Discovery of Selective Small Molecule Type III Phosphatidylinositol 4

Article pubs.acs.org/jmc

Discovery of Selective Small Molecule Type III Phosphatidylinositol 4‑Kinase Alpha (PI4KIIIα) Inhibitors as Anti Hepatitis C (HCV) Agents Anna L. Leivers,† Matthew Tallant,† J. Brad Shotwell,† Scott Dickerson,‡ Martin R. Leivers,† Octerloney B. McDonald,‡ Jeff Gobel,‡ Katrina L. Creech,‡ Susan L. Strum,‡ Amanda Mathis,†,§ Sabrinia Rogers,† Chris B. Moore,† and Janos Botyanszki*,† †

Infectious Diseases Medicines Discovery Unit, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, North Carolina 27709-3398, United States ‡ Platform Technology and Science, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, North Carolina 27709-3398, United States S Supporting Information *

ABSTRACT: Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients.1 We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure−activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.

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INTRODUCTION Hepatitis C virus (HCV), a positive-sense single-stranded RNA virus within the flaviviridae virus family, was identified almost a quarter century ago.2 An estimated 130−170 million people, about 3% of the world population, are chronically infected, and 80% of the infections will progress to liver cirrhosis which ultimately can lead to liver carcinoma.3 HCV infection is the most common cause of liver transplantation and poses a major health burden. After the recent approval of the HCV protease inhibitors (PIs) boceprevir4 and telaprevir,5 the standard of care for HCV infection now involves a triple combination therapy of a PI + pegylated interferon-α (INF α) + ribavirin. Although this regimen is more effective than the previous interferon−ribavirin only treatment, it is still less than ideal because of significant side effects attributed to both INF α and the protease inhibitors themselves.6 A safe, well tolerated, all oral drug regime with a high (>90%) cure rate remains an important scientific and societal goal. Traditional approaches toward developing an HCV treatment have mainly involved targeting the viral genome directly, including both structural and nonstructural viral proteins. Multiple molecules are in various phases of preclinical or clinical development.7 A complementary approach is to achieve therapeutically relevant inhibition of a host cellular component that is used by the HCV virus throughout its replication cycle. Inhibiting a host target (as opposed to a viral protein) could provide additional benefits including a higher barrier to viral resistance, activity against all the HCV genotypes, and activity © XXXX American Chemical Society

against other viruses that utilize the same cellular process. A potential challenge of interfering directly with a cellular protein is a higher likelihood of an on target related adverse phenotype. The most advanced example of inhibiting an HCV host target is the inhibition of cyclophilin B with cyclosporin A analogues. Cyclophilin B is a host factor necessary for HCV replication through the stimulation of NS5A’s binding to the RNA. The cyclosporin A analogues have been advanced to clinical evaluation8,9 and have proven to be effective inhibitors of HCV replication with an increased genetic barrier to resistance. Recently, a search for other host factors by multiple research groups10−13 suggested that type III phosphatidylinositol 4kinase alpha (PI4KIIIα) is an essential host factor for HCV replication. The relationship between PI4KIIIα and HCV replication was further strengthened when a small molecule that was previously thought to inhibit HCV replication through NS5A inhibition was recently shown to inhibit HCV replication through PI4KIIIα inhibition.14 PI4KIIIα is a lipid kinase that catalyzes the phosphorylation of phosphatidylinositol at the 4position. The product, phosphatidylinositol 4-phosphate (PI4P), is an intermediate toward the formation of phosphatidylinositol 4,5-diphosphate (PIP2) which also plays an important role in intracellular membrane trafficking. It was shown that siRNA silencing of the PI4KIIIα gene (PI4KCA) in Special Issue: HCV Therapies Received: May 26, 2013

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dx.doi.org/10.1021/jm400781h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Chemical starting point (pIC50 and pEC50). apIC50 = −log IC50. bpEC50 = −log EC50.

One of the most promising representative chemotypes that emerged from the HTS campaign was compound 1 (Figure 1), which showed high PI4KIIIα activity (pIC50 = 8.8). Although this molecule did show selectivity against PKs (data not shown), it suffered from the lack of selectivity against the other LKs we profiled. It was minimally selective against PI4KIIIβ (pIC50 = 7.7) and was almost equipotent to the entire family of PI3Ks (pIC50 all >8.6). Interestingly, the quinazoline core contained within compound 1 was a motif of a small molecule, a 4-anilinoquinazoline derivative that had been identified by another group as an inhibitor of PIK4IIIα.14 Initial optimization of 1 led to the development of compound 2 that had good activity against PI4KIIIα (pIC50 = 7.4) along with moderate-togood selectivity over PI4KIIIβ (pIC50 = 5.5) and PI3Kα, -β, -δ, -γ (pIC50 of 5.6, 5.0, 6.2, 6.2, respectively). This molecule also exhibited high in vitro potency against multiple HCV genotypes (GTs 1a, 1b, 2a). The key structural change that led to the improved PI4KIIIα selectivity was the incorporation of the 2amino functionality on the quinoline ring.21 The lead molecule 1 originated from an ATP competitive LK inhibitor library and was predicted to inhibit PI4KIIIα through binding to the ATP binding site. To confirm this assumption, we conducted an ATP competition study using compound 2. The study clearly indicated that 2 inhibits PI4KIIIα in an ATP competitive manner (see “Ki and Mode of Inhibition Study” in Supporting Information (section VIII)). In an attempt to gain insight into the SAR, a homology model was constructed from a proprietary PI3Kγ structure, since a crystal structure of PI4KIIIα was not available. The sequence homology of the two proteins allowed PI4KIIIα residue assignments to be made with high confidence for most of the ATP binding site with the exception of the P-loop containing a β sheet. To mitigate this, multiple P-loop models were constructed. The gross binding mode of 2, as illustrated in Figure 2, was predicted with high confidence. The aminoquinoline moiety is predicted to form hydrogen bonds to the hinge residue Ile1841. The sulfonamide hydrogen likely forms a hydrogen bond to the catalytic Lys1792. A water molecule, which is often conserved in PI3K structures, is also predicted to be present in PI4KIIIα and forms a hydrogen bond to the pyridyl nitrogen. The methoxy group is not expected to have a

Huh-7 cells prevents replication with 99% efficiency. It was proposed that PI4KIIIα plays an essential role in intracellular membrane alterations, specifically by regulating the NS5A phosphorylation which leads to modulating the morphology of the viral replication site leading to the formation of the functional HCV replication complex.10,15 On the basis of these results, GlaxoSmithKline initiated a program to explore the therapeutic use of PI4KIIIα inhibition for the treatment of HCV. In this report, we present the discovery of highly selective, small molecule PI4KIIIα inhibitors that effectively inhibit HCV replication in vitro.

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SCREENING HITS AND HOMOLOGY MODELING We conducted a focused high throughput screening (HTS) campaign of GlaxoSmithKline’s lipid kinase (LK) inhibitor collection against an HCV 1b replicon assay to identify a suitable chemical starting point. The collection contained molecules prepared for previous lipid kinase programs, in total over 10 000 molecules. The aim was to find molecules that inhibited HCV replication that were suitable for optimization and that might already exhibit selectivity versus protein kinases (PK). As PI4KIIIα belongs to a family of 19 phosphoinositide lipid kinases and it shares a sequence homology of the active site with PI4KIIIβ and PI3Ks,16,17 the active compounds from the HTS campaign were then tested for PI4KIIIα, PI4KIIIβ, PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ activity. This screen would allow us to find compounds selective for PI4KIIIα. Compounds with poor kinase selectivity could have adverse effects, especially since the closely related PI3Ks are common chemotherapeutic targets and are involved in cellular activities including proliferation, survival, and glucose homeostasis.18−20 In addition, we tested the most interesting molecules for other (type II) LKs and a panel of PKs. Overall, the inhibitors of PI4KIIIα from the HTS hits had decent to high selectivity against PKs but showed poor or no selectivity against type III PI4Kβ and type I PI3Ks. Thus, as the primary screening strategy, we measured PI4KIIIα activity (along with HCV inhibitory potency) and closely monitored the biochemical selectivity against PI4KIIIβ, PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ while occasionally testing the lead molecules for other (type II) LKs and a panel of PKs. B



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conditions using bis(pinacolato)diboron, potassium acetate, and PdCl2(dppf)−DCM in dioxane gave boronic esters 37−39. For late stage diversification of the headgroup, compound 32 was directly borylated to give compound 40. To explore a variety of cores (Scheme 2), 2-phenylacetonitrile (41) was used as a common intermediate. The reaction of 2-phenylacetonitrile and 2-amino-5-bromonicotinaldehyde with piperidine in dioxane at 100 °C gave an incomplete reaction; however, the addition of solid sodium hydroxide followed by heating at 80 °C gave complete conversion to 42. Treatment of the nitrile 41 and 5-amino-2chloroisonicotinaldehyde with sodium hydride gave the 1,7naphthyridine intermediate (43). The 1,5-naphthyridin-2amine intermediate (44) was synthesized through the cyclization of 2-phenylacetonitrile and 3-amino-6bromopicolinaldehyde in the presence of NaOH at 65 °C. The final cross-coupling reactions of the bromo or chloro intermediates (42−44) with compound 37 afforded compounds 4, 5, and 6 in moderate yields. The quinoxaline core (Scheme 3) was synthesized via a three-component cyclization reaction involving 4-bromo-1,2benzenediamine (45), benzaldehyde, and 1,1,3,3-tetramethylbutyl isocyanide. The primary adduct was oxidized with DDQ to give a mixture of 46a and 46b in about a 1:2 ratio. The two regioisomers were treated with HCl in dioxanes at 70 °C to give 47a and 47b, which were subsequently cross-coupled with compound 37 to afford 7 and 48, respectively. The isomers were separated by HPLC, and absolute structural assignments were made based on 13C chemical shifts and ab initio IR analysis. Synthesis of the quinazolinone derivatives (Scheme 4) was achieved through the cyclization of commercially available isothiocyanates with 2-aminobenzoic acids (49). Chlorination at position 2 was affected by refluxing in neat POCl3 in the presence of PCl5 to give intermediates 50−55. The 2-amino functionality was installed by displacement of the chlorine with (4-methoxyphenyl)methanamine in DMF, followed by cleavage of the para-methoxybenzyl (PMB) group by heating in neat TFA to give intermediates 56−61. Products 8, 9, and 11 were made by the cross-coupling reaction of 56 with the intermediates 37, 38, and 39 described in Scheme 1. Quinazolinone 56 (Y = Br) was borylated to give intermediate 62, which upon cross-coupling with intermediate 35 gave product 10. A cross-coupling reaction of 56 with 40 gave the diamino intermediate 63. It was possible to take advantage of the different reactivities of the 3-pyridyl and 2-quinazolinonylamino groups and incorporate a set of sulfonyl groups using the corresponding sulfonyl chlorides at the last step, allowing for late stage diversification of 63 to final products 12−19. Although N-arylquinazolinones were easily accessed from the aryl isothiocyanates, this route did not work using alkyl isothiocycanates. The initial cyclization with cyclopentyl isothiocyanate failed to give the desired product; therefore, an alternative route was devised (Scheme 5) based on a literature precedent.22 The O-phenylisourea intermediate 65 was formed from methyl 5-bromoanthranilate (64), which was treated with an alkylamine in hot isopropanol to give the corresponding tetrahydroquinazolines 66 and 67. Hydrolysis with concentrated HCl in DMSO gave the free amines 68 and 69 in quantitative yield. The “reverse” sulfonamide head was synthesized via an adaptation of a literature procedure23 (Scheme 6). Upon formation of a diazonium ion of 70, treatment with thionyl

Figure 2. Cutaway view of 2 bound to a PI4Kα homology model. Hydrogen bonds are shown in yellow. Image was created with Maestro molecular modeling software (Schrödinger LLC).

direct interaction with the protein: the model did not predict specific role in the inhibitors’ binding. The binding model led us to hypothesize that changes to both the phenylsulfonamide and morpholinophenyl moieties might impact selectivity over the PI3Ks. Changes to the core could impact binding deep within the pocket and could indirectly change the protein interactions made by the pyridyl moiety and sulfonamide headgroup. The morpholinophenyl group itself appeared quite solvent exposed. The model generally provided a significant framework for testing SAR hypotheses and evaluating findings. The 2-aminoquinoline series, represented by 2, was a step in the right direction with respect to selectivity; however, the potency against PI4KIIIα was significantly lower than the original hit (1) and the overall selectivity was still not satisfactory. In addition, the entire 2-aminoquinoline series suffered from poor DMPK properties (Table 2, entry 1). We hoped that further exploration of the SAR around the “head”, “linker”, “core”, and “tail” regions of the structure (Figure 3) would enable us to identify a molecule with pIC50 of >8.0 against PI4KIIIα, selectivity of >100× against other LKs and PKs, and a substantially improved DMPK profile.

Figure 3. Optimization strategy.

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CHEMISTRY The head sulfonamides 37−40 were synthesized as boronic ester building blocks (Scheme 1). Compounds 33−35 were made from sulfonylation of commercially available 5-bromo-3anilines (30−32) with 2,4-difluorobenzene-1-sulfonyl chloride in pyridine. Treatment of compound 33 with iodomethane gave the N-methylated sulfonamide 36. Standard borylation C



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Scheme 1. Synthesis of the Sulfonamide Head Groupsa

a Reagents and conditions: (a) 2,4-difluorobenzene-1-sulfonyl chloride, pyridine, room temperature, overnight, 72% yield; (b) MeI, K2CO3, DMF, room temperature, 48 h, 79% yield; (c) (BPin)2, KOAc, PdCl2(dppf)−DCM, dioxane, 80−100 °C, 2−18 h, 56−89% yield.

Scheme 2. Synthesis of the Alternative Coresa

Reagents and conditions: (a) 2-amino-5-bromonicotinaldehyde, piperidine, dioxane, 100 °C, 3 h, then NaOH, 80 °C, overnight, 74% yield; (b) 5amino-2-chloroisonicotinaldehyde, NaH, THF, room temperature, 30 min, 27% yield; (c) 3-amino-6-bromopicolinaldehyde, NaOH, dioxane, 65 °C, 2.5 h, 90% yield; (d) 37, KOAc or 2 M K2CO3, Pd(PPh3)2Cl2, dioxane, 120 °C, microwave, 1−1.75 h, 5−13% yield.

a

D



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Scheme 3. Synthesis of the Quinoxaline Corea

a

Reagents and conditions: (a) benzaldehyde, 1,1,3,3-tetramethylbutyl isocyanide, concentrated HCl, MeOH, room temperature, overnight, 100% yield, 72% purity; (b) DDQ, benzene, room temperature, overnight, 29.3% yield; (c) HCl, DCM, 70 °C, 94% yield; (d) 37, K2CO3, PdCl2(dppf)− DCM, dioxane/water, 80 °C, overnight, 69% yield.

Scheme 4. General Route to the Aryl Tail Derivatives of the Quinazolinone Corea

a

Reagents and conditions: (a) Ar(R) isothiocyanate, TEA, dioxane or EtOH, reflux, 6 h to overnight, 51−93% yield; (b) PCl5, POCl3, reflux, 7 h to overnight, 70−100% yield; (c) (4-methoxyphenyl)methanamine, DIEA, DMF, 80 °C, overnight to 48 h, 96−100% yield; (d) TFA, microwave, 140 °C, 8.0 against PI4KIIIα, selectivity of >100× against other LKs and PKs, and good DMPK profile, we decided to explore analogues E



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Scheme 5. Synthesis of 3-Alkyl Derivatives of the 2-Aminoquinazolinone Corea

a Reagents and conditions: (a) diphenyl cyanocarbodiimidate, pyridine, 50 °C, overnight, 64% yield; (b) R1-NH2, i-PrOH, 85 °C, 1 h, 58−100% yield; (c) concentrated HCl, DMSO, 100 °C, 1−2.5 h, 100% yield.

Scheme 6. Synthesis of the Reverse Sulfonamide Analoguesa

a Reagents and conditions: (a) SOCl2, water, Cu(I)Cl, HCl, NaNO2, −5 to 30 °C, overnight, 77% yield; (b) (1) 2,4-difluoroaniline, pyridine, DCM, room temperature, overnight, 93% yield; (2) NaOMe/MeOH, 50 °C, 4 h, 93% yield over two steps; (c) (BPin)2, KOAc, PdCl2(dppf)−DCM, dioxane, 95 °C, 2 h, 60% yield; (d) 56−61, 68, 69, Cs2CO3 or K2CO3 or KOAc, PdCl2(dppf)−DCM, dioxane/water, 80−100 °C, 1 h to overnight, 26−65% yield.

improved selectivity, and promising rat DMPK, we chose 8 as our new lead for optimization. Comprehensive SAR related to the “head”, “linker”, and “tail” around this new quinazolinone core of compound 8 was developed. The results of the head and linker optimization are summarized in Table 3. The nitrogen in the linker pyridine ring was crucial for high PI4KIIIα potency, as removing it (i.e., 9) resulted in a significant decrease (2.3 log). This is consistent with our model in which the pyridine-N forms an interaction with the backbone of the enzyme through a water bridge. Replacing the methoxy group with an amino group (10) slightly improved the PI4KIIIα activity and validated the model’s predictions that this group serves just as a space filling function. Interestingly, this change did not translate to HCV inhibition, as reflected by a significant decrease in the replicon activity (pIC50 = 7.8). N-Methylation of the sulfonamide (11) was not tolerated and decreased the PI4KIIIα potency over >1000× (3.4 log). These findings were consistent with the molecule’s predicted binding mode, which was thought to involve the sulfonamide hydrogen forming a H-bond with the catalytic Lys1792 (Figure 2, homology model). In order to decrease the molecular weight, we tried to minimize the headgroup by truncating parts of the molecule. In the case of the 2,4-difluorobenzenesulfonamide moiety, eliminating this group and replacing it with a methyl group (12) resulted in a 10-fold loss in PI4KIIIα potency (pIC50 = 7.7), although the selectivity was improved (5× to 10×). We wondered if slightly larger alkyl groups might restore PI4KIIIα potency; however, the isopropyl group (13) led to a further decrease in the potency of PI4KIIIα (pIC50 = 6.9) and a loss of

containing alternative cores (Table 1). We replaced the central quinoline ring of 3 with isosteric ring systems containing an additional heteroatom in hopes of creating a potential new interaction site with the target enzyme. In the case of the 1,8naphthyridine (4), this modification resulted in an almost 10fold decrease in PI4KIIIα potency (pIC50 = 6.2). Moving the nitrogen to the 7-position (5, pIC50 = 6.7) provided a compound with slightly lower PI4KIIIα potency relative to 3 and comparable selectivity (0.5−1.0 vs 0−1.1). In contrast, inserting the nitrogen at the 5-position (6, pIC50 = 5.1) was not well tolerated resulting in a 2 log decrease in potency. When we moved the nitrogen to the 4-position to give the quinoxaline core (7), a 1.3 log improvement in PI4KIIIα potency was observed (pIC50 = 8.3). The replicon activity and the selectivity was maintained. Given the high tolerability of the substitution at the 4-position for PI4KIIIα potency, a carbonyl derivative 8, a quinazolin-4-one core, was evaluated. A significant increase in PI4KIIIα potency was observed (pIC50 = 8.8), and the inhibition of HCV 1b replicon reached the picomolar level (pIC50 = 9.3). Compound 8 also offered a solid improvement in selectivity against PI3Ks (0.4−1.8). While the selectivity did improve, the absolute potencies against all PI3Ks were the highest within the alternative core series. The DMPK of 8 was also evaluated (Table 2). To our satisfaction, the low-dose rat DMPK study revealed that 8 had substantially better properties relative to the best molecule in the 2-amionquinoline series (2) in terms of exposure (AUC) and the percent bioavailability (% F) (10× and 2×, respectively). Also, the half-life has increased almost 3-fold (6.70 h vs 2.39 h). On the basis of the high PI4KIIIα potency, F



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Table 1. In Vitro Enzyme and Replicon Activity Profile of Analogues with Alternative Cores (pIC50 a)

a

pIC50 = −log IC50. bSelectivity range: smallest to highest differences of pIC50(PI4Kα) − pIC50(PIKx), assigned to 0 if negative.

substituted 8. Furthermore, an electron deficient heteroaromatic ring (19) proved to be the most potent analogue against PI4KIIIα in the series (pIC50 = 9.5). Although these molecules showed higher selectivity, the absolute potencies against the other lipid kinases were still high. The breakthrough toward improving selectivity came when the sulfonamide moiety of 8 was reversed to give 20. There was a minor decrease in PI4KIIIα potency (pIC50 of 8.2) but a significantly larger decrease in PI4KIIIβ (pIC50 of 5.5) and in PI3Kα,β,δ,γ activities (pIC50 of 6.0, 4.9, 6.3, 5.9, respectively), thereby improving the selectivity to the desirable range (1.9− 3.3). On the basis of the homology model (Figure 2), we theorized that reversing the sulfonamide the nature of the Hbond between the inhibitor and Lys1792 was likely weakened and this change was better tolerated by PI4KIIIα than by other lipid kinases. In the context of the new “reverse sulfonamide” structure, we investigated the tail region of the molecule with the goal of further optimization. The results of the tail SAR are summarized in Table 4. Replacing the phenyl group with smaller alkyl groups was not well tolerated. As exemplified with the ethyl derivative (21), a decrease in PI4KIIIα activity was observed (pIC50 = 7.4), and it was coupled with diminishing selectivity. Larger alkyl groups, like the tetrahydropyran (22), did maintain potency (pIC50 = 8.0) against PI4KIIIα, but the selectivity decreased below the starting molecule’s (20) range.

Table 2. Comparison of DMPK Parameters of 2 and 8 after Single Oral 5 mg/kg Solution Dose in Rat compd

AUC (ng·h/mL)

Cmax (ng/mL)

T1/2 (h)

Tmax (h)

F (%)

2 8

4996 60177

1392 3575

2.39 6.7

0.67 6

38 60

selectivity as well (0.2−1.6). An unsubstituted phenyl ring (14) improved the PI4KIIIα potency to the single digit nanomolar range (pIC50 = 8.6) and the initial selectivity was gained back. These molecules clearly suggested that an aromatic ring at this position is favorable for high PI4KIIIα potency and selectivity. To further explore the electronics and sterics around the aromatic ring, an electron-donating methyl group was introduced at various positions (15, 16, 17). In general, these changes were well tolerated, especially the 2-position (15) where the PI4KIIIα activity was slightly improved (pIC50 = 9.0). It was also the best in the series in terms of selectivity (1.4−2.6). A slight loss in potency and selectivity was observed placing the methyl group at the 3- or 4-position (pIC50 of 8.4 and 7.8, respectively). This trend was also observed in the corresponding replicon potencies as well (pIC50 of 9.4, 8.9, and 8.5, respectively). A similar exercise with electron-withdrawing substitutions was also conducted. The addition of a cyano group to the para position (18) resulted in activity on par with the 2,4-difluoroG



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Table 3. In Vitro Enzyme and Replicon Activity Profile of Analogues Optimized on the Linker and Head Regions (pIC50 a)

H



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Table 3. continued

a


Larger alkyl groups, like the tetrahydropyran (22), did maintain potency (pIC50 = 8.0) against PI4KIIIα, but the selectivity decreased compared with the starting lead molecule’s (20) range (1.4−2.6 vs 1.9−3.3, respectively). As the alkyl groups offered no significant improvement over the aryl groups, we optimized the aryl ring in hopes of finding greater improvements in activity and selevtivity. In the course of investigating substitutions on the tail phenyl ring, an electron withdrawing trifluoromethyl group was added to all possible positions (23, 24, 25). In general, the PI4KIIIα potency suffered, although the ortho isomer (23, pIC50 = 7.8) still retained significant activity. The selectivity showed the same trend: slight decrease and ortho position being the best. In fact, 23 displayed an excellent selectivity profile, reaching the target >100-fold selectivity against PI4KIIIβ and all PI3Ks with a selectivity range of 160- to 600-fold (2.2−2.8). The orthomethyl derivative (26) showed improved PI4KIIIα activity, suggesting that an electron donating substitution was favorable for potency, although this resulted in poorer selectivity against PI3Ks (1.3−2.3). To test the size aspect of substituting at the ortho position, the isopropyl analogue was synthesized. Displaying only marginally improved selectivity (1.5−2.7), this analogue (27) had a decreased PI4KIIIα activity (pIC50 = 7.5). Upon further examination of the SAR data, we concluded that 23 fulfilled our starting criteria (low nanomolar PI4KIIIα potency and >100-fold selectivity over PI4KIIIβ and PI3Kα,β,δ,γ); therefore, it became our candidate for further profiling. The structure of 23 suggested a possibility of atropisomerism. It appeared likely that the ortho-trifluoromethyl group could prevent the free rotation of the phenyl group because of the proximity of the amino and carbonyl groups in the

quinazolinone 2- and 4-positions, respectively. In such circumstances, one of the enantiomers could have a better fit within the chiral environment of the enzyme binding site which could translate to higher potency and better selectivity. To investigate this possibility, 23 was analyzed by means of chiral chromatography, and two distinct peaks were observed. The two enantiomers were separated to give 28 and 29 with a chiral purity of >99% for each. The absolute configurations of the enantiomers were determined by vibrational circular dichroism (VCD)24 and spectroscopy assigned as 28 (aS) and 29 (aR) (Figure 4). Upon heating at 80 °C in DMSO for 6 days, no interconversion or decomposition was observed suggesting that they would be thermodynamically stable in the conditions of a biological system. Table 5 summarizes the head-to-head comparison of the in vitro enzyme and replicon data of the isomers. As we expected, they had different profiles with 28 exhibiting 25× higher PI4KIIIα potency than 29 (pIC50 of 8.3 vs 6.9) and an improved selectivity range (2.7−3.2 vs 1.4−2.1) against the other lipid kinases. Comparing the anti-HCV activity, the difference in PI4KIIIα potencies was reflected in the replicon activities: 28 was about 1 order of magnitude more potent against all three genotypes measured (1a, 1b, 2a). We also compared the iv and po DMPK properties in rat of 28 and 29 (Table 6). The study revealed that 28 had acceptable properties, although the less potent 29 was superior in this aspect. In addition, we ran 28 against a 300-protein kinase panel in order to get a broad picture of the overall selectivity. It showed an extremely clean profile against all the measured PKs as well (see data in section VII of Supporting Information). On the basis of the above results, compound 28 was selected as a precandidate and was promoted for additional animal I



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Table 4. In Vitro Enzyme and Replicon Activity Profile of Analogues Optimized on the Tail Region (pIC50 a)

a


Figure 4. Resolution and Assignment of Atropisomers.

Table 5. Comparison of in Vitro Enzyme and Replicon Potencies of Racemic 23 and the Atropisomers 28 and 29 pIC50 a

a

pIC50 a b

compd

PI4Kα

PI4Kβ

PI3Kα

PI3Kβ

PI3Kδ

PI4Kγ

selectivity range

rep 1a

rep 1b

rep 2a

23 28 29

7.8 8.3 6.9

5.8 6.0 5.3

5.6 5.6 5.5

5.0 5.1 4.8

5.5 5.6 5.0

5.5 5.6 5.5

2.2−2.8 2.7−3.2 1.4−2.1

7.7 7.9 6.9

8.5 8.6 7.8

7.9 7.9 7.1


40 mg kg−1 day−1 had morbidity and mortality and test-articlerelated changes in the stomach, thymus, spleen, and clinical pathology parameters. A detailed description of the study will

studies. It had a maximum tolerated dose of 50 mg/kg, following single dose administration, and was poorly tolerated in the 14-day mouse toxicity study. Animals treated with 28 at J



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Table 6. DMPK Comparison of 28 and 29, Rats Single iv (1 mg/kg) and Oral (5 mg/kg) Administrationa compd

route

CL (mL min−1 kg−1)

T1/2 (h)

Vdss (L/kg)

29

iv po iv po

2.5

3.5 2.7 1.3 4.0

0.55

28 a

4.5

Cmax (ng/mL)

Tmax (h)

4429

3

1556

4.25

0.34

DNAUC (h kg ng mL−1 mg−1) 7113 9985 3695 3626

F (%) >100 98

Formulation: DMSO/cyclodextrin. tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-3-sulfonamide (37) (18.1 g, 89%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.11 (s, 1 H), 8.56 (d, J = 1.76 Hz, 1 H), 8.09 (d, J = 1.76 Hz, 1 H), 7.20−7.34 (m, 2 H), 7.00−7.09 (m, 1 H), 3.97 (s, 3 H), 1.28 (s, 12 H); LC−MS m/z = 427.20 (M + H)+. N-(5-(7-Amino-6-phenyl-1,8-naphthyridin-3-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (4). 2-Amino-5bromonicotinaldehyde (0.5g, 2.49 mmol), 2-phenylacetonitrile (41) (0.574 mL, 4.97 mmol), and piperidine (0.491 mL, 4.97 mmol) were dissolved in dioxane (10 mL) and heated to 100 °C for 3 h. The mixture was then treated with sodium hydroxide (0.099 g, 2.49 mmol) and was heated at 80 °C overnight. The solution was concentrated under reduced pressure and the residue was purified by silica gel chromatography (0−15% MeOH/DCM) to give 6-bromo-3-phenyl1,8-naphthyridin-2-amine (42) (0.549g, 74%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.74 (d, J = 2.54 Hz, 1 H), 8.39 (d, J = 2.74 Hz, 1 H), 7.87 (s, 1 H), 7.41−7.56 (m, 5 H), 6.63 (b.s., 2 H); LC−MS m/z = 300.2, 302.2 (M + H)+. A microwave vial was charged with 6-bromo-3-phenyl-1,8naphthyridin-2-amine (42) (0.2 g, 0.666 mmol), 2,4-difluoro-N-(2methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide (37) (0.284 g, 0.666 mmol), KOAc (0.196 g, 2.00 mmol), and 1,4-dioxane (10 mL). To the vial was then added Pd(PPh3)2Cl2 (0.047 g, 0.067 mmol), and the mixture was placed under a N2 atmosphere. The vial was heated to 100 °C in the microwave for 25 min and then heated for an additional hour at 120 °C. The solution was concentrated and the residue was prepurified by silica gel chromatography (0−5% MeOH/DCM) and was then purified by silica gel chromatography (100% EtOAc) to give N-[5-(7amino-6-phenyl-1,8-naphthyridin-3-yl)-2-(methyloxy)-3-pyridinyl]2,4-difluorobenzenesulfonamide (4) (18 mg, 5%) as a pale yellow solid. 1H NMR (400 MHz, MeOH-d4) δ ppm 8.85 (d, J = 2.5 Hz, 1 H), 8.23 (dd, J = 6.3, 2.4 Hz, 2 H), 8.02 (d, J = 2.1 Hz, 1 H), 7.89 (s, 1 H), 7.84 (td, J = 8.5, 6.2 Hz, 1 H), 7.50−7.56 (m, 4 H), 7.42−7.50 (m, 2 H), 7.14−7.23 (m, 1 H), 6.99−7.07 (m, 1 H), 4.88 (s, 1 H), 3.78 (s, 3 H); LC−MS m/z = 520.4 (M + H)+; HRMS calculated for C26H20N5O3F2S (M + 1)+, 520.1255; found 520.1255. N-(5-(2-Amino-3-phenyl-1,7-naphthyridin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (5). To a solution of 5-amino-2-chloroisonicotinaldehyde (344 mg, 2.20 mmol) and 2phenylacetonitrile (41) (258 mg, 2.20 mmol) in THF (15 mL) at room temperature was added 60% sodium hydride (264 mg, 6.60 mmol). The mixture was stirred for 30 min. The solvents were evaporated under reduces pressure and the residue was dissolved in EtOAc, washed with saturated NH4Cl, brine, dried over Na2SO4, and concentrated to give 6-chloro-3-phenyl-1,7-naphthyridin-2-amine (43) (0.150 g, 27%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.68 (s, 1 H), 7.87 (s, 1 H), 7.78 (s, 1 H), 7.44−7.61 (m, 5 H), 6.58 (br s, 2 H); LC−MS m/z = 256.2 (M + H)+. A microwave vial was charged with 6-chloro-3-phenyl-1,7naphthyridin-2-amine (43) (130 mg, 0.508 mmol), 2,4-difluoro-N(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3yl)benzenesulfonamide (37) (130 mg, 0.305 mmol), 2 M aquoeus K2CO3 (0.8 mL), and 1,4-dioxane (4 mL). The mixture was degassed, treated with Pd(PPh3)2Cl2 (35.7 mg, 0.051 mmol), and placed under a nitrogen atmosphere. The mixture was heated in the microwave at 120 °C for 1 h. The solution was concentrated under reduced pressure and prepurified by silica gel chromatography (0−40% EtOAc/DCM) and was then purified with reverse phase chromatography (0−10%ACN/ H2O) to give N-[5-(2-amino-3-phenyl-1,7-naphthyridin-6-yl)-2-(meth-

be reported in a subsequent publication (manuscript in preparation).

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CONCLUSION Targeting a host protein to achieve antiviral activity is a relatively unexplored area and carries the risk of higher likelihood of off-target activity. However, the potential benefits of wide-spectrum inhibition, higher barrier to resistance, and a novel mechanism of action are clear advantages of host−target inhibition. Starting with a focused HTS of a 10K lipid kinase library we targeted the host cell lipid kinase PI4KIIIα that was shown by siRNA knockout experiments to be vital for HCV replication. To determine if PI4KIIIα inhibition with a small molecule translates to an anti-HCV effect, we synthesized multiple series of inhibitors. The plotted PI4KIIIα vs replicon potencies clearly indicated a correlation (R2 = 0.79) that strongly supports the original hypothesis (section V of Supporting Information). Through extensive optimization, we developed a potent host enzyme inhibitor (28) that is the first extremely selective PI4KIIIα inhibitor against other lipid and protein kinases, coupled with the in vitro inhibition of HCV.

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EXPERIMENTAL SECTION

Compound Purities and Identity. All compounds underwent an immediate processing quality control protocol to confirm identity and determine relative purity immediately before processing in the enzyme and replicon assays. Analysis is by UPLC−MS with UV diode array detection to determine purity and MS used to confirm molecular weight. A Waters Acquity UPLC system comprising binary solvent manager, sample manager, PDA detector, Waters ZQ or SQD mass spectrometer, Waters Acquity evaporative light scattering detector or Polymer Laboratories evaporative light scattering detector was employed. Mobile phases were the following: acetonitrile + 0.1% formic acid; water + 0.1% formic acid. Wash solutions were the following: strong wash 100% acetonitrile + 0.1% formic acid; weak wash 50:50 acetonitrile/water + 0.1% formic acid. All individual lots of compounds tested ≥95% pure according to this protocol. N-(2,4-Difluorophenyl)-2-methoxy-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyridine-3-sulfonamide (37). A solution of 5-bromo-2-methoxypyridin-3-amine (30) (24.8 g, 117 mmol) in pyridine (45 mL) at room temperature was treated by the dropwise addition of 2,4-difluorobenzene-1-sulfonyl chloride (15.8 mL, 117 mmol) over 15 min. The mixture was stirred at room temperature overnight and was loaded onto a silica gel column and purified (0− 30% EtOAc/hexane) to give N-(5-bromo-2-methoxypyridin-3-yl)-2,4difluorobenzenesulfonamide (33) as a yellow solid (32 g, 72%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.46 (s, 1 H), 8.13 (d, J = 2.35 Hz, 1 H), 7.72−7.82 (m, 2 H), 7.58 (ddd, J = 10.65, 9.19, 2.44 Hz, 1 H), 7.23 (td, J = 8.50, 1.76 Hz, 1 H), 3.61 (s, 3 H); LC−MS m/z = 379.21, 381.20 (M + H)+. A solution of N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (33) (18g, 47.5 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl2,2′-bi(1,3,2-dioxaborolane) (15.7 g, 61.7 mmol), KOAc (14.0 g, 142 mmol) in 1,4-dioxane (100 mL) was treated with PdCl2(dppf)− CH2Cl2 (0.775 g, 0.949 mmol). The mixture was heated at 100 °C for 4 h and was then loaded onto silica gel column and purified (0−30% EtOAc/hexane) to give N-(2,4-difluorophenyl)-2-methoxy-5-(4,4,5,5K



Article

A flask containing the 35:65 mixture of 6-bromo-3-phenyl-2quinoxalinamine (47a) and 7-bromo-3-phenylquinoxalin-2-amine (47b) (0.086 g, 0.287 mmol), 2,4-difluoro-N-[2-(methyloxy)-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridinyl]benzenesulfonamide (37) (0.134 g, 0.315 mmol), K2CO3 (0.119 g, 0.860 mmol), PdCl2(dppf)−DCM (0.023 g, 0.029 mmol) in 1,4dioxane (5.97 mL) and water (5.97 mL) was heated at 80 °C, and the mixture was stirred overnight. After cooling to room temperature, the mixture was diluted with EtOAc and water. The combined organics were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by reverse phase chromatography (10−90% ACN/H2O) to give an unassigned 38:62 mixture of N-[5-(2-amino-3-phenyl-6-quinoxalinyl)-2-(methyloxy)-3pyridinyl]-2,4-difluorobenzenesulfonamide (7) and N-(5-(3-amino-2phenylquinoxalin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (48) (0.103 g, 69%) as a light yellow solid. LC−MS m/z = 520.2 (M + H)+. The isomers were separated on an Aglient preparative HPLC instrument using 7.5 mL/min 30% IPA in hexane on 10 mm ODH column to afford the first eluting isomer N-(5-(2amino-3-phenylquinoxalin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (7) (12 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (br s, 1 H), 8.44 (s, 1 H), 7.95−8.05 (m, 2 H), 7.72−7.88 (m, 4 H), 7.63−7.68 (m, 1 H), 7.50−7.62 (m, 4 H), 7.17−7.26 (m, 1 H), 6.69 (br s, 2 H), 3.66 (s, 3 H); LC−MS m/z = 520.3 (M + H)+; HRMS calculated for C26H20N5O3F2S (M + 1)+, 520.1255; found 520.1254. The second eluting isomer was N-(5-(3-amino-2-phenylquinoxalin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (48) (24 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.34 (br s, 1 H), 8.45 (s, 1 H), 7.98−8.01 (m, 1 H), 7.87−7.91 (m, 1 H), 7.74− 7.83 (m, 4 H), 7.61−7.66 (m, 1 H), 7.52−7.60 (m, 4 H), 7.17−7.26 (m, 1 H), 6.67 (br s, 2 H), 3.68 (s, 3 H); LC−MS m/z = 520.3(M + H)+; HRMS calculated for C26H20N5O3F2S (M + 1)+, 520.1255; found 520.1253. Absolute stereochemical assignments were made by using 13 C chemical shifts and using ab initio IR analysis. N-(5-(2-Amino-4-oxo-3-phenyl-3,4-dihydroquinazolin-6-yl)2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (8). A solution of isothiocyanatobenzene (22.7 mL, 190 mmol), 2amino-5-iodobenzoic acid (49, Y = I) (50 g, 190 mmol), and TEA (39.7 mL, 285 mmol) in 1,4-dioxane (750 mL) was maintained at reflux for 6 h. The mixture was cooled to room temperature, and solids were filtered. The solids were resuspended in Et2O and filtered to afford 6-iodo-3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (67.8 g, 178 mmol) as a white solid. 1H NMR (400 MHz, DMSOd6) δ ppm 13.10 (s, 1 H), 8.17 (d, J = 1.95 Hz, 1 H), 8.07 (dd, J = 8.63, 2.00 Hz, 1 H), 7.44−7.52 (m, 2 H), 7.37−7.44 (m, 1 H), 7.18− 7.30 (m, 3 H); LC−MS m/z = 380.83 (M + H)+. 6-Iodo-3-phenyl-2thioxo-2,3-dihydroquinazolin-4(1H)-one (34.8 g, 92 mmol) was added to POCl3 (205 mL, 2197 mmol) at room temperature. The mixture was treated with PCl5 (33.4 g, 160 mmol) in one portion and was stirred at room temperature for 15 min, then heated at 110 °C overnight. The mixture was allowed to cool to room temperature and was concentrated under reduced pressure. The residue was slurried in EtOAc, added portionwise to stirred ice−water, and stirred until all solids had dissolved. The organic phase was separated, washed with brine, dried over sodium sulfate, and concentrated. The residue was triturated by stirring in Et2O and then filtered to give 2-chloro-6-iodo3-phenylquinazolin-4(3H)-one (50, Y = I) (24.5 g, 70%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.38 (d, 1 H), 8.20 (dd, 1 H), 7.44−7.68 (m, 6 H); LC−MS m/z = 382.9 (M + H)+. DIEA (22.4 mL, 128 mmol) and 4-methoxybenzylamine (10.9 mL, 83 mmol) were dissolved in DMF (200 mL), and 2-chloro-6-iodo-3phenylquinazolin-4(3H)-one (50, Y = I) (24.5 g, 64.0 mmol) was added portionwise. The mixture was heated at 80 °C for 4 h and then cooled to room temperature. The mixture was concentrated to 75 mL and diluted with EtOAc. The organic phase was washed with 5% LiCl (2×), brine, dried over sodium sulfate, and concentrated. The residue was purified by silica chromatography (1:6:3 EtOAc/hexanes/DCM) to give the 6-iodo-2-((4-methoxybenzyl)amino)-3-phenylquinazolin4(3H)-one (29.7 g, 96%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.13 (d, 1 H), 7.85 (dd, 1 H), 7.50−7.67 (m, 3 H),

yloxy)-3-pyridinyl]-2,4-difluorobenzenesulfonamide (5) (20 mg, 13%) as a pale yellow oil. 1H NMR (400 MHz, chloroform-d) δ ppm 9.14 (s, 1 H), 8.60 (d, J = 1.8 Hz, 1 H), 8.40 (d, J = 1.8 Hz, 1 H), 7.84−7.96 (m, 1 H), 7.82 (s, 1 H), 7.78 (s, 1 H), 7.44−7.60 (m, 6 H), 6.87−6.99 (m, 2 H), 5.26 (br s, 2 H), 3.95 (s, 3 H); LC−MS m/z = 520.4 (M + H)+; HRMS calculated for C26H20N5O3F2S (M + 1)+, 520.1255; found 520.1254. N-(5-(6-Amino-7-phenyl-1,5-naphthyridin-2-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (6). A solution of 3-amino-6-bromopicolinaldehyde (0.13 g, 0.647 mmol), 2-phenylacetonitrile (41) (0.076 g, 0.647 mmol), and sodium hydroxide (0.078 g, 1.94 mmol) in 1,4-dioxane (10 mL) was heated at 65 °C for 2.5 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (0−30% EtOAc/hexanes) to give 6-bromo-3-phenyl-1,5-naphthyridin-2-amine (44) (0.175 g, 90%) as a yellow solid. LC−MS m/z = 300.2, 302.2 (M + H)+. A microwave vial was charged with 6-bromo-3-phenyl-1,5naphthyridin-2-amine (44) (170 mg, 0.566 mmol), 2,4-difluoro-N(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3yl)benzenesulfonamide (37) (241 mg, 0.566 mmol), KOAc (167 mg, 1.699 mmol), and 1,4-dioxane (10 mL). The solution was degassed, treated with Pd(PPh3)2Cl2 (39.7 mg, 0.057 mmol), placed under a nitrogen atmosphere, and heated in the microwave at 120 °C for 95 min. The mixture was washed with water and extracted into EtOAc. The organics were concentrated and prepurified by silica gel chromatography (0−100% EtOAc/DCM) and was then purified by reverse phase chromatography (10−100% ACN/H2O) to give N-[5(6-amino-7-phenyl-1,5-naphthyridin-2-yl)-2-(methyloxy)-3-pyridinyl]2,4-difluorobenzenesulfonamide (6) (20 mg, 7%) as a yellow solid. 1H NMR (400 MHz, chloroform-d) δ ppm 8.46 (d, J = 8.8 Hz, 1 H), 8.21 (d, J = 1.4 Hz, 1 H), 8.01 (d, J = 1.6 Hz, 1 H), 7.80−7.91 (m, 2 H), 7.65−7.74 (m, 2 H), 7.56 (d, J = 9.2 Hz, 1 H), 7.47 (dd, J = 5.2, 1.7 Hz, 3 H), 7.38 (br s, 1 H), 6.90−7.00 (m, 2 H), 6.57 (br s, J = 8.0 Hz, 2 H), 3.97 (s, 3 H); LC−MS m/z = 520.4 (M + H)+; HRMS calculated for C26H20N5O3F2S (M + 1)+, 520.1255; found 520.1257. N-(5-(2-Amino-3-phenylquinoxalin-6-yl)-2-methoxypyridin3-yl)-2,4-difluorobenzenesulfonamide (7). A solution of 4bromo-1,2-benzenediamine (45) (6.7 g, 35.8 mmol) was dissolved in degassed anhydrous MeOH (166 mL) and was purged with nitrogen. The solution was treated with concentrated HCl (2.94 mL, 35.8 mmol), followed by benzaldehyde (3.62 mL, 35.8 mmol) and 1,1,3,3-tetramethylbutyl isocyanide (6.24 mL, 35.8 mmol). The mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was partitioned between saturated NaHCO3 and CHCl3. The combined organics were washed with brine, dried over MgSO4, filtered, and concentrated to give 6-bromo-3-phenyl-N-(1,1,3,3-tetramethylbutyl)-1,4-dihydro-2quinoxalinamine (16.1 g, 100%, 72% purity) which was used without further purification. LC−MS m/z = 414.3, 416.3 (M + H)+. A solution of 6-bromo-3-phenyl-N-(1,1,3,3-tetramethylbutyl)-1,4-dihydro-2-quinoxalinamine (16.1 g, 38.8 mmol) in benzene (100 mL) was treated by the addition of DDQ (8.80 g, 38.8 mmol) in benzene (200 mL). The mixture was stirred at room temperature overnight. The dark solution was concentrated onto Celite and purified by silica gel chromatography (0−10% EtOAc/hexanes) to give an unassigned 38:62 mixture of 6-bromo-3-phenyl-N-(1,1,3,3-tetramethylbutyl)-2quinoxalinamine (46a) and 7-bromo-3-phenyl-N-(2,4,4-trimethylpentan-2-yl)quinoxalin-2-amine (46b) (4.68 g, 29%) as a yellow oil. LC− MS m/z = 412.3, 414.2 (M + H)+. The 38:62 mixture of 6-bromo-3-phenyl-N-(1,1,3,3-tetramethylbutyl)-2-quinoxalinamine (46a) and 7-bromo-3-phenyl-N-(2,4,4-trimethylpentan-2-yl)quinoxalin-2-amine (46b) (4.68 g, 11.4 mmol) in DCM (51.1 mL) was treated by the addition of 4 N in dioxanes HCl (33 mL) and was heated to 70 °C overnight. The solvents were evaporated under reduced pressure, and the residue was taken up in EtOAc and treated with saturated NaHCO3. The combine extracts were washed with brine, dried over Na2SO4, filtered, and concentrated to give an unassigned 35:65 mixture of 6-bromo-3-phenyl-2quinoxalinamine (47a) and 7-bromo-3-phenylquinoxalin-2-amine (47b) (3.20 g, 94%). LC−MS m/z = 300.1, 302.2 (M + H)+. L



Article

1 H), 6.31 (br s, 2 H), 5.09 (s, 2 H), 3.89 (s, 3 H); LC−MS m/z = 360.1 (M + H)+. A solution of MsCl (128 mg, 1.12 mmol) and 2-amino-6-[5-amino6-(methyloxy)-3-pyridinyl]-3-phenyl-4(3H)-quinazolinone (63) (50 mg, 0.139 mmol) in pyridine (1 mL) was stirred at room temperature for 1 day. The resulting mixture was treated with MeOH (1 mL) and concentrated. The residue was dissolved in DMF and purified by reverse phase chromatography (10−90% ACN/H2O) to obtain N-[5(2-amino-4-oxo-3-phenyl-3,4-dihydro-6-quinazolinyl)-2-(methyloxy)3-pyridinyl]methanesulfonamide (12) (17 mg, 28%) as a white solid: 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.07 (s, 3 H), 3.95 (s, 3 H), 6.37 (br s, 2 H), 7.29−7.42 (m, 3 H), 7.48−7.63 (m, 3 H), 7.87 (d, J = 2.24 Hz, 1 H), 7.92 (dd, J = 8.58, 2.34 Hz, 1 H), 8.05 (d, J = 2.24 Hz, 1 H), 8.29 (d, J = 1.76 Hz, 1 H), 9.38 (br s, 1 H); LC−MS m/z = 438.4 (M + H)+; HRMS calculated for C21H20N5O4S (M + 1)+, 438.1236; found 438.1237. 5-(2-Amino-4-oxo-3-phenyl-3,4-dihydroquinazolin-6-yl)-N(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (20). Step a. SOCl2 (14 mL, 192 mmol) was added dropwise over 60 min to water (83 mL) and cooled to 0 °C, maintaining the temperature of the mixture at 0−7 °C. The solution was allowed to warm to 18 °C over 17 h. Copper(I) chloride (51 mg, 0.515 mmol) was added to the mixture, and the resultant yellow-green solution was cooled to −3 °C using an acetone/ice bath. Step b. HCl (45 mL, 1480 mmol) was added, with agitation, to 5bromo-2-chloro-3-pyridinamine (70) (9.3 g, 44.8 mmol), maintaining the temperature of the mixture below 30 °C with ice cooling. The reaction mixture was cooled to −5 °C using an ice−acetone bath, and a solution of sodium nitrite (3.33 g, 48.3 mmol) in water (13 mL) was added dropwise over 15 min, maintaining the temperature of the reaction mixture between −5 to 0 °C. The resultant slurry was cooled to −2 °C and stirred for 10 min. Step c. The slurry from step b was cooled to −5 °C and added to the solution obtained from step a over 30 min, maintaining the temperature of the reaction mixture between −3 to 0 °C (the slurry from step b was maintained at −5 °C throughout the addition). As the reaction proceeded, a solid began to precipitate. When the addition was complete, the reaction mixture was agitated at 0 °C for 75 min. The suspended solid was filtered, washed with water, and dried under reduced pressure to give 5-bromo-2-chloro-3-pyridinesulfonyl chloride (71) (10 g, 77%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.52 (d, J = 2.54 Hz, 1 H), 8.30 (d, J = 2.54 Hz, 1 H). A solution of 5-bromo-2-chloro-3-pyridinesulfonyl chloride (71) (20.0 g, 68.7 mmol) in DCM (200 mL) was treated with pyridine (6.12 mL, 76 mmol) and 2,4-difluoroaniline (7.61 mL, 76 mmol). The mixture was stirred at room temperature overnight, and the volatiles were removed under reduced pressure. The residue was taken up in MeOH (200 mL), and 25% NaOMe/MeOH (200 mL, 896 mmol) solution was added. The mixture was heated at 50 °C for 4 h. The volatiles were removed under reduced pressure. To the dark oily residue icy water was added (400 mL) and was extracted with EtOAc (200 mL and 2 × 100 mL). The combined organics were washed with brine (2 × 100 mL), dried over MgSO4, and evaporated to give 5bromo-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (72) (24.26g, 93%). 1H NMR (400 MHz, DMSO-d6) δ = 10.27 (br s, 1 H), 8.48 (d, J = 2.4 Hz, 1 H), 8.06 (d, J = 2.4 Hz, 1 H), 7.24 (td, J = 6.2, 9.1 Hz, 1 H), 7.19−7.10 (m, 1 H), 7.00−6.89 (m, 1 H), 3.89 (s, 3 H); LC−MS m/z = 377.1, 379.1 (M − H)−. A flask was charged with 5-bromo-N-(2,4-difluorophenyl)-2methoxypyridine-3-sulfonamide (72) (13.12 g, 34.6 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (10.54 g, 41.5 mmol), KOAc (10.19 g, 104 mmol), PdCl2(dppf)−CH2Cl2 (2.83 g, 3.46 mmol), and 1,4-dioxane (173 mL). The mixture was degassed by bubbling nitrogen through for 15 min, and then the mixture was heated 95 °C for 2 h. The black liquid was filtered through a Celite pad and was evaporated to dryness. The black semisolid product was purified by silica gel chromatography (0−40% EtOAc/hexane) to give N-(2,4-difluorophenyl)-2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-3-sulfonamide (73) (8.89g, 60%) as a pale yellow solid. 1H NMR (400 MHz, DMSO-

7.34−7.46 (m, 2 H), 7.22 (d, 2 H), 7.09 (d, 1 H), 6.78−6.91 (m, 2 H), 6.46 (s, 1 H), 4.42 (d, 2 H), 3.70 (s, 3 H); LC−MS m/z = 484.1 (M + H)+. 6-Iodo-2-((4-methoxybenzyl)amino)-3-phenylquinazolin-4(3H)one (29.7 g, 61.5 mmol) was added to TFA (300 mL) and heated at reflux for 2 days. The mixture was cooled to room temperature and was concentrated. The residue was taken up in DCM and poured portionwise into an ice/saturated NaHCO3. The organic phase, with suspended solids, was separated and the aqueous phase extracted with DCM. The combined organic phases were diluted with Et2O to a ratio of 1:1 DCM/Et2O. The solids were collected by filtration, washed with Et2O, and dried under vacuum to give 2-amino-6-iodo-3-phenylquinazolin-4(3H)-one (56, Y = I) (10.2 g, 98% purity, 45%). The filtrate was concentrated and the residue slurried in 1:1 DCM/Et2O. The solids were collected and dried under vacuum to give additional 2amino-6-iodo-3-phenylquinazolin-4(3H)-one (56, Y = I) (7.9 g, 95% purity, 34%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.12 (d, J = 2.15 Hz, 1 H), 7.85 (dd, J = 8.63, 2.20 Hz, 1 H), 7.48−7.63 (m, 3 H), 7.30−7.39 (m, 2 H), 7.05 (d, J = 8.68 Hz, 1 H), 6.42 (br s, 2 H); LC− MS m/z = 363.90 (M + H)+. A solution of 2-amino-6-iodo-3-phenylquinazolin-4(3H)-one (56, Y = I) (0.150 g, 0.413 mmol), 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide (37) (0.194 g, 0.454 mmol), Cs2CO3 (0.404 g, 1.24 mmol), and PdCl2(dppf)-DCM (0.034 g, 0.041 mmol) in 1,4-dioxane (1.65 mL) and water (0.413 mL) was heated at 80 °C for 1 h. After cooling to room temperature, the solution was diluted with EtOAc and washed with water, brine, dried over Na2SO4, and concentrated. The residue was loaded onto Celite and purified by silica gel chromatography (50− 100% EtOAc/hexanes) and was triturated with Et2O to give N-(5-(2amino-4-oxo-3-phenyl-3,4-dihydroquinazolin-6-yl)-2-methoxypyridin3-yl)-2,4-difluorobenzenesulfonamide (8) (0.143 g, 65%) as a pale tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.30 (br s, 1 H), 8.33 (d, J = 2.15 Hz, 1 H), 8.02 (d, J = 2.34 Hz, 1 H), 7.89 (dd, J = 8.59, 2.34 Hz, 1 H), 7.85 (d, J = 2.34 Hz, 1 H), 7.72−7.80 (m, 1 H), 7.50− 7.64 (m, 4 H), 7.38 (d, J = 7.22 Hz, 2 H), 7.34 (d, J = 8.59 Hz, 1 H), 7.21 (t, 1 H), 6.39 (br s, 2 H), 3.65 (s, 3 H); LC−MS m/z = 536.14 (M + H)+; HRMS calculated for C26H20N5O4F2S (M + 1)+, 536.1204; found 536.1202. N-[5-(2-Amino-4-oxo-3-phenyl-3,4-dihydro-6-quinazolinyl)2-(methyloxy)-3-pyridinyl]methanesulfonamide (12). A solution of 5-bromo-2-(methyloxy)-3-pyridinamine (30) (Small Molecules, Inc., Hoboken, NJ) (5.0 g, 24.6 mmol), 4,4,4′,4′,5,5,5′,5′octamethyl-2,2′-bi-1,3,2-dioxaborolane (7.5 g, 29.6 mmol), PdCl2(dppf)−CH2Cl2 (2.0 g, 2.46 mmol), and KOAc (7.25 g, 73.9 mmol) in anhydrous dioxane (120 mL) was purged with nitrogen and heated to 100 °C for 18 h. The mixture was concentrated in vacuo. The residue was diluted with EtOAc (500 mL) and filtered through Celite. The filtrate was washed with cold water (250 mL), brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel chromatography (70−100% EtOAc/hexanes) to afford 2methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3amine (40) (4.38 g, 71%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.64 (d, J = 1.51 Hz, 1 H), 7.12 (d, J = 1.76 Hz, 1 H), 4.88 (s, 2 H), 3.87 (s, 3 H), 1.26 (s, 12 H); LC−MS m/z = 250.8 (M + H)+. A solution 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridin-3-amine (40) (4.38 g, 17.6 mmol), 2-amino-6-iodo-3phenyl-4(3H)-quinazolinone (56, Y = I) (5.8 g, 16.0 mmol), Cs2CO3 (15.6 g, 47.9 mmol), and PdCl2(dppf)−CH2Cl2 (1.30 g, 1.6 mmol) in THF (60 mL) and water (20 mL) was purged with nitrogen and heated to 65 °C for 1 h. The reaction was concentrated in vacuo, and the residue was diluted with ethyl acetate (500 mL) and filtered through Celite. The filtrate was washed with water, brine, dried over MgSO4, and concentrated in vacuo. The residue was triturated in hot ACN (60 mL), cooled to room temperature, and stirred for 30 min. The solids were filtered to give 2-amino-6-(5-amino-6-methoxypyridin-3-yl)-3-phenylquinazolin-4(3H)-one (63) (2.30 g, 40%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.00 (d, J = 2.3 Hz, 1 H), 7.85 (dd, J = 8.6, 2.3 Hz, 1 H), 7.68 (d, J = 2.1 Hz, 1 H), 7.46−7.64 (m, 3 H), 7.35−7.41 (m, 2 H), 7.31 (d, J = 8.4 Hz, 1 H), 7.18 (d, J = 2.1 Hz, M



Article

d6) δ 10.12 (s, 1H), 8.57 (d, J = 1.86 Hz, 1H), 8.10 (d, J = 1.76 Hz, 1H), 7.17−7.40 (m, 2H), 6.93−7.13 (m, 1H), 3.97 (s, 3H), 1.28 (s, 12H); LC−MS 344.89 m/z = (M + H)+. N-(2,4-Difluorophenyl)-2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-3-sulfonamide (73) (117 mg, 0.275 mmol), 2-amino-6-iodo-3-phenyl-4(3H)-quinazolinone (56) (100 mg, 0.275 mmol), PdCl2(dppf)-CH2Cl2 (22.49 mg, 0.028 mmol) were purged with nitrogen. The mixture was treated with 2 M sodium carbonate (500 μL, 1.00 mmol) and 1,4-dioxane (2 mL), and nitrogen was bubbled through the mixture. The mixture was heated at 80 °C for 2 h. After the mixture was cooled, silica gel was added and the volatiles were removed under reduced pressure. The residue was purified by column chromatography (DCM/EtOAc) to afford 5-(2-amino-4-oxo3-phenyl-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (20) (111 mg, 75%). 1H NMR (DMSOd6) δ: 10.19 (s, 1H), 8.76 (d, J = 2.3 Hz, 1H), 8.17 (d, J = 2.5 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.91 (dd, J = 8.7, 2.2 Hz, 1H), 7.47−7.69 (m, 4H), 7.29−7.41 (m, 4H), 6.97−7.11 (m, 1H), 6.42 (br s, 2H), 3.97 (s, 3H); LC−MS m/z = 536.4 (M + H)+. 5-(2-Amino-3-ethyl-4-oxo-3,4-dihydro-6-quinazolinyl)-N(2,4-difluorophenyl)-2-(methyloxy)-3-pyridinesulfonamide (21). Methyl 2-amino-5-bromobenzoate (64) (6.63 g, 28.8 mmol) in pyridine (53 mL) was treated with diphenyl cyanocarbodimidate (6.87 g, 28.8 mmol) and heated to 50 °C overnight. The mixture was concentrated under reduced pressure. The residue was taken up in DCM and 5% K2CO3 (200 mL). The aqueous portion was extracted with DCM. The combined organics were washed with brine (2×), dried over MgSO4, filtered, and concentrated to give an oil that began to solidify. The residue was then treated with 1:1 Et2O/hexanes (100 mL). The solids were filtered and rinsed with 1:1 Et2O/hexanes to give methyl 5-bromo-2-{[(cyanoamino)(phenyloxy)methylidene]amino}benzoate (65) as a white solid (6.95 g, 64%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.83 (br s, 1 H), 8.02 (d, J = 2.15 Hz, 1 H), 7.89 (dd, J = 8.60, 2.34 Hz, 1 H), 7.63 (d, J = 8.60 Hz, 1 H), 7.43−7.49 (m, 2 H), 7.30−7.37 (m, 1 H), 7.24 (d, J = 8.01 Hz, 2 H), 3.87 (s, 3 H); LC−MS m/z = 374.1, 376.1 (M + H)+. Methyl 5-bromo-2-(((cyanoimino)(phenoxy)methyl)amino)benzoate (65) (200 mg, 0.534 mmol) was placed in a sealed tube with i-PrOH (2.5 mL) and 2 M ethylamine in THF (0.267 mL, 1.07 mmol). The mixture was heated to 85 °C for 1 h. The solvents were removed under reduced pressure to give N-(6-bromo-3-ethyl-4-oxo3,4-dihydroquinazolin-2-yl)cyanamide (66) (164 mg, 100%) which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (br s, 1 H), 7.11−7.19 (m, 1 H), 6.70−6.80 (m, 1 H), 4.01 (q, J = 6.91 Hz, 2 H), 1.08−1.16 (m, 3 H); LC−MS m/z = 305.3, 307.3 (M + H)+. N-(6-Bromo-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)cyanamide (66) (156 mg, 0.534 mmol) was placed in a sealed tube followed by DMSO (1.79 mL) and concentrated HCl (3.56 mL). The mixture was heated to 100 °C for 1 h. The mixture was poured into water (20 mL), treated with K2CO3 until gas evolution ceased, and extracted with DCM (2 × 25 mL). The solvents were then removed under reduced pressure, and the residue was dissolved into EtOAc (25 mL), washed with water (12 mL), brine, dried over MgSO4, and concentrated to give 2-amino-6-bromo-3-ethylquinazolin-4(3H)-one (68) (0.143 g, 100%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.94 (d, J = 2.34 Hz, 1 H), 7.66 (dd, J = 8.74, 2.49 Hz, 1 H), 7.19 (br s, 2 H), 7.11 (d, J = 8.89 Hz, 1 H), 3.97−4.05 (m, 2 H), 1.10−1.21 (m, 3 H); LC−MS m/z = 268.3, 270.3 (M + H)+. PdCl2(dppf)−CH2Cl2 (9.14 mg, 0.011 mmol), KOAc (43.9 mg, 0.448 mmol), and N-(2,4-difluorophenyl)-2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-3-sulfonamide (73) (52.5 mg, 0.123 mmol) were combined with 2-amino-6-bromo-3-ethylquinazolin-4(3H)-one (68) (30 mg, 0.112 mmol) in dioxane (0.8 mL) and water (0.267 mL). The mixture was degassed and heated to 100 °C in a sealed tube. After 1 h, the mixture was cooled and the solution was filtered to remove any solids and purified by reverse phase chromatography (10−90% ACN/water) to give 5-(2-amino-3-ethyl4-oxo-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (21) (30 mg, 52%). 1H NMR (400 MHz,

DMSO-d6) δ ppm 8.72 (d, J = 2.1 Hz, 1 H), 8.10−8.23 (m, 2 H), 8.02 (d, J = 2.3 Hz, 1 H), 7.85 (dd, J = 8.7, 2.2 Hz, 1 H), 7.31 (td, J = 9.0, 6.2 Hz, 1 H), 7.24 (d, J = 8.6 Hz, 2 H), 7.17 (s, 2 H), 6.97−7.07 (m, 1 H), 3.99−4.09 (m, 2 H), 3.96 (s, 3 H), 1.17 (t, J = 6.9 Hz, 3 H); LC− MS m/z = 488.3 (M + H)+; HRMS calculated for C22H20N5O4F2S (M + 1)+, 488.1204; found 488.1200. 5-(2-Amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (23) and (aS)-5-(2-Amino-4-oxo-3-(2(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4difluorophenyl)-2-methoxypyridine-3-sulfonamide (28) and (aR)- 5-(2-Amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (29). A solution of 2-amino-5-iodobenzoic acid (49, Y = I) (35.0 g, 133 mmol), 1-isothiocyanato-2-(trifluoromethyl)benzene (24.1 mL, 160 mmol), and TEA (26.0 mL, 186 mmol) in EtOH (507 mL) was heated at reflux overnight. After the mixture was cooled to room temperature, a small amount of black solids were filtered, rinsed with EtOH, and the solids were discarded. The filtrate was concentrated and the brown residue was taken up in DCM and washed with saturated NaHCO3. Solids crashed out and were filtered and rinsed with DCM. The gray solids were taken up in EtOH and sonicated. The solids were filtered, rinsed with cold EtOH, and dried under reduced pressure at 45 °C overnight to give 6-iodo-2-thioxo-3(2-(trifluoromethyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (27.28 g, 46%). 1H NMR (400 MHz, DMSO-d6) δ ppm 13.28 (br s, 1 H), 8.16 (d, J = 1.95 Hz, 1 H), 8.04 (dd, J = 8.54, 1.51 Hz, 1 H), 7.75−7.85 (m, 2 H), 7.59−7.67 (m, 1 H), 7.50 (d, J = 7.80 Hz, 1 H), 7.20 (d, J = 8.68 Hz, 1 H); LC−MS m/z = 499.0 (M + H)+. A suspension of 6iodo-2-thioxo-3-(2-(trifluoromethyl)phenyl)-2,3-dihydroquinazolin4(1H)-one (15 g, 33.5 mmol) in POCl3 (74.9 mL, 803 mmol) was treated with PCl5 (11.15 g, 53.3 mmol), and the mixture was heated to reflux overnight. A second flask containing a suspension of 6-iodo-2thioxo-3-(2-(trifluoromethyl)phenyl)-2,3-dihydroquinazolin-4(1H)one (12.28 g, 27.4 mmol) in POCl3 (61.3 mL, 658 mmol) was treated with PCl5 (9.13 g, 43.8 mmol), and the mixture was heated to reflux overnight. The two reactions were concentrated under reduced pressure. The residues were taken up in EtOAc and combined. The combined organics were washed with water (3×), brine, dried over MgSO4, filtered, and concentrated to give 2-chloro-6-iodo-3-(2(trifluoromethyl)phenyl)quinazolin-4(3H)-one (51, Y = I) (26.1 g, 95%) as a light beige solid. LC−MS m/z = 450.8 (M + H)+. A solution of 2-chloro-6-iodo-3-(2-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (51, Y = I) (26.1 g, 57.9 mmol) and DIEA (15.2 mL, 87 mmol) in DMF (265 mL) was treated by the addition of 4-methoxybenzylamine (9.07 mL, 69.4 mmol) and was heated to 80 °C overnight. The mixture was diluted with EtOAc and washed with 5% LiCl (5×), brine, dried over MgSO4, filtered, and concentrated to give a dark brownish yellow solid. This residue was purified with silica gel chromatography (100% DCM) to give 6-iodo-2-((4methoxybenzyl)amino)-3-(2-(trifluoromethyl)phenyl)quinazolin4(3H)-one (14.34 g, 94% purity) as a solid. LC−MS m/z = 552.0 (M + H)+. The impure fractions (∼16 g) were purified by silica gel chromatography (10−30% then 50% EtOAc/hexanes) to give additional 6-iodo-2-((4-methoxybenzyl)amino)-3-(2(trifluoromethyl)phenyl)quinazolin-4(3H)-one (11.08 g). LC−MS m/z = 552.0 (M + H)+. The combined overall recovery was 25.4 g and 80% yield. A solution 6-iodo-2-((4-methoxybenzyl)amino)-3-(2(trifluoromethyl)phenyl)quinazolin-4(3H)-one (21.8 g, 39.5 mmol) in TFA (198 mL) was split among 12 microwave flasks and was heated to 140 °C in the microwave for 20 min. After cooling to room temperature, the portions were combined and the solvents were removed under reduced pressure and the residue was taken up in DCM. The solution was washed with saturated NaHCO3 (3×) during which solids formed, the solution/solid was washed with water, and the white solids were filtered to give 2-amino-6-iodo-3-(2(trifluoromethyl)phenyl)quinazolin-4(3H)-one (57, Y = I) (13.5 g, 79%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.11 (d, J = 1.95 Hz, 1 H), 7.94 (d, J = 7.82 Hz, 1 H), 7.84−7.90 (m, 2 H), 7.72−7.78 (m, 1 N



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H), 7.62 (d, J = 7.82 Hz, 1 H), 7.06 (d, J = 8.70 Hz, 1 H), 6.69 (br s, 2 H); LC−MS m/z = 432.0 (M + H)+. A mixture of 2-amino-6-iodo-3-(2-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (7.75 g, 18.0 mmol) (57, Y = I), N-(2,4difluorophenyl)-2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)pyridine-3-sulfonamide (8.43 g, 19.8 mmol) (73), PdCl2(dppf)− CH2Cl2 (1.47 g, 1.80 mmol), Cs2CO3 (17.6 g, 53.9 mmol) was treated with 1,4-dioxane (71.9 mL) and water (18.0 mL) and heated to 80 °C for 1 h. After cooling to room temperature, the mixture was diluted with EtOAc and water. The combine organics were washed with brine, dried over Na2SO4, filtered through a plug of silica, and concentrated to give dark brownish/black residue. The residue was taken up in DCM, loaded directly onto a silica gel column, and prepurified (1−3% MeOH/DCM) to give a peach solid (9.47 g) and the mixed fractions (4.378 g) as an orange solid. The residues, the peach solid (8.1 g) and the orange solid (4.378 g), were separately loaded onto silica gel and separately purified by silica gel chromatography (50−100% EtOAc/ hexane) to give a pale yellow solid foam (6 and 3 g), respectively. The batches were combined (9 g), and the yellow foam was sonicated in Et2O for 15 min. The solid was filtered off, washed with ether, and dried overnight at room temperature under high vacuum to give a white fluffy powder. The product was dried overnight under reduced pressure at 60 °C to give 5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (23) (8.1 g, 75%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.21 (br s, 1 H), 8.74 (s, 1 H), 8.17 (d, J = 2.54 Hz, 1 H), 8.01 (d, J = 2.34 Hz, 1 H), 7.85−7.98 (m, 3 H), 7.73−7.79 (m, 1 H), 7.62 (d, J = 7.80 Hz, 1 H), 7.26−7.36 (m, 2 H), 7.18−7.26 (m, 1 H), 6.97−7.05 (m, 1 H), 6.68 (br s, 2 H), 3.96 (s, 3 H); LC−MS m/z = 604.4 (M + H)+; HRMS calculated for C27H19N5O4F5S (M + 1)+, 604.1078; found 604.1077. Separation of 5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3sulfonamide (23) was performed by dissolving 5 g of material in 100 mL of CHCl3/MeOH (3:1) with a couple drops of formic acid, then separated by a preparative SFC method, 40% MeOH/CO2, 100 bar, 40 °C, 90 g/min, 220 nm, AD 25 cm × 3 cm, 83 mg/inj, to afford first eluting isomer (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide (28) (2.1 g, >99% ee): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.19 (br s, 1 H), 8.76 (d, J = 2.44 Hz, 1 H), 8.18 (d, J = 2.44 Hz, 1 H), 8.02 (d, J = 2.15 Hz, 1 H), 7.86−7.98 (m, 3 H), 7.73−7.81 (m, 1 H), 7.63 (d, J = 7.82 Hz, 1 H), 7.29−7.36 (m, 2 H), 7.20−7.28 (m, 1 H), 6.98−7.07 (m, 1 H), 6.67 (br s, 2 H), 3.98 (s, 3 H); LC−MS m/z = 604.2 (M + H)+. There was also a second eluting isomer (aR)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3sulfonamide (29) (2.22 g, >99% ee): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.18 (br s, 1 H), 8.75 (d, J = 2.34 Hz, 1 H), 8.17 (d, J = 2.44 Hz, 1 H), 8.01 (d, J = 2.15 Hz, 1 H), 7.86−7.98 (m, 3 H), 7.72−7.80 (m, 1 H), 7.59−7.66 (m, 1 H), 7.27−7.36 (m, 2 H), 7.18−7.27 (m, 1 H), 6.97−7.05 (m, 1 H), 6.67 (br s, 2 H), 3.97 (s, 3 H); LC−MS m/z = 604.2 (M + H)+); HRMS calculated for C27H19N5O4F5S (M + 1)+, 604.1078; found 604.1077. Absolute stereochemical assignments were made by VCD Analysis.24

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A.M.: Salix Pharmaceuticals Inc., 8510 Colonnade Center Drive, Raleigh, NC 27615 Author Contributions

Chemistry experiments: A.L.L., M.R.L., M.T., and J.B.. Modeling and compound design: S.D. Assay development and assays: O.B.M., J.G., and K.L.C. Enzymology experiments: S.L.S. DMPK experiments: A.M. and S.R. Contribution to or assistance in preparation of the manuscript: A.L.L., J.B., S.D., O.B.M., S.L.S., C.B.M., S.R., and J.B.S. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank George Adjabeng, Maosheng Duan, Xiaofei Li, Robert McFadyen, Wenyan Mo, Aniko Redman, and Jianjun Xue for the synthetic work; Douglas Minick and Timothy Spitzer for structure assignments; Rachel Schmidt, Eric Wentz, and Yang Zhao for separation and purification of key compounds; Zhi Hong, Robert Hamatake, Christopher Roberts, and Andrew Spaltenstein for overall contribution of project design.

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ABBREVIATIONS USED LK, lipid kinase; PI4KIIIα, type III phosphatidylinositol 4kinase α; PI4KIIIβ, type III phosphatidylinositol 4-kinase β; PI3Kα, type I phosphatidylinositol 3-kinase α; PI3Kβ, type I phosphatidylinositol 3-kinase β; PI3Kδ, type I phosphatidylinositol 3-kinase δ; PI3Kγ, type I phosphatidylinositol 3-kinase γ; PK, protein kinase

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ASSOCIATED CONTENT

S Supporting Information *

Procedures for enzyme and replicon assays, DMPK studies, and the synthesis of compounds not described under the Experimental Section. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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Discovery of Selective Small Molecule Type III Phosphatidylinositol 4

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