Discovery of DS68702229 as a Potent, Orally Available NAMPT (Nicotinamide Phosphoribosyltransferase) Activator

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD⁺) salvage pathway. Because NAD⁺ plays a pivotal role in energy metabolism and boosting NAD⁺ has positive effects on metabolic regulation, activation of NAMPT is an attractive therapeutic approach for the treatment of various diseases, including type 2 diabetes and obesity. Herein we report the discovery of 1-(2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea 12c (DS68702229), which was identified as a potent NAMPT activator. Compound 12c activated NAMPT, increased cellular NAD⁺ levels, and exhibited an excellent pharmacokinetic profile in mice after oral administration. Oral administration of compound 12c to high-fat diet-induced obese mice decreased body weight. These observations indicate that compound 12c is a promising anti-obesity drug candidate.

Introduction

Obesity is a global health concern, with its worldwide prevalence nearly tripling since 1975.¹⁾ In addition to its well-known association with disorders such as type 2 diabetes (T2D), hepatic steatosis, and atherosclerosis, obesity has been implicated in other diseases, such as colon, prostate, and hematologic malignancies, osteoarthritis, worsening autoimmune conditions, and lung diseases.^2–4) Weight gain is generally considered to be the result of an imbalance between total energy intake and expenditure. Although weight loss can be temporarily achieved through diet restriction and/or increased physical activity, many individuals tend to regain the weight they have lost over the long term because weight loss causes compensatory changes in energy expenditure.^5–7)

Nicotinamide adenine dinucleotide (NAD⁺) is a biologically important cellular factor implicated in many metabolic processes in cells.^8–12) NAD⁺ is known to have two modes of action: (1) as an electron-carrying coenzyme for oxidoreductases; and (2) as an ADP-donating cosubstrate for many enzymes. As a coenzyme, the nicotinamide moiety of NAD⁺ accepts hydride equivalents to form reduced nicotinamide adenine dinucleotide (NADH); both NAD⁺ and NADH have vital roles in many energy-producing processes, including glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation.^8–12) As a cosubstrate, NAD⁺ donates its ADP-ribose to certain enzymes, such as sirtuins (SIRTs), poly(ADP-ribose) polymerases (PARPs), and cADP-ribose (cADPR) synthases (CD38 and CD157).^8–12) In these reactions, the linkage between nicotinamide (NAM) and the ADP-ribosyl moieties of NAD⁺ is hydrolyzed, thus necessitating continuous replenishment of NAD⁺.¹⁰⁾ In mammals, the dominant NAD⁺ synthetic pathway is the salvage pathway, which converts NAM back to NAD⁺. The first step in this pathway, the conversion of NAM to NAM mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting step of the salvage pathway.^8–12)

Given its role as an intrinsic regulator of cell bioenergetics, increasing NAD⁺ levels has been shown to have several beneficial physiological effects similar to those achieved by diet or exercise.^8,9) In particular, NAMPT is considered an important enzyme in the regulation of NAD⁺ levels, with several preclinical studies suggesting that decreases in NAMPT are associated with metabolic disorders.^9,13,14) In one study, NAMPT overexpression in the skeletal muscle of C57BL6/J mice elicited increases in skeletal muscle NMN and NAD⁺ levels, which led to higher exercise endurance capacity compared with wild-type mice, along with an improvement in metabolic abnormalities.¹⁵⁾ Extrapolating from these results, pharmacological activation of NAMPT would be expected to modulate metabolism by increasing NAD⁺ levels. Therefore, NAMPT activators may be a promising approach for the treatment of metabolic disorders, including obesity and T2D.

We have previously described the acquisition of a series of urea-containing NAMPT activators, such as 2 derived from the high-throughput screening (HTS) hit 1. These compounds possess a (pyridin-4-yl-methyl)urea moiety and a triazolopyridine core structure¹⁶⁾ (Fig. 1). Compound 2 showed potent NAMPT activating activity in an NAMPT enzyme assay¹⁷⁾ and increased intracellular NAD⁺ levels in cell-based assays.¹⁸⁾ Compound 2 also exhibited attenuated direct inhibition (DI) of CYP, which is an issue that has also been observed with other triazolopyridine derivatives.¹⁶⁾ Although its low logD was beneficial for attenuation of CYP DI, compound 2 was not suitable for oral administration due to low membrane permeability. We therefore focused our structure–activity relationship (SAR) studies on screening bicyclic ring structures to find an alternative core structure. Herein we report further optimization of the series to develop an orally available potent NAMPT activator, DS68702229, in which benzoxazole with a (pyridin-4-ylmethyl)urea moiety was embedded.

Fig. 1. NAMPT Activators

Chemistry

The retrosynthesis of benzoxazole derivatives possessing the (pyridin-4-ylmethyl)urea moiety is shown in Fig. 2. The target molecules were synthesized from the corresponding 5-aminobenzoxazoles via several urea-forming methods. The intermediate 5-aminobenzoxazole derivatives were provided by using either one of two methods, namely palladium-catalyzed direct 2-arylation of the benzoxazoles (Method A) or condensation of 2-aminophenol derivatives with benzoyl chloride followed by cyclization under acidic conditions (Method B).

Fig. 2. Retrosynthesis of Benzoxazole Compounds Possessing a (Pyridin-4-ylmethyl)urea Moiety

Following the retrosynthesis, the compounds in Table 1 were prepared (Charts 1, 2). Chart 1 shows the synthesis of benzoxazole intermediates (benzoxazole intermediates 4a–c were commercially available). 2-Arylated compounds 4d and 4e were prepared from compound 3¹⁹⁾ by palladium-catalyzed direct 2-arylation via a deprotonative cross-coupling process (Method A).²⁰⁾ As for benzoxazole intermediates with substituents (F, Me, Cl) at 5-position (4f–h), the 6-position of 5f–h²¹⁾ was nitrated to give 6f–h, followed by reduction of the NO₂ group to an NH₂ group by hydrogenation to afford 4f–h. Synthesis of oxazolo[4,5-b]pyridine intermediate 4i was achieved using Method B; commercially available 7 was condensed with benzoic acid in polyphosphoric acid at 160 °C to give 6i, followed by NO₂ reduction to afford 4i. Regarding the benzoxazole intermediate with a nitrile group at 4-position (4k), commercially available 8 was brominated to give 9ka, followed by the construction of a benzoxazole structure (6ka) via Method B, a three-step procedure involving condensation of 9ka with benzoyl chloride, demethylation of the OMe group, and subsequent cyclization under acidic conditions. Subsequently, the nitrile group was introduced to 6ka via Pd-catalyzed coupling to give 6k, the NO₂ group of which was reduced using iron powder to give the intermediate 4k. As for 4j and 4l, which possess a methyl group or an amide group at the 4-position of the benzoxazole ring, the corresponding o-methoxyanilines (9j, 9la²²⁾) underwent a three-step procedure analogous to 9ka to provide benzoxazoles 6j and 6la. The methyl ester of 6la was converted to N,N-dimethylamide 6l via hydrolysis and amidation. Subsequently, the 6-bromine of 6j and 6l was converted to an NHBoc group by Pd-catalyzed amination to give 4j and 4l.²³⁾

Table 1. Exploration of Bicyclic Cores: EC₅₀ (µM)/E_max (%) Values

a) Percentages indicate the magnitude of the increase in NAD⁺ achieved with 1 µM of the compound relative to that achieved with 30 µM compound 1. b) Not determined. The NAMPT enzyme assay could not be performed because of the fluorescence of the compound. c) Not tested.

Chart 1. Reagents and Conditions

(a) Aryl bromide, NiXantphos, Pd(OAc)₂, Na(O-tBu), 1,2-dimethoxyethane, 17–30%. (b) Sulfuric acid, nitric acid, 0 °C, 49–76%. (c) [6f, g, i] H₂, Pd/C, EtOH, 67–71%. (d) [6h] H₂, PtO₂, EtOH, ethyl acetate, 48%. (e) [6k] Iron powder, NH₄Cl, EtOH, water, 80 °C, quant. (f) Benzoic acid, polyphosphoric acid, 160 °C. (g) N-Bromosuccinimide, dimethylformamide (DMF), 91%. (h) Benzoyl chloride, pyridine, tetrahydrofuran (THF), 60 °C. (i) BBr₃, CH₂Cl₂, from −78 °C to room temperature (r.t.). (j) Pyridinium p-toluenesulfonate, toluene, 120 °C, 32–69% over three steps. (k) Zinc cyanide, Pd(PPh₃)₄, DMF, 100 °C, 50%. (l) t-Butyl carbamate, t-butyl XPhos, Pd₂dba₃, Na(O-tBu), toluene, 76–94%. (m) 1 M NaOH (aq), THF, MeOH, r.t. (n) Dimethylamine (2.0 M solution in THF), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, N,N-diisopropylethylamine, DMF, 95% over two steps.

Chart 2. Reagents and Conditions

(a) 4 M HCl in 1,4-dioxane, CH₂Cl₂. (b) 4-Nitrophenyl [(pyridin-4-yl)methyl]carbamate, DIPEA, 1,4-dioxane, 50–80 °C, [12a, b, c, i, k] 25–65%, [12e, j, l] 19–85% over two steps. (c) Triphosgene, DIPEA, CH₂Cl₂, then 13, [12cb, cc, cd, f, g, h] 10–74%, [12d] 85% over two steps. (d) 4-Nitrophenyl chloroformate, toluene, 80 °C. (e) 1H-Pyrazol-4-ylmethylamine dihydrochloride, DIPEA, MeOH, 85% over two steps.

Chart 2 delineates the urea formation reactions of the benzoxazole intermediates to afford the final compounds 12a–l. Three methods were used for urea formation. The first was the carbamate method (Chart 2(a)). tert-Butoxycarbonyl (Boc)-protected 4e, 4j, and 4l were deprotected in acidic conditions and reacted with 4-nitrophenyl [(pyridin-4-yl)methyl]carbamate²⁴⁾ to provide 12e, 12j, and 12l. Similarly, compounds 4a–c,²⁵⁾4i, and 4k were converted to urea 12a–c, 12i, and 12k.

The second method was the triphosgene method (Chart 2(b)). Boc-protected 4d was deprotected and the aniline intermediate obtained was treated with triphosgene and coupled with the corresponding amine to provide 12d. Similarly, aniline derivatives 4c and 4f–h were converted to the corresponding urea derivatives 12cb, 12cc, 12cd, 12f, 12g, and 12h.

In the third method, the pyrazole derivative 12ca was synthesized by reacting the aniline 4c with commercially available 4-nitrophenyl chloroformate to afford the carbamate intermediate 14, which was coupled with 1H-pyrazol-4-ylmethylamine to give the final product 12ca (Chart 2(c)).

Results and Discussion

We initially focused our SAR studies on screening bicyclic ring structures (region B) in a search for an alternative core structure (Table 1). Our initial attempt started with the 2-phenyl-1H-benzoimidazole analog 12a, which, unfortunately, resulted in decrease of activity in the NAMPT enzyme assay compared to our previous lead compound 2. Conversion of the central ring to a benzoxazole (12b) led to recovery of activity in both NAMPT enzyme and cell-based assays. Notably, benzoxazole compound 12c, in which the positions of the oxygen and nitrogen atoms of the benzoxazole ring were exchanged compared with 12b, demonstrated higher NAMPT activation activity compared to 2. Compounds 12b and 12c were further profiled to assess their physicochemical and in vitro ADME (absorption, distribution, metabolism, and excretion) properties (Table 2). These compounds exhibited high membrane permeability compared to the prior triazolopyridine lead compound 2, which would be expected to be beneficial towards oral absorption. Of note, compound 12c showed a lower tendency of CYP DI compared to 12b, which was an issue widely observed in our previous triazolopyridine series.¹⁶⁾ Accordingly, we decided to fix the central region B to the benzoxazole ring of 12c for further derivatization.

Table 2. Physicochemical and In Vitro ADME Properties of Selected Benzoxazole Analogs

Cpd	logDa)	PAMPA permeability at pH 5.0/7.4 (nm/s)	Solubility at pH 1.2/6.8 (µg/mL)	Metabolic stabilityb) in human/mouse (%)	% CYP DIc) (1A2/2C9/2D6/3A4)
2	1.8	0.4/0.8	>680/18	97/83	15/42/16/25
12b	3.6	>50/>50	>680/0.6	85/83	63/39/16/6
12c	3.7	49.8/>50	640/0.1	83/86	28/21/12/22
12e	2.9	29.7/42.8	670/31	85/70	16/33/16/35
12ca	3.4	32.3/35.5	5.5/0.2	87/11	76/29/0/0
12i	3.1	28.8/>50	690/3.1	84/86	19/43/14/26
12j	4.1	>50/>50	670/0.2	NTd)/74	47/33/14/36
12k	3.7	>50/>50	74/0	58/64	46/29/12/14

a) The distribution coefficients (logD) were measured between 1-octanol and phosphate-buffered saline (pH 7.4). b) The percentage remaining of a 1-µM concentration of the compound after a 30-min reaction with human or mouse liver microsomes. c) The percentage inhibition by a 10-µM concentration of the compounds after a 10-min reaction with corresponding CYP isoforms. d) Not tested.

We decided to explore region A, B, and C within the benzoxazole template with an aim to consider whether further improvement of NAMPT activation activity, while concomitantly possessing a favorable ADME profile for oral absorption, was feasible within this template.

First, SAR exploration of the benzoxazole template was directed towards modification of the right-hand region C (Table 1). Installing substituents on the phenyl ring did not have a significant effect on NAMPT activation activity (data not shown), consistent with the results observed with our previous triazolopyridine series.¹⁶⁾ Subsequently, we screened several heteroaryl groups. Although installing a pyridine ring (12d) did not lead to significant improvements, installing an N-methylpyrazole structure (12e) resulted in comparable increases in intracellular NAD⁺ to those seen in our previously described triazolopyridine series.¹⁶⁾ Furthermore, various aliphatic groups were explored within this region, but generally resulted in impaired activity and suffered from instability in acidic medium.²⁶⁾

We then focused on the left-hand region A, the exposed pyridine ring. An intensive exploration around this left-hand region has been conducted previously; among the various heterocycles, 4-pyrazole and 5-oxazole were the only heterocycles that activated NAMPT, with the others exhibiting inhibitory or no activity.^27,28) 4-Pyrazole and 5-oxazole also activated NAMPT in our previous triazolopyridine series.¹⁶⁾ Consistent with the previous results, 4-pyrazole and 5-oxazole (12ca and 12cb) were also tolerated with the benzoxazole template, exhibiting equipotent NAMPT activation to the 4-pyridyl group. Interestingly, the benzoxazole template tolerated a slightly wider variation of heterocycles at this region, with 5-thiazole and 4-imidazole derivatives (12cc and 12cd) also exhibiting NAMPT activation.

Finally, we shifted our efforts to investigation of substituents on the central benzoxazole core in region B. Substitutions on the 5-position demonstrated that F substitution (12f) was tolerated in terms of NAMPT activation, but slightly larger substituents, such as methyl (12g) or Cl (12h), resulted in impaired activity, which suggested steric limitations around this position. In contrast, the 4-position seemed to be relatively tolerant of modification. For example, replacing the carbon atom with nitrogen was tolerated, with oxazolo[4,5-b]pyridine analog 12i being the most potent of the three possible oxazolopyridine isomers,²⁹⁾ exhibiting comparable cellular activity. Substitution was also tolerated; methyl (12j)- and nitrile (12k)-substituted analogs were equipotent to the non-substituted lead compound (12c), whereas a larger dimethylamide group (12l) had an adverse effect on potency.

The benzoxazole analogs with optimal activity in the cell-based assay were examined for their physicochemical and in vitro ADME properties³⁰⁾ (Table 2). Many of the benzoxazole compounds (12e, 12i, 12j, and 12k) retained the low tendency for CYP DI observed with 12c. Moreover, the benzoxazole compounds widely showed a higher membrane permeability compared to the prior triazolopyridine lead compound 2. Being aware of the low solubility at neutral pH observed with 12c, we investigated to see if the improvement of solubility was possible within this template. Incorporation of a pyrazole group in the right-hand side region C (12e) led to decrease in logD, which improved solubility at neutral pH and resulted in moderate membrane permeability. However, incorporation of a pyrazole ring in the left-hand side region A (12ca) was not beneficial, resulting in low solubility at both acidic and neutral pH, along with lower murine metabolic stability. Substitution on the central benzoxazole core structure (12i and 12j) showed a similar tendency of ADME properties as 12c being poorly soluble at neutral pH, whereas the nitrile analog (12k) additionally showed a relatively low solubility at acidic pH.

Several selected analogs were also examined for their in vivo pharmacokinetic properties in mice (Table 3). To consider the influence of low solubility at neutral pH on the absorption, oral suspensions of evaluation compounds were prepared using 0.5% methylcellulose without addition of any solubilizing agents and administered to mice. As a result, benzoxazole lead compound 12c exhibited the highest exposure as seen in the area under curve [AUC]. Compound 12e had a shorter half-life (t_½) than 12c, thus resulting in lower AUC than 12c. Moreover, compounds 12i, j had a lower maximum drug concentration (C_max) in addition to shorter t_½, resulting in a lower AUC than compound 12c. Obtained results suggested that low solubility of 12c at neutral pH (0.1 µg/mL in pH 7.4) was unlikely the critical issue of oral absorption, and 12c is highly likely well dissolved under the acidic condition (640 µg/mL in pH 1.2) in the stomach and subsequently absorbed in the intestine.

Table 3. Pharmacokinetic Parameters of Selected Compounds after Oral Administration (10 mg/kg) to C57BL/6N Mice^a)

a) Compounds were administered as a suspension in 0.5% methylcellulose.

Encouraged by the satisfactory overall profile of compound 12c, we evaluated the ability of 12c to boost NAD⁺ levels in mice (Fig. 3). Administration of a single oral dose (30 or 100 mg/kg) of compound 12c to diet-induced obese (DIO) mice elicited a significant increase in liver, gastrocnemius, and soleus tissue NAD⁺ levels compared with the vehicle control group.

Fig. 3. Effects of Compound 12c on Tissue NAD⁺ Levels in DIO Mice

Compound 12c, prepared in 0.5% methylcellulose, was orally administered to DIO mice at a dose of 30 or 100 mg/kg (Compound 12c_30 and Compound 12c_100, respectively). The NAD⁺ concentration (nmol/g) in different tissues was measured 24 h after administration. Gastro., gastrocnemius. Data are the mean ± S.E.M. (n = 4). * p < 0.05, ** p < 0.01 compared with the vehicle control group (Dunnett’s test).

Next, we examined the effect of compound 12c on body weight in order to assess its therapeutic potential as a treatment for obesity (Fig. 4). Oral administration of 30 mg/kg compound 12c once a day for 21 d resulted in a continuous and significant decrease in body weight compared with the vehicle control group. The compound was well tolerated, with no overt toxicity during the study. Because there was no difference in cumulative food intake between the two groups (data not shown), we hypothesize that the increases in NAD⁺ levels elicited by compound 12c led to enhanced energy metabolism, culminating in a reduction in body weight.

Fig. 4. Anti-obesity Effect of Compound 12c in DIO Mice

Compound 12c, prepared in 0.5% methylcellulose, was orally administered to 46-week-old male C57BL/6N DIO mice at a dose of 30 mg/kg once a day for 21 d. Data are the mean ± S.E.M. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the vehicle control group (Student’s t-test).

Conclusion

In summary, optimization of our prior lead compound 2 led to the discovery of a series of benzoxazole derivatives as potent NAMPT activators, culminating in the identification of compound 12c (DS68702229) as a promising lead compound. Compound 12c activated NAMPT in vitro and had an excellent pharmacokinetic profile in mice. This corresponded to a significant increase in tissue NAD⁺ levels in mice after the oral administration of compound 12c. Chronic oral dosing with compound 12c led to a robust decrease in body weight in DIO mice, demonstrating its use as a tool to explore the in vivo effects of pharmacological NAMPT activation. These results indicate that the use of NAMPT activators could be a promising approach for anti-obesity drugs.

Experimental

General

Unless noted otherwise, materials were obtained from commercial suppliers and used without further purification. ¹H-NMR spectra were recorded on a Unity Mercury Plus 400 or 500 spectrometer (Varian) and chemical shifts are given in ppm from tetramethylsilane (TMS) as an internal standard. Low-resolution mass spectroscopy was conducted on an Agilent Infinity 1260 LC/MS system. TLC was performed on Merck precoated TLC glass sheets with silica gel 60F254. Compounds were separated by column chromatography using Chromatorex Q-pack silica gel (Fuji Silysia Chemical, 30 µm). High-resolution (HR) MS was performed using an LC/MS system comprising a Waters Xevo Q-ToF MS and an Acquity UHPLC system. Elemental analyses were performed on a Microcorder JM10 and a Dionex ICS-1500 system. The purity of compounds was confirmed as >95% based on a diode array detector (DAD) signal area obtained using an Agilent Infinity 1260 LC/MS system. Conditions were; column, Develosil Combi-RP-5 2.0 mm ID ×50 mm L; gradient elution, 0.1% HCO₂H–H₂O/0.1% HCO₂H–MeCN = 98/2 to 0/100 (v/v); flow rate, 1.2 mL/min; UV detection, 254 nm; column temperature, 40 °C; ionization: atmospheric pressure chemical ionization (APCI)/electrospray ionization (ESI).

General Procedure for Urea Formation (12a, b, c, e, i, j, k, l) (general Procedure A; Carbamate Method)

Aniline (0.30 mmol) and (4-nitrophenyl)-N-(4-pyridylmethyl)carbamate (92 mg, 0.33 mmol) were suspended in 1,4-dioxane (2 mL), and then N,N-diisopropylethylamine (0.058 mL, 0.33 mmol) was added to the suspension. The mixture was stirred at 50 °C for 2 h. After cooling to r.t., the precipitated solid was collected by filtration and washed with diethyl ether to give the title compound (12a, b, c, e, i, j, k, l) as a solid.

1-(2-Phenyl-1H-benzimidazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12a)

Prepared according to general procedure A (yield: 25%). ¹H-NMR (dimethyl sulfoxide (DMSO)-d₆) δ: 4.36 (d, J = 6.2 Hz, 2H), 6.70 (t, J = 6.2 Hz, 1H), 7.05 (br s, 1H), 7.32 (d, J = 5.9 Hz, 2H), 7.43–7.57 (m, 4H), 7.88 (br s, 1H), 8.12 (d, J = 7.0 Hz, 2H), 8.52 (d, J = 5.9 Hz, 2H), 8.74 (br s, 1H), 12.69 (br s, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.8, 114.3, 122.0, 126.1, 128.9, 129.4, 130.3, 135.6, 149.4, 149.7, 150.6, 155.6; Anal. Calcd for C₂₀H₁₇N₅O: C, 63.37; H, 5.57; N, 18.48. Found C, 63.15; H, 5.44; N, 18.46. HRMS (ESI): Calcd for C₂₀H₁₇N₅O [M + H]⁺: 344.1511. Found 344.1497.

1-(2-Phenyl-1,3-benzoxazol-5-yl)-3-(pyridin-4-ylmethyl)urea (12b)

Prepared according to general procedure A (yield: 65%). ¹H-NMR (DMSO-d₆) δ: 4.36 (d, J = 6.3 Hz, 2H), 6.80 (t, J = 5.9 Hz, 1H), 7.30–7.37 (m, 3H), 7.57–7.69 (m, 4H), 7.96 (d, J = 2.0 Hz, 1H), 8.14–8.23 (m, 2H), 8.50–8.55 (m, 2H), 8.92 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.9, 108.4, 110.5, 116.6, 121.9, 126.5, 127.1, 129.2, 131.8, 137.7, 141.8, 145.3, 149.5, 149.5, 155.5, 162.6; Anal. Calcd for C₂₀H₁₆N₄O₂: C, 69.75; H, 4.68; N, 16.27. Found C, 69.51; H, 4.85; N, 16.25. HRMS (ESI): Calcd for C₂₀H₁₆N₄O₂ [M + H]⁺: 345.1352. Found 345.1355.

1-(2-Phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12c)

Prepared according to general procedure A (yield: 31%). ¹H-NMR (DMSO-d₆) δ: 4.37 (d, J = 6.0 Hz, 2H), 6.85 (t, J = 6.0 Hz, 1H), 7.23 (dd, J = 8.5, 2.0 Hz, 1H), 7.32 (d, J = 5.9 Hz, 2H), 7.58–7.62 (m, 3H), 7.66 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 1.7 Hz, 1H), 8.13–8.20 (m, 2H), 8.48–8.55 (m, 2H), 9.05–9.12 (m, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.8, 99.8, 115.6, 119.4, 121.9, 126.7, 126.8, 129.2, 131.3, 135.6, 138.6, 149.4, 150.7, 155.3, 161.2; Anal. Calcd for C₂₀H₁₆N₄O₂·0.27H₂O: C, 68.78; H, 4.77; N,16.04. Found C, 68.71; H, 4.95; N, 15.95. HRMS (ESI): Calcd for C₂₀H₁₆N₄O₂ [M + H]⁺: 345.1351. Found 345.1356.

1-(2-Phenyl[1,3]oxazolo[4,5-b]pyridin-6-yl)-3-(pyridin-4-ylmethyl)urea (12i)

Prepared according to general procedure A (yield: 65%).¹H-NMR (DMSO-d₆) δ: 4.38 (d, J = 6.0 Hz, 2H), 7.02 (t, J = 6.0 Hz, 1H), 7.32 (d, J = 5.9 Hz, 2H), 7.59–7.69 (m, 3H), 8.21 (dd, J = 7.8, 1.8 Hz, 2H), 8.41 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 2.2 Hz, 1H), 8.49–8.56 (m, 2H), 9.32 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.9, 107.7, 121.9, 126.1, 127.2, 129.3, 132.1, 135.3, 137.5, 142.8, 149.2, 149.4, 149.5, 155.3, 163.4; Anal. Calcd for C₁₉H₁₅N₅O₂·0.2H₂O: C, 65.40; H, 4.45; N, 20.07. Found C, 65.33; H, 4.42; N, 19.99. HRMS (ESI): Calcd for C₁₉H₁₅N₅O₂ [M + H]⁺: 346.1304. Found 346.1318.

1-(4-Cyano-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12k)

Prepared according to general procedure A (yield: 50%). ¹H-NMR (DMSO-d₆) δ: 4.37 (d, J = 6.1 Hz, 2H), 7.08 (t, J = 6.1 Hz, 1H), 7.28–7.35 (m, 2H), 7.59–7.69 (m, 3H), 7.83 (d, J = 2.0 Hz, 1H), 8.18–8.25 (m, 2H), 8.29 (d, J = 1.8 Hz, 1H), 8.49–8.54 (m, 2H), 9.36 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.9, 101.3, 105.0, 115.8, 119.0, 122.0, 125.6, 127.5, 129.4, 137.2, 132.4, 138.9, 149.2, 149.5, 150.9, 155.1, 163.5; Anal. Calcd for C₂₁H₁₅N₅O₂·0.247H₂O: C, 67.47; H, 4.18; N, 18.37. Found C, 67.74; H, 4.34; N, 18.47. HRMS (ESI): Calcd for C₂₁H₁₅N₅O₂ [M + H]⁺: 370.1304. Found 370.1311.

1-[2-(1-Methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-yl]-3-(pyridin-4-ylmethyl)urea (12e)

tert-Butyl [2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-yl]carbamate (4e) (45 mg, 0.14 mmol) was dissolved in dichloromethane (4 mL), and a 4-M solution of hydrochloric acid in 1,4-dioxane (1.0 mL, 4.0 mmol) was added. The mixture was stirred at r.t. overnight. A small amount of hexane was added to the solution, and the precipitated solid was collected by filtration to give crude 2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-amine hydrochloride (41 mg), which was used in the next reaction without further purification.

1-[2-(1-Methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-yl]-3-(pyridin-4-ylmethyl)urea (12e) was prepared according to general procedure A from crude 2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-amine hydrochloride (41 mg) obtained above (yield: 68% over two steps). ¹H-NMR (DMSO-d₆) δ: 4.28 (s, 3H), 4.37 (d, J = 6.2 Hz, 2H), 6.96 (t, J = 6.2 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 8.5, 2.0 Hz, 1H), 7.32 (d, J = 5.9 Hz, 2H), 7.64 (d, J = 2.2 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 1.7 Hz, 1H), 8.51 (d, J = 5.9 Hz, 2H), 9.27 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 8.34, 41.7, 45.2, 99.2, 108.6, 119.6, 122.0, 129.6, 134.8, 138.4, 139.3, 149.2, 149.8, 149.9, 153.6, 155.5; HRMS (ESI): Calcd for C₁₈H₁₆N₆O₂ [M + H]⁺: 349.1412. Found 349.1404.

1-(4-Methyl-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12j)

Crude 4-methyl-2-phenyl-1,3-benzoxazol-6-amine hydrochloride was obtained from tert-butyl (4-methyl-2-phenyl-1,3-benzoxazol-6-yl)carbamate (4j) following the same procedure as for the synthesis of crude 2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-amine hydrochloride described in the synthesis of 12e.

1-(4-Methyl-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12j) was prepared according to general procedure A from crude 4-methyl-2-phenyl-1,3-benzoxazol-6-amine hydrochloride obtained above (yield: 84% over two steps). ¹H-NMR (DMSO-d₆) δ: 2.53 (s, 3H), 4.36 (d, J = 5.9 Hz, 2H), 6.83 (t, J = 5.9 Hz, 1H), 7.07 (s, 1H), 7.31 (d, J = 5.9 Hz, 2H), 7.55–7.63 (m, 3H), 7.87 (s, 1H), 8.12–8.19 (m, 2H), 8.52 (d, J = 5.6 Hz, 2H), 8.97 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 16.3, 41.8, 97.2, 115.8, 121.9, 126.7, 126.7, 129.1, 129.4, 131.2, 135.1, 138.2, 149.4, 149.4, 150.4, 155.2, 160.5; Anal. Calcd for C₂₁H₁₈N₄O₂: C, 70.38; H, 5.06; N, 15.63. Found C, 70.19; H, 5.09; N, 15.51. HRMS (ESI): Calcd for C₂₁H₁₈N₄O₂ [M + H]⁺: 359.1508. Found 359.1521.

N,N-Dimethyl-2-phenyl-6-{[(pyridin-4-ylmethyl)carbamoyl]amino}-1,3-benzoxazole-4-carboxamide (12l)

Crude 6-amino-N,N-dimethyl-2-phenyl-1,3-benzoxazole-4-carboxamide hydrochloride was obtained from tert-butyl[4-(dimethylcarbamoyl)-2-phenyl-1,3-benzoxazol-6-yl]carbamate (4l) following the same procedure as for the synthesis of crude 2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-amine hydrochloride described in the synthesis of 12e.

N,N-Dimethyl-2-phenyl-6-{[(pyridin-4-ylmethyl)carbamoyl]amino}-1,3-benzoxazole-4-carboxamide (12l) was prepared according to general procedure A from crude 6-amino-N,N-dimethyl-2-phenyl-1,3-benzoxazole-4-carboxamide hydrochloride obtained above (yield: 19% over two steps). ¹H-NMR (DMSO-d₆) δ: 2.89 (s, 3H), 3.09 (s, 3H), 4.37 (d, J = 6.1 Hz, 2H), 6.92 (t, J = 6.1 Hz, 1H), 7.27–7.37 (m, 3H), 7.55–7.67 (m, 3H), 8.06 (d, J = 2.0 Hz, 1H), 8.17 (br dd, J = 7.9, 1.6 Hz, 2H), 8.52 (d, J = 6.1 Hz, 2H), 9.16 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 34.6, 38.8, 41.9, 100.5, 113.9, 122.0, 126.3, 127.1, 128.2, 129.2, 131.7, 132.3, 138.6, 149.4, 149.5, 150.5, 155.2, 161.8, 166.7; Anal. Calcd for C₂₃H₂₁N₅O₃·1.131H₂O: C, 63.39; H, 5.38; N, 16.07. Found C, 64.59; H, 5.47; N, 15.94. HRMS (ESI): Calcd for C₂₃H₂₁N₅O₃ [M + H]⁺: 416.1723. Found 416.1722.

General Procedure for Urea Formation (12d, cb, cc, cd, f, g, h) (General Procedure B; Triphosgene Method)

Aniline (0.30 mmol) and triethylamine (0.13 mL, 0.90 mmol) were dissolved in dichloromethane (3 mL) and cooled to 0 °C. Triphosgene (31 mg, 0.11 mmol) was then added to the solution. The mixture was stirred for 10 min. Arylmethylamine (0.33 mmol) was then added, and the mixture was stirred for 1 h. The reaction solution was poured into water (30 mL), followed by partition using ethyl acetate. The organic phase was washed with brine, then dried over magnesium sulfate and filtered. Then, the solvent was distilled off under reduced pressure to give the crude title compound. The crude product was purified by silica gel column chromatography to give the title compound (12cb, cc, cd, d, f, g, h) as a solid.

1-(1,3-Oxazol-5-ylmethyl)-3-(2-phenyl-1,3-benzoxazol-6-yl)urea (12cb)

Compound 12cb was prepared according to general procedure B (yield: 66%). ¹H-NMR (CD₃OD) δ: 4.50 (s, 2H), 7.06 (s, 1H), 7.18 (dd, J = 8.6, 2.0 Hz, 1H), 7.53–7.61 (m, 4H), 8.03 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H), 8.17–8.22 (m, 2H); ¹³C-NMR (DMSO-d₆) δ: 33.9, 99.8, 115.6, 119.5, 122.9, 126.6, 126.8, 129.2, 131.4, 135.7, 138.5, 150.3, 150.7, 151.5, 154.9, 161.3; Anal. Calcd for C₁₈H₁₄N₄O₃·0.117H₂O: C, 64.26; H, 4.26; N, 16.65. Found C, 64.15; H, 4.36; N, 16.69. HRMS (ESI): Calcd for C₁₈H₁₄N₄O₃ [M + H]⁺: 335.1144. Found 335.1147.

1-(2-Phenyl-1,3-benzoxazol-6-yl)-3-(1,3-thiazol-5-ylmethyl)urea (12cc)

Compound 12cc was prepared according to general procedure B (yield: 10%). ¹H-NMR (DMSO-d₆) δ: 4.55 (d, J = 5.9 Hz, 2H), 6.85 (t, J = 5.9 Hz, 1H), 7.22 (dd, J = 8.8, 2.0 Hz, 1H), 7.57–7.63 (m, 3H), 7.66 (d, J = 8.6 Hz, 1H), 7.80 (s, 1H), 8.09 (d, J = 2.0 Hz, 1H), 8.13–8.21 (m, 2H), 8.95–9.02 (m, 2H); Anal. Calcd for C₁₈H₁₄N₄O₂S·0.416H₂O: C, 60.41; H, 4.18; N, 15.65. Found C, 60.12; H, 3.90; N, 15.37. HRMS (ESI): Calcd for C₁₈H₁₄N₄O₂S [M + H]⁺: 351.0916. Found 351.0909.

1-(1H-Imidazol-4-ylmethyl)-3-(2-phenyl-1,3-benzoxazol-6-yl)urea (12cd)

Compound 12cd was prepared according to general procedure B (yield: 32%). ¹H-NMR (CD₃OD) δ: 4.37 (s, 2H), 6.99–7.04 (m, 1H), 7.17 (dd, J = 8.7, 1.9 Hz, 1H), 7.53–7.60 (m, 4H), 7.64 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H), 8.15–8.24 (m, 2H); ¹³C-NMR (DMSO-d₆) δ: 37.3, 99.4, 112.8, 115.3, 119.5, 126.7, 126.8, 129.2, 131.4, 135.0, 135.4, 138.3, 138.9, 150.8, 155.0, 161.1; Anal. Calcd for C₁₈H₁₅N₅O₂·0.676H₂O: C, 62.57; H, 4.77; N, 20.27. Found C, 62.45; H, 4.56; N, 20.42. HRMS (ESI): Calcd for C₁₈H₁₅N₅O₂ [M + H]⁺: 334.1304. Found 334.1298.

1-(5-Fluoro-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12f)

Compound 12f was prepared according to general procedure B (yield: 74%). ¹H-NMR (DMSO-d₆) δ: 4.40 (d, J = 5.9 Hz, 2H), 7.34–7.39 (m, 3H), 7.58–7.64 (m, 3H), 7.75 (d, J = 11.0 Hz, 1H), 8.14–8.17 (m, 2H), 8.50–8.54 (m, 3H), 8.79 (d, J = 2.9 Hz, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.8, 101.3, 105.7, 121.9, 126.4, 126.7, 126.9, 129.2, 131.7, 134.7, 134.8, 146.7, 149.0, 149.6, 154.5, 162.6; Anal. Calcd for C₂₀H₁₅N₄O₂F·0.806H₂O: C, 63.74; H, 4.44; N, 14.87; F, 5.04. Found C, 63.81; H, 4.53; N, 14.87; F, 4.98. HRMS (ESI): Calcd for C₂₀H₁₅N₄O₂F [M + H]⁺: 363.1257. Found 363.1259.

1-(5-Methyl-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12g)

Compound 12g was prepared according to general procedure B from 5-methyl-2-phenyl-1,3-benzoxazol-6-amine³¹⁾ (4g) (yield: 52%). ¹H-NMR (DMSO-d₆) δ: 2.35 (s, 3H), 4.39 (d, J = 5.9 Hz, 2H), 7.31–7.36 (m, 3H), 7.56–7.64 (m, 4H), 8.07 (s, 1H), 8.11–8.19 (m, 2H), 8.29 (s, 1H), 8.53 (d, J = 5.9 Hz, 2H); ¹³C-NMR (DMSO-d₆) δ: 18.4, 41.9, 102.0, 120.1, 122.1, 124.5, 126.7, 126.8, 129.2, 131.4, 136.1, 136.2, 149.0, 149.3, 149.6, 155.5, 161.4; Anal. Calcd for C₂₁H₁₈N₄O₂·0.429H₂O: C, 68.89; H, 5.19; N, 15.30. Found C, 68.79; H, 5.20; N, 15.36. HRMS (ESI): Calcd for C₂₁H₁₈N₄O₂ [M + H]⁺: 359.1508. Found 359.1487.

1-(5-Chloro-2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea (12h)

Compound 12h was prepared according to general procedure B (yield: 26%). ¹H-NMR (DMSO-d₆) δ: 4.40 (d, J = 6.1 Hz, 2H), 7.33–7.35 (m, 2H), 7.59–7.65 (m, 3H), 7.71 (t, J = 6.1 Hz, 1H), 7.95 (s, 1H), 8.15–8.19 (m, 2H), 8.43 (s, 1H), 8.51–8.57 (m, 3H); ¹³C-NMR (DMSO-d₆) δ: 41.8, 102.5, 118.8, 119.6, 122.0, 126.2, 127.1, 129.3, 131.9, 134.2, 136.3, 148.9, 149.3, 149.6, 155.0, 162.7; HRMS (ESI): Calcd for C₂₀H₁₅N₄O₂Cl [M + H]⁺: 379.0962. Found 379.0956.

1-[2-(Pyridin-3-yl)-1,3-benzoxazol-6-yl]-3-(pyridin-4-ylmethyl)urea (12d)

Crude 2-(pyridin-3-yl)-1,3-benzoxazol-6-amine hydrochloride was obtained from tert-butyl [2-(pyridin-3-yl)-1,3-benzoxazol-6-yl]carbamate (4d) following the same procedure as for the synthesis of crude 2-(1-methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-amine hydrochloride described in the synthesis of 12e.

The title compound was prepared according to general procedure B from crude 2-(pyridin-3-yl)-1,3-benzoxazol-6-amine hydrochloride obtained above (yield: 85% over two steps). ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.37 (d, J = 6.0 Hz, 2H), 6.87 (t, J = 6.0 Hz, 1H), 7.26 (dd, J = 8.6, 2.0 Hz, 1H), 7.29–7.33 (m, 2H), 7.60–7.66 (m, 1H), 7.70 (d, J = 8.6 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.49 (dt, J = 7.8, 2.2 Hz, 1H), 8.51–8.54 (m, 2H), 8.77 (dd, J = 4.7, 1.6 Hz, 1H), 9.13 (s, 1H), 9.31 (dd, J = 2.3, 0.8 Hz, 1H); ¹³C-NMR (DMSO-d₆) δ: 41.8, 99.7, 115.8, 119.7, 121.9, 123.0, 124.2, 134.2, 135.4, 139.0, 147.6, 149.4, 149.5, 150.8, 151.8, 155.3, 159.2; Anal. Calcd for C₁₉H₁₅N₅O₂·0.741H₂O: C, 63.62; H, 4.63; N, 19.52. Found C, 63.38; H, 4.36; N, 19.36. HRMS (ESI): Calcd for C₁₉H₁₅N₅O₂ [M + H]⁺: 356.1304. Found 346.1274.

1-(2-Phenyl-1,3-benzoxazol-6-yl)-3-(1H-pyrazol-4-ylmethyl)urea (12ca)

To a solution of 2-phenylbenzoxazol-6-ylamine (12c) (0.28 g, 1.3 mmol) in toluene (5 mL), 4-nitrophenyl chloroformate (0.26 g, 1.3 mmol) was added. The suspension was stirred at 80 °C for 1 h. The precipitate was filtered off to give the crude 4-nitrophenyl (2-phenyl-1,3-benzoxazol-6-yl)carbamate (14) (0.52 g), which was used in the next reaction without further purification.

Crude 4-nitrophenyl (2-phenyl-1,3-benzoxazol-6-yl)carbamate (14) (0.15 g) was added to a solution of 1H-pyrazol-4-ylmethylamine dihydrochloride (0.14 g, 0.80 mmol) and N,N-diisopropylethylamine (0.28 mL, 1.6 mmol) in methanol (3 mL) at 0 °C. The reaction mixture was warmed to 50 °C and stirred at that temperature for 30 min. After cooling to r.t., the precipitate was filtered off and washed with ethyl acetate and methanol to afford the title compound (94 mg, 71% over two steps) as a solid.

¹H-NMR (DMSO-d₆) δ: 4.19 (d, J = 5.5 Hz, 2H), 6.43 (t, J = 5.5 Hz, 1H), 7.17 (dd, J = 8.8, 2.0 Hz, 1H), 7.46 (br s, 1H), 7.55–7.69 (m, 5H) 8.10 (d, J = 2.0 Hz, 1H), 8.13–8.19 (m, 2H), 8.78 (s, 1H), 12.67 (br s, 1H); ¹³C-NMR (DMSO-d₆) δ: 33.7, 99.5, 115.4, 118.4, 119.5, 126.7, 126.8, 129.2, 131.4, 135.5, 138.8, 150.8, 155.0, 161.1; Anal. Calcd for C₁₈H₁₅N₅O₂·0.15H₂O: C, 64.33; H, 4.59; N, 20.84. Found C, 64.17; H, 4.71; N, 20.81. HRMS (ESI): Calcd for C₁₈H₁₅N₅O₂ [M + H]⁺: 334.1304. Found 334.1298.

tert-Butyl [2-(Pyridin-3-yl)-1,3-benzoxazol-6-yl]carbamate (4d)

tert-Butyl N-(1,3-benzoxazol-6-yl)carbamate¹⁹⁾ (3) (0.11 g, 0.47 mmol), 4,6-bis(diphenylphosphino)phenoxazine (40 mg, 0.072 mmol), and palladium (II) acetate (38 mg, 0.070 mmol) in ethylene glycol dimethyl ether (2 mL) were stirred at r.t. under an N₂ atmosphere for 5 min. 3-Bromopyridine (55 µL, 0.56 mmol) and sodium tert-butoxide (0.11 g, 1.12 mmol) were then added, and the reaction mixture was stirred at r.t. under an N₂ atmosphere for 8 h. Water was added to the reaction solution, followed by partition using ethyl acetate. The organic phase was washed with brine, then dried over sodium sulfate and filtered. Then, the solvent was distilled off under reduced pressure to give the crude title compound. The residue obtained was purified by silica gel column chromatography (ethyl acetate : hexane = 20 : 80 to 60 : 40) to give the title compound (43 mg, 30%) as a solid.

¹H-NMR (CDCl₃) δ: 1.55 (s, 9H), 6.88 (br s, 1H), 7.10 (dd, J = 8.6, 2.3 Hz, 1H), 7.46 (ddd, J = 7.9, 4.8, 1.0 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 8.05 (br s, 1H), 8.46 (dt, J = 8.0, 1.9 Hz, 1H), 8.75 (dd, J = 4.9, 1.8 Hz, 1H), 9.40–9.47 (m, 1H).

tert-Butyl [2-(1-Methyl-1H-pyrazol-5-yl)-1,3-benzoxazol-6-yl]carbamate (4e)

To a solution of tert-butyl 1,3-benzoxazol-6-ylcarbamate (3) (0.20 g, 0.85 mmol) in 1,2-dimethoxyethane (3 mL), 5-bromo-1-methylpyrazole (165 mg, 1.0 mmol), 4,6-bis(diphenylphosphino)phenoxazine (0.072 g, 0.13 mmol), and sodium tert-butoxide (0.21 g, 2.0 mmol) were added. The mixture was stirred at r.t. overnight. Then, 5-bromo-1-methylpyrazole (0.17 mg, 1.0 mmol), 4,6-bis(diphenylphosphino)phenoxazine (0.072 g, 0.13 mmol), and sodium tert-butoxide (0.21 g, 2.0 mmol) were added to the solution, and the mixture was stirred at 50 °C for 6 h. After cooling to r.t., the solvent was distilled off under reduced pressure to give the crude title compound. The crude product was purified by silica gel column chromatography (silica gel, ethyl acetate : hexane = 0 : 100 to 33 : 64) to give the title compound (45 mg, 17%) as a solid.

¹H-NMR (CDCl₃) δ: 1.56 (s, 9H), 4.39 (s, 3H), 6.68 (br s, 1H), 6.99 (d, J = 2.1 Hz, 1H), 7.09 (dd, J = 8.3, 2.0 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.99 (s, 1H). MS (ESI): m/z 315 [M + H]⁺.

5-Fluoro-6-nitro-2-phenyl-1,3-benzoxazole (6f)

5-Fluoro-2-phenyl-1,3-benzoxazole²¹⁾ (5f) (0.34 g, 1.6 mmol) was dissolved in concentrated sulfuric acid (2 mL) and then cooled to 0 °C. Nitric acid (0.20 mL, 5.0 mmol) was added to the solution and the mixture was stirred at 0 °C for 20 min. The reaction solution was poured into ice water (20 mL), followed by partitioning using dichloromethane. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and brine, then dried over magnesium sulfate and filtered. The solvent was then distilled off under reduced pressure to give the crude title compound. The crude product was purified by silica gel column chromatography (ethyl acetate : hexane = 3 : 97 to 35 : 65) to give the title compound (0.20 g, 49%) as a solid.

¹H-NMR (CDCl₃) δ: 7.55–7.68 (m, 5H), 8.26–8.31 (m, 1H), 8.36 (d, J = 6.1 Hz, 1H).

5-Fluoro-2-phenyl-1,3-benzoxazol-6-amine (4f)

5-Fluoro-6-nitro-2-phenyl-1,3-benzoxazole (6f) (0.20 g, 0.77 mmol) was dissolved in ethanol (4 mL) and ethyl acetate (6 mL), and 10% Pd/C (50% wet) (70 mg) was added to this solution. After purging of the reaction container with hydrogen, the mixture was stirred at 60 °C for 2 h. The reaction solution was filtered through Celite. Then, the filtrate was concentrated under reduced pressure to give the crude title compound. A small amount of ethyl acetate : dichloromethane (1 : 1) was then added. The precipitated solid was then collected by filtration to give the title compound (0.13 g, 71%) as a solid.

¹H-NMR (DMSO-d₆) δ: 5.51 (br s, 2H), 7.05 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 11.0 Hz, 1H), 7.54–7.60 (m, 3H), 8.06–8.12 (m, 2H).

5-Chloro-6-nitro-2-phenyl-1,3-benzoxazole (6h)

The title compound (0.32 g, 76%) was obtained from 5-chloro-2-phenyl-1,3-benzoxazole²¹⁾ (5h) (0.35 g, 1.5 mmol) following the same procedure as for the synthesis of 6f.

¹H-NMR (CDCl₃) δ: 7.54–7.68 (m, 3H), 7.91 (s, 1H), 8.19 (s, 1H), 8.25–8.31 (m, 2H).

5-Chloro-2-phenyl-1,3-benzoxazol-6-amine (4h)

5-Chloro-6-nitro-2-phenyl-1,3-benzoxazole (6h) (0.32 g, 1.2 mmol) was dissolved in ethanol (3 mL) and ethyl acetate (3 mL). Platinum (IV) oxide (30 mg) was added to this solution. After purging of the reaction container with hydrogen, the mixture was stirred at 50 °C for 1 h. The reaction solution was filtered through Celite. Then, the filtrate was concentrated under reduced pressure to give the crude title compound. A small amount of ethyl acetate : dichloromethane (1 : 1) was then added. Then, the precipitated solid was collected by filtration to give the title compound (0.13 g, 48%) as a solid.

¹H-NMR (CDCl₃) δ: 4.23 (br s, 2H), 6.97 (s, 1H), 7.45–7.53 (m, 3H), 7.66 (s, 1H), 8.13–8.20 (m, 2H).

6-Nitro-2-phenyl[1,3]oxazolo[4,5-b]pyridine (6i)

2-Amino-5-nitropyridin-3-ol (7) (0.30 g, 1.9 mmol) and benzoic acid (0.24 g, 1.9 mmol) were dissolved in polyphosphoric acid (3 mL), and the solution was heated at 160 °C for 4 h. The reaction solution was poured into ice water (20 mL), followed by partition using dichloromethane. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and brine, then dried over magnesium sulfate and filtered. The solvent was distilled off under reduced pressure to give the crude title compound. A small amount of diethylether (3 mL) was then added, and the precipitated solid was collected by filtration to give the title compound (0.26 g, 56%) as a solid.

¹H-NMR (DMSO-d₆) δ: 7.68–7.75 (m, 2H), 7.76–7.84 (m, 1H), 8.28–8.36 (m, 2H), 9.20 (d, J = 2.3 Hz, 1H), 9.44 (d, J = 2.3 Hz, 1H). MS (ESI): m/z 242 [M + H]⁺.

2-Phenyl[1,3]oxazolo[4,5-b]pyridin-6-amine (4i)

The title compound (0.15 g, 67%) was obtained from 6-nitro-2-phenyl[1,3]oxazolo[4,5-b]pyridine (6i) (0.26 g, 1.1 mmol) following the same procedure as for the synthesis of 4f.

¹H-NMR (CD₃OD) δ: 7.32 (d, J = 2.9 Hz, 1H), 7.52–7.62 (m, 4H), 7.97 (d, J = 2.3 Hz, 1H), 8.16–8.23 (m, 2H).

2-Bromo-6-methoxy-4-nitroaniline (9ka)

2-Methoxy-4-nitroaniline (8) (0.20 g, 1.2 mmol) in DMF (2 mL) was stirred in ice bath, and then NBS (0.23 g, 1.3 mmol) was added. The solution was stirred at r.t. for 3 h. The reaction solution was poured into saturated aqueous ammonium chloride solution (20 mL), followed by partitioning using ethyl acetate. The organic phase was washed with brine, then dried over magnesium sulfate and filtered. The solvent was then distilled off under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, ethyl acetate : hexane = 0 : 100 to 25 : 75) to give the title compound (0.27 g, 90%) as a solid.

¹H-NMR (CDCl₃) δ: 3.96 (s, 3H), 4.97 (br s, 2H), 7.62 (s, 1H), 8.09 (s, 1H).

4-Bromo-6-nitro-2-phenyl-1,3-benzoxazole (6ka)

2-Bromo-6-methoxy-4-nitro-aniline (9ka) (0.27 g, 1.1 mmol), benzoyl chloride (0.14 mL, 1.2 mmol) and pyridine (0.17 mL, 2.2 mmol) were suspended in THF (5 mL). The reaction mixture was stirred at r.t. for 16 h, and then at 60 °C for 6 h. Additional benzoyl chloride (0.14 mL, 1.2 mmol) was added, and the reaction was stirred at 60 °C for 18 h. The reaction was quenched with 1 M HCl (10 mL) and then extracted with dichloromethane. The residue was partially purified by silica gel column chromatography (silica gel, ethyl acetate : hexane = 0 : 100 to 40 : 60) to give crude N-(2-bromo-6-methoxy-4-nitro-phenyl)benzamide (200 mg), which was used in the next reaction without further purification.

Crude N-(2-bromo-6-methoxy-4-nitro-phenyl)benzamide (200 mg) obtained above was dissolved in dichloromethane (5 mL) and the solution was stirred at −78 °C. Boron tribromide (1 M in dichloromethane, 1.42 mL) was added to the solution at −78 °C. The reaction mixture was allowed to warm to r.t. while stirring, and stirred at r.t. for 3 h. After cooling in an ice bath, the reaction was quenched with saturated NaHCO₃ aqueous solution (6 mL) and then extracted with dichloromethane. The combined organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo to give crude N-(2-bromo-6-hydroxy-4-nitro-phenyl)benzamide (0.17 g), which was used in the next reaction without further purification.

Crude N-(2-bromo-6-hydroxy-4-nitro-phenyl)benzamide (0.17 g) obtained above and pyridinium p-toluenesulfonate (0.022 g, 0.088 mmol) were suspended in toluene (10 mL). The mixture was stirred at 130 °C for 1 h. After cooling to r.t., the reaction was quenched with water (10 mL) and then extracted with ethyl acetate. The combined organic phase was washed with saturated NaHCO₃ aqueous solution and brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Small amounts of hexane (1 mL) and dichloromethane (0.2 mL) were then added. Then, the precipitated solid was collected by filtration to give the title compound (0.13 g, 36% over three steps) as a solid.

¹H-NMR (CDCl₃) δ: 7.55–7.62 (m, 2H), 7.63–7.70 (m, 1H), 8.32–8.37 (m, 2H), 8.46 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 2.2 Hz, 1H). MS (ESI): m/z 319 [M + H]⁺.

6-Nitro-2-phenyl-1,3-benzoxazole-4-carbonitrile (6k)

4-Bromo-6-nitro-2-phenyl-1,3-benzoxazole (0.13 g, 0.39 mmol), zinc cyanide (98 mg, 0.82 mmol), and tetrakis(triphenylphosphine)palladium(0) (45 mg, 0.039 mmol) were dissolved in DMF (2 mL). The reaction mixture was stirred at 100 °C for 8 h. The reaction solution was poured into saturated aqueous ammonium chloride solution (20 mL), followed by partitioning using ethyl acetate. The organic phase was washed with brine, then dried over magnesium sulfate and filtered. The solvent was then distilled off under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, ethyl acetate : hexane = 0 : 100 to 30 : 70) to give the title compound (52 mg, 50%) as a solid.

¹H-NMR (CDCl₃) δ: 7.56–7.66 (m, 2H), 7.66–7.76 (m, 1H), 8.33–8.42 (m, 2H), 8.64 (d, J = 2.0 Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H).

6-Amino-2-phenyl-1,3-benzoxazole-4-carbonitrile (4k)

To a solution of 6-nitro-2-phenyl-1,3-benzoxazole-4-carbonitrile (52 mg, 0.20 mmol) and ammonium chloride (10 mg, 0.20 mmol) dissolved in ethanol, iron power (0.11 g) was added at 80 °C. The solution was stirred at 80 °C for 11 h. The reaction solution was filtered through Celite when still hot. Then, the filtrate was concentrated under reduced pressure to give the title compound (46 mg, quant.) as a solid.

¹H-NMR (CDCl₃) δ: 4.00 (br s, 2H), 6.96 (d, J = 2.2 Hz, 1H), 7.04–7.11 (m, 1H), 7.44–7.60 (m, 3H), 8.20–8.30 (m, 2H).

6-Bromo-4-methyl-2-phenyl-1,3-benzoxazole (6j)

The title compound (0.13 g, 36% over three steps) was obtained from 4-bromo-2-methoxy-6-methylaniline (9j) (0.40 g, 1.9 mmol) following the same procedure as for the synthesis of 6ka.

¹H-NMR (CDCl₃) δ: 2.64 (s, 3H), 7.30 (dd, J = 1.7, 0.7 Hz, 1H), 7.56–7.48 (m, 3H), 7.60–7.56 (m, 1H), 8.29–8.20 (m, 2H). MS (ESI): m/z 288 [M + H]⁺.

tert-Butyl (4-Methyl-2-phenyl-1,3-benzoxazol-6-yl)carbamate (4j)

6-Bromo-4-methyl-2-phenyl-1,3-benzoxazole (6j) (0.33 g, 1.1 mmol), tert-butyl carbamate (0.19 g, 1.6 mmol), 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (0.19 g, 0.45 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.10 g, 0.11 mmol), and sodium tert-butoxide (0.25 g, 2.6 mmol) were dissolved in toluene (12 mL) and then stirred at room temperature for 3 h. The reaction solution was poured into water (20 mL), followed by partitioning using dichloromethane. The organic phase was washed with brine, then dried over magnesium sulfate and filtered. Then, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (NH silica gel, ethyl acetate : hexane = 5 : 95 to 15 : 85) to give the title compound (0.35 g, 94%) as a solid.

¹H-NMR (CDCl₃) δ: 8.26–8.17 (m, 2H), 7.71 (br s, 1H), 7.50 (dd, J = 3.9, 2.7 Hz, 3H), 6.95 (s, 1H), 6.57 (br s, 1H), 2.62 (s, 3H), 1.54 (s, 9H). (ESI): m/z 325 [M + H]⁺.

Methyl 6-Bromo-2-phenyl-1,3-benzoxazole-4-carboxylate (6la)

The title compound (0.11 g, 32% over three steps) was obtained from methyl 2-amino-5-bromo-3-methoxy-benzoate²²⁾ (9la) (0.29 g, 1.1 mmol) following the same procedure as for the synthesis of 6ka.

¹H-NMR (CDCl₃) δ: 4.07 (s, 3H), 7.51–7.63 (m, 3H), 7.94 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 1.7 Hz, 1H), 8.30–8.38 (m, 2H). MS (ESI): m/z 332 [M + H]⁺.

6-Bromo-N,N-dimethyl-2-phenyl-1,3-benzoxazole-4-carboxamide (6l)

To a solution of methyl 6-bromo-2-phenyl-1,3-benzoxazole-4-carboxylate (6la) (0.30 g, 0.90 mmol) in THF (4 mL) and methanol (2 mL), 1 M NaOH solution (2 mL) was added at r.t. The mixture was stirred at r.t. for 1 h. Then, 1 M HCl (5 mL) and water (5 mL) were added to the solution, and the precipitate was filtered off and washed with water to give crude 6-bromo-2-phenyl-1,3-benzoxazole-4-carboxylic acid (0.28 g), which was used in the next reaction without further purification.

To a solution of crude 6-bromo-2-phenyl-1,3-benzoxazole-4-carboxylic acid (0.14 g) obtained above in DMF (2.5 mL), dimethylamine (2 M solution in THF, 340 µL, 0.68 mmol), N,N-diisopropylethylamine (77 mL, 0.45 mmol), and DMT-MM (0.17 g, 0.59 mmol) were added at r.t. The mixture was stirred at r.t. for 12 h. The reaction solution was poured into water (5 mL), followed by partitioning using ethyl acetate. The organic phase was washed with brine, then dried over magnesium sulfate and filtered. Then, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, ethyl acetate : hexane = 10 : 90 to 25 : 75) to give the title compound (0.15 g, 95% over two steps) as a solid.

¹H-NMR (CDCl₃) δ: 3.01 (s, 3H), 3.24 (s, 3H), 7.49–7.62 (m, 4H), 7.79 (d, J = 1.7 Hz, 1H), 8.22–8.28 (m, 2H). MS (ESI): m/z 345 [M + H]⁺.

tert-Butyl [4-(Dimethylcarbamoyl)-2-phenyl-1,3-benzoxazol-6-yl]carbamate (4l)

The title compound (0.12 g, 76%) was obtained from 6-bromo-N,N-dimethyl-2-phenyl-1,3-benzoxazole-4-carboxamide (6l) (0.15 g, 0.42 mmol) following the same procedure as for the synthesis of 4j.

¹H-NMR (CDCl₃) δ: 1.55 (s, 9H), 3.02 (s, 3H), 3.23 (s, 3H), 6.82 (br s, 1H), 7.17 (d, J = 2.0 Hz, 1H), 7.45–7.57 (m, 3H), 8.04–8.11 (m, 1H), 8.18–8.26 (m, 2H). MS (ESI): m/z 382 [M + H]⁺.

Biology. 1. Measurement of NAMPT Enzyme Activating Effect (In Vitro Cell-free Enzyme Assay)

The NAMPT enzyme assay was conducted in accordance with the method of Formentini et al.³²⁾ by chemically converting nicotinamide mononucleotide (NMN) produced by the NAMPT enzyme to a fluorescent substance and using the fluorescence intensity of this fluorescent substance as an index for the amount of NMN produced. Hereinafter, the procedures will be briefly described. The NAMPT enzyme reaction was carried out using polypropylene 384 well V shaped black plate (Greiner Bio One International GmbH). The NAMPT activity was measured in assay buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 50 mM NaCl, 5 mM MgCl₂, 1 mM TCEP, 0.1% Prionex, 0.005% Tween 20, 0.12 mM ATP, 5 µM nicotinamide (NAM), 6.25 µM phospho-ribosyl pyro-phosphate (PRPP), 0.04 U/mL pyrophosphatase and 2 ng/mL human NAMPT enzyme in the presence or absence of test compounds. After the 1-h incubation at 25 °C, the enzyme reaction was terminated by the addition of 5 µL of 2 M KOH and 5 µL of a 20% acetophenone solution. Then, 22 µL of 88% formic acid was added and the mixture was further incubated for 30 min in the dark. The NAMPT enzyme activity was calculated as the difference between fluorescence intensity (Ex 380 nm/Em 450 nm) from the reaction of a test compound treatment group and fluorescence intensity from control reaction free from the NAMPT enzyme.

  

The EC₅₀ of the tested compounds were calculated using GraphPad Prism (GraphPad Software, Inc.), and the Emax were calculated as percentage of the maximal activity achieved with 30 µM of compound 1.

2. Study on Effect of NAMPT Activator on the Intracellular NAD⁺ Level (In Vitro Cell-Based Assay)

HEK293A cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM (high glucose)) containing 10% fetal bovineserum (FBS), non-essential amino acid (NEAA), and antibiotic-antimycotic [reaction medium]. On the day before the test, HEK293A cells were inoculated at a ratio of 3.0 × 10⁴ cells/80 µL to 96 well poly-D-lysine coated plate and cultured overnight in a CO₂ incubator. On the day of the test, each test compound diluted with reaction medium to a concentration of 5 times the final concentration was added at a ratio of 20 µL/well, and the cells were further cultured for 3 h. After removal of the medium, the cells were lysed in 50 µL/well of Bicarbonate Base Buffer (100 mM sodium carbonate, 20 mM sodium bicarbonate, 100 mM nicotinamide, 20 mM Triton X-100, 1% dodecyl trimethyl-ammonium bromide (DTAB)). The NAD⁺ content in the cell extract was measured using NAD⁺/NADH-Glo assay kit according to the manufacturer’s instructions. Hereinafter, the procedures will be briefly described. 20 µL of the lysate was separated, and 10 µL of 0.4 N HCl was added thereto, followed by incubation at 60 °C for 15 min and subsequently at 25 °C for 10 min. The mixture was neutralized by the addition of 10 µL of 0.5 M Trizma base solution and then diluted 10-fold with distilled water. Fifteen microliters of the diluted sample was mixed with an equal amount (15 µL) of NAD⁺/NADH-Glo reagent in 384 well black plate, and the mixture was incubated for 30 min in the dark. Subsequently, the chemiluminescence intensity was measured.

3. Evaluation of NAMPT Activator on Tissue NAD⁺ Level in Diet-Induced Obese (DIO) Mice

Compound 12c prepared with 0.5% methylcellulose was orally administered at 30 or 100 mg/kg to 47-week-old male C57BL/6N DIO mice (high-fat loading obesity mouse models) having a pathological obese condition induced by purified high-fat diet (D12451) from 7 weeks of age. Twenty-four hours after the administration, each mouse was sacrificed by exsanguination. The liver, gastrocnemius, soleus, and brown adipose tissues were harvested. Each tissue was homogenized with a 10- or 100-fold amount of 2 M perchloric acid per its weight. Then, the samples were centrifuged, and the supernatant was neutralized with 5 M sodium hydroxide. After removal of potassium perchlorate, the NAD⁺ concentration in the supernatant of sample was measured using LC/MS.

4. Evaluation of Anti-obesity Effect of NAMPT Activator (DIO Mice)

Forty-five-week-old male C57BL/6N DIO mice were each acclimatized for 1 wk under individual housing. Then, the mice were divided into 2 groups each involving 6 mice with their weights, amounts of weight change, and food intakes during the acclimatization period as indexes. Each mouse was freely fed a purified high-fat diet (D12451) for 3 wk under individual rearing. Compound 12c prepared with 0.5% methylcellulose was orally administered at 30 mg/kg once a day for 3 wk from the start day of the test (a vehicle group was treated with 0.5% methylcellulose in the same way as above). The weight was compared between the test groups.

PAMPA, LogD, Solubility, and Metabolic Stability Assay

Each assay was performed as previously described.^33,34)

CYP Direct Inhibition Assay

The reversible inhibition by compounds on the specific activities of 4 CYP isoforms (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) were examined in the reaction mixture containing human liver microsome and a NADPH generating system. The microsomal protein concentration in the assay was 0.1 mg/mL. After CYP substrate was incubated at 37 °C for 10 min in the absence or presence of compounds (10 µmol/L), concentration of metabolites from CYP substrate was semi-quantitatively analyzed by LC-MS/MS and % inhibition was calculated by the following formula;

  

Pharmacokinetic Evaluation in Mice

The test compounds suspended with vehicle (0.5% (w/v) methyl cellulose 400 solution (Wako Pure Chemical Corporation, Osaka, Japan) were orally administered to 7-week-old male C57BL/6J mice at a dose of 10 mg/kg. Plasma samples were collected from the peripheral vein at several time points after dosing. The plasma concentration was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to obtain PK parameters via non-compartmental analysis.

Conflict of Interest

The authors declare no conflict of interest.

References and Notes

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