Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox

ABSTRACT

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 μM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

INTRODUCTION

Poxviridae is a family of large, double-stranded DNA viruses that contains a wide-range collection of infectious agents (1). These viruses infect a broad spectrum of organisms but have a limited cellular tropism. Within this family, the genus Orthopoxvirus contains several human pathogens, including monkeypox, cowpox, and variola virus, the causative agent of smallpox. When in circulation, smallpox was highly infectious with high rates of mortality (up to 30%) (2). It is estimated that the death toll from infection reached hundreds of millions (3). Smallpox was eradicated from circulation following a worldwide vaccination campaign led by the World Health Organization (WHO). Smallpox remains an agent of concern because of its potential use as a bioweapon along with recent experiments suggesting a direct path to reconstituting this pathogen (4). Two vaccines (vaccinia virus; ACAM2000, Jynneos) and two antivirals (TPOXX and TEMBEXA) are approved for use against smallpox by the U.S. Food and Drug Administration (FDA). Antivirals have only been tested on human cases of vaccinia, monkeypox, or cowpox infections as smallpox was eradicated before the development of these drugs. While prophylactic vaccination is highly effective, historic data suggest vaccination use in a post-exposure setting may be limited and should be provided within 3 days (5). Widespread prophylactic vaccination is unlikely, making antivirals targeting poxviruses the most likely first-line defense.

Orthopoxviruses, other than smallpox, continue to circulate worldwide. Worldwide orthopoxvirus immunity has waned following the near-universal cessation of vaccinia vaccination (6), and there has been a concomitant increase in the incidence of orthopoxviral disease. Particularly notable is monkeypox, whose incidence has increased markedly since 1970 (7) with reports in 15 countries (8, 9). Recent export cases highlight the global threat that the monkeypox virus (MPXV) poses. With more than 44,000 monkeypox cases reported worldwide at the time of submission (over 10,000 in the US alone), the use of FDA-approved smallpox antivirals to treat human monkeypox cases may play an important role in controlling the outbreak (10). Currently, more than 2,000 cases of MPXV are estimated annually in the area of endemicity of the Democratic Republic of Congo alone, with other countries reporting cases at increasing rates (11). Against this background of increasing infection and the need for treatment for orthopoxviruses, antiviral treatment is suggested to be more effective than vaccination in acute outbreaks (12).

To be prepared for the effective treatment of orthopoxviral disease, the WHO Advisory Committee on Variola Virus Research (ACVVR) has recommended having at least two antivirals stockpiled before the destruction of the remaining smallpox stocks. Of several investigational drugs used to treat cases of orthopoxvirus infection, two are approved by the FDA for use under the Animal Rule (13–15). Treatment of an orthopoxvirus infection with a single antiviral has resulted in resistance (16), suggesting a multitherapeutic treatment would be most effective. Multitherapeutic treatment has been reported to be more successful in animal models compared to single antiviral treatment (17). To this end, we have continued to develop a unique set of chemical compound inhibitors that potentially offer an orthogonal approach to limit poxviral replication with targets other than that of TPOXX or TEMBEXA.

We previously reported a novel, nonnucleosidal pyridopyrimidinone (PDPM), CMLDBU6128 (1) (Fig. 1), with broad spectrum antipoxviral activity and limited toxicity to host cells (18). CMLDBU6128 (1) was originally identified as an inhibitor of late poxviral gene expression in a medium-throughput (~2000 compound) screen using a dual-reporter recombinant vaccinia virus engineered to express the Cherry (red) fluorescent protein late in replication and the Venus (yellow) fluorescent protein early in replication. In this manuscript, we utilized the late Venus expressing vaccinia exclusively for follow-up compound screening. Here, we described our preliminary, exploratory medicinal chemistry efforts around this antiviral chemotype, and our work to further characterize the broad activity of improved PDPMs against orthopoxviruses, including variola virus. This work highlighted the capability of utilizing medicinal chemistry on an existing chemical structure as the basis for further development of novel therapeutics against orthopoxviruses.

FIG 1.

Chemical structure of CMLDBU6128 (1) and overview of exploratory PDPM analog libraries based on variation at C3 (R¹), C7 (R²), and N2 (R³). The red numbers represent sites of potential modifications described in this study.

RESULTS

Chemical synthesis and testing of a 23-member PDPM analog library.

Using CMLDBU6128 (1) as a starting point, we initially sought to explore the effects of minor structural modifications on antipoxviral activity, with the parallel goals of elucidating preliminary trends in structure-activity relationships (SAR) and identifying more potent inhibitors. We synthesized a 23-member exploratory library of analogs to probe modifications at three sites of diversification (Fig. 1; R¹, R², and R³). PDPMs are produced from the gold-catalyzed cyclization of alkyne-tethered dihydropyrimidinones (DHPMs) (5) (Fig. 2, Equation 1) (19). DHPMs, the products of the multicomponent Biginelli condensation (20–22), exhibit a wide array of biological activities (23) and are a privileged scaffold in medicinal chemistry (24, 25). The Biginelli reaction enables facile diversification at the PDPM C3 position (R¹) via variation of the original input aldehyde (3). Additional diversity can be accessed at the PDPM C6 position (R²) by varying the pendant group on the urea-tethered alkyne (2). In previous studies, we found that the gold-catalyzed cycloisomerization generally requires disubstituted (nonterminal) alkynes; however, C6-unsubstituted (R² = H) PDPMs (such as CMLDBU6128) can be obtained from trimethylsilylated precursors (6b-v), via Biginelli condensations with trimethylsilylpropargyl urea (2a), (Fig. 2, Equation 2), 2,4-pentanedione (4), and various aldehydes (3). The vinyl silane PDPM products can be subsequently protodesilylated with silver(I) fluoride to produce the R²-unsubstituted DHPMs (1) and (10 to 27) (see Table 1 and 2 for structures). Lastly, we introduced additional diversity at the N2 (R³) position via simple alkylation chemistry (compounds 8 to 10; Fig. 2, Equation 3). All the processes were robust and amenable to parallelization. Using this chemistry, we prepared a 23-member exploratory SAR library (6a, 6b, 7 to 27) for testing in our vaccinia reporter system (Table 1 and 2). Initial potency assessments were made using a single 8-point dose-response assay, with the highest potency inhibitors carried forward to validation experiments performed in quadruplicate.

FIG 2.

Synthesis of pyridopyrimidinones (PDPMs) used in this study via Biginelli multicomponent reaction followed by gold-catalyzed cycloisomerization (Equation 1). Variation of the input aldehyde (3) afforded a variety of PDPM analogs (1) and (10 to 27) (Equation 2). A limited number of N-alkylation reactions gave analogs (7 to 9) (Equation 3).

TABLE 1.

Early SAR exploring PDPM core substitutions at sites R¹ & R³

Entry	Compound	R1	R2	R3	Vaccinia LV IC50 (μM)
1	1	-H	-Ph	-H	~5.9
2	6a	-H	-Ph	-CH3	>10.0
3	6b	-H	-Ph	-Si(CH3)3	>10.0
4	7	-Me	-Ph	-H	~4.0
5	8	-Et	-Ph	-H	>10.0
6	9	-allyl	-Ph	-H	~10.0

TABLE 2.

Structure-activity relationships for R2 phenyl ring substitutions

Entry	Compound	X	Y	Z	Vaccinia LV IC50 (μM)
1	1	-H	-H	-H	~5.9
2	10	-F	-H	-H	>10.0
3	11	-Cl	-H	-H	1.7
4	12	-Br	-H	-H	0.3
5	13	-CH3	-H	-H	1.2
6	14	-OCH3	-H	-H	2.3
7	15	-CF3	-H	-H	1.8
8	16	-H	-F	-H	0.1
9	17	-H	-Cl	-H	9.0
10	18	-H	-Br	-H	1.4
11	19	-H	-CH3	-H	1.0
12	20	-H	-OCH3	-H	>10.0
13	21	-H	-CF3	-H	1.0
14	22	-H	-H	-F	6.4
15	23	-H	-H	-Cl	2.5
16	24	-H	-H	-Br	>10.0
17	25	-H	-H	-CH3	>10.0
18	26	-H	-H	-OCH3	1.1
19	27	-H	-H	-CF3	1.5

From the initial subset of CMLDBU6128 analogs (Table 1), we found that introduction of substituents at C6 (methylated 6a, trimethylsilylated 6b) fully eliminated activity, whereas N2-alkylation resulted in similar (compound 7) or suppressed (compounds 8 and 9) antiviral potency compared to the parental compound (1). Based on these observations, for efficiency and ease of parallel synthesis, we opted to focus most subsequent efforts on exploring C3 substitutions in the context of an N2/C6-unsubstituted scaffold (Table 2), which was most easily diversified.

We next explored methodical mono-substitution of the ortho (X), meta (Y), and para (Z) positions of the C3 phenyl ring at R¹ (compounds 10 to 27, Table 2). From this set, we observed broad tolerability for aryl ring substitution, identifying several compounds with comparable antipoxviral potency compared to (1). The inhibitory concentration to reduce 50% late venus (LV) expression (IC₅₀) values ranged in the single digit micromolar concentration. In addition, we identified two analogs with significantly improved potencies: ortho-brominated (12), and meta-fluorinated (16), with IC₅₀ values of 300 nM and 100 nM, respectively. Some limited nascent SAR trends also emerged, such as a preference for lipophilic methyl and/or halogen substituents versus the more polar methoxy group at the meta position, and an apparent strong sensitivity to the nature of the substituent at the para position. However, other SAR patterns are less clear, and more exhaustive medicinal chemistry exploration of this ring system is warranted.

A compiled summary of key physicochemical properties, activity, and ligand efficiency indices for each of the most potent inhibitors is provided in Table 3. Screening hit CMLDBU6128 (1) has a molecular weight of 268 Da and comports to the “Rule-of-three” (MW < 300 Da, log(P) ≤ 3, hydrogen bond-acceptors < 3) (26) typically applied in high-throughput screening (HTS) and fragment-based drug discovery (FBDD) to assess “lead-like” qualities of very small molecules (27–30). During optimization of low-molecular-weight molecules toward a more “drug-like” chemical space, molecular weight typically increases. Careful monitoring of such metrics as ligand efficiency (LE) (31) and lipophilic ligand efficiency (LLE or LipE) (32, 33) can help to determine whether gains in potency track with increased lipophilicity (33–36). From this analysis, potent poxviral inhibitors (12) and (16) exhibited improved (higher) values for LE and LLE compared to (1) and all other active analogs, suggesting that the observed potency gains are disassociated from lipophilicity increases, despite the apparent preference for lipophilic substituents. Based on these results, we progressed (16) and (12) as promising leads for further study.

TABLE 3.

Physicochemical properties and efficiency indices for 1 and active analogs

Entry	Compound	MW	H-Bond acceptors	Heavy atoms	CLogPa	pIC50	LEb	LLE/LipEc
1	1	268.1	2	20	1.3	5.2	0.36	3.9
2	11	302.8	2	21	1.9	5.8	0.38	3.9
3	12	347.2	2	21	2.1	6.5	0.43	4.4
4	13	282.3	2	21	1.9	5.9	0.39	4.0
5	16	286.3	2	21	1.5	7.0	0.47	5.5
6	18	347.2	2	21	2.1	5.9	0.39	3.8
7	19	282.3	2	21	1.9	6.0	0.40	4.1
8	21	336.3	2	24	2.2	6.0	0.35	3.8
9	23	302.8	2	21	1.9	5.6	0.37	3.7
10	27	336.3	2	24	2.2	5.8	0.34	3.6

^aCalculated using JChem version 20.16.0.12094 (ChemAxon, Ltd.).

^bLE = (1.4*pIC₅₀)/[heavy atoms].

^cLLE/LipE = pIC₅₀ – C log(P).

In Fig. 3, the dose-response curves for these compounds are shown. In experiments using a low multiplicity of infection (MOI = 0.1) of early venus expressing vaccinia virus, (12) (Fig. 3A) and (16) (Fig. 3B) inhibited the expression of virus gene expression with IC₅₀ values of 326 nM and 101 nM, respectively, an improvement of more than 10-fold over CMLDBU6128 (1). In experiments that used a higher initial MOI of 1, the observed IC₅₀s for these compounds were slightly higher, at 734 nM and 547 nM, respectively (Fig. 3C and D). When evaluated for cytotoxicity neither compound showed significant suppression of ATP pools at any concentration (Fig. 3A and B, dotted lines). As a comparison, we also tested the inhibitory activity of cidofovir and Molnupiravir against the vaccina virus in our reporter virus assay. Cidofovir is the active component of brincidovovir, a compound recently FDA approved to treat smallpox. Molnupiravir, a compound with emergency use authorization (EUA) approval for the treatment of COVID-19 was recently shown to have antipoxvirus activity (37). While both molnupiravir and cidofovir showed some efficacy (Fig. 3F), (12) and (16) compared favorability in their ability to reduce viral growth.

FIG 3.

Vaccinia virus gene expression in the presence of compounds (12) and (16). Late viral reporter expression (normalized) at 24 h postinfection (hpi) following infection with vaccinia virus LV at an MOI = 0.1 in Vero E6 cells in the presence of increasing concentrations of (12) (A) or (16) (B) is shown in closed circles. Dotted lines represent cell viability (normalized to DMSO control) and the same concentrations, determined at 24-h post drug treatment. Virus gene expression at 12 hpi at MOI = 1 in the presence of increasing concentrations of (12) (A) or (16) (B) is shown in (C) and (D), respectively. Error bars represent standard deviations from triplicate (B) or quadruplicate (A, C, and D) repeated conditions. A four-point (0.01 μM to 10 μM) dilution series shows plaque reduction in triplicate conditions with both compounds compared to no treatment (Tx) (E). LV reporter virus was compared to two demonstrated anti-poxviral drugs cidofovir and molnupiravir (F) in the same dilution series of (A to D) in Vero E6 cells.

As a separate method of assessing inhibitory capacity, we carried out a 4-point dilution curve using compounds (12) or (16) followed by plaque assays to determine the level of virus particle reduction. The results (Fig. 3E) showed that both compounds reduce infectious viral particle production, with (16) showing a particularly strong reduction at high concentrations. Based on these promising results against the vaccinia virus, compounds (12) and (16) were carried forward with compound (1) to characterize their activity against variola virus.

Ability of compounds to decrease variola virus growth.

To determine the effects of PDPM treatment against variola virus, we sought to determine the ability of the compounds to halt a single round of variola virus replication by infecting with a high MOI (MOI = 5) and sampling throughout 24 h postinfection (hpi). A549 cells were infected with either variola virus strain BSH74_sol or SLN68_258, primary clade I and II, respectively, allowing observation of broad variola virus inhibition. A reduction in viral replication was detected in as little as one hpi with the treatment of a PDPM (Fig. 4A). All three PDPMs reduced viral replication ranging from 87 to 92% for both viral clades (corresponding to a ~log-fold decrease in viral titer). Next, we sought to determine the ability of the compounds to halt variola virus spread by infecting with a low MOI (MOI = 0.1) and sampling at predetermined time points up to 72 hpi. The largest percent reduction was seen at 72 hpi against SLN68_258. Percent reduction ranged from 95% to 100% at 72 hpi across the compounds with compound (16) again having the highest percent reduction (Fig. 4B). The growth reduction assays indicate that in addition to inhibiting vaccinia, all PDPMs tested were able to decrease viral replication and spread of variola consistent with a conserved mechanism of anti-poxviral activity.

FIG 4.

Ability of compounds to decrease variola virus growth and spread. (A) The ability of the compounds to halt a single round of variola virus replication was observed by infecting A549 cells with an MOI = 5 and sampling up to 24 hpi. The starting inoculum varied by 13% and 26%, when comparing the compound-treated wells to the DMSO control, for BSH74_sol and SLN68_258, respectively. (B) The ability of the compounds to decrease variola virus spread was observed by infecting A549 cells at an MOI = 0.01 and sampling up to 72 hpi. The starting inoculum varied by 25 to 26%, for BSH74_sol and SLN68_258, respectively, compared to the DMSO control. All compounds were able to decrease viral spread which is evident by percent reduction calculations consistent with a conserved mechanism of anti-poxviral activity. Experiments were conducted in duplicate by titering on BSC-40 cells to calculate the PFU/mL. Titer counts were averaged, and the standard deviation was calculated and graphed using GraphPad.

DISCUSSION

Through the experiments shown here, we found that the small molecule CMLDBU6128 (1) discovered in a broad screening campaign as a poxvirus antiviral could be modified to create increasingly potent antipoxviral compounds ([12] and [16]) by adding substituents to pendant aromatic ring at R¹. These additions moved the IC₅₀ values from the micromolar into the nanomolar range. These inhibitors remain relatively low molecular weights of these PDPM inhibitors and showed excellent ligand efficiencies (LE) (31) and lipophilic ligand efficiencies (LLE) (32, 33), metrics that can be predictive of downstream ADME and safety profiles, as well as overall therapeutic tractability (28, 34–36). In addition to indicating good druglike properties, the low molecular weight and relatively high LE (>0.45) and LLE (>5) of the best-performing compound (16) also suggest the potential to further improve potency by way of molecular expansion/addition of binding elements to the parent scaffold.

A second key finding of this work is the ability of PDPMs like (1) and its derived analogs to limit variola virus replication and spread. It was encouraging that (1) and both derivatives (12) and (16) showed anti-variola virus activity against a strain from the primary 1 clade, the more pathogenic clade, and the successful in vitro results support in vivo testing to better gauge the efficacy of the compounds. The ability of these compounds to block variola virus activity is also consistent with earlier work suggesting that the DNA-dependent RNA polymerase (DdRp) is the target of PDPM activity (18). The vaccinia DdRp is one of the most highly conserved genes across the Orthopoxvirus genus (38). In our assays we noted that there was a decreased apparent efficacy of these compounds against variola compared to vaccinia inhibition; this is consistent with other reports that tested Tecovirmat (TPOXX) and found IC₅₀s for variola virus inhibition (39) that were higher than those seen for vaccinia and other poxviruses (40) although the relevancy of these findings requires further investigation as the mechanism for this difference is unknown. A potential explanation is the higher fidelity of the reporter-based assay for vaccinia virus as manipulation of variola virus is strictly prohibited to a nonreporter-based readout These minor differences in assays should be mentioned however comparing the results between assays does not obscure interpretation.

An exciting possibility raised by these studies is that combinatorial treatment with PDPMs and the FDA-approved smallpox therapeutics would lead to additive or synergistic antiviral activity. TPOXX targets poxvirus replication at assembly, while PDPMs attack replication earlier in the replication cycle. Combining the two compounds could lead to overlapping protection, where resistance mutants to one antiviral would be blocked by the second compound. Using both compounds could be highly important in the event of a purposeful smallpox release, but the combination could be equally important in the treatment of monkeypox, a virus that has reemerged on the African continent over the last decades. Combined use could limit the emergence of drug-resistant variants. Moving forward we hope to explore the expansion of the antiviral potential of 6128 by making chemical modifications to the core molecule. We additionally would like to move forward with drug testing against monkeypox with hopes of identifying molecules to move toward in vivo testing in a murine model.

From a limited exploratory library querying modifications to PDPM poxviral inhibitor CMLDBU6128 (1), we have mapped preliminary structure-activity relationships (SAR) and identified new analogs with comparable or improved potency against our vaccinia viral reporter system. The most promising of these PDPM compounds showed 10-fold improved potency and were carried forward into more therapeutically relevant assessments against variola virus (the causative agent of smallpox) where the anti-poxviral activity and irreversible mechanism of action were confirmed (data not shown). This suggests that the PDPM chemotype holds promise as an antiviral agent that is mechanistically orthogonal to the current arsenal of poxviral therapies. Importantly, this work sets the stage for our ongoing medicinal chemistry campaign to further optimize the scaffold for potency and DMPK properties, toward progression to future studies against orthopoxviruses such as monkeypox and variola in in vivo infection models.

MATERIALS AND METHODS

Biosafety committee approval.

All work involving the variola virus was carried out by the Centers for Disease Control (CDC). The experiments described here were approved by the Advisory Committee on Variola Virus Research (ACVVR) (https://www.who.int/groups/who-advisory-committee-on-variola-virus-research/) and were reviewed and approved at the CDC after careful review through the United States Government Policy for Oversight of Life Sciences Dual Use Research of Concern (DURC).

Chemical synthesis methods.

Synthetic protocols to produce (1), (6a), and (6b) were previously reported (18, 19). Compounds (10 to 27) were produced in parallel as a library, with limited purification and characterization of intermediates progressing from General Procedure A to General Procedure C. Full characterization data for final products are provided.

General methods.

¹H NMR spectra were recorded at 400 or 500 MHz at ambient temperature. ¹³C NMR spectra were recorded at 101 or 126 MHz at ambient temperature. Chemical shifts are reported in parts per million. Data for ¹H NMR are reported as follows: chemical shift, multiplicity (app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, hept = heptet, m = multiplet,), coupling constants, and integration. All ¹³C NMR spectra were recorded with complete proton decoupling. Analytical thin-layer chromatography was performed using 0.25 mm silica gel 60-F plates. Silica flash chromatography was performed using prepacked columns (SI-HC, puriFlash or Premium Universal, Yamazen) on either an Interchim puriFlash450 or Yamazen Smart Flash EPCLC W-Prep2XY system. All mass-guided preparative HPLC was performed using an acetonitrile/water gradient (mobile phase modified with 0.01% formic acid) on a Waters FractionLynx system equipped with a 600 HPLC pump, a micromass ZQ quadrupole, Waters 996 diode array, and Sedere Sedex 75 ELS detectors, using an XBridge Prep C18 5 μM OBD 19 mm diameter column of either 100 mm or 250 mm length. Isolated yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated. All reactions were carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise noted. Analytical ultra high performance liquid chromatography mass spectrometry (UPLC-MS) experiments were performed using a Waters Acquity (ultraperformance liquid chromatography) with a binary solvent manager, SQ mass spectrometer, Waters 2996 PDA (photodiode array) detector, and evaporative light scattering detector (ELSD). All microwave experiments were performed on a CEM Discover microwave reactor, using a sealed 10 or 35 mL vessel with temperatures monitored by an external sensor. SiliaPrep Thiourea and SiliaPrep Thiol solid-phase extraction (SPE) cartridges were purchased from SiliCycle (Quebec City, QC, Canada). All compounds tested in biological assays were determined to be >95% pure by UPLC-MS-ELSD analysis.

1-(3-[trimethylsilyl]prop-2-yn-1-yl)urea (2a) in a 500 mL round-bottomed flask, urea (42.8 g, 713 mmol, 20 equiv) was dissolved in 216 mL glacial acetic acid. The suspension was stirred for a minimum of 5 h to ensure saturation of the solution. 3-(trimethylsilyl)propionaldehyde (4.5 g, 35.6 mmol, 1.0 equiv) was then added, followed by chlorotrimethylsilane (5.4 mL, 42.8 mmol, 1.2 equiv.) The reaction was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (16.6 g, 78.4 mmol, 2.2 equiv) was added carefully in small portions over 15 min, and the reaction was stirred at room temperature for 18 h. The reaction was then quenched with the addition of methanol (25 mL). Approximately half of the acetic acid was removed via rotary evaporation. When the solution volume was reduced by approximately half, the remaining solution was partitioned between ether (100 mL) and deionized water (100 mL), and the aqueous layer was extracted three times with 150 mL of diethyl ether. The combined organics were then washed seven times with 100 mL of deionized water. Saturated aqueous sodium bicarbonate was next added slowly and carefully in 20 mL portions, to neutralize the residual acetic acid. Sodium bicarbonate washes were repeated carefully, with frequent venting, in 20 mL portions until gas evolution ceased (~6 × 20 mL washes), and then in 100 mL portions until aqueous washings were alkaline as indicated by pH paper (~2 × 100 mL washes). After neutralization, the organic fraction was dried over sodium sulfate, filtered, and condensed to give a yellow oil. Trituration with cold hexanes gave (2a) a fluffy white solid (3.42 g, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ = 6.27 (t, J = 5.5 Hz, 1H), 5.60 (s, 2H), 3.80 (d, J = 5.5 Hz, 2H), 0.12 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆) δ = 158.1, 105.2, 85.4, 29.7, −0.2. HRMS [M+H]⁺, calcd. for C₇H₁₅N₂OSi, 171.0954; found 171.0949.

General procedure A.

Alkylation of CMLDBU6128 (1) To a 1-dram vial equipped with a magnetic stir-bar were added CMLDBU6128 (1) (0.1 mmol), Cs₂CO₃ (0.15 mmol, 1.5 equiv), and Ag₂O (0.05 mmol, 0.5 equiv). The vial was fitted with a rubber septum, evacuated, and backfilled with nitrogen. Anhydrous DMF (0.5 mL, 0.2 M) was added, followed by the desired alkyl or allyl iodide (0.5 mmol, 5 equiv). The reaction was stirred for 18 h. Upon completion, the reaction mixture was transferred via pipette to a 1 g (6 mL) SiliaPrep Thiol SPE cartridge to remove residual silver, eluting with 6 mL ethyl acetate and 6 mL methanol. The filtrate was then condensed and purified by mass-guided preparative HPLC (acetonitrile/water) to give the N-alkylated PDPM product.

Full characterization data for PDPMs 7 to 9:

4-acetyl-2-methyl-3-phenyl-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (7) was synthesized using CMLDBU6128 (1) (0.1 mmol) and iodomethane (0.5 mmol) according to General procedure A. (7) (15.1 mg, 0.053 mmol, 53% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.37 – 7.07 (m, 6×H), 6.50 – 6.44 (m, 1H), 5.23 (s, 1H), 4.30 – 4.22 (m, 1H), 3.57 – 3.48 (m, 1H), 2.95 (s, 3H), 2.51 – 2.32 (m, 2H), 2.22 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.55, 152.91, 141.83, 139.95, 135.98, 129.08, 128.43, 127.13, 121.90, 112.01, 61.62, 39.09, 34.51, 30.95, 24.42.; HRMS [M+H]⁺, calcd. for C₁₇H₁₉N₂O₂, 283.1447; found, 283.1448.

4-acetyl-2-ethyl-3-phenyl-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (8) was synthesized using CMLDBU6128 (1) (0.1 mmol) and iodoethane (0.5 mmol) according to General procedure A. (8) (4.1 mg, 0.014 mmol, 14% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.34 – 7.23 (m, 5H), 7.15 – 7.10 (m, 1H), 6.50 – 6.43 (m, 1H), 5.37 (s, 1H), 4.32 – 4.23 (m, 1H), 3.84 – 3.73 (m, 1H), 3.55 – 3.46 (m, 1H), 3.12 – 3.02 (m, 1H), 2.50 – 2.32 (m, 2H), 2.28 (s, 3H), 1.12 (t, J = 7.2 Hz, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.38, 152.71, 141.93, 141.32, 136.00, 128.99, 128.22, 127.00, 121.94, 112.81, 58.63, 41.92, 39.05, 31.16, 24.45, 13.16.; HRMS [M+H]⁺, calcd. for C₁₈H₂₁N₂O₂, 297.1603; found, 297.1598.

4-acetyl-2-allyl-3-phenyl-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (9) was synthesized using CMLDBU6128 (1) (0.1 mmol) and iodoethane (0.5 mmol) according to General procedure A. 9 (9.9 mg, 0.032 mmol, 32% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.36 – 7.20 (m, 6×H), 6.51 – 6.44 (m, 1H), 5.82 – 5.69 (m, 1H), 5.31 (s, 1H), 5.30 – 5.25 (m, 1H), 5.24 (t, J = 1.5 Hz, 1H), 4.69 – 4.61 (m, 1H), 4.32 – 4.22 (m, 1H), 3.57 – 3.48 (m, 1H), 3.43 – 3.34 (m, 1H), 2.53 – 2.32 (m, 2H), 2.22 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.60, 152.79, 141.85, 140.25, 136.00, 132.93, 129.07, 128.39, 127.27, 121.96, 118.38, 112.25, 57.73, 48.18, 39.21, 30.88, 24.44.; HRMS [M+H]⁺, calcd. for C₁₉H₂₁N₂O₂, 309.1603; found, 309.1603.

General procedure B: Biginelli condensation.

The Biginelli condensation was adapted from the procedure of Ryabukhin, et al. (41) Briefly, trimethylsilylproprargylurea (2a) (1.0 equiv), aldehyde (3) (1.0 equiv) and 2,4-pentanedione (1.0 equiv) were combined in anhydrous DMF (0.5 M). Chlorotrimethylsilane (5.0 equiv) was added in one portion and the reaction vessel was sonicated for 1 h and allowed to stand at room temperature overnight. The reaction progress was tracked by UPLC-MS over 1 to 5 days. Upon completion, the solution was diluted 20-fold with deionized water and sonicated for 1 h. If a solid precipitated, the water was filtered or decanted and the residue was isolated by filtration, dissolved in ethyl acetate, dried over sodium sulfate, concentrated, and carried on to the next step without further purification. If an oil precipitated, the water was decanted, and the oil was triturated with either ether/hexanes or purified by flash column chromatography (ethyl acetate/hexanes) if trituration was unsuccessful.

General procedure C: gold-catalyzed cycloisomerization.

The crude dihydropyrimidinone obtained from General procedure B was placed in a reaction vessel under nitrogen. 1,2-Dichloroethane was added to produce a 0.1 M solution. Auric acid (1.0 equiv) was added in one portion and the reaction was heated to 80°C overnight. The reaction mixture was then cooled and passed through a 1 g (6 mL) SiliaPrep Thiourea SPE cartridge, eluting with three volumes of ethyl acetate. The filtrate was condensed and carried on to the next step without further purification.

General procedure D: silver-mediated protodesilylation.

The dried residue obtained from General procedure C was dissolved in a 10:2:2:1 mixture of THF:methanol:DMSO:water to give a final concentration of 0.2 M. Ammonium fluoride (5.0 equiv) was added and the reaction vessel was wrapped in aluminum foil to exclude light. Silver(I) fluoride (2.0 equiv) was added in the dark and the reaction was stirred at room temperature for 48 h, with reaction progress tracked by UPLC-MS-ELSD. Upon completion, the reaction mixture was transferred via pipette to a 1 g (6 mL) SiliaPrep Thiol SPE cartridge to remove residual silver, eluting with ~20 mL ethyl acetate. If black silver precipitate remained visible in the eluent, the eluent was subjected to an additional pass-through fresh Si-Thiol cartridges until it ran clear. The filtrate was then condensed and purified using either flash column chromatography (ethyl acetate/hexanes) or mass-guided preparative HPLC (acetonitrile/water) to give the final PDPM product.

Full characterization data for PDPMs 10 to 27:

4-acetyl-3-(2-fluorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (10) Obtained from (2a), 2-fluorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.53 – 7.45 (m, 1H), 7.34 – 7.28 (m, 1H), 7.20 – 7.13 (m, 1H), 7.13 – 7.05 (m, 2H), 6.56 – 6.48 (m, 1H), 5.70 (d, J = 3.4 Hz, 1H), 5.60 (s, 1H), 4.23 – 4.14 (m, 1H), 3.50 – 3.41 (m, 1H), 2.49 – 2.28 (m, 2H), 2.13 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.88, 160.62 (d, J = 245.7 Hz), 152.88, 143.40, 136.53, 130.44 (d, J = 8.5 Hz), 128.43 (d, J = 13.5 Hz), 127.82 (d, J = 3.7 Hz), 124.98 (d, J = 3.4 Hz), 121.80, 116.19 (d, J = 21.5 Hz), 108.52, 48.71 (d, J = 3.5 Hz), 38.43, 30.04, 24.20.; ¹⁹F NMR (470 MHz, CDCl₃) δ −119.75 – −119.84 (m).; HRMS [M+H]⁺, calcd. for C₁₆H₁₆FN₂O₂, 287.1196; found, 287.1191.

4-acetyl-3-(2-chlorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (11) Obtained from (2a), 2-chlorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.57 – 7.50 (m, 1H), 7.43 – 7.38 (m, 1H), 7.29 – 7.15 (m, 3H), 6.61 – 6.41 (m, 1H), 5.92 (d, J = 3.4 Hz, 1H), 5.77 (d, J = 3.4 Hz, 1H), 4.19 – 4.06 (m, 1H), 3.46 (m, 1H), 2.52 – 2.26 (m, 2H), 2.04 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.91, 152.60, 143.59, 138.02, 136.55, 133.01, 130.36, 129.99, 127.98, 127.95, 121.75, 108.47, 51.63, 38.36, 29.80, 24.13. HRMS [M+H]⁺, calcd. for C₁₆H₁₆ClN₂O₂, 303.0900; found, 303.0907.

4-acetyl-3-(2-bromophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (12) Obtained from (2a), 2-bromobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.62 – 7.51 (m, 2H), 7.31 – 7.23 (m, 1H), 7.22 – 7.13 (m, 2H), 6.56 – 6.48 (m, 1H), 5.92 (d, J = 3.5 Hz, 1H), 5.74 (d, J = 3.4 Hz, 1H), 4.19 – 4.08 (m, 1H), 3.52 – 3.43 (m, 1H), 2.47 – 2.30 (m, 2H), 2.02 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.92, 152.43, 143.52, 139.53, 136.56, 133.63, 130.30, 128.64, 128.24, 123.35, 121.75, 108.63, 54.05, 38.37, 29.82, 24.12.; HRMS [M+H]⁺, calcd. for C₁₆H₁₆BrN₂O₂, 347.0395; found, 346.0393.

4-acetyl-3-(o-tolyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (13) Obtained from (2a), o-tolualdehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.43 – 7.37 (m, 1H), 7.23 – 7.12 (m, 4H), 6.53 – 6.45 (m, 1H), 5.61 (d, J = 2.8 Hz, 1H), 5.53 – 5.49 (m, 1H), 4.13 – 4.05 (m, 1H), 3.63 – 3.54 (m, 1H), 2.46 (s, 3H), 2.44 – 2.36 (m, 2H), 2.02 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.38, 152.63, 142.22, 139.32, 135.78, 134.80, 131.50, 128.60, 127.29, 126.96, 121.79, 110.61, 51.85, 38.41, 30.01, 24.23, 19.13.; HRMS [M+H]⁺, calcd. for C₁₈H₁₉N₂O₂, 283.1447; found, 283.1440.

4-acetyl-3-(2-methoxyphenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (14) Obtained from (2a), 2-methoxy benzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.60 – 7.53 (m, 1H), 7.31 – 7.24 (m, 1H), 7.09 – 7.04 (m, 1H), 6.93 – 6.84 (m, 2H), 6.52 – 6.44 (m, 1H), 5.89 (d, J = 2.9 Hz, 1H), 5.65 (d, J = 3.2 Hz, 1H), 4.19 – 4.11 (m, 1H), 3.89 (s, 3H), 3.43 – 3.34 (m, 1H), 2.46 – 2.27 (m, 2H), 2.06 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.56, 156.88, 153.47, 143.50, 136.05, 129.74, 128.63, 126.63, 121.97, 121.02, 110.78, 108.45, 55.49, 49.27, 38.28, 29.71, 24.19.; HRMS [M+H]⁺, calcd. for C₁₇H₁₉N₂O₃, 299.1396; found, 299.1403.

4-acetyl-3-(2-[trifluoromethyl]phenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (15) Obtained from (2a), 2-(trifluoromethyl)benzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.75 – 7.70 (m, 1H), 7.60 – 7.53 (m, 1H), 7.52 – 7.47 (m, 1H), 7.47 – 7.40 (m, 2H), 6.58 – 6.51 (m, 1H), 5.79 (d, J = 3.1 Hz, 1H), 5.42 (s, 1H), 4.17 – 4.09 (m, 1H), 3.62 – 3.53 (m, 1H), 2.53 – 2.34 (m, 2H), 2.02 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.08, 151.94, 143.31, 139.56, 136.52, 133.55, 128.82 (d, J = 25.7 Hz), 127.22 (d, J = 30.0 Hz), 126.95 (q, J = 6.0 Hz), 125.73, 123.55, 121.78, 109.15, 50.98 (d, J = 1.9 Hz), 38.46, 30.13, 24.17.; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.97.; HRMS [M+H]⁺, calcd. for C₁₇H₁₆F₃N₂O₂, 337.1164; found, 337.1165.

4-acetyl-3-(3-fluorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (16) Obtained from (2a), 3-fluorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.32 – 7.22 (m, 2H), 7.06 – 7.00 (m, 1H), 6.99 – 6.90 (m, 2H), 6.73 – 6.69 (m, 1H), 6.53 – 6.45 (m, 1H), 5.37 (d, J = 3.5 Hz, 1H), 4.19 – 4.09 (m, 1H), 3.51 – 3.42 (m, 1H), 2.45 – 2.30 (m, 2H), 2.17 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.87, 163.21 (d, J = 247.4 Hz), 153.26, 144.86 (d, J = 6.1 Hz), 142.33, 136.32, 130.72 (d, J = 8.1 Hz), 122.14 (d, J = 2.9 Hz), 121.71, 115.24 (d, J = 21.1 Hz), 113.61 (d, J = 22.0 Hz), 111.16, 53.88 (d, J = 1.8 Hz), 38.38, 30.62, 24.14.; ¹⁹F NMR (470 MHz, CDCl₃) δ −111.63 – −111.89 (m).; HRMS [M+H]⁺, calcd. for C₁₆H₁₆FN₂O₂, 287.1196; found 287.1198.

4-acetyl-3-(3-chlorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (17) Obtained from (2a), 3-chlorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.31 – 7.22 (m, 4H), 7.19 – 7.12 (m, 1H), 6.55 – 6.48 (m, 1H), 5.83 – 5.79 (m, 1H), 5.37 (d, J = 3.4 Hz, 1H), 4.26 – 4.15 (m, 1H), 3.55 – 3.46 (m, 1H), 2.51 – 2.31 (m, 2H), 2.20 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.73, 152.83, 144.34, 142.29, 136.47, 135.15, 130.58, 128.66, 126.86, 124.74, 121.72, 111.33, 54.27, 38.47, 30.84, 24.21.; HRMS [M+H]⁺, calcd. for C₁₆H₁₆ClN₂O₂, 303.0900; found, 303.0901.

4-acetyl-3-(3-bromophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (18) Obtained from (2a), 3-bromobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.43 – 7.37 (m, 2H), 7.30 – 7.26 (m, 1H), 7.22 – 7.14 (m, 2H), 6.55 – 6.47 (m, 1H), 6.19 (d, J = 3.5 Hz, 1H), 5.36 (d, J = 3.5 Hz, 1H), 4.22 – 4.13 (m, 1H), 3.54 – 3.45 (m, 1H), 2.49 – 2.33 (m, 2H), 2.19 (s, 3H).; 13C NMR (126 MHz, CDCl₃) δ 195.76, 152.99, 144.58, 142.36, 136.46, 131.53, 130.82, 129.81, 125.17, 123.29, 121.73, 111.18, 54.09, 38.46, 30.80, 24.20.; HRMS [M+H]⁺, calcd. for C₁₆H₁₆BrN₂O₂, 347.0395; found, 347.0389.

4-acetyl-3-(m-tolyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (19) Obtained from (2a), m-tolualdehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.36 – 7.29 (m, 1H), 7.23 – 7.17 (m, 1H), 7.09 – 7.01 (m, 3H), 6.50 – 6.43 (m, 1H), 5.88 (s, 1H), 5.33 (d, J = 3.1 Hz, 1H), 4.18 – 4.07 (m, 1H), 3.56 – 3.47 (m, 1H), 2.46 – 2.33 (m, 2H), 2.32 (s, 3H), 2.14 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.43, 152.95, 142.18, 141.76, 139.03, 135.81, 129.25, 129.16, 127.34, 123.64, 121.90, 111.40, 54.95, 38.39, 30.54, 24.20, 21.62.; HRMS [M+H]⁺, calcd. for C₁₈H₁₉N₂O₂, 283.1447; found 283.1448.

4-acetyl-3-(3-methoxyphenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (20) Obtained from (2a), 3-methoxybenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.36 – 7.30 (m, 1H), 7.26 – 7.18 (m, 1H), 6.87 – 6.77 (m, 3H), 6.50 – 6.42 (m, 1H), 6.17 (s, 1H), 5.32 (d, J = 3.3 Hz, 1H), 4.18 – 4.11 (m, 1H), 3.76 (s, 3H), 3.53 – 3.43 (m, 1H), 2.46 – 2.30 (m, 2H), 2.14 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.27, 160.24, 153.08, 143.78, 141.98, 135.91, 130.33, 121.87, 118.80, 113.41, 112.65, 111.19, 55.32, 54.71, 38.36, 30.50, 24.17.; HRMS [M+H]⁺, calcd. for C₁₇H₁₉N₂O₃, 299.1396; found, 299.1403.

4-acetyl-3-(3-[trifluoromethyl]phenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (21) Obtained from (2a), 3-(trifluoromethyl)benzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.56 – 7.51 (m, 2H), 7.49 – 7.42 (m, 2H), 7.28 – 7.21 (m, 1H), 6.57 – 6.49 (m, 1H), 6.13 (d, J = 3.6 Hz, 1H), 5.47 (d, J = 3.6 Hz, 1H), 4.24 – 4.15 (m, 1H), 3.52 – 3.43 (m, 1H), 2.49 – 2.34 (m, 2H), 2.22 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.82, 153.24, 143.63, 142.74, 136.91, 131.79 (q, J = 32.5 Hz), 130.24 – 130.10 (m), 130.07, 125.58 – 125.45 (m), 123.75 – 123.61 (m), 121.89, 111.73, 54.35, 43.07, 38.72, 31.17, 24.45.; ¹⁹F NMR (470 MHz, CDCl₃) δ −62.64.; HRMS [M+H]⁺, calcd. for C₁₇H₁₆F₃N₂O₂, 337.1164; found, 337.1170.

4-acetyl-3-(4-fluorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (22) Obtained from (2a), 4-fluorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.30 – 7.21 (m, 3H), 7.05 – 6.97 (m, 2H), 6.53 – 6.46 (m, 1H), 5.79 (s, 1H), 5.38 (d, J = 3.3 Hz, 1H), 4.19 – 4.13 (m, 1H), 3.51 (ddd, J = 12.8, 8.8, 5.3 Hz, 1H), 2.48 – 2.33 (m, 2H), 2.17 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.00, 162.67 (d, J = 247.5 Hz), 152.87, 141.88, 138.26 (d, J = 3.3 Hz), 136.18, 128.40 (d, J = 8.3 Hz), 121.76, 116.18 (d, J = 21.9 Hz), 111.76, 54.14, 38.45, 30.72, 24.22.; 19F NMR (470 MHz, CDCl₃) δ −113.47 – −113.69 (m).; HRMS [M+H]⁺, calcd. for C₁₆H₁₆FN₂O₂, 287.1196; found, 287.1193.

4-acetyl-3-(4-chlorophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (23) Obtained from (2a), 4-chlorobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.31 – 7.27 (m, 2H), 7.27 – 7.23 (m, 1H), 7.23 – 7.18 (m, 2H), 6.53 – 6.47 (m, 1H), 5.98 (d, J = 3.2 Hz, 1H), 5.37 (d, J = 3.4 Hz, 1H), 4.21 – 4.13 (m, 1H), 3.53 – 3.44 (m, 1H), 2.47 – 2.34 (m, 2H), 2.19 (s, 3H).; 13C NMR (126 MHz, CDCl₃) δ 195.87, 153.02, 142.09, 140.89, 136.27, 134.18, 129.36, 128.01, 121.71, 111.57, 53.90, 38.42, 30.74, 24.18.; HRMS [M+H]⁺, calcd. for C₁₆H₁₆ClN₂O₂,303.0900; found, 303.0908.

4-acetyl-3-(4-bromophenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (24) Obtained from (2a), 4-bromobenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.48 – 7.42 (m, 2H), 7.26 – 7.22 (m, 1H), 7.18 – 7.12 (m, 2H), 6.54 – 6.46 (m, 1H), 5.93 (s, 1H), 5.36 (d, J = 3.4 Hz, 1H), 4.21 – 4.14 (m, 1H), 3.52 – 3.43 (m, 1H), 2.48 – 2.33 (m, 2H), 2.19 (s, 3H).; ¹³C NMR (126 MHz, cdcl₃) δ 195.82, 152.96, 142.09, 141.40, 136.33, 132.37, 128.32, 122.37, 121.71, 111.65, 54.03, 38.45, 30.82, 24.21.; HRMS [M+H]⁺, calcd. for C₁₆H₁₆BrN₂O₂, 347.0395; found, 347.0396.

4-acetyl-3-(p-tolyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (25) Obtained from (2a), p-tolualdehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.34 – 7.28 (m, 1H), 7.19 – 7.10 (m, 4H), 6.50 – 6.43 (m, 1H), 5.57 (d, J = 3.0 Hz, 1H), 5.33 (d, J = 3.1 Hz, 1H), 4.19 – 4.08 (m, 1H), 3.57 – 3.48 (m, 1H), 2.44 – 2.36 (m, 2H), 2.32 (s, 3H), 2.14 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 196.41, 152.96, 141.68, 139.33, 138.30, 135.77, 129.96, 126.58, 121.90, 111.56, 54.70, 38.39, 30.51, 24.20, 21.22.; HRMS [M+H]⁺, calcd. for C₁₈H₁₉N₂O₂, 283.1447; found, 283.1451.

4-acetyl-3-(4-methoxyphenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (26) Obtained from (2a), 4-methoxybenzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.34 – 7.28 (m, 1H), 7.25 – 7.11 (m, 2H), 6.90 – 6.82 (m, 2H), 6.50 – 6.43 (m, 1H), 5.66 (d, J = 3.0 Hz, 1H), 5.32 (d, J = 3.1 Hz, 1H), 4.18 – 4.09 (m, 1H), 3.78 (s, 3H), 3.56 – 3.50 (m, 1H), 2.44 – 2.33 (m, 2H), 2.13 (s, 3H).;13C NMR (126 MHz, CDCl₃) δ 196.46, 159.67, 152.94, 141.56, 135.73, 134.49, 127.94, 121.89, 114.59, 111.62, 55.42, 54.42, 38.38, 30.45, 24.19.; HRMS [M+H]⁺, calcd. for C₁₇H₁₉N₂O₃, 299.1396; found, 299.1367.

4-acetyl-3-(4-[trifluoromethyl]phenyl)-2,3,7,8-tetrahydro-1H-pyrido[1,2-c]pyrimidin-1-one (27) Obtained from (2a), 4-(trifluoromethyl)benzaldehyde, and 2,4-pentanedione in three steps using General procedures B-D. ¹H NMR (500 MHz, CDCl₃) δ 7.61 – 7.55 (m, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.24 – 7.18 (m, 1H), 6.56 – 6.49 (m, 1H), 6.12 – 6.08 (m, 1H), 5.48 (d, J = 3.6 Hz, 1H), 4.24 – 4.15 (m, 1H), 3.51 – 3.42 (m, 1H), 2.47 – 2.33 (m, 2H), 2.23 (s, 3H).; ¹³C NMR (126 MHz, CDCl₃) δ 195.55, 153.07, 146.29, 142.39, 136.61, 130.54 (q, J = 32.2 Hz), 126.94, 126.21 (q, J = 3.7 Hz), 121.63, 111.79, 53.92, 42.82, 38.49, 30.99, 24.21.; ¹⁹F NMR (470 MHz, CDCl₃) δ −62.65.; HRMS [M+H]⁺, calcd. for C₁₇H₁₆F₃N₂O₂, 337.1164; found, 337.1169.

Strain selection.

For the vaccinia virus work, all work was performed in a biosafety level 2 laboratory with the viruses being modified from the western reserve (WR) strain. This vaccinia strain possesses a late gene expression Venus protein that is used to quantify fluorescence using a Tecan Spark (Mannedorf, Switzerland) as described previously and abbreviated throughout as LV (18, 42). All work with live variola virus was conducted within a biosafety level 4 laboratory following guidelines and approvals from the WHO ACVVR. At the time of this work, there were 49 complete genomic sequences available in GenBank for the variola virus. Because the presumed target for the PDPM CMLDBU6128 (1) is the viral RNA polymerase large subunit gene J6R (18), we evaluated all 49 amino acid sequences using BioEdit Sequence Alignment Editor (Ibis Biosciences, CA). Five amino acid changes were identified (data not shown); however, none of these matched the identified changes in the vaccinia virus that conferred resistance (18). Based on these results, a representative from primary clade I and primary clade II were chosen for testing against the PDPMs, BSH74_sol, and SLN68_258, respectively.

Cells.

For growth, A549 cells (Division of Scientific Resources, CDC) were plated in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher, MA) with 10% fetal bovine serum supplemented with 100 units/mL of Penicillin and 100 μL/mL of Streptomycin. For the vaccinia virus work experiments were performed in African green monkey kidney (Vero E6) cells under similar cultured conditions using DMEM with no antibiotic and supplemented with l-glutamine and 7% fetal bovine serum. For infection or compound treatment, the conditions remained the same except that 2% fetal bovine serum was used instead of 10%. For the MTT assay, the conditions remained the same except phenol red-free DMEM was used (Sigma-Aldrich, MO).

Toxicity determination in Vero E6 cells.

To determine the Vero E6 toxicity of the compounds used in the vaccinia assay, the CellTiter-Glo luminescence assay (Madison, WI). Briefly Vero E6 cells were seeded and grown overnight to near confluence. Cells were then treated in triplicate with either 12, 16, similar concentrations of DMSO or no compound. After 24 h of incubation, the CellTiter-Glo reagent is mixed and 100 μL of the solution is added to each well at room temperature. Following a 30 min room temperature incubation with mixing, luminescence is read on a BMG Labtech Omega and exported to GraphPad for analysis. Data are presented as averages of all concentrations although there is no significant concentration-dependent difference between treatments.

Vaccinia IC₅₀ determination.

Vero E6 cells were plated at a concentration to provide ~100% confluence within 24 h. Compounds were diluted in DMEM containing 7% FBS and titrated from 33 μM in a series of 3-fold dilutions to 0.0001 μM and added to cells for 30 min before infection. After 30 min Vaccinia expressing a late gene Venus reporter was added to the 96-well plate and left for 12h (MOI = 1) or 24h (MOI = 0.1) before being quantified as described later.

Variola virus time courses.

A549 cells (Division of Scientific Resources, CDC) were plated at 1.5× 10⁶ cells/well in a 6-well tissue culture treated plate and incubated overnight at 37.0°C with 6% CO₂. Crude virus preparations were sonicated at 100% output in an ice-cold water bath for 1.5 min. For each time course, cells were infected for 1 h with variola virus strains BSH74_sol or SLN68_258 at an MOI = 0.1 or an MOI = 5.0 in media containing 12.5 μM DMSO or a PDPM compound. Afterward, the inoculum was removed and fresh media containing +/− the appropriate compound was added to the wells. For the MOI = 5, the cells and supernatant were harvested at 0, 1, 2, 4, 8, and 24 hpi. For the MOI of 0.1, cells and supernatant were harvested at 1, 24, 48, and 72 hpi. Each time course was completed in duplicate. Samples were frozen at −80°C. Titrations were performed on BSC-40 cells as previously described (43).

Fluorescent reading of early Venus expressing vaccinia.

Cells were infected at a low or high MOI (0.1 or 1.0) and the virus could propagate in the presence of drug until peak fluorescence was observed by microscopy in cells lacking drug treatment. The low MOI infection exhibited peak fluorescence from 24 to 48 h and the high MOI peaked in fluorescence at 12 to 30 h postinfection. For reading the 96-well plates were read on a Tecan Spark (Mannedorf, Switzerland) with the wavelength settings being from 515 to 536 nm. The z-position as well as the optimal gain was determined internally on a plate-by-plate basis. For data analysis, each drug concentration was read in quadruplicate and normalized to untreated fluorescence following subtraction of the mock fluorescence. Rows normalized were utilized if peak fluorescence was observed at exceedingly low concentration. For IC₅₀ calculations a variable slope four parameter inhibitor response curve was generated using GraphPad.