Structure-Based Optimization of Small Molecule Human Galactokinase Inhibitors

Abstract

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure–activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC₅₀ < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

Graphical Abstract

INTRODUCTION

Classic galactosemia is a rare and potentially lethal inborn error of metabolism caused by deficient galactose-1 phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism.^1–4 As a result of GALT deficiency, galactose-1 phosphate (gal-1P) produced by galactokinase (GALK1, gene code GALK1) accumulates, and UDP-galactose and UDP-glucose become deficient in patient cells.^5–7 If untreated, classic galactosemia can result in severe disease in the neonatal period, including liver failure, coagulopathy, coma, and death.^8,9 With galactosemia being included in newborn screening panels in the United States and with prompt removal of galactose from the diet, most patients can survive the neonatal toxicity. However, many well-treated newborns continue to develop complications such as primary ovarian insufficiency (POI), ataxia, speech dyspraxia, and intellectual deficits.^10–12 The precise causes of these organ-specific complications remain largely unknown, but there is a strong association with the intracellular accumulation of toxic galactose metabolites such as gal-1P. Studies have shown that patients on a galactose-restricted diet are never really “galactose-free” as a significant amount of galactose is found in nondairy foodstuffs such as vegetables and fruits.^13–15 More importantly, galactose is produced endogenously from the natural turnover of glycolipids and glycoproteins. Using isotopic labeling, Berry et al. demonstrated that a 50 kg adult male could produce up to 2 g of galactose per day.^16,17 Once galactose is formed intracellularly, it is converted to gal-1P by GALK1 (Figure 1), and in GALT-deficient patients’ cells in vitro, gal-1P is concentrated more than one order of magnitude above normal even without galactose in the culture media.^5,18–20

Figure 1.

Leloir pathway for galactose metabolism. GALK1: galactose-1-phosphate kinase, GALT: galactose-1-phosphate uridylyltransferase, GALE: UDP-galactose 4-epimerase, and UDP: uridine diphosphate.

Accumulation of gal-1P is regarded as the major, if not sole, factor for the chronic complications seen in patients with classic galactosemia, as suggested by both clinical observations and experimental results from yeast and fruit fly models. First, patients with inherited deficiency of GALK1, who do not produce gal-1P, show much milder and even benign phenotypes. With the exception of cataracts, patients often do not experience the neurologic and ovary complications seen in GALT-deficient patients.^21,22 Second, while gal7 (i.e., GALT-deficient) mutant yeast stops propagating upon galactose challenge, a ga17 ga11 double mutant (i.e., GALT- and GALK1-deficient) strain is no longer sensitive to galactose (i.e., GALK1 deletion rescues the GALT knockout phenotype).^23–25 In addition, a recent study of a Drosophila model of classic galactosemia identified the galactokinase gene as a genetic modifier of the neurological defects, and co-removal of the Drosophila GALK1 corrected these defects.^26,27 Based on these observations, the therapeutic hypothesis has emerged that inhibiting GALK1 activity with a safe small-molecule inhibitor, in conjunction with dietary therapy, might prevent the sequelae of chronic gal-1P exposure in patients with classic galactosemia.^21,28

Although GALK1 phosphorylates galactose, a six-carbon monosaccharide, it does not belong to the sugar kinase family. GALK1 is an archetype of the GHMP kinase family (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase), characterized by a distinct structure compared to other kinase families.^28,29 All members of the GHMP kinase family have three conserved motifs (I, II, and III). Motif II is the most conserved one with a typical sequence of Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala and is involved in nucleotide binding and catalytic process. The three-dimensional structure of human GALK1 with bound α-D-galactose and Mg-AMPPNP revealed a unique active site geometry associated with substrate recognition.^30–33 A number of site-directed mutations known to give rise to type II (GALK1-deficient) galactosemia have been investigated and provided valuable insights in understanding the GALK1 biology at the molecular/structural levels for structure-based drug development.^31,34–38

Through quantitative high-throughput screening (qHTS) using a functional biochemical assay that measured the ability of recombinant human galactokinase to phosphorylate galactose, we previously reported a series of tetrahydroquinazolin-5(1H)-ones that were characterized as selective inhibitors of human galacotokinase.³⁹ We had developed the structure–activity relationship (SAR) around the chemotype to develop the lead compound NCGC00238624 (1) (Figure 2), which showed IC₅₀ of 7.69 and 13.67 μM against the human and mouse recombinant GALK1, respectively, and lowered gal-1P levels in primary fibroblast cells derived from patients with classic galactosemia. In addition, compound 1 was inactive against GALK2 and showed negligible cross-reactivity against other kinases in a KINOMEScan screen (Eurofins) at 10 μM (see dendrogram in the Supporting Information).

Figure 2.

Alignment of co-crystal structure of compound 1 and GALK1 (PDB 7RDI) with 1WUU. (a) The human GALK1 crystal structure 1WUU is colored magenta with the substrate and co-factor colored orange. Lys252Ala-Glu253Ala-GALK1-compound 1 co-crystal structure is colored cyan with compound 1 colored green. The red circle indicates the two surface entropy reduction mutants. The blue circle highlights the loop region containing Ser230 and Leu231. (b) Chemical structure of compound 1 with biochemical potency.

Here, we describe a co-crystal structure of the lead inhibitor with human GALK1 and the use of structure-guided drug design to further improve the modest biochemical potency of the chemical series. Medicinal chemistry optimization provided compounds with enzyme inhibition at <100 nM concentrations, acceptable in vitro drug-like properties and pharmacokinetics, and activity in patient cells.

RESULTS AND DISCUSSION

Co-crystallization of Modified Human GALK1 with Inhibitors.

Human GALK1 was previously co-crystallized with AMP-PNP and galactose (PDB 1WUU).^30,31

To support our structure-based inhibitor studies, we attempted to crystallize GALK1 following the published protocol. We met with limited success, and our efforts were ultimately rewarded after modification of two residues of the recombinant protein. The two residues were identified by surface entropy reduction analysis and substituted with alanine. The resulting Lys252Ala-Glu253Ala-GALK1 protein was purified and concentrated to 18 mg/mL for crystallization. The introduction of these two mutations to the protein did not affect the K_M for either ATP or galactose, or the V_max of the enzyme (data not shown). Further characterization showed that known GALK1 inhibitors also inhibited this modified GALK1 with the same potency as for the wild-type enzyme (data not shown). We successfully co-crystallized this double mutant Lys252Ala-Glu253Ala-GALK1 with bound galactose and ADP. These crystals were used as seeds for co-crystallization with compounds 1 (NCGC00238624) and 38 (PDB 7RDI).

The overall structure of Lys252Ala-Glu253Ala-GALK1 was nearly identical to the previously reported human GALK1 crystal structure (PDB 1WUU) (Figure 2a). The root-mean-square deviation (RMSD) between these two structures was 1.23 Å, indicating that the surface entropy reduction mutations did not significantly affect the enzyme structure (Figure 2a, red circle). In contrast to the previously reported GALK1 structure (1WUU), where the loop region containing residues Ser230 and Leu231 (Figure 2a, blue circle) was disordered, this region in the Lys252Ala-Glu253Ala-GALK1-ADP co-crystal is ordered.

Our previous studies identified compound 1 as an ATP competitive inhibitor for human GALK1, and the Lys252Ala-Glu253Ala-GALK1-compound 1 co-crystal structure showed that it occupied the ATP binding pocket of the enzyme. The benzoxazole ring in compound 1 occupied the area where the adenine ring of ATP binds. Because we could not determine unambiguously the orientation of the benzoxazole ring from this structure, the orientation was arbitrarily assigned (Figure 2b).

The 4-chloro-1H-pyrazole at the 4-position of the core occupied a pocket surrounded by Arg105, Trp106, and Asp83 (Figure 3). The chloro appeared to engage in a halogen-acid provided an attractive opportunity to further improve the potency by installing functional groups on cyclohexanone that could extend into the binding groove. To that end, we devised a strategy to open the cyclohexanone ring and substitute it with amides that would maintain the H-bond interaction with Arg105 and facilitate additional interactions.

Figure 3.

Binding pose of compound 1 (PDB 7RDI). Compound 1, green. GALK1 protein, cyan. The molecular surface of the binding pocket is depicted as a mesh contour. Red dotted lines indicate possible interactions between the small molecule and GALK1 (see text).

A closer look at the benzoxazole binding pocket (Figure 3) also revealed the presence of Ser144, Thr61, and Ser131 near the 6′- and 7′-positions. We hypothesized that the placement of a hydrogen bond donor at the 7′-position, especially a NH₂ group that could mimic the same functionality in adenine, could potentially result in hydrogen bonding with these residues. Our structural models predicted distances of 2.6, 3.5, and 3.7 Å between the nitrogen of the NH₂ and the oxygens of Thr61, Ser131, and Ser144, respectively, with the Thr61 showing the shortest distance and better angle. The presence of a second small hydrophobic pocket formed by the γ-carbon of Thr61, β-carbon of Ser131, backbone carbon of Val130, and β-carbon of Val129 suggested that the placement of a non-bulky lipophilic substitution at the 6′-position might improve the inhibitor binding through hydrophobic interaction and van der Waals contact.

We initiated a medicinal chemistry campaign with the sequence of reactions shown in Scheme 1 to synthesize the analogs shown in Table 1. We decided to substitute the chloropyrazole in compound 1 with an ortho-chloro phenyl bioisostere because the chloropyrazole limited the scope of the synthesis with poor yields; we also wanted to reduce the nitrogen atom count within the chemotype to enhance drug-like properties. A simple unsubstituted phenyl group in analog 2 maintained GALK1 inhibition in the biochemical assay (Table 1). Decorating the phenyl ring systematically with a halogen such as a fluorine gave analogs 3–5, which displayed slightly improved potencies compared to 2. A similar strategy with a chlorine atom led to the discovery that while 2-chloro substituted 6 was favorable, the 3- and 4-Cl substitutions in 7 and 8 led to a complete loss of activity. Probing the chemical space with a hydroxyl group, which is capable of being a hydrogen bond donor as well as an acceptor, showed that the 2- and 4-OH groups in phenols 9 and 10 were tolerated. Placement of an ester at the 3- and 4-positions in 12 and 13, and not the 2-position (11), was modestly tolerated. A carboxylate at the 3- and 4-positions in 15 and 16, but not the 2-position in 14, showed >5-fold improvement in potency compared to unsubstituted phenyl 2. Placement of a hydroxyl with a methylene spacer from the phenyl ring showed that the 2- and 3-benzyl alcohols 17 and 18 retained potency, while the 4-benzyl derivative 19 lost potency.

Scheme 1.

General Sequence of Reactions to Obtain Compound Analogs 2–66

Table 1.

SAR at Amide^a

Comp#	R	GALK1 IC50 (μM)	GALK1 pIC50 ± SD
2	Ph	10.86	4.98 ± 0.03
3	2-F-Ph	7.69	5.11 ± 0.01
4	3-F-Ph	6.85	5.18 ± 0.08
5	4-F-Ph	7.69	5.11 ± 0.13
6	2-Cl-Ph	4.32	5.38 ± 0.03
7	3-Cl-Ph	inactive
8	4-Cl-Ph	inactive
9	2-OH-Ph	6.85	5.16 ± 0.05
10	4-OH-Ph	3.85	5.43 ± 0.20
11	2-CO2Me-Ph	inactive
12	3-CO2Et-Ph	3.85	5.41 ± 0.01
13	4-CO2Me-Ph	4.32	5.35 ± 0.03
14	2-CO2H-Ph	34.35	4.46 ± 0.23
15	3-CO2H-Ph	2.43	5.58 ± 0.06
16	4-CO2H-Ph	1.53	5.81 ± 0.17
17	2-CH2OH-Ph	3.85	5.40 ± 0.03
18	3-CH2OH-Ph	3.85	5.38 ± 0.03
19	4-CH2OH-Ph	17.21	4.76 ± 0.2
20	2-OH-Bn	6.85	5.20 ± 0.03
21	2-CH2OH-Bn	6.11	5.20 ± 0.06
22	3-CH2OH-Bn	4.85	5.33 ± 0.05
23	4-CH2OH-Bn	6.85	5.18 ± 0.05
24	3-CO2H-Bn	7.69	5.03 ± 0.10
25	4-CO2H-Bn	3.43	5.43 ± 0.03
26	2-Py	4.32	5.36 ± 0.01
27	3-Py	6.85	5.18 ± 0.03
28		3.43	5.43 ± 0.03
29		5.44	5.28 ± 0.16
30		1.72	5.80 ± 0.03
31		15.34	4.80 ± 0.08
32		1.09	5.98 ± 0.03
33		1.37	5.85 ± 0.03
34		3.43	5.46 ± 0.05
35		1.08	5.96 ± 0.01

^aIC₅₀ values are an average of N = 3. The pIC₅₀ is also included to indicate standard deviation (SD).

We then shifted focus from the phenyl amides to benzyl amides in 20–25. While the 2-hydroxybenzyl derivative 20 was modest, the benzyl alcohols in 2-, 3-, and 4-positions in 21 to 23 were tolerated; this was a contrast to the set of benzyl alcohols 17–19 where substitution at the 4-position (19) led to a decrease in potency. Similarly, based on the potency improvements observed in carboxylates 15 and 16, we explored the homologous congeners 24 and 25 to find that 25 was 2-fold better than 24. This systematic study with the first subset of compounds 2–25 revealed that carboxylate derivative 16 had the best improvement in activity compared to compound 2.

Next, we moved to investigate heterocyclic replacements of the phenyl ring in compound 2. To that end, 2- and 3-pyridyl replacements 26 and 27 showed modest improvements in potency, as did the thiadiazoles 28 and 29. Heterocycles with homologation, such as compounds 30–33, revealed that the imidazole 32 and pyrazole 33 with a 3-methyl substitution provided ~7–9-fold better activity than 2. The combination of a carboxylic acid attached to a heterocycle led to the synthesis of compounds 34 and 35, and the pyridyl acid 35 displayed an IC₅₀ of 1.08 μM and a 10-fold potency improvement over 2.

We then explored the SAR around the benzoxazole portion that was occupying the adenine pocket. Having observed the residues surrounding this tight pocket, we decided to methodically explore single substitutions beginning with the small fluorine atom that was systematically installed at the 4-position through to the 7-position in compounds 36–39. This led to the discovery of the 6-F derivative 38 with better activity than lead 35, as predicted by the structural study (Figure 3). A similar scan on the benzoxazole, from the 4- to 7-position, with a chlorine atom in compounds 40–43 did not show any improvements over unsubstituted 35, but with a methyl group in 44–47, yielded the 7-methyl congener 47 that was equipotent to 35. Substitution with the methoxy group did not seem favorable in compounds 48–50 with up to a 20-fold loss in activity. Placement of an amino group at positions 4, 5, and 7 led to the discovery of the 7-amino aniline 53 that showed a benchmark IC₅₀ of 220 nM, a 5-fold potency improvement over 35, which was also predicted by the structural study (Figure 3). Thus, exploration of the benzoxazole uncovered compounds 38 and 53 with submicromolar biochemical potency against GALK1.

Using 38 as a template, we executed a brief study of the 2-chlorophenyl substituent at the 4-position of the dihydropyrimidine core (Table 3). While a bromine atom replacement for the chlorine was tolerated in analog 54, a variety of small substituents at the 4-position were explored in analogs 55–58. In this subset, the 2-choloro-4-methyl congener 57 stood out with slightly better activity than 2-chloro 38. Large substitutions were tolerated at the 4-position such as phenyl 59, pyrazole 60, morpholino 61, imidazole 62, piperadine 63, pyrrolidine 64, methylpyridine 65, and methylpyrimidine 66. Notable analogs in this subset were compounds 62 and 66 with IC₅₀’s of 120 and 110 nM, respectively. Although this study was important to appreciate the tolerance of large substitutions on the 4-position at the core, these analogs were not progressed further due to their increased molecular weight.

Table 3.

SAR at the 4-Position of the Dihydropyrimidine Core^a

Compound	R	GALK IC50 (μM)	GALK1 pIC50 ± SD
38		0.68	6.20 ± 0.03
54		0.54	6.25 ± 0.03
55		0.86	6.05 ± 0.03
56		1.09	5.98 ± 0.03
57		0.39	6.41 ± 0.05
58		0.43	6.35 ± 0.03
59		3.43	5.46 ± 0.01
60		0.77	6.11 ±0.01
61		0.43	6.35 ± 0.03
62		0.12	6.93 ±0.12
63		1.22	5.90 ± 0.03
64		1.09	5.96 ±0.05
65		0.34	6.46 ± 0.01
66		0.11	6.95 ± 0.03

^aIC₅₀ values are an average of N = 3. The pIC₅₀ is also included to indicate standard deviation (SD).

Analysis of PAMPA (parallel artificial membrane permeability assay) passive permeability revealed that the compounds shown in Table 3 had very poor permeability, probably due to the presence of the carboxylic acid (carboxylate in solution at neutral pH). Thus, we sought bioisostere replacements for the carboxylate functionality. SAR studies on the amide at the 5-position of the dihydropyrimidine core (Table 1) has shown pyrazole 33 to be equipotent to pyridyl acid 35. To that end, we proceeded to synthesize the pyrazole analogs shown in Table 4; these also contained the favorable 2-choloro-4-methyl di-substitution instead of just the 2-chloro substitution at the 4-position of the dihydropyrimide core (Table 3).

Table 4.

SAR of 6′-F and 7′-NH2 Benzoxazole with Chirality at the C4-Position of the Dihydropyrimidine Core: Biochemical Activities in Human and Mouse GALK, Human and Mouse Microsome Stability, Solubility, and PAMPA^a

compd	C4	R1	R2	human GALK IC50 (μM)	human GALK1 pIC50 ± SD	mouse GALK IC50 (μM)	MLM T1/2 (min)	HLM T1/2 (min)	Solubility (μg/mL)	PAMPA (10−6 cm/s)
67	±	H	Me	0.22	6.68 ± 0.03	2.17	11	32	8.3	315
(R)-67	R	H	Me	0.13	6.92 ± 0.03	1.58	7	37	17	661
(S)-67	S	H	Me	12.19	4.93 ± 0.03	30.61	ND	ND	ND	ND
68	±	F	Me	0.048	7.30 ± 0.03	0.69	14	47	1.3	485
69	±	H	Et	0.17	6.76 ± 0.05	3.06	ND	120	2.4	632
70	±	F	Et	0.048	7.31 ± 0.05	0.61	14	8	<1.0	410
(R)-70	R	F	Et	0.068	7.15 ± 0.06	0.97	7	12	<1.0	773
(S)-70	S	F	Et	1.53	5.83 ± 0.03	10.86	12	10	2.1	407

^aMLM: mouse liver microsome, HLM: mouse liver microsome, PAMPA: parallel artificial membrane permeability assay, and ND: no data. The PIC₅₀ is also included to indicate standard deviation (SD).

The SAR study described in Table 2 had also revealed that pronounced improvements in GALK1 inhibition was possible by the incorporation of either a 6′-fluoro and 7′-amino at the benzoxazole, as evidenced by 38 and 53, respectively. To understand the structural basis on this improvement in potency, we tried to co-crystalize these two compounds (38 and 53) with hGALK1 but only succeeded with compound 38. The co-crystal structure showed that compound 38 also bound to the ATP-binding site of the enzyme and its binding pose overlapped with compound 1 (Figure 4a, PDB 7RCN). For instance, the fluoro substitution at the C6 position of the benzoxazole ring of compound 38 completely overlapped with the C6-hydrogen in compound 1 (Figure 4b). The central dihydropyrimidine and the ortho-chloro aryl group overlapped and occupied the same pocket (Figure 4a). The decahydroquinazoline ring in compound 1 was opened in compound 38, but the hydrogen bond between the carbonyls and Arg105 remained intact, and Arg105 moved closer to the carbonyl group in the crystal structure with compound 38 (Figure 4c). In addition, the amide nitrogen made a hydrogen bond with Tyr109 (Figure 4c), which explained why moving from the closed ring to open amide structures led to better activity. The isonicotinic acid extended outside of the active center of the enzyme and was solvent exposed (Figure 4a). Because the crystal structure demonstrated that the benzoxazole ring’s binding conformation did not change upon the substitution of fluoride at the C6 position with no changes to the pocket around the C7 pocket in compound 38 (Figure 4b), we envisioned that combining C6 fluoride substitution and C7 amine substitution in one molecule would result in better hGALK1 inhibitors.

Table 2.

SAR at Benzoxazole^a

compound	R	GALK, IC50 (μM)	GALK1, pIC50 ± SD
35	benzoxazole	1.08	5.96 ± 0.01
36	4-F benzoxazole	3.85	5.41 ± 0.05
37	5-F benzoxazole	3.43	5.50 ± 0.03
38	6-F benzoxazole	0.68	6.20 ± 0.03
39	7-F benzoxazole	1.72	5.75 ± 0.03
40	4-Cl benzoxazole	5.44	5.26 ± 0.01
41	5-Cl benzoxazole	2.43	5.58 ± 0.06
42	6-Cl benzoxazole	1.93	5.71 ± 0.01
43	7-Cl benzoxazole	1.93	5.7 ± 0.03
44	4-Me benzoxazole	9.68	5.03 ± 0.08
45	5-Me benzoxazole	1.37	5.86 ± 0.01
46	6-Me benzoxazole	5.44	5.26 ± 0.01
47	7-Me benzoxazole	0.97	6.03 ± 0.03
48	5-OMe benzoxazole	6.85	5.18 ± 0.03
49	6-OMe benzoxazole	21.67	4.71 ± 0.05
50	7-OMe benzoxazole	21.67	4.66 ± 0.01
51	4-NH2 benzoxazole	21.67	4.71 ± 0.01
52	5-NH2 benzoxazole	7.69	5.13 ± 0.03
53	7-NH2 benzoxazole	0.22	6.63 ± 0.03

^aIC₅₀ values are an average of N = 3. The pIC₅₀ is also included to indicate standard deviation (SD).

Figure 4.

Co-crystal structure of compound 38 (PDB 7RCN). (a) Overview of the active site of hGALK1 in the aligned co-crystal structures of compound 38 (blue) and compound 1 (cyan and green). (b) Close-up view of alignment at benzoxazole rings of the two co-crystal structures. Red circle indicates the fluoride atom of compound 38. (c) Hydrogen bonds formed between compounds 1 (blue dotted lines) and 38 (red dotted lines) with Asp83, Arg105, and Tyr109 of the enzyme.

To incorporate both C6-F and C7-NH₂ substitutions in the benzoxazole, in conjunction with a pyrazole replacement of the pyridyl acid, we developed a modified synthetic route, shown in Scheme 2. Condensation of 2,2,6-trimethyl-4H-1,3-dioxin-4-one 71 with (1-methyl-1H-pyrazol-4-yl)methanamine 72P provided a β-ketoamide 73K that was further condensed with 2-chloro-4-methylbenzaldehyde 75D and thiourea. This produced a 2-thioxo-tetrahydropyrimidine 84A that was derivatized with methyl 2-amino-6-fluorobenzo[d]oxazole-7-carboxylate 85B using a SN_Ar reaction with mercuric acetate. The methyl ester 86A was hydrolyzed to the acid 89, which was subjected to a Curtis rearrangement with diphenylphosphonic azide 90 in the presence of t-butanol to produce a N-boc aniline that could be deprotected with trifluoracetic acid to reveal the amine at the 7′-position. This sequence of synthetic steps led to compound 68 with a 6′-F-7′-NH₂ di-substitution. We also synthesized compound 67, which had the 7′-NH₂ group alone; this analog had an IC₅₀ of 0.22 μM. Using chiral HPLC, we separated the enantiomers at C4 and found that (R)-67 (IC₅₀ = 130 nM) was almost 10-fold more potent than (S)-67. In comparison, analog 68 recorded a benchmark IC₅₀ of 54 nM against GALK1. Unfortunately, we were unable to resolve the enantiomers at C4 of compound 68 using HPLC despite evaluating eight different chiral columns. We performed a similar study with the ethylated pyrazoles 69 and 70. While des-fluoro 69 had a GALK1 IC₅₀ of 170 nM, the incorporation of the 6′-F in 70 led to a 4-fold improvement in GALK1 inhibition. In this set, attempts at separation were successful only with analog 70 and not 69. As expected, (R)-70 was more potent (18-fold) than (S)-70. We noticed that racemic analog 70 appeared to be more potent than (R)-70; we think that this is a technical artifact. Analogs in Table 4 were also evaluated for their drug-like properties. Generally, most analogs had acceptable passive permeability and a marked improvement over the acids in Table 3 but poor solubility. Moderate stability in mouse liver microsomes (MLMs) was observed for compounds 68 and 70. The human liver microsome (HLM) stability seemed the best for compounds 68 and 69. We also evaluated activity against mouse GALK1 and observed a 2.5- to 18-fold reduction in the inhibitory potency of compounds in the series. Nevertheless, analogs 68, 70, and (R)-70 maintained submicromolar inhibition.

Scheme 2.

Modified Synthetic Route to Obtain Compounds with 6′-Fluoro and 7′-Amino at the Benzoxazole (Exemplified with Compound 68)

With the discovery of highly potent human GALK1 inhibitors, we decided to analyze the series for its ability to reduce gal-1P, the metabolite produced by GALK1, after a galactose challenge in fibroblasts from a patient with classic galactosemia. To that end, we show in Figure 5 that exemplar analog 68 (NCATS-SM4487) was able to reduce gal-1P/protein levels in dose dependence and to ~50% of control at 3 μM (Figure 5).

Figure 5.

Dose-dependent reduction of Gal-1P in patient fibroblasts by analog 68 (NCATS-SM4487).

Despite being racemic, we selected compound 68 (NCATS-SM4487), which had the best combination of MLM and HLM stability data (Table 4), for preliminary pharmacokinetic (PK) experiments in female CD1 mice (Figure 6). A single-dose 3 mpk IV study indicated a C_o of 8.25 μM drug concentration in plasma with a short half-life of ~0.4 h with compound levels below LLQ after 2 h (Table 5). A 30 mpk PO study led to a C_max of 7.02 μM and AUC_inf of 15.96 h·μM with a bioavailability of 33%. We also evaluated a single 50 mg/kg intraperitoneal (IP) dose and found favorable plasma concentrations above 1 μM up to ~7 h after dosing. This was promising as we intend to use IP dosing in pharmacodynamic and efficacy studies that are planned in the immediate future. Compound 68 shows 98.74% protein binding in mixed-gender CD-1 mouse plasma, and this might also a play a role in PK/PD studies.

Figure 6.

Concentrations of NCATS-SM4487 (analog 68) in female CD1 mouse plasma after administration of compound formulated in 5% NMP, 25% PEG 300, 70% of a 15% w/w solution of Kolliphor HS 15 in water at 0.6, 3, and 5 mg/mL for 3 mpk IV, 30 mpk PO, and 50 mpk IP studies, respectively.

Table 5.

PK Parameters from IV, PO, and IP Dosing Studies Shown in Figure 6

	route	dose (mpk)	Cl_obs (mL/min/kg)	t1/2 (h)	tmax (h)	Co (μM)	AUClast (h·μM)	AUCinf (h·μM)	AUClast/D (h·mg/mL)	Vssobs (L/kg)
mean	IV	3	19.9	0.41	NA	8.25	4.66	4.81	812	0.652
SD			0.9	0.01	NA	1.52	0.22	0.21	38	0.052
	route	dose	t1/2 (h)	tmax (h)	C max	AUClast (h·μM)	AUCinf (h·μM)	AUClast/D (h·mg/mL)	F (%)
mean	PO	30	2.27	0.67	7.02		15.96	16.06	278	33
SD			1.75	0.29	2.32		5.23	5.16	91	11
mean	IP	50	1.70	1.333	21.86		80.36	80.36	841
SD			0.05	0.58	0.88		9.49	9.49	99

CONCLUSIONS

In summary, using a structure-guided strategy, we substantially expanded the SAR of our previous chemotype (compound 1) to discover small molecules that can inhibit human galactokinase biochemically at <100 nM, with >100-fold improvement in potency. A key racemic compound from this series was able to reduce galactose-1-phosphate in patient fibroblasts and had promising exposure in mice, paving the way for evaluation in animal models of galactosemia. We believe that this new probe molecule represents significant progress in the development of a small molecule therapeutic approach for galactosemia, especially for the management of long-term complications. We aim to carry out long-term animal dosing experiments and observe gal-1P reductions in specific tissues to test this therapeutic hypothesis. If successful, these compounds will help us decipher gal-1P’s role in the chronic complications associated with the disease.

MATERIALS AND METHODS: BIOLOGY

Purification of K252A-E253A-GALK1.

Human GALK1 cDNA was cloned to the pET21D vector. The Surface Entropy Reduction prediction (SERp) server (http://service.mbi.ucla.edu/sSER) identified Lys252 and Glu253 as potential high surface entropy residues, which were mutated to alanine through site-directed mutagenesis. The plasmid was transfected to E. coli HMS174, and a single colony was picked and grown in 2 mL of LB media overnight at 37 °C. The next day, 200 μL of the overnight culture was inoculated into 1 L of LB media and cultured at 37 °C in shaker flasks at 150 rpm until the O.D. reached 0.9, at which point 1 mM of IPTG was added and the culture was shifted to 19 °C. The bacteria were further cultured overnight before harvesting by centrifugation. The pellet was stored in −80 °C.

The pellet was thawed in ice-cold protein lysis buffer containing 50 mM sodium phosphate buffer at pH 8, 100 mM galactose, 5% glycerol, 300 mM NaCl, and 20 mM imidazole. Cells were disrupted by combining lysozyme/DNase treatment and sonication. The lysate was cleared by centrifugation, and the supernatant was incubated for 1 h with nickel beads that had been pre-equilibrated with the lysis buffer. The beads were subsequently washed with the lysis buffer, and the protein was eluted with the elution buffer, which comprised the lysis buffer made up with 200 mM imidazole. The protein elution was loaded to the SD200 sizing column in 50 mM HEPES at pH 8, 100 mM galactose, 5% glycerol, and 200 mM NaCl. The fractions containing the target protein were pooled together and were concentrated to 18 mg/mL using an Amicon spin concentrator with 10 kDa molecular weight cutoff. The purity of the final protein solution was around 90% as judged by Coomassie-stained SDS-PAGE.

Co-crystallization of GALK1 and Compounds.

Lys252Ala-Glu253Ala-GALK1 in the purification buffer containing 100 mM galactose was co-crystallized with ADP (10 to 20 mM) by sitting drop vapor diffusion. The initial crystallization conditions were screened with the Hampton Research Phosphate kit (https://hamptonresearch.com/documents/growth_101/22.pdf) with the concentration of the buffer ranging from 1.0 to 3.2 M Na/K and pH from 5.8 to 7.6. Crystals grew in the buffer from 1.8 to 2.6 M Na/K and pH from 6.0 to 6.8, and a fine grid of these conditions was used for further crystallization efforts. The ratios of protein solution to crystallization solution in drops were 1:2, 1:1, and 2:1, all of which produced crystals.

Lys252Ala-Glu253Ala-GALK1 was co-crystallized with inhibitors by seeding with ADP complex crystals 1 day after the drops were set in the same way as with ADP but with the compound in place of ADP. The protein/compound ratio used was in the range 1:2 to 1:10, where the compounds were dissolved in 2.5% or less DMSO. Crystals typically appeared within 7 days of seeding and grew over a period of 1 to 2 weeks. Crystals were harvested into 2.4 M Na/K at pH 6.4, suspended in a loop, and cryo-cooled by plunging into liquid nitrogen. Data were collected at the SSRL synchrotron and indexed, integrated, and scaled with HKL2000.⁴⁰ The structures were determined by molecular replacement using the program Phaser⁴¹ in the phenix software suite.⁴² Model building was performed using COOT⁴³ and model refinement with REFMAC5.⁴⁴

The PDB ID codes are 7RCL (ADP), 7RCM (ADP), 7RCN (compound 38), and 7RDI (compound 1).

GALK1 Biochemical Assay.

The in vitro GALK1 activity assay was performed as previously described by coupling the activity of recombinant human GALK1 to the Kinase-Glo Plus luminescent ATP detection kit (Promega) using ATP depletion as a measure of GALK1 turnover.³⁹ Specifically, 3 μL/well of ATP substrate solution (35 μM ATP) in the assay buffer (20 mM HEPES pH 8.0, 5 mM MgCl₂, 60 mM NaCl, 1 mM DTT, and 0.01% BSA final concentration) was dispensed into 1536-well assay plates (Greiner, white solid-bottom medium-binding plates). Twenty-three nanoliters of the compound (solubilized in DMSO) was transferred to the assay plates using a Kalypsys 1536-well pintool. One microliter per well of the GALK-galactose solution (5 nM GALK1 and 100 μM galactose) in the assay buffer was then added, yielding a final reaction volume of 4 μL/well. Following a 1-h room temperature incubation, 4 μL of the Kinase-Glo Plus detection reagent was added to provide an ATP-dependent luminescent readout (final assay volume: 8 μL/well). Luminescence was detected using a ViewLux plate reader (PerkinElmer, Waltham, MA) after a10-min incubation using a 1 s exposure time and 2× binning.

All concentration response curves (CRCs) were fitted using in-house developed software (http://ncgc.nih.gov/pub/openhts/). Curves were categorized into four classes: complete response curves (Class 1), partial curves (Class 2), single-point actives (Class 3), and inactives (Class 4).

Cellular Gal-1P Accumulation Assay.

The Gal-1P level was measured using the alkaline phosphatase coupled method as previously described.³⁹ Fibroblasts derived from GALT-deficient patients were maintained in a galactose-free DMEM supplemented with 10% fetal bovine serum (FBS). Inhibitors were added to the medium at designated concentrations and incubated at 37 °C for 2 to 4 h. Galactose was subsequently added to reach 0.05% in the medium (a “galactose challenge”). After 4 h of challenge, cells were collected and washed with PBS twice. Then, the cells were disrupted in 300 μL of an ice-cold hypotonic buffer containing 25 mM Tris–HCl (pH 7.4), 25 mM NaCl, 0.5 mM EDTA, and protease inhibitor cocktail (Roche, #11697498001). The lysates were passed five times through a 30-gauge needle and centrifuged for 20 min at 16,000g and 4 °C. A small portion of supernatant was saved for protein concentration measurement. The gal-1P concentration was normalized to the protein concentration.

Kinetic Solubility Assay.

Pion’s patented μSOL assay was used for kinetic solubility determination. In this assay, the classical saturation shake-flask solubility method was adapted as previously described.⁴⁵ Test compounds were prepared in 10 mM DMSO stock and diluted to a final drug concentration of 150 μM in the aqueous solution (pH 7.4, 100 mM phosphate buffer). Samples were incubated at room temperature for 6 h and vacuum-filtered using a Tecan Te-Vac to remove any precipitates. The concentration of the compound in the filtrate was measured via UV absorbance (λ: 250–498 nm). The unknown drug concentration was determined by comparing the fully solubilized reference plate that contained 17 μM of the compound dissolved in spectroscopically pure n-propanol. All compounds were tested in duplicate. The kinetic solubility (μg/mL) of compounds was calculated using the μSOL Evolution software (NEED reference). The three controls used were albendazole (low solubility), phenazolpyridine (moderate solubility), and furosemide (high solubility).⁴⁶

Rat Liver Microsome Stability Assay.

Single time point microsomal stability was determined in a 96-well HTS format. Sample preparation was automated using a Tecan EVO 200 robot. A high-resolution LC/MS (Thermo Q Exactive) instrument was used to measure the percentage of the compound remaining after incubation using a previously described method.⁴⁷ Six standard controls were tested in each run: buspirone and propranolol (for short half-life), loperamide and diclofenac (for short to medium half-life), and carbamazepine and antipyrine (for long half-life). DMSO stock solutions (10 mM) of the drugs were first diluted to 10 μM in 1:2 MeCN/DI H₂O and then further diluted to 1 μM in the assay buffer. Briefly, the incubation consisted of 0.5 mg/mL microsomal protein, 1.0 μM drug concentration, and an NADPH regeneration system (containing 0.650 mM NADP+, 1.65 mM glucose 6-phosphate, 1.65 mM MgCl₂, and 0.2 U/mL G6PDH) in 100 mM phosphate buffer at pH 7.4. The incubation was carried out at 37 °C for 15 min.⁴⁸ The reaction was quenched by adding 555 μL of acetonitrile (~1:2 ratio) containing 0.28 μM albendazole (internal standard). Sample acquisition and data analysis were done using a previously described method.⁴⁷

Parallel Artificial Membrane Permeability Assay (PAMPA).

The stirring double-sink PAMPA method (patented by pION Inc.) was employed to determine the permeability of compounds via PAMPA as published before.⁴⁹ The PAMPA lipid membrane consisted of an artificial membrane of a proprietary lipid mixture and dodecane (Pion Inc.), optimized to predict gastrointestinal tract (GIT) passive permeability. The lipid was immobilized on a plastic matrix of a 96-well “donor” filter plate placed below a 96-well “acceptor” plate. A pH 7.4 solution was used in both donor and acceptor wells. The test articles, stocked in 10 mM DMSO solutions, were diluted to 0.05 mM in aqueous buffer (pH 7.4), and the concentration of DMSO was 0.5% in the final solution. During the 30-min permeation period at room temperature, the test samples in the donor compartment were stirred using the Gutbox technology (Pion Inc.) to reduce the aqueous boundary layer. The test article concentrations in the donor and acceptor compartments were measured using a UV plate reader (Nano Quant, Infinite 200 PRO, Tecan Inc., Mannedorf, Switzerland). Permeability calculations were performed using the Pion Inc. software and were expressed in units of 10⁻⁶ cm/s. Compounds with low or weak UV signal were analyzed using high-resolution LC/MS (Thermo QExactive). The three controls used were ranitidine (low permeability), dexamethasone (moderate permeability), and verapamil (high permeability).

Mouse Pharmacokinetic Studies.

Studies were conducted by Pharmaron. Fed male CD1 mice (sourced from Si Bei Fu Laboratory Animal Technology Co. Ltd.), approximately 6–8 weeks of age and with weight of approximately 25–30 g, were dosed with analog 68 at 50 mpk dose IP, 30 mpk PO, and mpk IV. The formulations were solutions of analog 68 in 5% NMP, 25% PEG 300, and 70% of a 15% w/w solution of Kolliphor HS 15 in water at 0.6, 3, and 5 mg/mL for the IV, PO, and IP studies, respectively. These were prepared prior to dosing a cohort of N = 24 mice. Plasma was collected from N = 3 mice at 5, 15, and 30 min and 1, 2, 4, 8, and 24 h postdose. Approximately 0.200 mL of blood was collected via heart puncture at each time point. Blood samples were then transferred into plastic microcentrifuge tubes containing heparin–Na as anticoagulant. Samples were then centrifuged at 4000g for 5 min at 4 °C to obtain plasma. Plasma samples were then stored in polypropylene tubes, quickly frozen, and kept at −75 °C until analyzed by LC/MS/MS. The following pharmacokinetic parameters were calculated for plasma and brain: T_max, C_max, and AUC_24h. Animals were also monitored during the in-life phase by once-daily cageside observations; no adverse clinical signs were noted.

Use of Animal Subjects.

All animal studies included as part of this manuscript were performed in accordance with institutional guidelines as defined by Institutional Animal Care and Use Committee (IACUC).

EXPERIMENTAL SECTION: CHEMISTRY

General Methods for Chemistry.

All air- or moisture-sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide (DMF), acetonitrile, methanol, and triethylamine were purchased from Sigma-Aldrich. Flash silica gel chromatography was conducted with CombiFlash (Teledyne ISCO) systems.

Preparative purification was performed on a Waters Autopurification HPLC system (Waters Corporation, Milford, MA, U.S.A.) equipped with a binary gradient module pump, HPLC pumps, photodiode array detector, QDa mass detector, and a sample manager. Purification was performed with either of following methods:

1. Method acidic standard gradient: acidic method using an Agilent XDB-C18 PrepHT (5 μm, 30 × 100 mm) with mobile phase HPLC-grade water with 0.1% trifluoroacetic acid (TFA) (solvent A) and HPLC-grade acetonitrile with 0.1% TFA (solvent B);

2. Method basic standard gradient: basic method using a Waters XBridge Prep C18 OBD (5 μm, 30 × 75 mm) with mobile phase HPLC-grade water with 0.1% NH₄OH (solvent C) and HPLC-grade acetonitrile with 0.1% NH₄OH (solvent D).

The selected purification gradient was determined by analytical HPLC retention time with the gradient ranging from a potential minimum of 10% solvent B or D to a potential maximum of 100% solvent B or D, respectively, at a flow rate of 41 mL/min and a total run time of 11 min. Fraction collection was triggered by UV detection (220 nM).

The purity of all analogs tested in the biological assays was ≥95% as determined on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA) using a 7-min gradient of 4 to 100% acetonitrile (containing 0.025% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) with an 8-min run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 μm, 3 × 75 mm) was used at a temperature of 50 °C using an Agilent Diode Array Detector. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode.

Chiral Separation.

Chiral analysis was performed on an Agilent 1200 series LC system (Agilent Technologies, Wilmington, DE, U.S.A.) equipped with a binary pump, a photodiode array detector, and a PDR-Chiral advanced laser polarimeter. Chiral screening was conducted using eight chiral columns and two solvent systems followed by further refinement of the method with the selected column. Chiral separation was performed on an Agilent 1200 series chromatography LC system (Agilent Technologies, Wilmington, DE, U.S.A.) equipped with a binary pump and a photodiode array detector using a Chiralpak IA column (20 μm, 5 × 50 cm) at a flow rate of 35 mL/min. Mobile phase 1 was composed of acetonitrile and ethanol (20/80). Mobile phase 2 was composed of acetonitrile, ethanol, and diethylamine (20/80/0.02).

NMR Spectroscopy.

¹H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with nondeuterated solvent (DMSO-d₅ peak at 2.50 ppm) as internal standard for DMSO-d₆ solutions. ¹H and ¹³C NMR spectra were recorded using a Varian 400 (100) MHz VNMRS spectrometer equipped with a 5 mm PFG AutoX dual broadband probe. Chemical shifts are reported in δ (ppm) units using 1H (residual d₅-DMSO) and 13C signals from d₆-DMSO (2.50 and 39.51, respectively) as the internal standard. Data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, sext = sextet, sept = septet, m = multiplet, and br = broad), coupling constant.

Synthetic Procedures.

Method 1: Ethyl 3-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (Table 1, Compound 12).

Step 1: A mixture of 2,2,6-trimethyl-4H-1,3-dioxin-4-one 71 (0.30 mL, 2.28 mmol) and ethyl 3-aminobenzoate 72A (0.377 g, 2.28 mmol) in p-xylene (2.3 mL) was sealed and heated at 150 °C for 30 min in a Biotage microwave. The solvent was removed. The crude product 73A was used in the next reaction without further purification. MS m/z (M + H⁺) 250.1.

Step 2: The mixture of ethyl 3-(3-oxobutanamido)benzoate 73A (0.052 g, 0.209 mmol), 2-chlorobenzaldehyde 75 (0.026 mL, 0.229 mmol), and 1-(benzo[d]oxazol-2-yl)guanidine 74 (0.037 g, 0.209 mmol) was sealed in microwave vial and heated at 170 °C for 2 h. The solvent was removed. MeOH was added to the residue. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method acidic standard gradient) to afford ethyl 3-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate 12 as a TFA salt. MS m/z (M + H⁺) 530.1.

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-phenyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 2).

This compound was prepared via method 1 using 3-oxo-N-phenylbutanamide 73B (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-phenyl-1,4-dihydropyrimidine-5-carboxamide 2 as a TFA salt. MS m/z (M + H⁺) 458.1.

2-(Benzo[d]oxazol-2-ylamino)-N-(2-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 3).

This compound was prepared via method 1 using N-(2-fluorophenyl)-3-oxobutanamide 73C (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(2-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 3 as a TFA salt. MS m/z (M + H⁺) 476.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.32–10.26 (m, 1H), 10.15 (d, J = 1.8 Hz, 1H), 9.65 (s, 1H), 7.57–7.48 (m, 3H), 7.42–7.34 (m, 4H), 7.25–7.07 (m, 5H), 6.23–5.96 (m, 1H), 2.28 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N-(3-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 4).

This compound was prepared via method 1 using N-(3-fluorophenyl)-3-oxobutanamide 73D (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(3-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 4 as a TFA salt. MS m/z (M + H⁺) 476.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.31 (d, J = 2.9 Hz, 1H), 10.21–10.13 (m, 1H), 10.10 (s, 1H), 7.51 (qd, J = 7.4, 7.0, 1.7 Hz, 3H), 7.43–7.28 (m, 6H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.10 (td, J = 7.7, 1.3 Hz, 1H), 6.89–6.82 (m, 1H), 6.14–6.02 (m, 1H), 2.22 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N-(4-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 5).

This compound was prepared via method 1 using N-(4-fluorophenyl)-3-oxobutanamide 73E (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(4-fluorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 5 as a TFA salt. MS m/z (M + H⁺) 476.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 10.13 (d, J = 1.8 Hz, 1H), 9.95 (s, 1H), 7.58–7.47 (m, 4H), 7.42–7.33 (m, 4H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.14–7.08 (m, 3H), 6.08 (dd, J = 2.9, 1.1 Hz, 1H), 2.22 (d, J = 0.9 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N,4-bis(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 6).

This compound was prepared via method 1 using N-(2-chlorophenyl)-3-oxobutanamide 73F (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-N,4-bis(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 6 as a TFA salt. MS m/z (M + H⁺) 492.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.32 (d, J = 3.1 Hz, 1H), 10.17 (s, 1H), 9.41 (s, 1H), 7.57–7.50 (m, 2H), 7.49 (dd, J = 8.0, 1.6 Hz, 1H), 7.45 (dd, J = 8.0, 1.5 Hz, 1H), 7.42–7.33 (m, 4H), 7.31–7.25 (m, 1H), 7.18 (tt, J = 7.7, 1.6 Hz, 2H), 7.10 (td, J = 7.7, 1.3 Hz, 1H), 6.11 (dd, J = 3.0, 1.2 Hz, 1H), 2.35 (d, J = 0.9 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N-(3-chlorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 7).

This compound was prepared via method 1 using N-(3-chlorophenyl)-3-oxobutanamide 73G (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(3-chlorophenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 7 as a TFA salt. MS m/z (M + H⁺) 492.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.30 (d, J = 2.8 Hz, 1H), 10.17 (d, J = 1.9 Hz, 1H), 10.07 (s, 1H), 7.74 (t, J = 2.0 Hz, 1H), 7.50 (ddd, J = 7.4, 5.1, 1.7 Hz, 2H), 7.44 (ddd, J = 8.3, 2.0, 1.0 Hz, 1H), 7.42–7.33 (m, 4H), 7.31 (t, J = 8.1 Hz, 1H), 7.18 (td, J = 7.6, 1.2 Hz, 1H), 7.13–7.07 (m, 2H), 6.08 (dd, J = 3.0, 1.1 Hz, 1H), 2.23 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 8).

This compound was prepared via method 1 using N-(4-chlorophenyl)-3-oxobutanamide 73H (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 8 as a TFA salt. MS m/z (M + H⁺) 492.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 10.15 (d, J = 1.8 Hz, 1H), 10.02 (s, 1H), 7.60–7.55 (m, 2H), 7.53–7.47 (m, 2H), 7.42–7.30 (m, 6H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.14–7.07 (m, 1H), 6.15–6.01 (m, 1H), 2.22 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N-(2-hydroxyphenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 9).

This compound was prepared via method 1 using 2-aminophenol 72B as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(2-hydroxyphenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 9 as a TFA salt. MS m/z (M + H⁺) 474.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.35–10.26 (m, 1H), 10.19–10.11 (m, 1H), 9.74 (s, 1H), 8.90 (s, 1H), 7.62 (dd, J = 8.0, 1.6 Hz, 1H), 7.59–7.49 (m, 2H), 7.43–7.33 (m, 4H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.10 (td, J = 7.7, 1.3 Hz, 1H), 6.96–6.88 (m, 1H), 6.82 (dd, J = 8.0, 1.4 Hz, 1H), 6.72 (td, J = 7.6, 1.5 Hz, 1H), 6.07 (dd, J = 3.1, 1.1 Hz, 1H), 2.34 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-N-(4-hydroxyphenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 10).

This compound was prepared via method 1 using 4-aminophenol 72C as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-N-(2-hydroxyphenyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 10 as a TFA salt. MS m/z (M + H⁺) 474.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 10.06 (s, 1H), 9.65 (s, 1H), 9.17 (s, 1H), 7.53 (dd, J = 7.6, 1.9 Hz, 1H), 7.49 (dd, J = 7.8, 1.6 Hz, 1H), 7.46–7.24 (m, 6H), 7.20–7.13 (m, 1H), 7.09 (td, J = 7.7, 1.4 Hz, 1H), 6.71–6.60 (m, 2H), 6.05 (d, J = 3.0 Hz, 1H), 2.20 (s, 3H).

Methyl 2-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (Table 1, Compound 11).

This compound was prepared via method 1 using methyl 2-aminobenzoate 72D as the starting material in step 1 to afford methyl 2-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate 11 as a TFA salt. MS m/z (M + H⁺) 516.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.72 (s, 1H), 10.38 (t, J = 2.5 Hz, 1H), 10.30 (d, J = 1.9 Hz, 1H), 8.16 (dd, J = 8.4, 1.1 Hz, 1H), 7.87 (dd, J = 8.0, 1.6 Hz, 1H), 7.56 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H), 7.51 (dd, J = 7.6, 1.6 Hz, 1H), 7.46–7.43 (m, 1H), 7.43–7.37 (m, 3H), 7.35 (dd, J = 7.3, 2.1 Hz, 1H), 7.21–7.13 (m, 2H), 7.11 (td, J = 7.7, 1.3 Hz, 1H), 6.02 (dd, J = 3.1, 1.1 Hz, 1H), 3.79 (s, 3H), 2.39 (d, J = 0.8 Hz, 3H).

Methyl 4-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (Table 1, Compound 13).

This compound was prepared via method 1 using methyl 4-aminobenzoate 72E as the starting material in step 1 to afford methyl 4-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate 13 as a TFA salt. MS m/z (M + H⁺) 516.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.32 (d, J = 2.9 Hz, 1H), 10.22 (s, 1H), 10.19 (d, J = 1.8 Hz, 1H), 7.91–7.85 (m, 2H), 7.73–7.67 (m, 2H), 7.50 (ddd, J = 9.5, 7.8, 1.6 Hz, 2H), 7.44–7.30 (m, 4H), 7.17 (td, J = 7.6, 1.3 Hz, 1H), 7.13–7.07 (m, 1H), 6.10 (dd, J = 3.1, 1.1 Hz, 1H), 3.81 (s, 3H), 2.24 (d, J =0.9 Hz, 3H).

Method 2: 2-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic Acid (Table 1, Compound 14).

To a solution of methyl 2-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (50 mg, 0.097 mmol) (Table 2, compound 11) in THF (1 mL) was added LiOH (0.388 mL, 0.194 mmol) (0.5 M). The mixture was stirred at room temperature for 2 days. The solvent was removed. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method acidic standard gradient) to afford 2-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic acid 14 as a TFA salt. MS m/z (M + H⁺) 502.1.

3-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic Acid (Table 1, Compound 15).

This compound was prepared via method 2 using ethyl 3-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydro pyrimidine-5-carboxamido)benzoate (Table 2, compound 12) as the starting material to afford 3-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic acid 15 as a TFA salt. MS m/z (M + H⁺) 502.1, ¹H NMR (400 MHz, DMSO-d₆) δ 12.94 (s, 1H), 10.31 (d, J = 2.8 Hz, 1H), 10.15 (d, J = 1.8 Hz, 1H), 10.07 (s, 1H), 8.18 (t, J = 1.8 Hz, 1H), 7.79 (ddd, J = 8.2, 2.2, 1.1 Hz, 1H), 7.60 (ddd, J = 7.7, 1.6, 1.1 Hz, 1H), 7.56–7.47 (m, 2H), 7.43–7.30 (m, 5H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.13–7.07 (m, 1H), 6.10 (dd, J = 2.8, 1.1 Hz, 1H), 2.24 (d, J = 0.8 Hz, 3H).

4-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic Acid (Table 1, Compound 16).

This compound was prepared via method 2 using ethyl 4-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (Table 2, compound 13) as the starting material to afford 4-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoic acid 16 as a TFA salt. MS m/z (M + H⁺) 502.1, 1H NMR (400 MHz, DMSO-d₆) δ 12.69 (s, 1H), 10.32 (s, 1H), 10.18 (d, J = 2.1 Hz, 2H), 7.88–7.83 (m, 2H), 7.69–7.64 (m, 2H), 7.51 (ddd, J = 11.6, 7.7, 1.6 Hz, 2H), 7.43–7.31 (m, 4H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.14–7.07 (m, 1H), 6.19–6.04 (m, 1H), 2.24 (d, J = 0.8 Hz, 3H).

Method 3: 2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 17).

To a solution of methyl 2-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)benzoate (60.0 mg, 0.116 mmol) (Table 2, compound 11) in THF (1 mL) was added dropwise LiBH₄ (0.070 mL, 0.140 mmol) (2.0 M in THF). The mixture was stirred at room temperature for 2 days. The solvent was removed. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method acidic standard gradient) to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 17 as a TFA salt. MS m/z (M + H⁺) 488.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.34–10.28 (m, 1H), 10.15 (d, J = 1.8 Hz, 1H), 9.42 (s, 1H), 7.52 (ddd, J = 7.3, 5.5, 1.8 Hz, 2H), 7.48–7.43 (m, 1H), 7.43–7.31 (m, 6H), 7.22–7.15 (m, 2H), 7.14–7.07 (m, 2H), 6.05 (d, J = 2.8 Hz, 1H), 4.33 (q, J = 13.9 Hz, 2H), 2.33 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(3-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 18).

This compound was prepared via method 3 using ethyl 3-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)-benzoate (Table 2, compound 12) as the starting material to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(3-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 18 as a TFA salt. MS m/z (M + H⁺) 488.1.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 10.10 (d, J = 1.7 Hz, 1H), 9.88 (s, 1H), 7.54 (dt, J = 6.0, 1.9 Hz, 2H), 7.49 (dd, J = 7.7, 1.4 Hz, 1H), 7.45–7.31 (m, 6H), 7.24–7.14 (m, 2H), 7.10 (td, J = 7.7, 1.3 Hz, 1H), 6.96 (ddd, J = 7.6, 1.8, 1.0 Hz, 1H), 6.09 (dd, J = 3.1, 1.1 Hz, 1H), 4.43 (s, 2H), 2.22 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 19).

This compound was prepared via method 3 using ethyl 4-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)-benzoate (Table 2, compound 13) as the starting material to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-(hydroxymethyl)phenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 19 as a TFA salt. MS m/z (M + H⁺) 488.1.

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-hydroxybenzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 20).

This compound was prepared via method 1 using 2-(aminomethyl)phenol 72F as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-hydroxybenzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 20 as a TFA salt. MS m/z (M + H⁺) 488.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.22 (s, 1H), 9.99 (s, 1H), 9.47 (s, 1H), 8.27 (t, J = 5.8 Hz, 1H), 7.50 (ddd, J = 11.9, 6.1, 3.6 Hz, 2H), 7.37 (td, J = 10.5, 9.1, 5.9 Hz, 4H), 7.16 (t, J = 7.6 Hz, 1H), 7.13–7.04 (m, 1H), 7.00 (td, J = 7.6, 1.8 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.69–6.63 (m, 1H), 6.55 (t, J = 7.4 Hz, 1H), 6.02 (d, J = 2.8 Hz, 1H), 4.19 (d, J = 5.8 Hz, 2H), 2.19 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 21).

This compound was prepared via method 1 using methyl 2-(aminomethyl)benzoate hydrochloride 72G as the starting material in step 1 and method 3 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(2-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 21 as a TFA salt. MS m/z (M + H⁺) 502.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 9.98 (s, 1H), 8.29 (t, J = 5.8 Hz, 1H), 7.54–7.50 (m, 1H), 7.50–7.46 (m, 1H), 7.38 (dt, J = 5.8, 2.3 Hz, 3H), 7.36–7.30 (m, 2H), 7.16 (td, J = 7.7, 1.1 Hz, 2H), 7.09 (td, J = 7.8, 1.2 Hz, 1H), 7.02 (td, J = 7.5, 1.3 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.03 (d, J = 2.7 Hz, 1H), 4.47 (s, 3H), 4.29 (dd, J = 5.8, 2.7 Hz, 2H), 2.18 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(3-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 22).

This compound was prepared via method 1 using methyl 3-(aminomethyl)benzoate 72H as the starting material in step 1 and method 3 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(3-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 22 as a TFA salt. MS m/z (M + H⁺) 502.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.24 (s, 1H), 9.97 (s, 1H), 8.41 (t, J = 6.0 Hz, 1H), 7.52 (dd, J = 5.8, 3.4 Hz, 1H),7.47 (dd, J = 5.9, 3.5 Hz, 1H), 7.36 (ddd, J = 13.8, 7.7, 2.6 Hz, 4H), 7.19–7.12 (m, 3H), 7.08 (td, J = 7.7, 1.3 Hz, 1H), 7.02 (s, 1H), 6.87–6.82 (m, 1H), 6.02 (d, J = 2.8 Hz, 1H), 4.38 (s, 2H), 4.26 (d, J = 5.9 Hz, 2H), 3.83 (s, 1H), 2.19 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 23).

This compound was prepared via method 1 using methyl 4-(aminomethyl)benzoate hydrochloride 72I as the starting material in step 1 and method 3 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-N-(4-(hydroxymethyl)benzyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 23 as a TFA salt. MS m/z (M + H⁺) 502.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 9.97 (s, 1H), 8.38 (t, J = 6.0 Hz, 1H), 7.55–7.50 (m, 1H), 7.50–7.45 (m, 1H), 7.38 (ddd, J = 7.0, 3.0, 1.9 Hz, 3H), 7.36–7.31 (m, 1H), 7.19–7.11 (m, 3H), 7.11–7.05 (m, 1H), 6.95 (s, 1H), 6.94 (s, 1H), 6.02 (d, J = 2.8 Hz, 1H), 4.43 (s, 2H), 4.24 (d, J = 6.0 Hz, 3H), 2.18 (s, 3H).

3-((2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)methyl)benzoic Acid (Table 1, Compound 24).

This compound was prepared via method 1 using methyl 3-(aminomethyl)benzoate 72H as the starting material in step 1 and method 2 to afford 3-((2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)-methyl)benzoic acid 24 as a TFA salt. MS m/z (M + H⁺) 516.1. ¹H NMR (400 MHz, DMSO-d₆) δ 12.85 (s, 1H), 10.21 (s, 1H), 9.95 (d, J = 1.7 Hz, 1H), 8.45 (t, J = 5.9 Hz, 1H), 7.74 (dt, J = 9.1, 1.4 Hz, 2H), 7.50–7.44 (m, 1H), 7.44–7.40 (m, 1H), 7.36–7.25 (m, 5H), 7.18 (dt, J = 7.8, 1.5 Hz, 1H), 7.13 (td, J = 7.6, 1.2 Hz, 1H), 7.06 (td, J = 7.7, 1.3 Hz, 1H), 6.00–5.95 (m, 1H), 4.36–4.22 (m, 2H), 2.17 (d, J = 0.9 Hz, 3H).

4-((2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)methyl)benzoic Acid (Table 1, Compound 25).

This compound was prepared via method 1 using methyl 4-(aminomethyl)benzoate hydrochloride 72I as the starting material in step 1 and method 2 to afford 4-((2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)methyl)benzoic acid 25 as a TFA salt. MS m/z (M + H⁺) 516.1, ¹H NMR (400 MHz, DMSO-d₆) δ 12.83 (s, 1H), 10.21 (s, 1H), 10.00 (s, 1H), 8.46 (t, J = 5.9 Hz, 1H), 7.79–7.71 (m, 2H), 7.59–7.51 (m, 1H), 7.51–7.43 (m, 1H), 7.42–7.36 (m, 3H), 7.34 (dd, J = 7.9, 1.1 Hz, 1H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 7.12–7.03 (m, 3H), 6.04 (dd, J = 2.9, 1.2 Hz, 1H), 4.46–4.21 (m, 2H), 2.20 (d, J = 0.9 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 26).

This compound was prepared via method 1 using pyridin-2-amine 72J as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxamide 26 as a TFA salt. MS m/z (M + H⁺) 459.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.52 (s, 1H), 10.27 (t, J = 2.3 Hz, 1H), 10.18 (d, J = 1.8 Hz, 1H), 8.28 (ddd, J = 5.0, 1.9, 0.9 Hz, 1H), 7.87 (dt, J = 8.5, 1.0 Hz, 1H), 7.74 (ddd, J = 8.8, 7.3, 1.9 Hz, 1H), 7.57 (dd, J = 7.6, 1.9 Hz, 1H), 7.47 (dd, J = 7.8, 1.5 Hz, 1H), 7.41–7.26 (m, 4H), 7.15 (td, J = 7.6, 1.2 Hz, 1H), 7.11–7.05 (m, 2H), 6.12 (d, J = 2.8 Hz, 1H), 2.23 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(pyridin-3-yl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 27).

This compound was prepared via method 1 using 3-oxo-N-3-pyridinylbutanamide 73I (from commercial sources) as the starting material in step 2 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(pyridin-3-yl)-1,4-dihydropyrimidine-5-carboxamide 27 as a TFA salt. MS m/z (M + H⁺) 459.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (d, J = 2.9 Hz, 1H), 10.32 (s, 1H), 10.25 (s, 1H), 8.86 (d, J = 2.5 Hz, 1H), 8.35 (d, J = 5.0 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.58–7.46 (m, 3H), 7.38 (dtd, J = 20.4, 9.3, 8.5, 5.6 Hz, 4H), 7.18 (t, J = 7.6 Hz, 1H), 7.15–7.06 (m, 1H), 6.10 (d, J = 2.9 Hz, 1H), 2.26 (s, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(1,3,4-thiadiazol-2-yl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 28).

This compound was prepared via method 1 using 1,3,4-thiadiazol-2-amine 72K as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(1,3,4-thiadiazol-2-yl)-1,4-dihydropyrimidine-5-carboxamide 28 as a TFA salt. MS m/z (M + H⁺) 466.0.

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(5-methyl-1,3,4-thiadiazol-2-yl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 29).

This compound was prepared via method 1 using 5-methyl-1,3,4-thiadiazol-2-amine 72L as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-(5-methyl-1,3,4-thiadiazol-2-yl)-1,4-dihydropyrimidine-5-carboxamide 29 as a TFA salt. MS m/z (M + H⁺) 480.1, ¹H NMR (400 MHz, DMSO-d₆) δ 12.31 (s, 1H), 10.50–10.19 (m, 2H), 7.50 (dd, J = 7.1, 1.9 Hz, 1H), 7.47–7.30 (m, 5H), 7.22–7.15 (m, 1H), 7.11 (td, J = 7.6, 1.3 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 2.56 (s, 3H), 2.31 (d, J = 11.7 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-5-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 30).

This compound was prepared via method 1 using (1-methyl-1H-imidazol-5-yl)methanamine 72M as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-5-yl)-methyl)-1,4-dihydropyrimidine-5-carboxamide 30 as a TFA salt. MS m/z (M + H⁺) 476.1.

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-2-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 31).

This compound was prepared via method 1 using (1-methyl-1H-imidazol-2-yl)methanamine 72N as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-2-yl)-methyl)-1,4-dihydropyrimidine-5-carboxamide 31 as a TFA salt. MS m/z (M + H⁺) 476.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.29 (d, J = 2.7 Hz, 1H), 10.13 (d, J = 1.8 Hz, 1H), 8.48 (t, J = 5.2 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.51–7.46 (m, 1H), 7.39 (ddd, J = 7.9, 1.2, 0.6 Hz, 1H), 7.35 (qd, J = 2.0, 1.2 Hz, 1H), 7.34–7.31 (m, 3H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.13–7.07 (m, 1H), 5.93 (dd, J = 3.1, 1.0 Hz, 1H), 4.56–4.43 (m, 2H), 3.59 (s, 3H), 2.22 (d, J = 0.8 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 32).

This compound via prepared via method 1 using (1-methyl-1H-imidazol-4-yl)methanamine 72O as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-imidazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide 32 as a TFA salt. MS m/z (M + H⁺) 476.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.19 (d, J = 2.7 Hz, 1H), 9.96 (s, 1H), 8.20 (t, J = 5.7 Hz, 1H), 7.52–7.48 (m, 1H), 7.47–7.42 (m, 1H), 7.40–7.31 (m, 4H), 7.27 (d, J = 0.9 Hz, 1H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 7.12 (d, J = 0.8 Hz, 1H), 7.09 (td, J = 7.7, 1.3 Hz, 1H), 5.97 (dd, J = 2.9, 1.1 Hz, 1H), 4.07 (d, J = 5.7 Hz, 2H), 3.72 (s, 3H), 2.15 (d, J = 1.0 Hz, 3H).

2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 1, Compound 33).

This compound was prepared via method 1 using (1-methyl-1H-pyrazol-4-yl)methanamine 72P as the starting material in step 1 to afford 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide 33 as a TFA salt. MS m/z (M + H⁺) 476.2.

4-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)picolinic Acid (Table 1, Compound 34).

This compound was prepared via method 1 using methyl 4-aminopicolinate 72Q as the starting material in step 1 and method 2 to afford 4-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)picolinic acid 34 as a TFA salt. MS m/z (M + H⁺) 503.1. ¹H NMR (400 MHz, DMSO-d₆) δ 10.53 (s, 1H), 10.33 (s, 1H), 10.28 (s, 1H), 8.50 (d, J = 5.7 Hz, 1H), 8.24 (d, J = 2.1 Hz, 1H), 7.79 (dt, J = 5.8, 1.6 Hz, 1H), 7.47 (ddt, J = 7.7, 5.1, 1.3 Hz, 2H), 7.43–7.28 (m, 5H), 7.16 (tt, J = 7.7, 1.2 Hz, 1H), 7.09 (tt, J = 7.6, 1.2 Hz, 1H), 6.10 (d, J = 3.0 Hz, 1H), 2.25 (s, 3H).

2-(2-(Benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 1, Compound 35).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and method 2 to afford 2-(2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 35 as a TFA salt. MS m/z (M + H⁺) 503.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.58 (s, 1H), 10.66 (s, 1H), 10.31 (s, 1H), 10.17 (d, J = 1.8 Hz, 1H), 8.47 (dd, J = 5.1, 0.9 Hz, 1H), 8.43 (dd, J = 1.5, 0.9 Hz, 1H), 7.60 (dd, J = 7.6, 1.8 Hz, 1H), 7.53–7.45 (m, 2H), 7.45–7.30 (m, 4H), 7.18 (td, J = 7.6, 1.2 Hz, 1H), 7.14–7.06 (m, 1H), 6.20–6.13 (m, 1H), 2.27 (d, J = 0.9 Hz, 3H).

Synthesis of Substituted 1-(Benzo[d]oxazol-2-yl)guanidine Intermediates.

Method 4: 1-(4-Fluorobenzo[d]oxazol-2-yl)guanidine 74A.

To a suspension of 2-amino-3-fluorophenol 81A (1 g, 7.87 mmol) and dicyandiamide 82 (0.661 g, 7.87 mmol) in EtOH (4.85 mL) was added HCl (0.646 mL, 7.87 mmol). The mixture was sealed in a microwave tube and heated at 90 °C for 30 min. After cooling to room temperature, the solid was filtered. The solid was stirred in water (10 mL) and 10% KOH (2.5 mL) at 100 °C for 2 h. After cooling, the solid was filtered and dried.

1-(5-Fluorobenzo[d]oxazol-2-yl)guanidine 74B.

This compound was prepared via method 4 using 2-amino-4-fluorophenol 81B as the starting material to afford 1-(5-fluorobenzo[d]oxazol-2-yl)guanidine 74B.

1-(6-Fluorobenzo[d]oxazol-2-yl)guanidine 74C.

This compound was prepared via method 4 using 2-amino-5-fluorophenol 81C as the starting material to afford 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C.

1-(7-Fluorobenzo[d]oxazol-2-yl)guanidine 74D.

This compound was prepared via method 4 using 2-amino-6-fluorophenol 81D as the starting material to afford 1-(7-fluorobenzo[d]oxazol-2-yl)guanidine 74D.

1-(4-Chlorobenzo[d]oxazol-2-yl)guanidine 74E.

This compound was prepared via method 4 using 2-amino-3-chlorophenol 81E as the starting material to afford 1-(4-chlorobenzo[d]oxazol-2-yl)guanidine 74E.

1-(5-Chlorobenzo[d]oxazol-2-yl)guanidine 74F.

This compound was prepared via method 4 using 2-amino-4-chlorophenol 81F as the starting material to afford 1-(5-chlorobenzo[d]oxazol-2-yl)guanidine 74F.

1-(6-Chlorobenzo[d]oxazol-2-yl)guanidine 74G.

This compound was prepared from method 4 via 2-amino-5-chlorophenol 81G as the starting material to afford 1-(6-chlorobenzo[d]oxazol-2-yl)guanidine 74G.

1-(7-Chlorobenzo[d]oxazol-2-yl)guanidine 74H.

This compound was prepared via method 4 using 2-amino-6-chlorophenol 81H as the starting material to afford 1-(7-chlorobenzo[d]oxazol-2-yl)guanidine 74H.

1-(4-Methylbenzo[d]oxazol-2-yl)guanidine 74I.

This compound was prepared via method 4 using 2-amino-3-methylphenol 81I as the starting material to afford 1-(4-methylbenzo[d]oxazol-2-yl)guanidine 74I.

1-(5-Methylbenzo[d]oxazol-2-yl)guanidine 74J.

This compound was prepared via method 4 using 2-amino-4-methylphenol 81J as the starting material to afford 1-(5-methylbenzo[d]oxazol-2-yl)guanidine 74J.

1-(6-Methylbenzo[d]oxazol-2-yl)guanidine 74K.

This compound was prepared via method 4 using 2-amino-5-methylphenol 81K as the starting material to afford 1-(6-methylbenzo[d]oxazol-2-yl)guanidine 74K.

1-(7-Methylbenzo[d]oxazol-2-yl)guanidine 74L.

This compound was prepared via method 4 using 2-amino-6-methylphenol 81L as the starting material to afford 1-(7-methylbenzo[d]oxazol-2-yl)guanidine 74L.

1-(5-Methoxybenzo[d]oxazol-2-yl)guanidine 74M.

This compound was prepared via method 4 using 2-amino-4-methoxyphenol hydrochloride 81M as the starting material to afford 1-(5-methoxybenzo[d]oxazol-2-yl)guanidine 74M.

1-(6-Methoxybenzo[d]oxazol-2-yl)guanidine 74N.

This compound was prepared via method 4 using 2-amino-5-methoxyphenol hydrochloride 81N as the starting material to afford 1-(6-methoxybenzo[d]oxazol-2-yl)guanidine 74N.

1-(7-Methoxybenzo[d]oxazol-2-yl)guanidine 74O.

This compound was prepared via method 4 using 2-amino-6-methoxyphenol hydrochloride 81O as the starting material to afford 1-(7-methoxybenzo[d]oxazol-2-yl)guanidine 74O.

1-(4-Nitrobenzo[d]oxazol-2-yl)guanidine 74P.

This compound was prepared via method 4 using 2-amino-3-nitrophenol 81P as the starting material to afford 1-(4-nitrobenzo[d]oxazol-2-yl)guanidine 74P.

1-(5-Nitrobenzo[d]oxazol-2-yl)guanidine 74Q.

This compound was prepared via method 4 using 2-amino-4-nitrophenol 81Q as the starting material to afford 1-(5-nitrobenzo[d]oxazol-2-yl)guanidine 74Q.

1-(7-Nitrobenzo[d]oxazol-2-yl)guanidine 74R.

This compound was prepared via method 4 using 2-amino-6-nitrophenol 81R as the starting material to afford 1-(7-nitrobenzo[d]oxazol-2-yl)guanidine 74R.

2-(4-(2-Chlorophenyl)-2-((4-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 36).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(4-fluorobenzo[d]oxazol-2-yl)guanidine 74A as the starting material in step 2 followed by method 2. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method basic standard gradient) to afford 2-(4-(2-chlorophenyl)-2-((4-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 36 as an ammonium salt. MS m/z (M + H⁺) 521.1.

2-(4-(2-Chlorophenyl)-2-((5-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 37).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(5-fluorobenzo[d]oxazol-2-yl)guanidine 74B as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((5-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 37 as an ammonium salt. MS m/z (M + H⁺) 521.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.53 (s, 1H), 10.62 (s, 1H), 10.18 (s, 2H), 8.43 (d, J = 5.1 Hz, 1H), 8.38 (s, 1H), 7.56 (dd, J = 7.6, 1.9 Hz, 1H), 7.50–7.44 (m, 2H), 7.42–7.28 (m, 2H), 7.18 (dd, J = 8.9, 2.6 Hz, 1H), 7.04 (d, J = 9.9 Hz, 1H), 6.95–6.82 (m, 1H), 6.14 (s, 1H), 2.23 (s, 3H).

2-(4-(2-Chlorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 38).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 38 as a TFA salt. MS m/z (M + H⁺) 521.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.59 (s, 1H), 10.65 (s, 1H), 10.18 (d, J = 4.1 Hz, 2H), 8.47 (dd, J = 5.0, 0.9 Hz, 1H), 8.42 (dd, J = 1.5, 0.9 Hz, 1H), 7.59 (dd, J = 7.6, 1.8 Hz, 1H), 7.53–7.47 (m, 2H), 7.44–7.29 (m, 4H), 7.02 (ddd, J = 10.2, 8.6, 2.6 Hz, 1H), 6.20–6.13 (m, 1H), 2.26 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((7-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 39).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(7-fluorobenzo[d]oxazol-2-yl)guanidine 74D as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((7-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 39 as an ammonium salt. MS m/z (M + H⁺) 521.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.62 (s, 1H), 10.68 (s, 1H), 10.24 (d, J = 1.8 Hz, 1H), 10.16 (t, J = 2.4 Hz, 1H), 8.46 (dd, J = 5.1, 0.9 Hz, 1H), 8.42 (t, J = 1.1 Hz, 1H), 7.60 (dd, J = 7.6, 1.9 Hz, 1H), 7.53–7.46 (m, 2H), 7.43–7.30 (m, 2H), 7.25–7.12 (m, 2H), 7.02 (ddd, J = 10.5, 8.0, 1.3 Hz, 1H), 6.26–6.08 (m, 1H), 2.27 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((4-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 40).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(4-chlorobenzo[d]oxazol-2-yl)guanidine 74E as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((4-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 40 as an ammonium salt. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.61 (s, 1H), 10.70 (s, 1H), 10.51 (t, J = 2.4 Hz, 1H), 10.26 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 5.1, 0.8 Hz, 1H), 8.43 (t, J = 1.1 Hz, 1H), 7.62 (dd, J = 7.5, 1.9 Hz, 1H), 7.53 (dd, J = 7.7, 1.5 Hz, 1H), 7.49 (dd, J = 5.1, 1.5 Hz, 1H), 7.43–7.32 (m, 3H), 7.26 (dd, J = 8.2, 0.9 Hz, 1H), 7.11 (t, J = 8.1 Hz, 1H), 6.22 (d, J = 3.0 Hz, 1H), 2.30 (d, J = 0.8 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((5-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 41).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(5-chlorobenzo[d]oxazol-2-yl)guanidine 74F as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((5-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 41 as an ammonium salt. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.67 (s, 1H), 10.23 (d, J = 1.8 Hz, 1H), 10.21 (t, J = 2.4 Hz, 1H), 8.46 (dd, J = 5.1, 0.8 Hz, 1H), 8.42 (t, J = 1.2 Hz, 1H), 7.59 (dd, J = 7.5, 1.9 Hz, 1H), 7.53–7.47 (m, 2H), 7.43 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 4.1 Hz, 1H), 7.41–7.30 (m, 2H), 7.12 (dd, J = 8.5, 2.1 Hz, 1H), 6.20–6.15 (m, 1H), 2.26 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((6-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 42).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-chlorobenzo[d]oxazol-2-yl)guanidine 74G as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((6-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 42 as an ammonium salt. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.67 (s, 1H), 10.20 (dt, J = 4.9, 2.0 Hz, 2H), 8.47 (dd, J = 5.1, 0.8 Hz, 1H), 8.42 (t, J = 1.1 Hz, 1H), 7.60 (dd, J = 7.6, 1.9 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.50 (dd, J = 2.7, 1.5 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 7.41–7.31 (m, 3H), 7.21 (dd, J = 8.4, 2.1 Hz, 1H), 6.17 (d, J = 2.8 Hz, 1H), 2.26 (s, 3H).

2-(4-(2-Chlorophenyl)-2-((7-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 43).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 with 1-(7-chlorobenzo[d]oxazol-2-yl)guanidine 74H as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((7-chlorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 43 as an ammonium salt. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.61 (s, 1H), 10.69 (s, 1H), 10.22 (d, J = 1.8 Hz, 1H), 10.13 (t, J = 2.3 Hz, 1H), 8.47 (dd, J = 5.1, 0.9 Hz, 1H), 8.42 (t, J = 1.1 Hz, 1H), 7.60 (dd, J = 7.6, 1.9 Hz, 1H), 7.51 (dd, J = 2.7, 1.5 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 7.42–7.31 (m, 3H), 7.21–7.16 (m, 2H), 6.21–6.16 (m, 1H), 2.27 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((4-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 44).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(4-methylbenzo[d]oxazol-2-yl)guanidine 74I as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((4-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 44 as an ammonium salt. MS m/z (M + H⁺) 517.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.61 (s, 1H), 10.65 (s, 1H), 10.63 (s, 1H), 10.14 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 5.0, 0.9 Hz, 1H), 8.44–8.42 (m, 1H), 7.63 (dd, J = 7.6, 1.9 Hz, 1H), 7.53 (dd, J = 7.7, 1.5 Hz, 1H), 7.49 (dd, J = 5.1, 1.5 Hz, 1H), 7.40 (td, J = 7.5, 1.5 Hz, 1H), 7.35 (td, J = 7.5, 1.9 Hz, 1H), 7.24–7.18 (m, 1H), 7.04–6.97 (m, 2H), 6.19 (dd, J = 3.0, 1.0 Hz, 1H), 2.45 (d, J = 0.8 Hz, 3H), 2.31 (d, J = 0.8 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((5-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 45).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(5-methylbenzo[d]oxazol-2-yl)guanidine 74 J as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((5-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 45 as an ammonium salt. MS m/z (M + H⁺) 517.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.58 (s, 1H), 10.60 (s, 1H), 10.32 (s, 1H), 10.12 (d, J = 1.8 Hz, 1H), 8.43 (dd, J = 5.0, 0.8 Hz, 1H), 8.39 (t, J = 1.1 Hz, 1H), 7.57 (dd, J = 7.6, 1.9 Hz, 1H), 7.51–7.43 (m, 2H), 7.39–7.27 (m, 2H), 7.23 (d, J = 8.1 Hz, 1H), 7.17–7.14 (m, 1H), 6.90–6.85 (m, 1H), 6.15–6.11 (m, 1H), 2.32 (s, 3H), 2.24 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((6-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 46).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-methylbenzo[d]oxazol-2-yl)guanidine 74 K as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((6-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 46 as an ammonium salt. MS m/z (M + H⁺) 517.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.63 (s, 1H), 10.27 (d, J = 2.8 Hz, 1H), 10.12 (d, J = 1.8 Hz, 1H), 8.47 (dd, J = 5.1, 0.8 Hz, 1H), 8.45–8.39 (m, 1H), 7.59 (dd, J = 7.6, 1.9 Hz, 1H), 7.53–7.46 (m, 2H), 7.44–7.29 (m, 2H), 7.27–7.20 (m, 2H), 7.03–6.96 (m, 1H), 6.16 (d, J = 2.8 Hz, 1H), 2.35 (s, 3H), 2.27 (d, J = 0.8 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((7-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 47).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(7-methylbenzo[d]oxazol-2-yl)guanidine 74 L as the starting material in step 2 followed by method 2 to afford 2-(4-(2chlorophenyl)-2-((7-methylbenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 47 as an ammonium salt. MS m/z (M + H⁺) 517.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.64 (s, 1H), 10.27 (s, 1H), 10.10 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 5.1, 0.9 Hz, 1H), 8.42 (dd, J = 1.5, 0.9 Hz, 1H), 7.59 (dd, J = 7.6, 1.8 Hz, 1H), 7.50 (dd, J = 3.1, 1.4 Hz, 1H), 7.48 (d, J = 1.5 Hz, 1H), 7.38 (td, J = 7.5, 1.5 Hz, 1H), 7.33 (td, J = 7.6, 1.9 Hz, 1H), 7.18 (ddd, J = 7.8, 1.2, 0.7 Hz, 1H), 7.06 (t, J = 7.7 Hz, 1H), 6.92 (dt, J = 7.5, 1.1 Hz, 1H), 6.29–6.06 (m, 1H), 2.36 (s, 3H), 2.28 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((5-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 48).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(5-methoxybenzo[d]oxazol-2-yl)guanidine 74 M as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((5-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 48 as an ammonium salt. MS m/z (M + H⁺) 533.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.61 (s, 1H), 10.63 (s, 1H), 10.25 (d, J = 2.7 Hz, 1H), 10.15 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 5.1, 0.8 Hz, 1H), 8.41 (t, J = 1.2 Hz, 1H), 7.59 (dd, J = 7.6, 1.9 Hz, 1H), 7.54–7.47 (m, 2H), 7.44–7.31 (m, 2H), 7.29 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.66 (dd, J = 8.8, 2.6 Hz, 1H), 6.18–6.14 (m, 1H), 3.76 (s, 3H), 2.26 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((6-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 49).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-methoxybenzo[d]oxazol-2-yl)guanidine 74N as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((6-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 49 as an ammonium salt. MS m/z (M + H⁺) 533.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.62 (s, 1H), 10.20 (s, 1H), 10.10 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 5.1, 0.9 Hz, 1H), 8.42 (dd, J = 1.5, 0.8 Hz, 1H), 7.59 (dd, J = 7.6, 1.9 Hz, 1H), 7.52–7.46 (m, 2H), 7.42–7.29 (m, 2H), 7.26 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 6.77 (dd, J = 8.6, 2.4 Hz, 1H), 6.18–6.12 (m, 1H), 3.75 (s, 3H), 2.27 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chlorophenyl)-2-((7-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 50).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(7-methoxybenzo[d]oxazol-2-yl)guanidine 74O as the starting material in step 2 followed by method 2 to afford 2-(4-(2-chlorophenyl)-2-((7-methoxybenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 50 as an ammonium salt. MS m/z (M + H⁺) 533.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.57 (s, 1H), 10.62 (s, 1H), 10.24 (s, 1H), 10.13 (d, J = 1.8 Hz, 1H), 8.44 (dd, J = 5.1, 0.8 Hz, 1H), 8.39 (t, J = 1.1 Hz, 1H), 7.57 (dd, J = 7.6, 1.8 Hz, 1H), 7.49–7.44 (m, 2H), 7.33 (dtd, J = 20.2, 7.4, 1.6 Hz, 2H), 7.07 (t, J = 8.1 Hz, 1H), 6.94 (dd, J = 7.9, 0.9 Hz, 1H), 6.75 (dd, J = 8.3, 1.0 Hz, 1H), 6.29–6.02 (m, 1H), 3.86 (s, 3H), 2.24 (d, J = 0.9 Hz, 3H).

Method 5: 2-(2-((4-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 51).

Step 1: To a suspension of methyl 2-(4-(2-chlorophenyl)-6-methyl-2-((4-nitrobenzo[d]oxazol-2-yl)amino)-1,4-dihydropyrimidine-5-carboxamido)isonicotinate 83 (47 mg, 0.084 mmol) (this compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R in step 1 and 1-(4-nitrobenzo[d]oxazol-2-yl)guanidine 74P in step 2) in EtOH (0.4 mL) and water (0.4 mL) was added ammonium chloride (8.95 mg, 0.167 mmol) followed by iron (23.35 mg, 0.418 mmol). The mixture was stirred at 90 °C for 2 h. After cooling, EtOAc was added and the reaction mixture was passed through Celite. The solvent was evaporated. The crude product was used in next reaction without purification. MS m/z (M + H⁺) 532.2.

Step 2: This compound was prepared via method 2 using the above compound as the starting material to afford 2-(2-((4-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 51 as a TFA salt. MS m/z (M + H⁺) 518.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.62 (s, 1H), 10.71 (s, 1H), 10.28 (s, 1H), 10.14 (s, 1H), 8.45 (dd, J = 5.1, 0.9 Hz, 1H), 8.42 (dd, J = 1.5, 0.9 Hz, 1H), 7.56 (dd, J = 7.6, 1.9 Hz, 1H), 7.48 (t, J = 1.6 Hz, 1H), 7.46 (dd, J = 4.2, 1.5 Hz, 1H), 7.36 (td, J = 7.5, 1.6 Hz, 1H), 7.31 (td, J = 7.5, 1.9 Hz, 1H), 6.87 (t, J = 7.9 Hz, 1H), 6.76 (s, 1H), 6.57 (s, 1H), 6.12–6.06 (m, 1H), 2.85 (s, 2H), 2.23 (d, J = 0.8 Hz, 3H).

2-(2-((5-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 52).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(5-nitrobenzo[d]oxazol-2-yl)guanidine 74Q as the starting material in step 2 followed by method 5 to afford 2-(2-((5-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 52 as a TFA salt. MS m/z (M + H⁺) 518.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H), 10.28 (s, 1H), 10.18 (s, 1H), 8.45 (dd, J = 5.1, 0.9 Hz, 1H), 8.40 (t, J = 1.2 Hz, 1H), 7.58 (dd, J = 7.6, 1.9 Hz, 1H), 7.48 (t, J = 1.7 Hz, 1H), 7.47 (t, J = 1.2 Hz, 1H), 7.41–7.27 (m, 3H), 7.10 (s, 1H), 6.84 (s, 1H), 6.13 (d, J = 2.9 Hz, 1H), 3.09–2.78 (m, 2H), 2.25 (s, 3H).

2-(2-((7-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 2, Compound 53).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(7-nitrobenzo[d]oxazol-2-yl)guanidine 74R as the starting material in step 2 followed by method 5 to afford 2-(2-((7-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 53 as a TFA salt. MS m/z (M + H⁺) 518.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.62 (s, 1H), 10.69 (s, 1H), 10.22 (s, 1H), 10.08 (s, 1H), 8.45 (dd, J = 5.1, 0.8 Hz, 1H), 8.40 (dd, J = 1.5, 0.9 Hz, 1H), 7.57 (dd, J = 7.6, 1.8 Hz, 1H), 7.48 (d, J = 1.5 Hz, 1H), 7.46 (dd, J = 3.2, 1.5 Hz, 1H), 7.37 (td, J = 7.5, 1.5 Hz, 1H), 7.31 (td, J = 7.6, 1.9 Hz, 1H), 6.89 (t, J = 7.9 Hz, 1H), 6.67–6.60 (m, 1H), 6.50–6.43 (m, 1H), 6.15–6.10 (m, 1H), 2.99 (s, 2H), 2.34–2.09 (m, 3H).

2-(4-(2-Bromophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 54).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-bromobenzaldehyde 75A as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-bromophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 54 as a TFA salt. MS m/z (M + H⁺) 567.1, ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 10.31 (s, 1H), 10.16 (d, J = 1.8 Hz, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.39 (s, 1H), 7.66 (dd, J = 8.0, 1.2 Hz, 1H), 7.62 (dd, J = 7.8, 1.7 Hz, 1H), 7.48 (dd, J = 5.0, 1.5 Hz, 1H), 7.46–7.33 (m, 3H), 7.26 (ddd, J = 7.9, 7.3, 1.7 Hz, 1H), 7.03 (ddd, J = 10.2, 8.6, 2.5 Hz, 1H), 6.10 (dd, J = 3.0, 1.0 Hz, 1H), 2.27 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chloro-4-fluorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 55).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-fluorobenzaldehyde 75B as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-fluorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 55 as a TFA salt. MS m/z (M + H⁺) 539.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.61 (s, 1H), 10.68 (s, 1H), 10.20 (d, J = 1.8 Hz, 1H), 10.16 (t, J = 2.4 Hz, 1H), 8.47 (dd, J = 5.0, 0.9 Hz, 1H), 8.42 (dd, J = 1.5, 0.8 Hz, 1H), 7.62 (dd, J = 8.8, 6.2 Hz, 1H), 7.54–7.47 (m, 2H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H), 7.36 (dd, J = 8.6, 5.0 Hz, 1H), 7.27 (td, J = 8.5, 2.7 Hz, 1H), 7.03 (ddd, J = 10.1, 8.6, 2.5 Hz, 1H), 6.15–6.09 (m, 1H), 2.26 (d, J = 0.8 Hz, 3H).

2-(4-(2,4-Dichlorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 56).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2,4-dichlorobenzaldehyde 75C as the starting materials in step 2 followed by method 2 to afford 2-(4-(2,4-dichlorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 56 as a TFA salt. MS m/z (M + H⁺) 555.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.62 (s, 1H), 10.67 (s, 1H), 10.22 (d, J = 1.8 Hz, 1H), 10.16 (d, J = 2.9 Hz, 1H), 8.47 (dd, J = 5.1, 0.8 Hz, 1H), 8.42 (t, J = 1.1 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 5.1, 1.5 Hz, 1H), 7.47 (dd, J = 8.4, 2.1 Hz, 1H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H), 7.36 (dd, J = 8.6, 5.0 Hz, 1H), 7.03 (ddd, J = 10.2, 8.6, 2.6 Hz, 1H), 6.15–6.07 (m, 1H), 2.25 (d, J = 0.9 Hz, 3H).

2-(4-(2-Chloro-4-methylphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 57).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-methylbenzaldehyde 75D as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-methylphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 57 as a TFA salt. MS m/z (M + H⁺) 535.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.57 (s, 1H), 10.61 (s, 1H), 10.11 (d, J = 2.0 Hz, 2H), 8.44 (dd, J = 5.0, 0.9 Hz, 1H), 8.39 (dd, J = 1.5, 0.9 Hz, 1H), 7.46 (dd, J = 5.0, 1.5 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.37 (dd, J = 8.6, 2.5 Hz, 1H), 7.31 (dd, J = 8.6, 5.0 Hz, 1H), 7.29 (dd, J = 1.7, 0.8 Hz, 1H), 7.17–7.12 (m, 1H), 6.99 (ddd, J = 10.2, 8.6, 2.5 Hz, 1H), 6.10 (d, J = 2.5 Hz, 1H), 2.22 (d, J = 0.9 Hz, 6H).

2-(4-(2-Chloro-4-methoxyphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 58).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-methoxybenzaldehyde 75E as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-methoxyphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 58 as a TFA salt. MS m/z (M + H⁺) 551.1, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.62 (s, 1H), 10.14 (d, J = 1.8 Hz, 1H), 10.11 (t, J = 2.3 Hz, 1H), 8.47 (dd, J = 5.0, 0.9 Hz, 1H), 8.43 (dd, J = 1.5, 0.8 Hz, 1H), 7.53–7.46 (m, 2H), 7.40 (dd, J = 8.6, 2.5 Hz, 1H), 7.35 (dd, J = 8.6, 5.0 Hz, 1H), 7.07 (d, J = 2.6 Hz, 1H), 7.02 (ddd, J = 10.1, 8.6, 2.5 Hz, 1H), 6.93 (dd, J = 8.7, 2.6 Hz, 1H), 6.11 (dd, J = 2.8, 1.1 Hz, 1H), 3.74 (s, 3H), 2.25 (d, J = 0.9 Hz, 3H).

Method 6: 2-(4-(3-Chloro-[1,1′-biphenyl]-4-yl)-2-((6fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 59).

Step 1: To a solution of methyl 2-(3-oxobutanamido)isonicotinate 73J (0.250 g, 1.06 mmol) (this compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1), 3-chloro-[1,1′-biphenyl]-4-carbaldehyde 75F (0.229 g, 1.058 mmol) and thiourea (0.081 g, 1.058 mmol) in acetonitrile (5.0 mL) and DMF (2.5 mL) were added dropwise to TMS-Cl (0.135 mL, 1.058 mmol) at room temperature. The mixture was stirred at 80 °C overnight. The reaction mixture was poured onto crushed ice and stirred until all ice had melted. The solid was filtered and dried. The crude product 84 was used in the next reaction without further purification. MS m/z (M + H⁺) 493.1.

Step 2: The mixture of methyl 2-(4-(3-chloro-[1,1′-biphenyl]-4-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido)isonicotinate 84 (0.43 g, 0.87 mmol), 6-fluorobenzo[d]oxazol-2-amine 85 (0.199 g, 1.308 mmol), and mercuric acetate (0.334 g, 1.047 mmol) in DCM (5 mL) and DMF (1 mL) in the sealed tube was stirred at 80 °C for 72 h. After the reaction was completed, the mixture was filtered through Celite and washed with EtOAc. The filtrate was concentrated. The crude product 86 was purified by flash silica gel chromatography (30–80%, EtOAc/hex). MS m/z (M + H⁺) 611.2.

Step 3: To a solution of methyl 2-(4-(3-chloro-[1,1′-biphenyl]-4-yl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinate 86 (0.21 g, 0.34 mmol) in THF (3 mL) was added LiOH (1.375 mL, 0.687 mmol) (0.5 M). The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed. HCl (1 N) was added to the residue. The solid was filtered, washed with water, and dried to afford 2-(4-(3-chloro-[1,1′-biphenyl]-4-yl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 59 as a HCl salt. MS m/z (M + H⁺) 597.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.58 (s, 1H), 10.64 (s, 1H), 10.18 (d, J = 3.7 Hz, 2H), 8.41 (d, J = 8.8 Hz, 2H), 7.77–7.71 (m, 1H), 7.65 (d, J = 1.6 Hz, 1H), 7.62 (d, J = 1.3 Hz, 3H), 7.48–7.29 (m, 6H), 7.05–6.95 (m, 1H), 6.16 (d, J = 2.6 Hz, 1H), 2.25 (s, 3H).

2-(4-(2-Chloro-4-(1H-pyrazol-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 60).

This compound was prepared from method 6 using 2-chloro-4-(1H-pyrazol-1-yl)benzaldehyde 75G as the starting material in step 1 to afford 2-(4-(2-chloro-4-(1H-pyrazol-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 60 as a HCl salt. MS m/z (M + H⁺) 587.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.56 (s, 1H), 10.67 (s, 1H), 10.19 (s, 1H), 10.15 (s, 1H), 8.52 (d, J = 2.6 Hz, 1H), 8.45 (d, J = 5.1 Hz, 1H), 8.42–8.40 (m, 1H), 7.97 (d, J = 2.6 Hz, 1H), 7.81 (dd, J = 8.6, 2.4 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.50–7.44 (m, 1H), 7.38 (dd, J = 8.5, 2.6 Hz, 1H), 7.34 (dd, J = 8.7, 4.9 Hz, 1H), 7.05–6.95 (m, 1H), 6.51 (t, J = 1.7 Hz, 1H), 6.15 (d, J = 2.9 Hz, 1H), 2.25 (s, 3H).

2-(4-(2-Chloro-4-morpholinphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 61).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-morpholinbenzaldehyde 75H as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-morpholinphenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 61 as a TFA salt. MS m/z (M + H⁺) 606.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.57 (s, 1H), 10.08 (s, 1H), 10.05 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.41 (s, 1H), 7.47 (d, J = 5.1 Hz, 1H), 7.41–7.27 (m, 3H), 6.98 (dd, J = 18.8, 10.2 Hz, 2H), 6.87 (d, J = 8.5 Hz, 1H), 6.05 (s, 1H), 3.68 (t, J = 5.0 Hz, 4H), 3.08 (t, J = 5.0 Hz, 4H), 2.21 (s, 3H).

2-(4-(2-Chloro-4-(1H-imidazol-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 62).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-(1H-imidazol-1-yl)benzaldehyde 75I as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-(1H-imidazol-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 62 as a TFA salt. MS m/z (M + H⁺) 587.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.71 (s, 1H), 10.24 (d, J = 4.4 Hz, 2H), 9.47 (s, 1H), 8.45 (dd, J = 5.1, 0.7 Hz, 1H), 8.41 (dd, J = 1.4, 0.9 Hz, 1H), 8.19 (t, J = 1.7 Hz, 1H), 8.03–8.01 (m, 1H), 7.77 (t, J = 1.7 Hz, 1H), 7.75 (d, J = 1.9 Hz, 2H), 7.47 (dd, J = 5.2, 1.5 Hz, 1H), 7.38 (dd, J = 8.5, 2.5 Hz, 1H), 7.33 (dd, J = 8.6, 5.0 Hz, 1H), 7.01 (ddd, J = 10.1, 8.5, 2.5 Hz, 1H), 6.17 (d, J = 2.8 Hz, 1H), 2.27 (s, 3H).

2-(4-(2-Chloro-4-(piperidin-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 63).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-(piperidin-1-yl)benzaldehyde 75J as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-(piperidin-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 63 as a TFA salt. MS m/z (M + H⁺) 604.3, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.56 (s, 1H), 10.07 (s, 1H), 10.04 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.42 (s, 1H), 7.47 (d, J = 5.1 Hz, 1H), 7.37 (dd, J = 8.6, 2.1 Hz, 1H), 7.35–7.27 (m, 2H), 7.00 (t, J = 9.1 Hz, 1H), 6.92 (s, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.04 (s, 1H), 3.13 (d, J = 5.8 Hz, 4H), 2.21 (s, 3H), 1.50 (d, J = 8.3 Hz, 6H).

2-(4-(2-Chloro-4-(pyrrolidin-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6methyl-1,4dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 64).

This compound was prepared via method 1 using methyl 2-aminoisonicotinate 72R as the starting material in step 1 and 1-(6-fluorobenzo[d]oxazol-2-yl)guanidine 74C and 2-chloro-4-(pyrrolidin-1-yl)benzaldehyde 75K as the starting materials in step 2 followed by method 2 to afford 2-(4-(2-chloro-4-(pyrrolidin-1-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 64 as a TFA salt. MS m/z (M + H⁺) 590.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.58 (s, 1H), 10.49 (s, 1H), 10.03 (s, 1H), 10.00 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.42 (s, 1H), 7.46 (d, J = 5.1 Hz, 1H), 7.36 (dd, J = 8.6, 2.2 Hz, 1H), 7.30 (t, J = 7.6 Hz, 2H), 7.02–6.95 (m, 1H), 6.50 (d, J = 1.8 Hz, 1H), 6.47–6.41 (m, 1H), 6.05 (s, 1H), 3.14 (s, 4H), 2.20 (s, 3H), 1.90–1.82 (m, 4H).

Method 7: 2-(4-(2-Chloro-4-(6-methylpyridin-3-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 65).

In a microwave tube were placed 2-(4-(4-bromo-2-chlorophenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 87 (50 mg, 0.083 mmol) (this compound was prepared via method 6 using 4-bromo-2-chlorobenzaldehyde 75L as the starting material in step 1), (6-methylpyridin-3-yl)boronic acid 88A (22.8 mg, 0.167 mmol), potassium carbonate (69.1 mg, 0.500 mmol), and the PdCl₂(dppf)-CH₂Cl₂ adduct (6.81 mg, 8.34 μmol). Then, dioxane (0.6 mL) and water (0.2 mL) were added and stirred at 80 °C for 3 h. After being cooled to room temperature, EtOAc (3 mL) and 1 N HCl (3 mL) were added to the mixture. The organic layer was separated, washed with brine, dried over MgSO₄, and concentrated. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method acidic standard gradient) to afford 2-(4-(2-chloro-4-(6-methylpyridin-3-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 65 as a TFA salt. MS m/z (M + H⁺) 612.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H), 10.72 (s, 1H), 10.23 (s, 2H), 8.94 (d, J = 2.3 Hz, 1H), 8.45 (dd, J = 5.1, 0.8 Hz, 1H), 8.42 (dd, J = 1.5, 0.8 Hz, 2H), 7.93 (d, J = 1.9 Hz, 1H), 7.75 (dd, J = 8.1, 1.9 Hz, 1H), 7.66 (dd, J = 8.3, 4.1 Hz, 2H), 7.49–7.44 (m, 1H), 7.39 (dd, J = 8.6, 2.5 Hz, 1H), 7.33 (dd, J = 8.6, 5.0 Hz, 1H), 7.01 (ddd, J = 10.1, 8.6, 2.5 Hz, 1H), 6.18 (d, J = 2.7 Hz, 1H), 2.58 (s, 3H), 2.26 (s, 3H).

2-(4-(2-Chloro-4-(2-methylpyrimidin-5-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic Acid (Table 3, Compound 66).

This compound was prepared via method 7 using (2-methylpyrimidin-5-yl)boronic acid 88B as the starting material to afford 2-(4(2-chloro-4-(2-methylpyrimidin-5-yl)phenyl)-2-((6-fluorobenzo[d]oxazol-2-yl)amino)-6-methyl-1,4-dihydropyrimidine-5-carboxamido)isonicotinic acid 66 as a TFA salt. MS m/z (M + H⁺) 66.2, ¹H NMR (400 MHz, DMSO-d₆) δ 13.58 (s, 1H), 10.70 (s, 1H), 10.21 (d, J = 3.0 Hz, 2H), 8.98 (s, 2H), 8.45 (dd, J = 5.1, 0.8 Hz, 1H), 8.43–8.39 (m, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.74 (dd, J = 8.1, 1.9 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.47 (dd, J = 5.1, 1.5 Hz, 1H), 7.38 (dd, J = 8.5, 2.5 Hz, 1H), 7.33 (dd, J = 8.7, 5.0 Hz, 1H), 7.00 (ddd, J = 10.1, 8.6, 2.5 Hz, 1H), 6.17 (d, J = 2.7 Hz, 1H), 2.61 (s, 3H), 2.26 (s, 3H).

Method 8: 2-((7-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound 67).

The mixture of 4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-2-((7-nitrobenzo[d]oxazol-2-yl)amino)-1,4-dihydropyrimidine-5-carboxamide 86A (80 mg, 0.150 mmol) (this compound was prepared via method 1 using (1-methyl-1H-pyrazol-4-yl)methanamine 72P in step 1 and via method 6 using 2-chloro-4-methylbenzaldehyde 75D as the starting material in step 1), ammonium chloride (16.00 mg, 0.299 mmol), and iron (41.8 mg, 0.748 mmol) in EtOH (1 mL) and water (1.000 mL) was stirred at 80 °C for 2 h. After cooling, EtOAc was added, and the reaction mixture was passed through Celite. The solvent was evaporated. The crude product was purified by flash silica gel chromatography (0–20%, MeOH/EtOAc) to afford 2-((7-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide 67. MS m/z (M + H⁺) 505.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.16 (s, 1H), 9.68 (s, 1H), 8.13 (t, J = 5.7 Hz, 1H), 7.32–7.22 (m, 3H), 7.11 (d, J = 7.5 Hz, 2H), 6.80 (t, J = 7.9 Hz, 1H), 6.51 (d, J = 7.7 Hz, 1H), 6.35 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 2.7 Hz, 1H), 5.11 (s, 2H), 4.03 (t, J = 5.9 Hz, 2H), 3.69 (s, 3H), 2.24 (s, 3H), 2.12 (s, 3H).

(R)-2-((7-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound (R)-67).

This compound was prepared via method 8 followed by chiral separation to afford (R)-2-((7-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (R)-67. MS m/z (M + H⁺) 505.2, 1H NMR (400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 9.68 (s, 1H), 8.13 (t, J = 5.7 Hz, 1H), 7.32–7.22 (m, 3H), 7.11 (d, J = 6.3 Hz, 2H), 6.80 (t, J = 7.9 Hz, 1H), 6.51 (dd, J = 7.8, 1.1 Hz, 1H), 6.35 (dd, J = 8.0, 1.1 Hz, 1H), 5.87 (t, J = 1.9 Hz, 1H), 5.10 (s, 2H), 4.11–3.95 (m, 2H), 3.69 (s, 3H), 2.24 (s, 3H), 2.12 (s, 3H).

(S)-2-((7-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound (S)-67).

This compound was prepared via method 8 followed by chiral separation to afford (S)-2-((7-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (S)-67. MS m/z (M + H⁺) 505.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 9.68 (s, 1H), 8.14 (t, J = 5.7 Hz, 1H), 7.31–7.22 (m, 3H), 7.15–7.07 (m, 2H), 6.80 (t, J = 7.8 Hz, 1H), 6.51 (dd, J = 7.8, 1.0 Hz, 1H), 6.35 (dd, J = 8.0, 1.1 Hz, 1H), 5.90–5.84 (m, 1H), 5.10 (s, 2H), 4.11–3.95 (m, 2H), 3.69 (s, 3H), 2.24 (s, 3H), 2.12 (s, 3H).

Method 9: 2-((7-Amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound 68).

Step 1: The mixture of 4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide 84A (0.68 g, 1.74 mmol) (this compound was prepared via method 1 using (1-methyl-1H-pyrazol4-yl)methanamine 72P as the starting material in step 1 and via method 6 using 2-chloro-4-methylbenzaldehyde 75D as the starting material in step 1), methyl 2-amino-6-fluorobenzo[d]oxazole-7-carboxylate 85A (0.367 g, 1.74 mmol), and mercuric acetate (0.834 g, 2.62 mmol) in DCM (10 mL) and DMF (2 mL) in the sealed tube was stirred at 80 °C for 72 h. The reaction was diluted with EtOAc, filtered through Celite, and washed with EtOAc. The filtrate was concentrated. EtOAc was added to the residue, and the organic layer was washed with sat. aq. NaHCO₃ (2×) and brine, dried over MgSO₄, and concentrated. DCM was added to residue. The solid was filtered and washed with DCM. The filtrate was purified by flash silica gel chromatography (0–20%, MeOH/EtOAc) to give the desired product 86A (0.11 g, 11%). MS m/z (M + H⁺) 566.0.

Step 2: To a solution of methyl 2-((4-(2-chloro-4-methylphenyl)-6-methyl-5-(((1-methyl-1H-pyrazol-4-yl)methyl)carbamoyl)-1,4-dihydropyrimidin-2-yl)amino)-6-fluorobenzo[d]oxazole-7-carboxylate 86A (100 mg, 0.177 mmol) in THF (1.5 mL) was added LiOH (0.707 mL, 0.353 mmol, 0.5 M in water). The mixture was stirred at room temperature for 4 h. The solvent was evaporated under vacuum. Water was added to the residue. The mixture was extracted with EtOAc, and the water layer pH was adjusted to 2 with 1 N HCl. The solid was filtered and dried. The crude product 89 was used in the next reaction without further purification (44 mg, 37%). MS m/z (M + H⁺) 552.2.

Step 3: To a solution of 2-((4-(2-chloro-4-methylphenyl)-6-methyl-5-(((1-methyl-1H-pyrazol-4-yl)methyl)carbamoyl)-1,4-dihydropyrimidin-2-yl)amino)-6-fluorobenzo[d]oxazole-7-carboxylic acid 89 (44 mg, 0.080 mmol) in 1,4-dioxane (1 mL) were added 2-methylpropan-2-ol (0.061 mL, 0.638 mmol), TEA (0.044 mL, 0.319 mmol), and diphenyl phosphorazidate 90 (0.018 mL, 0.084 mmol) at room temperature. The mixture was stirred at 100 °C for 5 h. Upon cooling, the cloudy mixture was filtered and washed with EtOAc. The filtrate was evaporated under vacuum. To a solution of the residue (40.0 mg, 0.064 mmol) in DCM (1 mL) was added TFA (0.4 mL, 5.19 mmol). The mixture was stirred at room temperature for 2 h. The solvent was removed. The crude mixture was diluted with DMSO and purified by reverse phase chromatography (method acidic standard gradient) to afford 2-((7-amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide 68 as a TFA salt. MS m/z (M + H⁺) 523.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.01 (s, 1H), 9.74 (s, 1H), 8.15 (t, J = 5.8 Hz, 1H), 7.31–7.26 (m, 2H), 7.24 (s, 1H), 7.11 (d, J = 9.1 Hz, 2H), 6.83 (dd, J = 12.2, 8.5 Hz, 1H), 6.46 (dd, J = 8.5, 3.9 Hz, 1H), 5.90–5.84 (m, 1H), 4.03 (t, J = 5.2 Hz, 2H), 3.69 (s, 3H), 2.24 (s, 3H), 2.11 (s, 3H); NH₂ did not show in the NMR.

2-((7-Aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound 69).

This compound was prepared via method 8 using (1-ethyl-1H-pyrazol-4-yl)methanamine 72S as the starting material to afford 2-((7-aminobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-6-methyl-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-1,4-dihydropyrimidine-5-carboxamide 69 as a TFA salt. MS m/z (M + H⁺) 519.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.19–10.14 (m, 1H), 9.68 (s, 1H), 8.15 (t, J = 5.7 Hz, 1H), 7.32–7.23 (m, 3H), 7.11 (d, J = 6.3 Hz, 2H), 6.80 (t, J = 7.9 Hz, 1H), 6.51 (dd, J = 7.8, 1.1 Hz, 1H), 6.35 (dd, J = 8.0, 1.1 Hz, 1H), 5.91–5.85 (m, 1H), 5.11 (s, 2H), 4.04 (t, J = 5.3 Hz, 2H), 3.97 (q, J = 7.2 Hz, 2H), 2.24 (s, 3H), 2.12 (s, 3H), 1.25 (t, J = 7.3 Hz, 3H).

2-((7-Amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound 70).

This compound was prepared via method 9 using (1-ethyl-1H-pyrazol-4-yl)methanamine 72S as the starting material to afford 2-((7-amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide 70 as a TFA salt. MS m/z (M + H⁺) 537.2, ¹H NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 1H), 9.73 (s, 1H), 8.16 (t, J = 5.7 Hz, 1H), 7.32–7.27 (m, 2H), 7.26 (s, 1H), 7.12 (d, J = 4.0 Hz, 2H), 6.83 (dd, J = 12.2, 8.5 Hz, 1H), 6.46 (dd, J = 8.5, 3.9 Hz, 1H), 5.91–5.86 (m, 1H), 4.04 (dd, J = 5.6, 3.1 Hz, 2H), 3.97 (q, J = 7.2 Hz, 2H), 2.24 (s, 3H), 2.11 (s, 3H), 1.25 (t, J = 7.3 Hz, 3H); NH₂ did not show in the NMR.

(R)-2-((7-Amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound (R)-70).

This compound was prepared via method 9 followed by chiral separation to afford (R)-2-((7-amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (R)-70. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, Methanol-d₄) δ 7.31 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 2.9 Hz, 2H), 7.19 (s, 1H), 7.16–7.06 (m, 1H), 6.80 (dd, J = 12.0, 8.6 Hz, 1H), 6.59 (dd, J = 8.5, 4.0 Hz, 1H), 6.02–5.97 (m, 1H), 4.17 (s, 2H), 4.04 (q, J = 7.3 Hz, 2H), 2.29 (s, 3H), 2.17 (d, J = 1.3 Hz, 3H), 1.35 (t, J = 7.3 Hz, 3H); 3x(NH) and NH₂ did not show in the NMR.

(S)-2-((7-Amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (Table 4, Compound (S)-70).

This compound was prepared via method 9 followed by chiral separation to afford (S)-2-((7-amino-6-fluorobenzo[d]oxazol-2-yl)amino)-4-(2-chloro-4-methylphenyl)-N-((1-ethyl-1H-pyrazol-4-yl)methyl)-6-methyl-1,4-dihydropyrimidine-5-carboxamide (S)-70. MS m/z (M + H⁺) 537.1, ¹H NMR (400 MHz, Methanol-d₄) δ 7.30 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 2.4 Hz, 2H), 7.19 (s, 1H), 7.09 (dd, J = 8.1, 1.8 Hz, 1H), 6.80 (td, J = 9.5, 8.6, 1.9 Hz, 1H), 6.59 (dd, J = 8.6, 4.0 Hz, 1H), 6.00 (s, 1H), 4.17 (s, 2H), 4.04 (q, J = 7.3 Hz, 2H), 2.29 (s, 3H), 2.17 (s, 3H), 1.35 (t, J = 7.3 Hz, 3H); 3x(NH) and NH₂ did not show in the NMR.

ABBREVIATIONS USED

ADP

adenosine diphosphate

AMP-PNP

adenylyl imidodiphosphate

ATP

adenosine triphosphate

DMSO

dimethyl sulfoxide

DMEM

Dulbecco’s modified Eagle’s medium

EtOAc

ethyl acetate

GALT

galactose-1 phosphate uridylyltransferase

GALK1

galactokinase 1

gal-1P

galactose-1-phosphate

GHMP

galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate

HLM

human liver microsome

hGALK1

human galactokinase 1

IC₅₀

half-maximal inhibitory concentration

KOH

potassium hydroxide

mGALK

mouse galactokinase 1

MeOH

methanol

MLM

mouse liver microsome

MLPCN

Molecular Libraries Probe Production Centers Network

MS

mass spectroscopy

NCATS

National Center for Advancing Translational Sciences

NMR

nuclear magnetic resonance

PAMPA

parallel artificial membrane permeability assay

qHTS

quantitative high-throughput screen

RMSD

root-mean-square deviation

RLM

rat liver microsome

SAR

structure–activity relationship

SN_Ar

substitution nucleophilic aromatic

UDP

uridine diphosphate