Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5

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Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5methylpyrrolo[1, 2-f][1, 2, 4]triazine-6-carbonyl) (propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (BMS-751324), a Clinical Prodrug of p38# MAP Kinase Inhibitor Chunjian Liu, James Lin, John Hynes, Hong Wu, Stephen Todd Wrobleski, Shuqun Lin, T. G. Murali Dhar, Vivekananda Murthy Vrudhula, Jung-Hui Sun, Sam Chao, Rulin Zhao, Bei Wang, Bang-Chi Chen, Gerry Everlof, Christoph Gesenberg, HJ Zhang, Punit H. Marathe, Kim W. McIntyre, Tracy L. Taylor, Kathleen M. Gillooly, David J. Shuster, Murray McKinnon, John H. Dodd, Joel C. Barrish, Gary L. Schieven, and Katerina Leftheris J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00839 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Journal of Medicinal Chemistry

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Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1, 2-f][1, 2, 4]triazine-6-carbonyl)(propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (BMS751324), a Clinical Prodrug of p38α MAP Kinase Inhibitor

Chunjian Liu*, James Lin, John Hynes, Hong Wu, Stephen T. Wrobleski, Shuqun Lin, T. G. Murali Dhar, Vivekananda M. Vrudhula, Jung-Hui Sun, Sam Chao, Rulin Zhao, Bei Wang, Bang-Chi Chen, Gerry Everlof, Christoph Gesenberg, Hongjian Zhang, Punit H. Marathe, Kim W. McIntyre, Tracy L. Taylor, Kathleen Gillooly, David J. Shuster, Murray McKinnon, John H. Dodd, Joel C. Barrish, Gary L. Schieven, and Katerina Leftheris

Research & Development, Bristol-Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543

* To whom correspondence should be addressed: Tel: +1 609 252 3682. Fax: +1 609 252 7410. email: [email protected].

ABSTRACT In search for prodrugs to address the issue of pH dependent solubility and exposure associated with 1 (BMS-582949), a previously disclosed phase II clinical p38α MAP kinase inhibitor, a structurally novel clinical prodrug, 2 (BMS-751324), featuring a carbamoylmethylene linked promoiety containing hydroxyphenyl acetic acid (HPA) derived ester and phosphate functionalities, was identified. Prodrug 2 was not only stable but also water soluble under both acidic and neutral conditions. It was effectively bio-converted into parent drug 1 in vivo by alkaline phosphatase and esterase in a stepwise manner, providing higher exposure of 1 compared to its direct administration, especially within higher dose ranges. In a rat LPS-induced TNFα pharmacodynamic model and a rat adjuvant arthritis model, 2

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demonstrated similar efficacy to 1. Most importantly, it was shown in clinical studies that prodrug 2 was indeed effective in addressing the pH dependent absorption issue associated with 1.

INTRODUCTION p38α MAP kinase plays a crucial role in regulating the biosynthesis of many inflammatory cytokines including TNFα and IL-1β.1 Biological agents such as etanercept and infliximab have demonstrated that blockage of TNFα function is effective in the treatment of autoimmune diseases including rheumatoid arthritis (RA).2 Thus, small molecule p38α inhibitors have garnered tremendous attention for the treatment of autoimmune and inflammatory diseases.3 We have previously disclosed 1 (BMS-582949) (Figure 1) as a Phase II clinical p38α inhibitor for the treatment of RA.4 An important consideration for the development of 1 was its pH dependent solubility, which was determined to be 0.280 mg/mL at pH 1.2 but only 0.003 mg/mL at pH 6.5. When 1 was dosed in humans with the H2 blocker famotidine, which would cause the stomach pH to increase, the exposure dropped by 70%. Since over 50% of RA patients take famotidine or other H2 blockers, this pH dependent solubility/exposure will likely limit the potential use of 1 in RA patients. As a result, a prodrug program was launched. To minimize any additional developmental complications that a prodrug may incur, our objective was to identify a suitable prodrug that should address the pH dependent aqueous solubility/exposure issue, be chemically stable in the pH 1-7 range, and be enzymatically converted to the parent drug before or during absorption to minimize systemic circulation of the prodrug. Also, the parent drug exposure upon prodrug administration should be comparable to or higher than what was observed with the parent drug itself. In addition, the prodrug in vivo by-products resulting from its bio-conversion should be safe. Our investigation ultimately led to the discovery of 2 (BMS-751324) that met all the afore-mentioned criteria and was advanced to clinical development.



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RESULTS AND DISCUSSIONS Our initial desire was to synthesize and evaluate methylene phosphate prodrugs of 1, since methylene phosphate prodrugs are among the most successful water soluble prodrugs in literature. For example, Fosphenytoin5 is a FDA approved methylene phosphate prodrug of Phenytoin for the treatment of seizures. Methylene phosphate prodrugs of the Syk kinase inhibitor (Fostamatinib)6 and the HIV-1 attachment inhibitor (BMS-663749)7 were evaluated in clinical trials. The parent drug 1 contains two amide NH groups and one diaryl NH that can be considered as logical handles for promoiety installation. However, prodrugs derived from amide NH and biaryl NH functionalities are not wellprecedented in the literature. After a great deal of effort, we were able to prepare all three possible isomeric methylene phosphate prodrugs 3, 4, and 5 (Scheme 1, 2, and 3, respectively). As shown in Scheme 1, reaction of pyrrolotriazine carboxylic acid 64 with thionyl chloride and subsequently with N((phenylthio)methyl)propan-1-amine 9 provided N-phenylthiomethyl amide 7. Reagent 9 was prepared, according to a literature procedure for similar compounds,8 by reacting 1,3,5-tripropyl-1,3,5-triazinane 8 with benzenethiol in the presence of 1 N HCl in diethyl ether. Treatment of 7 with thionyl chloride, followed by phosphoric acid and N,N-diisopropylethylamine, afforded target prodrug 3. For the preparation of 4, benzoic acid 104 was coupled with N-(2,4,6trimethoxybenzyl)cyclopropanamine 15, prepared from 2,4,6-trimethoxybenzaldehyde 14 by reductive amination, to supply amide 11 (Scheme 2). Compound 11 was treated with two equivalents of sodium hydride, followed by one equivalent of chloromethyl methyl sulfide in the presence of sodium iodide to provide 12. The 2,4,6-trimethoxybenzyl group in 12 was removed by trifluoroacetic acid (TFA), giving rise to 13, which was reacted with sulfuryl dichloride, followed by tetrabutylammonium dihydrogen phosphate and an ion exchange process to afford the phosphate prodrug 4.


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To synthesize 5, the parent drug 1 was directly treated with sodium hydride and chloromethyl methyl sulfide to provide methyl sulfide 16 as a major product (Scheme 3). The regiochemistry of 16 was determined by NMR studies. The exchangeable cyclopropylamide N-H doublet near 6.4 ppm of 1 was not observed after the alkylation while the C6-amide N-H remained as a triplet near 5.9 ppm in CDCl3. The aniline N-H was also still observed as an exchangeable singlet near 8.3 ppm. Methylenephosphate 5 was then obtained from 16 as previously described for the transformation of 7 to 3 in Scheme 1.

With 3, 4, and 5 in hand, we began our prodrug evaluations with solution stability tests (Table 1). Methylene phosphate prodrugs 3, 4, and 5 were found to be highly stable at pH 7.4, as their half lives (t1/2) at 37 oC were determined to be 54, 20, and 257 h, respectively. Unfortunately, they were all highly unstable under acidic conditions, with the t1/2 values at pH 1.0 and 3.1 being much shorter than 0.25 h. In fact, attempts to isolate the free forms of 3, 4, and 5 were unsuccessful due to their instability at low pH ranges. Methylene phosphate prodrugs 3, 4, and 5 can be related to N-acyloxymethyl prodrugs of amides. The latter is noticed to exhibit poor aqueous stability due to the elimination of an acyloxy group, generating an N-acylimine, which is then hydrolyzed to the parent amide.9 For phosphate prodrugs 3, 4, and 5, it may be reasonable to propose that the rapid degradation under acidic conditions is initiated by the elimination of a dihydrogen phosphoryloxy group. However, at pH 7.4, the dihydrogen phosphate will remain deprotonated and the deprotonated dihydrogen phosphoryloxy becomes a less strong leaving group.

We then turned our attention to carbamoylmethylene phosphate prodrugs represented by 1710 (Figure 2). Contrary to methylene phosphate prodrugs, carbamoyl methylene phosphate 17 was unstable under neutral conditions although it was stable at pH 1.0. We believed that the instability of 17 under neutral conditions was likely due to an intramolecular nucleophilic attack of the phosphate oxygen anion to the ACS Paragon Plus Environment


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carbamoyl carbonyl. The idea of having a structurally rigid promoiety to prevent any intramolecular nucleophilic reactions led to the identification of fumaric acid derived prodrug 18.10 Prodrug 18 proved to be stable under both acidic and neutral conditions, and displayed a significantly improved aqueous solubility profile. Encouraged by these results, we further prepared p-hydroxyphenyl acetic acid (HPA) derived carbamoylmethylene ester prodrug 2, where intramolecular nucleophilic reactions of the phosphate oxygen anion to the carbamoyl carbonyl are unlikely to occur. The other reason for selecting HPA as the promoiety template in 2 is that HPA is an endogenous by-product of tyrosine metabolism11 and a typical metabolite of ingested dietary phenolic acids.12 The synthesis of prodrug 2 is outlined in Scheme 4. Parent drug 1 was reacted with di-tert-butyl dicarbonate in the presence of 4dimethylaminopyridine (DMAP) to afford 19; the fact that carbamylation occurred on the pyrrolotriazine ring nitrogen instead of the anilino nitrogen was revealed by single crystal x-ray crystallography.10 Treatment of 19 with two equivalents of lithium bis(trimethylsilyl)amide, followed by one equivalent of chloromethyl chloroformate, led to the formation of chloromethyl carbamate 20, regioselectively. The regioselectivity was confirmed by the x-ray structure of 18,10 which was derived from 20. Chloromethyl carbamate 20 was converted to iodomethyl carbamate 21 by Finkelstein reaction. Interestingly, the Boc group of 20 was simultaneously removed under the reaction conditions. Heating 21 with silver carboxylate 26 (Scheme 5) in toluene led to the formation of 22. Debenzylation of 22 under standard hydrogenation conditions provided the target prodrug 2. Silver carboxylate 26 was prepared from commercially available methyl 2-(4-hydroxyphenyl)acetate 23. Reaction of 23 with dibenzyl chlorophosphite, generated in situ from carbon tetrachloride and dibenzyl phosphite in the presence of N,N-diisopropylethylamine and DMAP,13 gave phosphate 24. Hydrolysis of 24 with lithium hydroxide provided carboxylic acid 25, which was converted into silver salt 26 with sodium hydroxide, followed by silver nitrate.


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As anticipated, prodrug 2 was found to be stable under both acidic and neutral conditions, with its t1/2 values at 37 oC determined to be 21 and 37 h at pH 1.1 and 7.4, respectively. Furthermore, it was highly soluble in water, as its aqueous solubility was measured to be 0.10 mg/mL and 3.03 mg/mL at pH 1.2 and 6.7, respectively (Table 2).

After achieving the desired chemical stability and aqueous solubility, prodrug 2 was then evaluated in vivo for its bio-conversion into parent drug 1. Thus, 2 was orally dosed as a methocel suspension to rats at 1, 10, and 100 mpk (equivalents to 1), and the exposures of 1 in plasma were measured at different time points over 24 h. The obtained Cmax, AUC0-24, and bioavailability (BA) values for 1 are shown in Table 3. The results from direct administration of 1 (as a methocel nano suspension) are incorporated in the table for comparison. When dosed at 1 mpk, 2 provided 1 with a Cmax of 0.4 µM, AUC0-24 of 3.1 µM*h, and bioavailability (BA) of 41%. These PK parameters were comparable to what were obtained from the direct administration of 1 at the same dose. When the dose of 2 was escalated to 10 and 100 mpk, the Cmax of 1 dose-responsively increased to 18 and 122 µM, AUC0-24 increased to 78 and 875 µM*h, and BA improved to 103 and 115%, respectively. The fact that the BA at 10 and 100 mpk is significantly higher than that at 1 mpk suggests that drug metabolizing enzymes and/or drug transporters may have been saturated at the higher doses. In contrast, due to solubility-limiting absorption, the exposures after direct dose of 1 at 10 and 100 mpk did not significantly increase, and the BA decreased to 31 and 4.3%, respectively.

Prodrug 2 was also studied for its bio-conversion into parent drug 1 in Cynomolgus monkeys, where direct administration of 1 gave a BA of only16% even with a PEG 400 solution dose at 10 mpk (Table 4). When 2 was administrated as a methocel suspension at 10 mpk (equivalent to 1), the exposure of 1 was significantly higher and the BA improved to 51%. When the dose of 2 was increased to 30 mpk



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(equivalent to 1), the exposure of 1 nearly dose-proportionally increased and the BA remained high (69%).

The promising exposure of 1 observed after administration of prodrug 2 warranted examinations of the pharmacodynamic effect of 2 versus 1 in the rat LPS-induced TNFα model. Male Lewis rats were treated with compounds one hour prior to IP injection of LPS. Prodrug 2 and parent drug 1 were dosed as a methocel suspension and a PEG 300 solution, respectively. Ninety minutes after the LPS injection, blood samples were obtained and the serum TNF levels were determined. As shown in Figure 3, administration of prodrug 2 inhibited LPS-induced TNFα production in rats in a very similar dosedependent manner as parent 1. Its ED50, expressed as its parent equivalent (0.89 mg), was virtually identical to what was obtained for 1 (0.88 mg). It is understandable that at the low doses, especially when dosed as a PEG 300 solution, the parent drug and prodrug can be equally efficacious because the parent compound may not necessarily show solubility-limiting absorption at the low doses.

Table 5 shows the efficacy observed after administration of prodrug 2 versus parent drug 1 in the rat adjuvant arthritis model. In this study, complete Freund’s adjuvant was given to male Lewis rats on day one and the rats’ immune responses were allowed to develop for 10 days. On day 11, the rats started to receive compounds orally twice a day, and paw swelling was measured periodically. Prodrug 2 and parent drug 1 were dosed as a methocel suspension and a PEG 300 solution, respectively. At equivalent doses, prodrug 2 provided efficacy comparable to what was observed with 1 itself, inhibiting paw swelling by 39 ±15% and 46 ± 23% at doses of 1 and 3 mpk (equivalents to 1), versus 27 ± 31% and 35 ± 25% for 1, respectively. Comparison of the exposures obtained from the administrations of prodrug 2 and parent drug 1 suggested that at the low doses with PEG 300 as the vehicle, the parent drug did not show solubility-limiting absorption in this study.


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Administration of 2 with famotidine in rat has demonstrated that the prodrug can indeed be effective in addressing the pH dependent absorption issue associated with parent drug 1.14

Prodrug 2 contains a novel promoiety, carbamoylmethylene linked HPA derived ester and phosphate functionalities. To understand the mechanism by which the prodrug is bio-converted into its parent, 2 was incubated with commercially available human placental alkaline phosphatase (ALP) and porcine liver esterase at 37 oC, separately. It was found that 2 was rapidly hydrolyzed by ALP to give intermediate 27 (Scheme 6). After incubation with ALP for 0.25 h, only 12.5% of 2 remained (Table 6). When the HALT phosphatase inhibitor cocktail was added in the process, the hydrolysis was significantly inhibited. No hydrolysis was detected after 0.25 h, and 64.5% of 2 remained unchanged after 1 h. Interestingly, porcine liver esterase was found completely incapable of hydrolyzing 2 when incubated for 1 h. However, the esterase was highly effective in hydrolyzing intermediate 27, since only 8.2% of 27 remained after incubation with the esterase for 0.25 h (Table 7). Without the esterase, 27 stayed largely unchanged in the testing buffer (potassium phosphate), indicating that chemically 27 is relatively stable. It should be mentioned that during in vivo studies, neither 2 nor 27 was detected in systemic circulation. This is likely due to the fact that 2 and 27 are rapidly hydrolyzed by ALP and esterase, respectively, at the apical side of enterocytes, and that neither of them is permeable enough to be absorbed through the enterocyte. Consistent with this hypothesis, when 2 and 27 were loaded on the apical side of Caco-2 cells and incubated at 37 oC for 2 h, prodrug 2, intermediate 27 and parent 1 were found on the apical side, but 1 was the only compound detected on the basal side. Based on these in vitro and in vivo studies, it can be proposed that prodrug 2 is bio-converted into parent drug 1 in a stepwise manner, first by ALP to intermediate 27 and then by esterase to 1 most probably at the brush border in the intestine. Both steps most likely occur without 2 and 27 being absorbed. Hydrolysis of 27 by esterases presumably yields p-hydroxyphenyl acetic acid and 28, which decomposes to parent drug 1, carbon dioxide, and formaldehyde (Scheme 6).15 ACS Paragon Plus Environment


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In summary, to address the issue of pH dependent solubility and absorption associated with the clinical p38α inhibitor 1, a prodrug strategy was implemented. We were able to synthesize methylene phosphate prodrugs from each of the three NH groups in 1 despite the fact that prodrugs derived from amide NH and biaryl NH groups were not well-precedented. Unfortunately, they were all found to be highly unstable under acidic conditions. Exploration of carbamoylmethylene phosphate prodrugs led to the discovery of 2 as a clinical prodrug of 1. Prodrug 2 is stable and more soluble than 1 under both acidic and neutral conditions. It provided significantly improved exposures of parent 1 in vivo compared to the administration of 1 itself, especially within higher dose ranges. It demonstrated similar efficacy to 1 in the rat LPS-induced TNFα model and the rat adjuvant arthritis model. Most importantly, prodrug 2 did not show pH dependent exposure of 1 when administrated with famotidine in rat and human (clinical data not disclosed yet). In vitro and in vivo studies suggest that 2 is bio-converted into 1 in a stepwise fashion. ALP hydrolyzes 2 to intermediate 27, which is then hurther converted by esterases. Both steps take place without 2 and 27 being absorbed, resulting in neither of them being present in systemic circulation.

Experimental Section Chemistry. All reagents were purchased from commercial sources and used without further purification unless otherwise noted. All reactions involving air or moisture sensitive reagents were performed under an inert atmosphere. Proton and carbon magnetic resonance (1H and 13C NMR) spectra were recorded either on a Bruker Avance 400 or a JEOL Eclipse 500 spectrometer and are reported in ppm relative to the reference solvent of the sample in which they were run. HPLC and LCMS analyses were conducted using a Shimadzu LC-10AS liquid chromatograph and a SPDUV-vis detector at 220 or 254 nm with the MS detection performed with a Micromass Platform LC spectrometer. HPLC analyses were performed using the following conditions: Ballistic YMC S5 ODS 4.6 mm x 50 mm column with a binary solvent ACS Paragon Plus Environment

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system where solvent A = 10% methanol, 90% water, 0.2% phosphoric acid, and solvent B = 90% methanol, 10% water, and 0.2% phosphoric acid, flow rate = 4 mL/min, linear gradient time = 4 min. All final compounds had an HPLC purity of 95% or better unless specifically mentioned. LCMS analyses were performed using the following conditions: Phenomenex 5 µm C184.6 mm x 50 mm column with a binary solvent system where solvent A = 10% methanol, 90% water, 0.1% trifluoroacetic acid, and solvent B = 90% methanol, 10% water, and 0.1% trifluoroacetic acid, flow rate = 4 mL/min, linear gradient time = 2 min. Preparative reverse-phase HPLC purifications were performed using the following conditions: Ballistic YMC S5 ODS 20 mm x 100 mm column with a binary solvent system where solvent A = 10% methanol, 90% water, 0.1% trifluoroacetic acid, and solvent B = 90% methanol, 10% water, and 0.1% trifluoroacetic acid, flow rate = 20 mL/min, linear gradient time = 10 min.

4-((5-(Cyclopropylcarbamoyl)-2-methylphenyl)amino)-5-methyl-N-((phenylthio)methyl)-Npropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide (7). To 4-((5-(cyclopropylcarbamoyl)-2methylphenyl)amino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid 64 (713 mg, 1.95 mmol) was added SOCl2 (6 mL). The mixture was stirred for 3 h, and then the volatiles were removed under vacuum. To the residue was added heptane (20 mL), the resulting suspension was stirred for 30 min. The corresponding acid chloride (809 mg) was collected as a white solid by filtration and dried under high vacuum. To a suspension of the acid chloride (617 mg, 1.47 mmol) and intermediate 9 (270 mg, 1.47 mmol) in dichloromethane (10 mL) at 0 °C was added N,N-diisopropylethylamine (DIPEA) (1.0 mL, 5.9 mmol). The mixture was stirred at rt for 18 h and then diluted with dichloromethane (5 mL). Insoluble solids were removed by filtration, and the filtrate was concentrated under vacuum and purified directly on silica gel (50 - 100% ethyl acetate/dichloromethane) to furnish the title compound (405 mg, 77% yield) as a white solid. LCMS (M + H)+ = 529.29. N-((Phenylthio)methyl)propan-1-amine hydrochloride (9). To a solution of 1 N HCl in diethyl ether (6.6 mL, 6.6 mmol) at -35 oC was added dropwise 1,3,5-tri-n-propyl triazine 8 (427 mg, 2 mmol) in ACS Paragon Plus Environment


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diethyl ether (3 mL). The mixture was stirred for 10 minutes before benzene thiol (0.65 mL, 6.4 mmol) was added. The entire mixture was stirred for 5 min, then allowed to warm to room temperature and stirred for 8 h. The title compound (1.10 g, 84% yield) was collected as a white solid by filtration and rinsed with diethyl ether. The product was contaminated with n-propylamine hydrochloride. 1H NMR (chloroform-d, 400 MHz): δ 9.94 (br s, 2H), 7.62 (d, J = 1.6 Hz, 2H), 7.36 (m, 3H), 4.39 (s, 2H), 2.96 (br s, 2H), 1.18 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). Diisopropylethylamonium (4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5-methyl-Npropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamido)methyl dihydrogenphosphate (3). This compound was prepared according to the procedure described for 5. LCMS (M + H)+ = 517.04. 1H NMR (methanol-d4, 400 MHz): δ 7.83 (m, 1H), 7.61 (m, 2H), 7.28 (m, 2H), 5.04 (br s, 2H), 3.60 (m, 4H), 3.13 (m, 2H), 2.74 (br s, 1H), 2.56 (s, 3H), 2.24 (s, 3H), 1.65 (m, 2H), 1.27 (m, 15H), 0.90 (m, 3H), 0.70 (m, 2H), 0.53 (m, 2H). 4-((5-(Cyclopropyl(2,4,6-trimethoxybenzyl)carbamoyl)-2-methylphenyl)amino)-5-methyl-Npropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide (11). A mixture of 4-methyl-3-((5-methyl-6(propylcarbamoyl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)amino)benzoic acid 104 (0.93 g, 2.53 mmol), 1hydroxybenzotriazole (HOBt) (0.40 g, 2.9 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.61 g, 3.2 mmol) in DMF (8 mL) was stirred at rt for 30 min. Then, a solution of intermediate 15 (0.95 g, 4.0 mmol) and diisopropylethylamine (0.46 mL, 2.7 mmol) in DMF (1 mL) was added. The mixture was stirred at rt for 1 h and then slowly added to a well-stirred solution of saturated sodium bicarbonate and water (50/50, 75 mL). The title compound (1.22 g, 78% yield) was collected as a white solid by suction filtration, washed with water, and dried under vacuum. LCMS (M + H)+ = 587.33. 1H NMR (DMSO-d6, 400MHz,): δ 8.55 (s, 1H), 8.13 - 8.05 (m, 2H), 7.77 (s, 1H), 7.63 (s, 1H), 7.37 - 7.24 (m, 2H), 6.28 - 6.19 (m, 2H), 4.53 (br s, 2H), 3.77 (s, 3H), 3.74 (s, 6H), 3.18 (q, J = 6.3 Hz, 2H), 2.82 (s, 3H), 2.68 (m, 1H), 2.25 (s, 3H), 1.59 - 1.46 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H), 0.51 0.34 (m, 3H). ACS Paragon Plus Environment

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4-((5-(Cyclopropyl(2,4,6-trimethoxybenzyl)carbamoyl)-2methylphenyl)((methylthio)methyl)amino)-5-methyl-N-propylpyrrolo[2,1-f][1,2,4]triazine-6carboxamide (12). A mixture of 11 (470 mg, 0.80 mmol), NaI (120 mg, 0.80 mmol), and NaH (60% oil dispersion, 70 mg, 1.76 mmol), in THF (5 mL) were stirred at rt for 15 min. Then, chloromethyl methyl sulfide (0.07 mL, 0.90 mmol) was added. The resulting mixture was stirred at rt for 1.5 h. The reaction was quenched with crushed ice and extracted with ethyl acetate (3 x 15 mL). The combined organic extract was washed with brine, dried over sodium sulfate, and concentrated in vacuo to give a yellow foam (860 mg). The crude product was further purified by flash chromatography using silica gel column eluted with 2% methanol/dichloromethane to afford the title compound (173 mg, 33% yield) as a light cream solid. LCMS (M + H)+ = 647.24. 1H NMR (DMSO-d6, 500 MHz): δ 8.14 (s, 1H), 8.10 (s, 1H), 7.90 (br s, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.31 (m, 1H), 6.83 (br s , 1H), 6.16 (s, 2H), 4.90 - 5.10 (br m, 2H), 4.36 (m, 2H), 3.78 (s, 3H), 3.60 (s, 6H), 3.30 (m, 1H), 3.01 (m, 2H), 2.33 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.39 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H), 0.40-0.10 (m, 4H). 4-((5-(Cyclopropylcarbamoyl)-2-methylphenyl)((methylthio)methyl)amino)-5-methyl-Npropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide (13). To a solution of 12 (310 mg, 0.48 mmol) in dichloromethane (5 mL) at rt was added TFA (0.40 mL), and the resulting solution was stirred for 10 min. The mixture was poured into saturated aqueous sodium bicarbonate (15 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with brine, dried over sodium sulfate, and concentrated in vacuo. The residue (a tan solid) was triturated with dichloromethane (1 mL), ethyl acetate (1 mL) and hexanes (6 mL) to afford the title compound (208 mg, 91% yield) as a pale solid. LCMS (M + H)+ = 467.27. 1H NMR (400MHz, chloroform-d) δ 8.01 (s, 1H), 7.76 (s, 1H), 7.60 (dd, J = 7.6, 1.5 Hz, 1H), 7.49 (br s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 6.22 (br s, 1H), 5.72 (br. t, 1H), 5.02 (br s, 2H), 3.24 (m, 2H), 2.77 (m, 1H), 2.25 (s, 3H), 1.80 (s, 3H), 1.66 - 1.51 (m, 5H), 0.87 (t, J = 6.6 Hz, 3H), 0.85 (m, 2H), 0.52 (m, 2H).



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Sodium ((5-(cyclopropylcarbamoyl)-2-methylphenyl)(5-methyl-6-(propylcarbamoyl)pyrrolo[2,1f][1,2,4]triazin-4-yl)amino)methyl hydrogenphosphate (4). To a slurry of 13 (100 mg, 0.21 mmol) in dichloromethane (2 mL) at 0 °C was added sulfuryl chloride (1.0 M in dichloromethane, 0.19 mL) slowly, and the resulting mixture was stirred at 0 °C for 10 min. Additional sulfuryl chloride (1.0 M in dichloromethane, 0.040 mL) was added, and the mixture was stirred for another 10 min. The resulting mixture was then added via cannula into a flask containing a well-stirred mixture of tetrabutylammonium dihydrogen phosphate (713 mg, 2.1 mmol) and 4 angstrom molecular sieves in dichloromethane (2 mL) at 0°C. After being stirred at 0 °C for 30 min and rt for 1 h, the mixture was concentrated in vacuo and the resulting tan foam was partitioned between saturated aqueous sodium bicarbonate (5 mL) and ethyl acetate (30 mL). The layers were separated and the aqueous portion was extracted with ethyl acetate (2 x 5 mL). The aqueous portion containing the product was further concentrated in vacuo at rt to remove any residual organic solvents. The aqueous layer was loaded onto a C18 reverse phase column and eluted with a gradient of 0-20% acetonitrile in water. Fractions containing the uv-active product were combined and lyophilized to afford a tetrabutylammonium hydrogen phosphate intermediate (54 mg) as a pale solid. LCMS (M + H)+ = 517.04. The tetrabutylammonium hydrogen phosphate was dissolved in water (4 mL) and stirred with ion exchange resin (6 g of Dowex 500WX8-100, prewashed with water, methanol and water, then treated with 1N aqueous sodium hydroxide for 1 h, washed with water to neutral pH) for 10 min. The resin was removed by filtration and washed with water (2 x 2 mL). The aqueous filtrate was purified on a C18 reverse phase column eluted with a gradient of 0-20% acetonitrile in water. The fractions containing the uv-active product were combined and lyophilized to afford the title compound (19 mg, 16% yield) as a pale solid. LCMS (M + H)+ = 517.04. 1H NMR (methanol-d4, 500 MHz) ): δ 8.03 (s, 1H), 8.01 (s, 1H), 7.88 (s, 1H), 7.72 (d, J = 9.9 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 5.78 (d, J = 4.4 Hz, 2H), 3.26 (t, J = 6.9 Hz, 2H), 2.89 - 2.75 (m, 1H), 2.18 (s, 3H), 1.88 (br s, 3H), 1.68 - 1.52 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H), 0.81 0.74 (m, 2H), 0.71 - 0.63 (m, 2H). ACS Paragon Plus Environment

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N-(2,4,6-Trimethoxybenzyl)cyclopropanamine (15). To a solution of 2,4,6-trimethoxybenzaldehyde 14 (10.0 g, 51.0 mmol) and cyclopropylamine (5.3 mL, 76.0 mmol) in dichloromethane at 0 oC was added sodium triacetoxyborohydride in one portion, followed by acetic acid (15 mL) dropwise. The mixture was stirred at 0 oC for 2 h and at rt for 1 h. To the mixture at 0 oC was slowly added aqueous sodium hydroxide (6 N, 70 mL), and the mixture was stirred at 0 oC for 1 h. Then, aqueous HCl (1 N, 20 mL) was added and the mixture was stirred for 1h. Two layers were separated and the aqueous portion was extracted with dichloromethane (2 x 100 mL). The combined extract was dried over sodium sulfate and concentrated in vacuo to give a clear oil as the crude product. The crude pruduct was further purified by silica gel column chromatography eluting with 10% methanol in dichloromethane to afford the title compound (12.0 g, 95% yield) as a nearly clear oil, which solidified upon standing. LCMS (M + H)+ = 238.2. 1H NMR (DMSO-d6, 400 MHz): δ 6.22 (s, 2H), 4.24 (br s, 1H)), 3.77 (s, 9H), 3.70 (s, 2H), 2.04 (m, 1H), 0.36 (m, 2H), 0.26 (m, 2H). 4-((5-Cyclopropyl((methylthio)methyl)carbamoyl)-2-methylphenyl)amino)-5-methyl-Npropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide (16). To a solution of 1 (0.50 g, 1.2 mmol) in DMF (4 mL) at rt was added sodium hydride (60% dispersion in mineral oil, 0.25 g, 6.2 mmol). The mixture was stirred for 15 min before chloromethyl methyl sulfide (0.15 mL, 1.84 mmol) was added. The mixture was stirred at rt for 1.5 h, cooled in an ice bath, diluted with ethyl acetate (30 mL), and water (20 mL) was dropwise added. The resulting layers were separated and the aqueous portion was extracted with ethyl acetate (2 x 20 mL). The combined extract was diluted with hexanes (~ 20 mL), washed with water (5 x 15 mL) and brine (15 mL), and dried over anhydrous sodium sulfate. After solvent was removed under vacuum, the residue was purified by flash chromatography on silica gel using 60 to 70% ethyl acetate in hexanes to afford the title compound (0.20 g, 35% yield) as a tan solid. LCMS (M + H)+ = 467.0. 1H NMR (chloroform-d, 400 MHz): δ 8.38 (s, 1H), 7.88 (s, 1H), 7.75 (s, 1H), 7.37 - 7.28 (m, 3H), 5.90 (br s, 1H), 4.76 (s, 2H), 3.43 (m, 2H), 3.06 (m, 1H), 2.94 (s, 3H), 2.42 (s, 3H), 2.27 (s, 3H), 1.67 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H), 0.79 (m, 2H), 0.59 (m, 2H). ACS Paragon Plus Environment


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(N-Cyclopropyl-4-methyl-3-((5-methyl-6-(propylcarbamoyl)pyrrolo[2,1-f][1,2,4]triazin-4yl)amino)benzamido)methyl dihydrogen phosphate (5). To a solution of 16 (150 mg, 0.32 mmol) in dichloromethane (1.5 mL) at rt was added thionyl chloride (47 µL, 0.64 mmol). The mixture was stirred at rt for 2 h, then transferred via pipette into a well-stirred mixture of 85% H3PO4 (0.26 mL, 3.85 mmol) and diisopropylethylamine (3.3 mL, 19.3 mmol) in acetonitrile (4.5 mL). The mixture was stirred at rt for 15 min and then concentrated on a rotary evaporator. The resulting solids were partitioned between water (7 mL) and ethyl acetate (30 mL). The layers were separated, and the aqueous portion was extracted with ethyl acetate (2 x 10 mL). The resulting aqueous layer containing the product was purified by chromatography on a C-18 column (2 x 2 cm) eluting initially with water followed by a gradient elution from 5 to 15% aqueous acetonitrile. Fractions containing the uv-active product were lyophilized to afford the title compound (25 mg, 12% yield) as an off-white solid. LCMS (M + H)+ = 517.04. 1H NMR (methanol-d4, 400 MHz): δ 8.10 (s, 1H), 7.75 (s, 1H), 7.58 (s, 1H), 7.27 (m, 2H), 5.06 (m, 2H), 3.62 (m, 2H), 3.20 - 3.30 (m, 4H), 2.89 (m, 1H), 2.73 (s, 3H), 2.20 (s, 3H), 1.53 (m, 2H), 1.27 (m, 15H), 0.89 (t, J = 7.4 Hz, 3H), 0.71 (m, 4H). tert-Butyl 4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)imino)-5-methyl-6(propylcarbamoyl)pyrrolo[2,1-f][1,2,4]triazine-3(4H)-carboxylate (19). To a solution of 1 (4.06 g, 10.0 mmol) in DMF (34 mL) at 0 oC were added Boc anhydride (4. 37 g, 20. 0 mmol) and DMAP (0.26 g, 2.1 mmol). After 5 min. , the cold bath was removed, and the mixture was stirred at room temperature for 10 min. and then at 60 oC for 75 min. Upon cooling to rt, the mixture was slowly poured into water (300 mL). The precipitate was collected by suction filtration, triturated with hexanes, and dried to give the title compound (3.52 g, 70% yield) as a light yellow solid. LCMS (M + H)+ = 507.19. 1H NMR (DMSO-d6) δ 8.17 (d, J = 4.0 Hz, 1H), 8.11 (s, 1H), 7.96 (t, J = 5.6 Hz, 1H), 7.86 (s, 1H), 7.42 (dd, J = 6.8, 1.5 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 1.4 Hz, 1H), 3.17 (m, 2H), 2.82 (m, 1H), 2.66 (s, 3H), 2.23 (s, 3H), 1.52 (m, 2H), 1.09 (s, 9H), 0.90 (t, J = 7.4 Hz, 3H), 0.69 (m, 2H), 0.52 (m, 2H); 13C


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NMR (DMSO-d6) δ 166.63, 163.13, 146.61, 146.03, 137.57, 133.65, 132.78, 132.36, 129.93, 122.00, 120.83, 120.02, 118.54, 117.15, 114.46, 85.52, 40.09, 26.30, 22.66, 22.23, 18.00, 11.21, 5.51. Anal. Calcd for C27H34N6O4: C, 64.01; H, 6.76; N, 16.58. Found: C, 63.98; H, 6.71; N, 16.58. tert-Butyl 6-(((chloromethoxy)carbonyl)(propyl)carbamoyl)-4-(5-(cyclopropylcarbamoyl)-2methylphenylimino)-5-methylpyrrolo[1,2-f][1,2,4]triazine-3(4H)-carboxylate (20). To a solution of 19 (10.00 g, 19.7 mmol) in THF (200 mL) at -78 oC was added lithium bis(trimethylsilyl)amide (1M in THF, 40.0 mL, 40.0 mmol) over 30 min. The cold bath was removed, and the reaction mixture was allowed to warm to -45 oC and stirred at the temperature for 30 min. The mixture was then cooled to -78 o

C again before chloromethyl carbonochloridate (2.0 mL, 22.5 mmol) in THF (4.4 mL) was added. One

hour later, the reaction mixture was diluted with ethyl acetate and water and allowed to warm to rt. Layers were separated, and the organic layer was washed with brine and dried over Na2SO4. After the solvent was removed under vacuum, the residue was applied to silica gel chromatography to afford the title compound as a yellow foam (9.00 g, 76% yield). LCMS (M + H)+ = 599. 39. 1H NMR (DMSO-d6) δ 8.15 (d, J = 3.8 Hz, 1H), 8.13 (s, 1H), 7.79 (s, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.28 (m, 2H), 5.89 (s, 2H), 3.66 (d, J = 7.4 Hz, 2H), 2.81 - 2.78 (m, 1H), 2.55 (s, 3H), 2.22 (s, 3H), 1.65 - 1.60 (m, 2H), 1.07 (s, 9H), 0.88 (t, J = 7.4 Hz, 3H), 0.69-0.65 (m, 2H), 0.52-0.48 (m, 2H); 13C NMR (DMSO-d6) δ 166.75, 152.33, 146.61, 146.03, 138.52, 133.55, 132.96, 132.56, 130.15, 123.18, 122.37, 120.77, 119.17, 117.70. 114.60, 85.91, 71.39, 47.29, 26.46, 22.82, 21.75, 18.15, 11.15, 10.99, 5.69. Anal. Calcd for C29H35ClN6O6: C, 58.14; H, 5.88; Cl, 5.91; N, 14.02. Found: C, 58.20; H, 5.73; Cl, 6.01; N, 14.02. Iodomethyl 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1, 2-f][1, 2, 4] triazine-6-carbonyl(propyl)carbamate (21). A mixture of 20 (7.45 g, 12.4 mmol) and sodium iodide (1.62 g, 49.8 mmol) in acetone (115 mL) was heated at relux for 130 min. The insoluble material generated during the reaction was removed by suction filtration. The filtrate was concentrated to remove acetone. The residue was diluted with ethyl acetate (550 mL), washed with saturated sodium thiosulfate ACS Paragon Plus Environment


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solution (2 x 100 mL) and brine (100 mL), and dried over anhydrous MgSO4. Removal of solvent under vacuum provided crude product 21 (6.65 g) as pale yellow solid, which was directly used in the next step. ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1, 2-f][1, 2, 4] triazine-6carbonyl)(propyl) carbamoyloxy)methyl-2-(4-(bis(benzyloxy)phosphoryloxy)phenyl)acetate (22). A mixture of 21 (6.65g, ≤ 11.3 mmol) and silver 2-(4 (bis(benzyloxy)phosphoryloxy)phenyl)acetate 26 (10.0 g, 19.2 mmol) in toluene (450 mL) was heated at 65 oC for 30 min. The solid phase was removed by suction filtration through Celite 545. The filtrate was concentrated and the residue was subjected to chromatography (silica gel, 20-60% ethyl acetate/dichloromethane) to provide the title compound (3.05 g, 31% over 2 steps) as a pale yellow solid. LCMS (M + H)+ = 875.21. 1H NMR (chloroform-d) δ 8.32 (d, J = 1.2 Hz, 1H), 7.89 (s, 1H), 7.65 (s, 1H), 7.55 (dd, J = 7.9, 1.7 Hz, 1H), 7.34 - 7.28 (m, 11H), 7.25 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.41 (br s, 1H), 5.73 (s, 2H), 5.09 (s, 2H), 5.07 (s, 2H), 3.77 (t, J = 7.5 Hz, 2H), 3.57 (s, 2H), 2.90 (m, 1H), 2.72 (s, 3H), 2.36 (s, 3H), 1.68 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H), 0.86 (m, 2H), 0.63 (m, 2H); 13C NMR (CDCl3) δ 169.72, 168.21, 167.79, 154.74, 153.65, 149.87, 148.71, 135.63, 135.33, 134.84, 133.38, 130.98, 130.58, 129.65, 128.67, 128.60, 128.01, 124.55, 123.38, 120.77, 120.26, 119.90, 113.46, 113.06, 80.82, 69.98, 47.80, 39.97, 23.17, 22.29, 18.35, 11.87, 11.28, 6.77. Anal. Calcd for C46H47N6O10P: C, 63.15; H, 5.41; N, 9.60; P, 3.54. Found: C, 63.29; H, 5.27; N, 9.56; P, 3.31. ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1, 2-f][1, 2, 4]triazine-6carbonyl)(propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (2). A mixture of 22 (2.50 g, 2.86 mmol) and 10% Pd/C (0.50 g) in MeOH (200 mL) and THF (50 mL) was stirred under H2, provided with a H2 balloon, for 40 min. The solid phase was removed by suction filtration through Celite 545. The filtrate was concentrated under vacuum to dryness. To the residue was added 5% methanol/ethyl acetate (50 mL), and the heterogeneous mixture was stirred at rt for 30 min. The title compound (1.79 g, 28% yield over 3 steps) was collected as a white solid by suction filtration. LCMS ACS Paragon Plus Environment

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(M + H)+ = 695. 42. 1H NMR (2:1 of DMSO-d6/methanol-d4) δ 7.72 (d, J = 1.7 Hz, 1H), 7.68 (s, 1H), 7.59 (s, 1H), 7.57 (dd, J = 7.7, 1.7 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 5.65 (s, 2H), 3.64 (t, J = 7.2 Hz, 2H), 3.57 (s, 2H), 2.78 (m, 1H), 2.62 (s, 3H), 2.20 (s, 3H), 1.57 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H), 0.67 (m, 2H), 0.54 (m, 2H); 13C NMR (2:1 of DMSOd6/methanol-d4) δ169.55, 168.07, 167.25, 152.95, 150.3, 145.35, 137.78, 137.18, 132.28, 129.96, 129.88, 128.87, 124.36, 120.07, 119.97, 119.64, 115.48, 113.23, 80.19, 46.83, 38.56, 22.28, 21.39, 16.78, 10.37, 10.05, 4.87. Anal. Calcd for C32H35N6O10P•0.72 H2O: C, 54.32; H, 5.19; N, 11.88; P, 4.38. Found: C, 54.30; H, 4.96; N, 11.86; P, 4.24. Methyl 2-(4-(bis(benzyloxy)phosphoryloxy)phenyl)acetate (24). To a solution of methyl 2-(4hydroxyphenyl)acetate 23 (1.89 g, 11.4 mmol) in acetonitrile (90 mL) at -10 oC was added CC14 (5.5 mL, 57.0 mmol), DIPEA (4.16 mL, 23.9 mmol) and DMAP (139 mg, 1.14 mmol). The mixture was stirred for 10 mins before dibenzyl phosphite (3.7 mL, 16.5 mmol) was slowly added to keep the temperature below -10 oC. The resulting mixture was stirred at -10 oC for l h, quenched with aqueous K2HPO4 (0.5 M, 20 mL) in acetonitrile (60 mL), allowed to warm to rt, and extracted with ethyl acetate (3 x 150 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, and concentrated under vacuum. The residue was subjected to chromatography (120 g silica gel, 30-50% ethyl acetate/hexanes) to give the title compound (4.3 g, 88% yield) as a colorless oil. 2-(4-(Bis(benzyloxy)phosphoryloxy)phenyl)acetic acid (25). To a solution of 24 (11.65 g, 27.3 mmol) in THF (110 mL) and MeOH (36 mL) at 0 oC was added a solution of lithium hydroxide (0.981 g, 41.0 mmol) in water (38 mL) over 15 min. The mixture was stirred at 0 oC for 2 h before it was acidified with 1 N HCl to pH 2. The mixture was concentrated under vacuum and then extracted with ethyl acetate (3 x 60 mL). The combined extract was washed with brine and dried over anhydrous MgSO4. Removal of solvent under vacuum provided the title compound (11.25 g, 100% yield) as a colorless oil. 1H NMR (DMSO-d6) δ 12.38 (br s, 1H), 7.38 (m, 10H), 7.26 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 5.15



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(d, J = 8.3 Hz, 4H), 3.57 (s, 2H); 13C NMR (DMSO-d6) δ 172.97, 149.24, 142.46, 135.98, 132.47, 131.22, 128.88, 128.36, 120.13, 69.74, 48.97. Silver 2-(4-(bis(benzyloxy)phosphoryloxy)phenyl)acetate (26). To a suspension of 25 (11.25 g, 27.28 mmol) in water (350 mL) at 0 oC was added sodium hydroxide solution (1.00 M, 29.5 mL, 29.5 mmol) over 10 min. The mixture was stirred at 0 oC for 30 min before a solution of AgNO3 (5.33 g, 31.4 mmol) in water (30 mL) was added over 10 min. The resulting mixture was stirred at 0 oC for 40 min. To the mixture was added diethyl ether (50 mL), and the title compound (12.6 g, 89% yield) was collected as a grey solid by suction filtration. Bio-conversion of 2 into 1 in rats. After overnight fast, three groups of male Sprague-Dawley rats (219-277 g) (N = 3 per group) received prodrug 2 by oral gavage (1, 10 and 100 mg/kg). Serial blood samples (~0.3 mL) were obtained from the jugular vein pre-dose and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, and 24 h post-dose. Plasma samples, obtained by centrifugation at 4 °C (1500-2000xg), were stored at -20 °C until analysis by LC/MS/MS. Bio-conversion of 2 into 1 in monkeys. After overnight fast, three male cynomolgus monkeys (4.2 to 5.2 kg) received prodrug 2 by oral gavage (10 or 30 mg/kg, 5 mL/kg of methocel suspension), with a one-week washout between dosing. Serial blood samples (~0.3 mL) were collected from a femoral artery pre-dose and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, and 24 h post-dose, and centrifuged at 4 °C (15002000xg) to obtain plasma. Plasma samples were stored at -20 °C until analysis by LC/MS/MS. Rat LPS-Induced TNFα inhibition. Male Lewis rats (Harlan; 200-250g) were treated with control vehicle or compound 1 h prior to IP injection with 10ug lipopolysaccharide (LPS, E. coli O111:B4; Sigma) in 1.0 mL sterile saline. Animals were dosed orally with prodrug 2 as an aqueous suspension in 0.75% Methocel, 0.1% Tween 80, whereas 1 was given to the animals orally utilizing PEG 300 as a vehicle. The different vehicles were used because of the differences in the solubility of the two compounds. Ninety minutes after LPS injection, a blood sample was obtained and serum was separated.


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Serum TNF levels were determined by commercial enzyme-linked immunosorbent assay (ELISA; BioSource). Rat adjuvant arthritis model. Male Lewis rats (Harlan, 175-225g) were immunized SC at the base of the tail with 0.1ml complete Freund’s adjuvant containing 10mg/ml M. butyricum. Seven to 10 days later, baseline (pre-disease) measurements of hind paw volume were determined by volume displacement plethysmometry (Ugo Basile, Italy). Compounds were administered BID in 0.5 mL vehicle beginning on day 11. Animals were dosed orally with prodrug 2 as an aqueous suspension in 0.75% Methocel, 0.1% Tween 80, whereas 1 was given to the animals orally utilizing PEG 300 as a vehicle. Paw volume measurements were repeated 3 times per week for the remainder of the study. In vitro hydrolysis of 2 by alkaline phosphatase. Human placental ALP (Sigma, St. Louis, MO; Cat. #: P3895) (1 unit/mL) was incubated with 2 (10 µM) in tris buffer (89 mM, pH 7.5) at 37 oC in a final volume of 1 mL. Reactions (N = 3) were initiated with the addition of the test compound and aliquots (100 µL) were taken at 0 (immediately after mixing), 15, 30, and 60 min. For inhibition studies, HALT phosphatase inhibitor cocktail (10 µL) (Pierce, Rockford, IL; Cat. #: GH100187) was added with 100 x dilution prior to the addition of test compounds. Incubation mixtures were collected into Eppendorf tubes containing 10 mM EDTA (20 µL). After vortexing, acetonitrile (200 µL) was added and the mixture was centrifuged at 4 oC for 5 min with a bench top centrifuge. After centrifugation, samples were collected, transferred to a 96-well sample plate, and analyzed immediately using LC/MS/MS. In vitro hydrolysis of 2 and 27 by esterase. Porcine liver esterase (Sigma, St. Louis, MO; Cat. #: E3019) was incubated with either prodrug 2 (10 µM) or intermediate 27 (10 µM) in potassium phosphate buffer (89 mM, pH 7.4) at 37 oC in a final volume of 1 mL. Reactions were initiated with the addition of test compounds and aliquots (200 µL) were taken at 0, 15, 30 and 60 min. Acetonitrile (200 µL) was added, and the mixture was centrifuged at 4 oC for 5 min with a bench top centrifuge. After centrifugation, samples were collected, and transferred to a 96-well sample plate, and analyzed immediately using LC/MS/MS. ACS Paragon Plus Environment


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All studies involving animals were reviewed and approved by the BMS Institutional Animal Care and Use Committee.

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Figure 1. p38α inhibitor 1 and its clinical prodrug 2

H N

HN O

N N

nPr NH

O

N

1 (BMS-582949)

H N

HN O nPr N O

N N O

N

O OPO(OH) 2

O O 2 (BMS-751324)

Scheme 1a

a

Reagent and conditions: (a) (i) SOCl2, rt, 3 h, 98%, (ii) 9, N,N-diisopropylethylamine, CH2Cl2, rt, 18 h, 77%; (b) (i) SOCl2, rt, 2 h, (ii) 85% H3PO4, N,N-

diisopropylethylamine, rt, 15 min, 21%; (c) 1 N HCl in diethyl ether, PhSH, rt, 8 h, 84%.


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Scheme 2a

a

Reagent and conditions: (a) 15, HOBt, EDCI, N,N-diisopropylethylamine, DMF, rt, 1 h, 78%; (b) NaH, MeSCH2Cl, NaI, THF, rt, 1.5 h, 33%; (c) TFA,

CH2Cl2, rt, 10 min, 91%; (d) (i) SO2Cl2, CH2Cl2, 0 oC, (ii) tetrabutylammonium dihydrogen phosphate, 4Å molecular sieves, 0 oC for 0.5 h, and then rt for 1 h, 33% over 2 steps, (iii) Dowex500WX8-100, NaOH, 50%; (e) c-PrNH2, NaBH(OAc)3, HOAc, CH2Cl2, 0 oC for 2 h, and then rt for 1 h, 95%.



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Scheme 3a

a

Reagent and conditions: (a) NaH, MeSCH2Cl, NaI, DMF, rt, 1.5 h, 35%; (b) (i) SOCl2, rt, 2 h, (ii) 85% H3PO4, N,N-diisopropylethylamine, MeCN, rt, 15

min, 12% over 2 steps. Table 1. Solution stability profiles of 3, 4, and 5 at 37 oC compd 3 4

Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5

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