₁₀F₃N₂O₃⁺ [M+H]⁺, 323.0638; found 323.0626 (Δ 3.7 ppm).

1,4-dihydroxy-6-(4-(methylthio)phenyl)-1,8-naphthyridin-2(1H)-one (4c-8)

This compound was synthesized according to the procedure described above. Yield over two steps (22 mg, 25 %) as an off-white solid; HPLC purity 100 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s, 1H), 9.01 – 8.95 (d, J = 2.4 Hz, 1H), 8.44 – 8.39 (d, J = 2.4 Hz, 1H), 7.79 – 7.71 (m, 2H), 7.44 – 7.36 (m, 2H), 6.00 (s, 1H), 2.55 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 159.4, 148.4, 147.5, 139.1, 139.0, 132.8, 130.4, 129.7, 127.6, 126.9, 111.5, 99.6, 15.1; HRMS (m/z) calculated for C₁₅H₁₁N₂O₃S⁻ [M−H]⁻, 299.0496; found 299.0504 (Δ 2.6 ppm).

6-(2,5-dimethoxyphenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-9)

This compound was synthesized according to the procedure described above. Yield over two steps (18 mg, 20 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.69 (s, 1H), 8.79 – 8.75 (d, J = 2.3 Hz, 1H), 8.33 – 8.29 (d, J = 2.3 Hz, 1H), 7.14 – 7.07 (d, J = 8.9 Hz, 1H), 7.01 – 6.94 (m, 2H), 5.97 (s, 1H), 3.78 (s, 3H), 3.75 (s, 1H); 13C NMR (101 MHz, DMSO-d₆) δ 160.3, 159.4, 159.4, 154.0, 151.8, 150.8, 147.8, 147.7, 132.6, 128.0, 126.8, 116.4, 114.7, 113.7, 110.4, 110.4, 99.3, 56.7, 56.0; HRMS (m/z) calculated for C₁₆H₁₅N₂O₅⁺ [M+H]⁺, 315.0975; found 315.0971 (Δ 1.3 ppm).

6-(3-fluoro-4-methoxyphenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-10)

This compound was synthesized according to the procedure described above. Yield over two steps (25 mg, 27 %) as an off-white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 9.04 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H), 7.80 (dd, J = 12.8, 2.3 Hz, 1H), 7.66 (dt, J = 8.6, 1.5 Hz, 1H), 7.37 (t, J = 8.8 Hz, 1H), 6.08 – 6.03 (s, 1H), 3.98 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2, 159.4, 156.7 (d, J_CF = 2.0 Hz), 151.2, 149.6, 148.0, 147.6 (d, J_CF = 10.0 Hz), 146.6, 129.7, 128.9 (d, J_CF = 2 Hz), 123.5 (d, J_CF = 3.0 Hz), 115.0, 114.7 (d, J_CF = 19.2 Hz), 111.0, 99.5, 56.6 (d, J_CF = 3.1 Hz; HRMS (m/z) calculated for C₁₅H₁₂FN₂O₄⁺ [M+H]⁺, 303.0772; found 303.0776 (Δ 1.3 ppm).

1,4-dihydroxy-6-(thiophen-3-yl)-1,8-naphthyridin-2(1H)-one (4c-11)

This compound was synthesized according to the procedure described above. Yield over two steps (20 mg, 27 %) as an off-white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.77 (s, 1H), 9.07 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 2.3 Hz, 1H), 8.10 (dd, J = 2.8, 1.5 Hz, 1H), 7.76 – 7.68 (m, 2H), 5.98 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 159.4, 149.2, 147.7, 137.8, 129.3, 128.2, 126.5, 125.8, 122.2, 111.1, 99.5; HRMS (m/z) calculated for C₁₂H₉N₂O₃S⁺ [M+H]⁺, 261.0328; found 261.0327 (Δ 0.4 ppm).

3-(5,8-dihydroxy-7-oxo-7,8-dihydro-1,8-naphthyridin-3-yl) benzamide (4c-12)

This compound was synthesized according to the procedure described above. Yield over two steps (15 mg, 18 %) as an off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 9.05 (dd, J = 8.9, 2.4 Hz, 1H), 8.54 (d, J = 2.4 Hz, 0.2H), 8.46 (d, J = 2.4 Hz, 0.7H), 8.27 (apparent t, J = 1.8 Hz, 1H), 8.09 – 7.89 (m, 2H), 7.71 – 7.57 (dt, J = 22.6, 7.7 Hz, 1H), 6.00 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.1, 167.6, 160.3, 159.5, 149.8, 148.4, 137.2, 136.8, 135.6, 132.2, 131.6, 130.3, 130.1, 129.7, 129.3, 127.7, 126.1, 111.1, 99.6; HRMS (m/z) calculated for C₁₅H₁₀N₃O₄⁻ [M−H]⁻, 296.0677; found 296.0686 (Δ 3.0 ppm).

6-(3-fluoro-4-methylphenyl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-13)

This compound was synthesized according to the procedure described above. Yield over two steps (18 mg, 24 %) as an off-white solid; HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s, 1H), 8.96 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 7.75 (dd, J = 7.4, 2.4 Hz, 1H), 7.64 (ddd, J = 7.9, 4.9, 2.5 Hz, 1H), 7.28 (dd, J = 9.7, 8.5 Hz, 1H), 5.99 (s, 1H), 2.34 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.2, 159.4, 149.6, 148.1, 133.0, 132.9, 130.5 (d, J_CF = 2 Hz), 130.0, 129.4, 126.5 (d, J_CF = 8 Hz), 125.6 (d, J_CF = 17.0 Hz), 116.2 (d, J_CF = 22.2 Hz), 111.0, 99.5, 14.7 (d, J_CF = 3 Hz); HRMS (m/z) calculated for C₁₅H₁₀FN₂O₃⁻ [M−H]⁻, 285.0681; found 285.0686 (Δ 1.4 ppm).

6-bromo-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (4c-14)

Yield (22 mg, 33 %); HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR 11.87 (s, 1H), 8.80 (d, J = 2.3 Hz, 0.5H), 8.74 (d, J = 2.4 Hz, 0.8H), 8.55 (d, J = 2.3 Hz, 0.6H), 8.33 (d, J = 2.4 Hz, 0.9H), 6.97 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1, 158.4, 156.9, 155.7, 151.9, 151.9, 147.8, 146.4, 134.6, 134.3, 113.0, 112.7, 112.5, 111.9, 100.1, 98.9; HRMS (m/z) calculated for C₈H₄BrN₂O₃⁻ [M−H]⁻, 254.9411; found 254.9428 (Δ 6.6 ppm).

Characterizations for subtype 5 analogs

5-([1,1’-biphenyl]-3-yl)-1,4-dihydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (5–1)

This compound was synthesized according to the procedure described above. Yield (25 mg, 51 %); ¹H NMR (400 MHz, DMSO-d₆) δ 9.79 (s, 1H), 8.65 (d, J = 4.9 Hz, 1H), 7.76 – 7.58 (m, 5H), 7.47 (dd, J = 10.7, 5.5 Hz, 4H), 7.38 (dt, J = 10.1, 8.1 Hz, 5H), 7.32 – 7.24 (m, 3H), 7.18 (d, J = 4.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.4, 156.0, 149.7, 149.7, 148.8, 142.3, 140.4, 139.6, 132.9, 131.7, 129.8 129.4, 129.2, 128.5, 128.4, 128.0, 127.9, 127.3, 127.1, 126.1, 122.1, 114.1, 109.0; HRMS (m/z) calculated for C₂₆H₁₉N₂O₃⁺ [M + H]⁺, 407.1390; found 407.1389 (Δ 0.2 ppm).

1,4-dihydroxy-5-(4-(methylthio)phenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (5–2)

This compound was synthesized according to the procedure described above. Yield (95 mg, 53 %); ¹H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 8.61 (dd, J = 16.6, 5.0 Hz, 1H), 7.54 – 7.40 (m, 5H), 7.40 – 7.28 (m, 2H), 7.26 – 7.21 (m, 2H), 7.10 (d, J = 5.1 Hz, 1H), 6.84 (m, 2H), 6.80 (s, 1H), 2.53 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 192.3, 167.3, 153.4, 153.0, 151.2, 140.0, 137.2, 133.8, 131.7, 129.9, 129.6, 128.3, 126.6, 125.8, 122.2, 113.7, 85.5, 14.7; HRMS (m/z) calculated for C₂₁H₁₇N₂O₃S⁺ [M + H]⁺, 377.0955; found 377.0954 (Δ 0.3 ppm).

5-([1,1’-biphenyl]-3-yl)-1,4-dihydroxy-1,8-naphthyridin-2(1H)-one (5–3)

This compound was synthesized according to the procedure described above. Yield (0.13 g, 57 %); HPLC purity 100 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.28 (s, 1H), 9.61 (s, 1H), 8.65 (d, J = 5.0 Hz, 1H), 7.75 – 7.67 (m, 3H), 7.64 (t, J = 1.7 Hz, 1H), 7.55 – 7.42 (m, 3H), 7.37 (tdd, J = 7.6, 2.2, 1.2 Hz, 2H), 7.21 (d, J = 5.0 Hz, 1H), 5.88 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.8, 159.1, 140.4, 140.3, 140.2, 139.8, 139.6, 129.4, 129.4, 128.6, 128.5, 128.1, 128.1, 127.9, 127.8, 127.3, 127.3, 127.3 127.0, 126.6, 121.7, 110.0, 100.0; HRMS (m/z) calculated for C₂₀H₁₅N₂O₃₊ [M+H]⁺, 331.1077; found 331.1076 (Δ 0.3 ppm)

1,4-dihydroxy-5-(4-(methylthio)phenyl)-1,8-naphthyridin-2(1H)-one (5–4)

This compound was synthesized according to the procedure described above. Yield (80 mg, 58 %); HPLC purity 95 % ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR (400 MHz, DMSO-d₆) δ 11.14 (s, 1H), 8.61 (d, J = 4.9 Hz, 1H), 7.34 – 7.24 (m, 4H), 7.05 (d, J = 4.9 Hz, 1H), 5.84 (s, 1H), 2.52 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.7, 159.3, 151.0, 148.6, 147.8, 138.7, 136.7, 129.6, 125.0, 121.5, 109.1, 99.7, 15.0; HRMS (m/z) calculated for C₁₅H₁₁N₂O₃S⁻ [M−H]⁻, 299.0496; found 299.0508 (Δ 4.0 ppm)

6-([1,1’-biphenyl]-3-yl)-1-hydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (6–1)

This compound was synthesized according to the procedure described above. Yield (65 mg, 53 %); HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (d, J = 2.2 Hz, 1H), 8.74 (d, J = 2.2 Hz, 1H), 8.20 (s, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.85 – 7.71 (m, 7H), 7.64 (t, J = 7.7 Hz, 1H), 7.56 – 7.38 (m, 7H); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.6, 149.2, 147.5, 141.7, 140.4, 137.5, 136.2, 135.5, 134.3, 133.1, 131.1, 130.4, 129.5, 129.3, 128.9, 128.7, 128.2, 127.5, 126.9, 126.2, 125.6, 115.0; HRMS (m/z) calculated for C₂₆H₁₉N₂O₂⁺ [M+H]⁺, 391.1441; found 391.1438 (Δ 0.7 ppm).

1-hydroxy-6-(3-(methylthio)phenyl)-3-phenyl-1,8-naphthyridin-2(1H)-one (6–2)

This compound was synthesized according to the procedure described above. Yield (55 mg, 57 %); HPLC purity 99 %; ¹H NMR (400 MHz, DMSO-d₆) δ ¹H NMR (400 MHz, DMSO) δ 9.01 (d, J = 2.3 Hz, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.19 (s, 1H), 7.80 – 7.74 (m, 4H), 7.55 – 7.40 (m, 5H), 2.55 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.5, 148.8, 147.3, 138.9, 136.2, 134.7, 134.2, 133.1, 133.1, 130.6, 129.3, 128.9, 128.7, 127.5, 127.0, 115.0, 15.1; HRMS (m/z) calculated for C₂₁H₁₇N₂O₂S⁺ [M+H]⁺, 361.1005; found 361.0990 (Δ 4.2 ppm).

DHN subtype 7 analogs 7–1 – 7–5 were obtained as side products from the Suzuki reaction while synthesizing intermediate 12b. Compound 7–6 was obtained as a side product from the condensation reaction to yield intermediate 11b from 10b. Compounds 7–7 and 7–8 resulted from the over-reduction of the benzyl group in intermediate 12a with Pd/C.

Below is the characterization of compounds for subtype 7.

3-ethyl-4-hydroxy-6-(6-methoxynaphthalen-2-yl)-1,8-naphthyridin-2(1H)-one (7–1)

Yield (8 mg, 12 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s, 1H), 10.46 (s, 1H), 8.92 (d, J = 2.2 Hz, 1H), 8.60 (dd, J = 14.9, 3.7 Hz, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.00 – 7.86 (m, 2H), 7.43 – 7.36 (m, 1H), 7.23 (dd, J = 9.0, 2.4 Hz, 1H), 3.92 (s, 3H), 2.63 (d, J = 7.4 Hz, 2H), 1.03 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.3, 158.0, 156.2, 150.1, 148.6, 136.6, 134.1, 132.4, 130.2, 130.1, 129.3, 129.2, 128.1, 125.6, 125.5, 124.4, 119.7, 114.9, 111.3, 106.3, 55.7, 17.0, 13.6; HRMS (m/z) calculated for C₂₁H₁₉N₂O₃⁺ [M+H]⁺, 347.1391; found 347.1390 (Δ 0.3 ppm).

3-ethyl-6-(3-fluoro-4-methoxyphenyl)-4-hydroxy-1,8-naphthyridin-2(1H)-one (7–2)

Yield (12 mg, 20 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.81 (s, 1H), 10.47 (s, 1H), 8.80 (dd, J = 2.4, 0.9 Hz, 1H), 8.41 (d, J = 2.4 Hz, 1H), 7.71 (dt, J = 12.8, 2.4 Hz, 1H), 7.57 (dtd, J = 8.5, 2.5, 1.1 Hz, 1H), 7.32 (m, 1H), 3.94 (s, 3H), 2.64 (q, J = 7.3 Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.3, 156.2, 153.6, 151.2, 148.3 (d, J_CF = 19.2 Hz), 147.4 (q, J_CF = 11.1 Hz), 147.3, 130.4 (d, J_CF = 11.1 Hz), 129.4, 129.0, 128.7 (d, J_CF = 2.1 Hz), 128.3 (q, J_CF = 2 Hz), 114.9, 114.6 (q, J_CF = 19.2 Hz), 111.1, 56.6, 16.9, 13.5; HRMS (m/z) calculated for C₁₇H₁₆FN₂O₃⁺ [M+H]⁺, 315.1135; found 315.1139 (Δ 1.3 ppm).

3-ethyl-4-hydroxy-6-(4-(trifluoromethyl)phenyl)-1,8-naphthyridin-2(1H)-one (7–3)

Yield (15 mg, 25 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (s, 1H), 10.59 (s, 1H), 8.93 (q, J = 2.5 Hz, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.05 (t, J = 5.0 Hz, 2H), 7.91 (m, 2H), 2.66 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.4, 156.2, 148.8 (d, J_CF = 10 Hz), 141.5, 129.8, 128.9 (q, J_CF = 8.0 Hz), 128.5 (q, J_CF = 32.2 Hz), 128.4, 128.9, 127.7, 126.4 (q, J_CF = 2 Hz), 126.1, 115.0, 111.4, 16.9, 13.5; HRMS (m/z) calculated for C₁₇H₁₄F₃N₂O₂⁺ [M+H]⁺, 335.1004; found 335.1003 (Δ 0.15 ppm).

3-ethyl-6-(2-fluorophenyl)-4-hydroxy-1,8-naphthyridin-2(1H)-one (7–4)

Yield (15 mg, 28 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.89 (s, 1H), 10.55 (s, 1H), 8.70 (t, J = 1.9 Hz, 1H), 8.46 (dd, J = 2.3, 1.3 Hz, 1H), 7.70 (td, J = 7.8, 1.7 Hz, 1H), 7.57 – 7.47 (m, 1H), 7.47 – 7.36 (m, 2H), 7.33 – 7.25 (m, 0H), 2.64 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.4, 160.9, 158.4, 156.0, 149.9, 149.9, 148.3, 131.6 (d, J_CF = 2.0 Hz), 131.2 (d, J_CF = 2.0 Hz), 130.5 (q, J_CF = 9.2 Hz), 129.4, 128.7, 125.8, 125.7 (d, J_CF = 9.1 Hz), 125.4 (d, J_CF = 13.1 Hz), 125.0, 116.7 (d, J_CF = 22.2 Hz), 114.9, 111.1, 16.9, 13.5; HRMS (m/z) calculated for C₁₆H₁₄FN₂O₂⁺ [M+H]⁺, 285.1034; found 285.1034 (Δ 0 ppm).

3-ethyl-4-hydroxy-6-(4-phenoxyphenyl)-1,8-naphthyridin-2(1H)-one (7–5)

Yield (10 mg, 15 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.63 (s, 1H), 8.75 (s, 1H), 8.50 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.43 (dd, J = 8.6, 7.3 Hz, 3H), 7.16 – 7.04 (m, 5H), 2.57 (d, J = 7.4 Hz, 3H), 1.05 (t, J = 7.3 Hz, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.4, 157.2, 157.0, 156.9, 148.2, 148.1, 132.7, 131.0, 130.9, 130.6, 130.3, 129.3, 129.1, 129.0, 128.8, 128.8, 128.7, 124.2, 119.6, 119.3, 29.5, 29.2, 13.6; HRMS (m/z) calculated for C₂₂H₁₇N₂O₃⁻ [M−H]⁻, 357.1245; found 357.1255 (Δ 2.8 ppm).

6-bromo-3-ethyl-4-hydroxy-1,8-naphthyridin-2(1H)-one (7–6)

Yield (55 mg, 6 %) as off-white solid; ¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 10.58 (s, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.39 (d, J = 2.4 Hz, 1H), 2.57 (q, J = 7.4 Hz, 2H), 1.02 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.2, 155.1, 150.4, 147.7, 133.5, 115.5, 113.0, 111.9, 17.0, 13.4; HRMS (m/z) calculated for C₁₀H₈BrN₂O₂⁻ [M−H]⁻, 266.9775; found 266.9786 (Δ 4.1 ppm).

6-(3,4-dimethoxyphenyl)-4-hydroxy-3-phenyl-1,8-naphthyridin-2(1H)-one (7–7)

Yield (15 mg, 32 %) as white solid; HPLC purity 96 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.90 – 11.85 (s, 1H), 8.88 – 8.83 (d, J = 2.3 Hz, 1H), 8.58 – 8.53 (d, J = 2.3 Hz, 1H), 7.48 – 7.38 (m, 4H), 7.38 – 7.25 (m, 3H), 7.13 – 7.07 (d, J = 8.4 Hz, 1H), 3.91 – 3.86 (s, 3H), 3.84 – 3.80 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6, 149.8, 149.3, 149.2, 148.3, 131.6, 130.2, 129.4, 128.1, 127.5, 119.3, 112.9, 110.8, 56.2, 56.1; HRMS (m/z) calculated for C₂₂H₁₇N₂O₄⁻ [M−H]⁻, 373.1194; found 373.1217 (Δ 6.3 ppm).

4-hydroxy-3-phenyl-6-(p-tolyl)-1,8-naphthyridin-2(1H)-one (7–8)

Yield (41 mg, 87 %) as white solid; HPLC purity 98 %; ¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 10.53 (s, 1H), 8.87 – 8.82 (d, J = 2.3 Hz, 1H), 8.60 – 8.52 (d, J = 2.3 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.45 – 7.29 (m, 7H), 2.37 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.7, 157.1, 149.1, 148.6, 137.7, 134.3, 133.5, 131.6, 130.3, 130.1, 129.6, 128.2, 127.5, 126.8, 114.1, 111.2, 21.2; HRMS (m/z) calculated for C₂₁H₁₇N₂O₂⁺ [M + H]⁺, 329.1285; found 329.1281 (Δ 1.2 ppm).

Biology

FRET-based Mpr activity assays

Substrate and enzyme (Mpr bearing an N-terminal maltose binding protein fusion) for biochemical assays were prepared as previously described³³. All FRET assays described below were carried out in a buffer composed of 20 mM Tris-HCl (pH 8.5 at 22 °C), 50 mM NaCl, 10 mM MgCl₂, 5% v/v glycerol, 5 mM β-mercaptoethanol, 0.0025% v/v Tween 20, 0.5% DMSO (“assay buffer” hereafter). Reactions were assembled in black 384-well plates (Corning 3575) using final concentrations of 5 nM FRET substrate and 10 nM Mpr. For each FRET assay, fluorescence emission at 535 nm (excitation at 485 nm) was measured using a SpectraMax i3 plate reader (Molecular Devices) for 15 min at 37 °C after the addition of substrate to initiate the reactions. The rate of fluorescence increase was determined from the linear phase of each timecourse and expressed as a percentage of the rate observed in the presence of a matched concentration of DMSO (vehicle). All assays were performed twice in duplicate.

Initial screening of analogs was performed at a final concentration of 50 μM compound. Stock solutions of each compound were prepared in DMSO and working dilutions were prepared in assay buffer immediately before use. Compounds were combined with Mpr in the assay plate and incubated for 15 min at 37 °C before adding substrate to initiate the assay.

Compounds that produced >50% apparent inhibition of Mpr in initial screening were tested to ensure that they did not directly quench 6-FAM fluorescence. Compounds (50 μM final concentration) were diluted in assay buffer, added to the assay plate, and incubated for 15 min at 37 °C. The 6-FAM-labeled substrate oligonucleotide (5 nM final concentration) was added, and fluorescence emission was recorded after 0, 15, and 30 min incubation at 37 °C. Compounds that suppressed 6-FAM fluorescence by more than three standard deviations relative to the vehicle mean (~20% inhibition) were excluded from further analysis.

For assays to determine inhibitor potency, compounds were serially diluted in assay buffer and incubated with Mpr for 15 min at 37 °C before addition of substrate. Inhibition curves were fit in GraphPad Prism (version 10.6) using a four-parameter inhibition model with the upper plateau fixed at 100% and the lower plateau fixed at 0%. If −1.0 was contained within the 95% confidence interval for the fitted Hill slope, then the slope was fixed at −1.

All compounds selected for concentration-response analysis were also screened for the possibility of non-specific Mpr inhibition mediated by colloidal aggregation⁴⁰. Compounds were diluted in assay buffer to 1.67x their target concentration (IC₇₅ - IC₈₀ final concentration) and split into two aliquots. One aliquot was centrifuged for 20 min at 13,000 g prior to addition to the assay plate; the other aliquot was incubated for 20 min at room temperature before adding to the assay plate. Compounds were incubated with Mpr for 15 min at 37 °C prior to substrate addition. Mean inhibition produced by centrifuged and non-centrifuged compound stocks was compared using a two-tailed t-test, and compounds were flagged if this method found a significant difference between the two mean values.

Antiviral assays

The half maximal effective concentration (EC₅₀) for inhibition of viral replication was determined by using a recombinant VACV encoding secreted Gaussia luciferase under a viral late promoter (vLGluc)³⁸ using the protocol described elsewhere³⁷. Briefly, human foreskin fibroblasts (HFFs) were grown in 96-well plates and infected with vLGluc at an MOI of 0.01in the presence of either DMSO or varying concentrations of serially diluted test compounds. After 24 h post-infection the Gaussia luciferase activity was measured from the cell supernatant using a luminometer. The EC₅₀ value was determined by non-linear regression analysis with the “log(inhibitor) vs. normalized response – variable slope” model using GraphPad Prism (version 10.3.1).

Cytotoxicity Assays

The 50% cytotoxic concentration (CC₅₀) was determined with a CCK-8 assay following the manufacturer’s instructions. Briefly, HFFs were seeded in 96-well plates and treated with DMSO or indicated compounds at various concentrations. After 24 h incubation, CCK-8 reagent (10 μL) was added to each well, and the cells were incubated for 3 h at 37 °C. The absorbance at 450 nm was measured for each well using a BioTek Cytation 5 imaging reader. CC₅₀ values were determined by non-linear regression analysis with the “log(inhibitor) vs. normalized response – variable slope” model using GraphPad Prism (version 10.3.1).

Computational methods

To model the structure of Mpr in the absence of reported experimental structure, we used AlphaFold 3⁴¹ to obtain structural model using the same procedure outlined in our previous work³³ with the only difference of putting 4 Mg cofactors instead of 2 to mimic the biologically relevant number of cofactors. The AlphaFold structures were prepared using the protein preparation workflow⁴² in Schrödinger Maestro, which includes filling of missing proteins and optimization of the side chain network. The resulting hydrogen bond network was optimized, and the heavy atoms were converged to root mean squared deviation of 0.3 Å using the OPLS4 force field⁴³.

Molecular docking of the ligands (3 and 5–1) was performed using the AlphaFold 3 model as described above. The position of the citric acid placeholder was used as the center of the binding site. The receptor grid was generated with box size of edge length 10Å centered at the placeholder ligand. The ligand structures were generated using LigPrep at pH of 7.4 ± 2. (LigPrep, Schrödinger, LLC, New York, NY, 2025.) The ligands were docked to the receptor grid using Glide (Glide, Schrödinger, LLC, New York, NY, 2025.) with van der Waals radii scaling factor of 0.8⁴⁴. Molecular visualization was performed with PyMOL (PyMOL Molecular Graphics System, Schrödinger, LLC, New York, NY, 2025.).

Relative binding free energy calculation was performed with FEP+ (FEP+, Schrödinger, LLC, New York, NY, 2025.) Web Service from the Schrödinger Suite 2025–3⁴⁵. The docked structures of 3 and 5–1 were used as the initial structure for free energy calculation. The OPLS4 force field was used to model the system⁴³. The number of λ windows was set to be 12. For each window, the production MD was run for 5 ns for complex, solvent, and vacuum legs in the μVT ensemble. The complex system was built with SPC water model with buffer size of 5Å.

ADME assays

Aqueous solubility assay

The aqueous solubility of each analyte was quantitatively assessed in Dulbecco’s Phosphate-Buffered Saline (DPBS, pH 7.2). Initially, each compound was dissolved in dimethyl sulfoxide (DMSO) to achieve a 100 mM stock solution. Subsequently, 5 μL of this stock was added to 495 μL of DPBS to form a supersaturated solution. The resulting suspension was equilibrated at ambient temperature for 2 h which is then underwent filtration using a 0.45 μm PVDF syringe filter. The filtrate was subjected to quantitative analysis by liquid chromatography-mass spectrometry (LC-MS/MS) employing an Agilent 1260 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled to an AB Sciex QTrap 5500 mass spectrometer (AB Sciex LLC, Toronto, ON, Canada).

Plasma stability assay

Plasma stability of compounds was conducted by incubating each test compound at a final concentration of 1 μM in human plasma (Innovative Research, Novi, MI, USA), which was diluted to 80% with 0.1 mol/L potassium phosphate buffer (pH 7.4) and maintained at 37°C. At predetermined intervals (0, 1, 3, 6, and 24 h), 50 μL aliquots of the plasma mixture were withdrawn and immediately quenched with 200 μL of acetonitrile containing 0.1% formic acid. The resulting mixtures were subjected to vortex mixing, followed by centrifugation at 15,000 rpm for 5 min (Thermo Scientific Sorvall ST 8R, NI, Germany). Supernatants were subsequently collected and analyzed by LC-MS/MS for determination of the in vitro plasma half-life (t_1/2).

Microsomal stability assay

Microsomal stability (Phase I) assays were performed in triplicate utilizing commercially sourced human liver microsomes (Sekisui XenoTech, Kansas City, KS, USA), with nicotinamide adenine dinucleotide phosphate (NADPH) provided as a metabolic cofactor. In brief, test compounds were spiked at a final concentration of 1 μM into reaction mixtures comprising liver microsomal protein (0.5 mg/mL final concentration) and MgCl₂ (1 mM final concentration) in 0.1 M potassium phosphate buffer (pH 7.4). The metabolic reactions were initiated by the addition of NADPH (1 mM final concentration) and maintained at 37°C. Negative control reactions lacking NADPH were ran in parallel to evaluate possible chemical instability or NADPH-independent degradation. In-house standard (verapamil) served as a positive control to validate microsomal activity and incubation conditions. At predetermined time points, 50 μL aliquots were withdrawn and quenched with 200 μL acetonitrile containing 0.1% formic acid, followed by vortex-mixing and centrifugation at 15,000 rpm for 5 min at 4°C. Supernatants were subjected to LC-MS/MS analysis to determine the in vitro metabolic half-life (t_1/2).

Parallel artificial membrane permeability assay (PAMPA)

To predict passive transcellular membrane permeability, selected compounds was assessed utilizing the artificial membrane of Corning^® BioCoat^™ Pre-coated PAMPA Plate System (catalog No. 353015, Corning, Glendale, AZ, USA). The plate assembly stored at −20°C were equilibrated to room temperature for 30 min prior to use. Permeability assays were proceeded according to the manufacturer’s instructions. In brief, the 96-well filter plate, coated with lipids, served as the permeation acceptor, while a corresponding 96-well receiver plate functioned as the permeation donor. Test compounds were prepared by diluting 10 mM DMSO stock solutions to a final concentration of 10 μM in 10% methanol in DPBS. A volume of 300 μL of the compound solution was dispensed into each well of the receiver plate, and 200 μL of 10% methanol in DPBS was added to each well of the pre-coated filter plate. The filter plate was then aligned with the receiver plate, and the assembly was kept at ambient temperature without agitation for 5 h. Upon completion of the incubation, the plates were separated, and compound concentrations in both donor and acceptor wells were quantified by LC-MS/MS analysis. Permeability of the compounds were calculated using Eq. (1):

$$Pe=\left\{-ln\left[1-C_A\left(t\right)/C_{eq}\right]/\left[A\times \left(1/V_D+1/V_A\right)\times t\right]\right.$$ (1)

where $\mathrm{A}$ = filter area (0.3 cm²), ${\mathrm{V}}_{\mathrm{D}}$ = donor well volume (0.3 mL), ${\mathrm{V}}_{\mathrm{A}}$ = acceptor well volume (0.2 mL), $t$ = incubation time (seconds), ${\mathrm{C}}_{\mathrm{A}}\left(t\right)$ = compound concentration in acceptor well at time t, ${\mathrm{C}}_{\mathrm{D}}\left(t\right)$ = compound concentration in donor well at time $t$, and ${\mathrm{C}}_{\mathrm{e}\mathrm{q}}$ was calculated using Eq. (2):

$$C_{eq}=\left[C_D\left(t\right)\times V_D+C_A\left(t\right)\times V_A\right]/\left(V_D+V_A\right)$$ (2)

A cutoff criteria of $Pe$ value at 1.5 × 10⁻⁶ cm/s was used to classify the compounds into high and low permeability according to the literature report of this PAMPA plate system ⁴⁶.

Supplementary Material

ASSOCIATED CONTENT

Supporting Information Available. ¹H ,¹³C NMR spectra, HRMS, and HPLC traces of subtypes 4, 5, 6 and 7, PDB coordinates for docked compounds 3 and 5–1, molecular formula strings (CSV). This material is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS USED

ADME

absorption, distribution, metabolism, and excretion

PAMPA

Parallel artificial membrane permeability assay (PAMPA)

FP

fluorescence polarization

Fpr

fowlpox resolvase

FRET

Förster resonance energy transfer

FQ

fluorescence quenching

AG

aggregation

HFFs

human foreskin fibroblasts

DHN

1-hydroxy-1,8-napthyridinone

SAR

structure-activity relationship

SI

selectivity index

HJ

Holliday junction

Mpr

mpox resolvase

MPXV

mpox virus

RNHL

ribonuclease H-like

VACV

vaccinia virus

VARV

variola virus