Discovery of Novel, Highly Potent, and Selective Quinazoline-2

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Discovery of Novel, Highly Potent, and Selective Quinazoline-2carboxamide-Based Matrix Metalloproteinase (MMP)-13 Inhibitors without a Zinc Binding Group Using a Structure-Based Design Approach Hiroshi Nara,* Kenjiro Sato, Takako Naito, Hideyuki Mototani, Hideyuki Oki, Yoshio Yamamoto, Haruhiko Kuno, Takashi Santou, Naoyuki Kanzaki, Jun Terauchi, Osamu Uchikawa, and Masakuni Kori Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan S Supporting Information *

ABSTRACT: Matrix metalloproteinase-13 (MMP-13) has been implicated to play a key role in the pathology of osteoarthritis. On the basis of X-ray crystallography, we designed a series of potent MMP-13 selective inhibitors optimized to occupy the distinct deep S1′ pocket including an adjacent branch. Among them, carboxylic acid inhibitor 21k exhibited excellent potency and selectivity for MMP-13 over other MMPs. An effort to convert compound 21k to the mono sodium salt 38 was promising in all animal species studied. Moreover, no overt toxicity was observed in a preliminary repeat dose oral toxicity study of compound 21k in rats. A single oral dose of compound 38 significantly reduced degradation products (CTX-II) released from articular cartilage into the joint cavity in a rat MIA model in vivo. In this article, we report the discovery of highly potent, selective, and orally bioavailable MMP-13 inhibitors as well as their detailed structure−activity data.

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INTRODUCTION Osteoarthritis (OA) is a primarily noninflammatory, degenerative joint disease characterized by progressive cartilage degradation that leads to pain and reduced mobility in affected joints. Current treatments are limited to symptomatic relief with nonsteroidal anti-inflammatory drugs (NSAIDs) or selective cyclooxygenase-2 (COX-2) inhibitors, intra-articular injections of hyaluronic acid, or surgical joint replacement. In addition, the COX-2 inhibitors have recently raised concerns regarding cardiovascular side effects,1 resulting in the withdrawal of coxibs (COX-2 inhibitors). Therefore, there is a significant unmet need for disease-modifying osteoarthritis drugs (DMOADs) that may be able to alter disease progression and that do not have any major side effects. The matrix metalloproteinases (MMPs) are a family of structurally related zinc-containing endopeptidases that are involved in the progression of OA. Among them, MMP-13 (collagenase-3) shows a substrate specificity favoring degradation of cartilage type II collagen,2,3 the main structural © XXXX American Chemical Society

component of the cartilage matrix, and is expressed at higher levels by OA chondrocytes than by normal chondrocytes,3,4 suggesting a crucial role in the destruction of articular cartilage during the advancement of OA. In preclinical testing, MMP inhibitors have shown inhibitory activity toward the destruction of cartilage in some animal models of OA.5 However, most clinical trials of broad-spectrum MMP inhibitors have been discontinued due to concerns of dose-limiting toxicity (skin rash and musculoskeletal side effects (MSS) characterized by joint stiffness and pain). While a number of hypotheses have been proposed for the cause of MSS, including the inhibition of specific MMPs such as MMP-1 or MMP-14 or of the related sheddases, the pharmacological basis for this side effect remains unknown.6−8 Therefore, considerable interest has been directed toward potent inhibitors of MMP-13 with a high degree of Received: June 28, 2014

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dx.doi.org/10.1021/jm500981k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structure of HTS lead 37 and MMP-13 selective inhibitor 21k.

selectivity over other MMPs, which may avoid undesirable side effects. Many of the MMP inhibitors reported to date are a class of the zinc binding inhibitors, which possess a motif capable of chelating the catalytic zinc ion9,10 and substituents recognizing subsites of the target enzyme, but there are very few studies of a non-zinc binding type of MMP-13 selective inhibitors.11−14 A new class of MMP inhibitors that does not possess a zinc chelating group and that binds effectively to an MMP-13 enzyme would be expected to have improved physicochemical properties and potentially increased selectivity. High-throughput screening (HTS) of an MMP-13 inhibition assay afforded a moderately potent (IC50 = 12 nM) quinazoline-2-carboxamide 37 (Figure 1), which was successfully cocrystallized with the MMP-13 catalytic domain. On the basis of the three-dimensional structure of the protein target, we applied a structure-based approach for designing MMP-13 selective inhibitors. For the inhibition of MMPs, an interaction often observed involves hydrogen bonding between the inhibitor and the main chain amino acid residues flanking the catalytic site, specifically Leu185 and Ala186. Herein, we report a series of quinazoline-2-carboxamides with greatly improved in vitro potency and bioavailability. On the basis of X-ray crystallography of the complex of lead compound 37 with MMP-13, we designed and synthesized a new series of potent MMP-13 selective inhibitors that are involved in interactions with both the distinct deep S1′ pocket and the adjacent side pocket. Among them, 4-[2-({6-fluoro-2-[({[3-(methyloxy)phenyl]methyl}amino)carbonyl]-4-oxo-3,4-dihydroquinazolin5-yl}oxy)ethyl]benzoic acid (21k) exhibited excellent potency (IC50 = 0.0039 nM) and selectivity (greater than 41 000-fold) over other MMPs (MMP-1, 2, 3, 7, 8, 9, 10, and 14 and TACE). X-ray analysis of the complex of 21k with MMP-13 confirmed that the compound is buried deep in the S1′ pocket by forming a β-sheet type interaction with hydrogen bonding to the enzyme’s backbone (Thr245 and Thr247) spanning the S1′ pocket, which extends into an additional S1′ side pocket (S1″ pocket) of MMP-13 without interacting with the catalytic zinc (Figure 2). Results from the in vitro safety assessment of these compounds (effect on hERG current, inhibition of the CYP isoforms, ATP as a marker for cell viability, phototoxicity, and mutagenic side effects) are also promising. Encouraged by these in vitro profiles, we next conducted further pharmacokinetic evaluation and in vivo toxicological assessment of 21k. This compound, however, showed low oral bioavailability in all tested species (rats, guinea pigs, rabbits, beagle dogs, and cynomolgus monkeys), likely due to poor solubility. In an attempt to improve the poor bioavailability, we selected the sodium salt of compound 38 to examine the effects of the MMP-13 selective inhibitor on articular cartilage in vivo; the

Figure 2. Schematic representation of binding interaction of 21k and MMP-13, as determined by X-ray crystallography. Inhibitor 21k occupies the S1′ site and its side pocket (S1″ pocket) of MMP-13 and does not undergo coordination with the catalytic zinc. Hydrogenbonding and ionic interactions of 21k with MMP-13 are depicted as dashed lines.

monoiodoacetate (MIA) induced OA model was utilized. In this experiment, 38 was protective against cartilage damage induced by MIA. Compound 38 showed significant inhibition (69%) of cartilage damage at a dose of 10 mg/kg po, comparable to that of the broad spectrum hydroxamate-type MMP inhibitor 39 (87% inhibition), and is considered to be a good candidate for continued study. In this article, the design, synthesis, and biological activity of novel non-zinc binding inhibitors are described.

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CHEMISTRY We have demonstrated that the key intermediate 4-oxo-3,4dihydroquinazoline-2-carboxylic acid ethyl esters 8 could be readily prepared from anthranilic acid amides 6 via a two-step acylation/cyclization sequence (method A, Scheme 1) or anthranilic acid esters 15 via a single-step cyclization (method B, Scheme 2). Oxazines prepared from substituted anthranilic acids 1a−d and triphosgene underwent aminolysis with ammonia to give the desired anthranilic acid amides 6d, 6h, 6j, and 6l in reasonable yields. Ortho-nitrobenzoic acid 2a was converted with oxalyl chloride into acyl chloride, which was reacted with ammonia to afford the carboxamide. Reduction of the nitro group of the nitrobenzamide gave anthranilic acid amide 6b. Nitrobenzonitriles 5a−c were synthesized from dinitrobenzonitriles 3a−b by condensing them with alkali metal alkoxides. Reaction of nitrobenzonitriles 5a−c with hydrazine over Raney nickel simultaneously converted the nitro group to the amine and the cyano group to the carboxamide to provide B



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Scheme 1. Synthesis of 4-Oxo-3,4-dihydroquinazoline-2-carboxamide Derivatives 9a−m (Method A)a

a Reagents and conditions: (a) (1) triphosgene, THF, 50 °C; (2) NH3, H2O, 50 °C, 13−75% over 2 steps; (b) (1) (COCl)2, DMF, THF, 0 °C to rt; NH3, H2O, rt, 56%; (2) H2, Pd on carbon, MeOH, rt, 99% (for 6b); (c) (1) BnBr, K2CO3, rt, 98%; (2) KOH, MeOH, H2O, reflux, 84%; (3) (COCl)2, DMF, THF, 0 °C to rt; NH3, H2O, rt, 93%; (4) Fe powder, NH4Cl, EtOH, H2O, reflux, 100% (for 6i); (d) MeONa, MeOH, reflux, 38− 99% (for 5a,c); (e) 2-phenylethanol, NaH, DMF, 90 °C, 32% (for 5b); (f) NaNO2, HCl, H2O, 0 °C to rt; CuCN, NaCN, toluene, H2O, 0 °C to reflux; (g) NH2NH2·H2O, Raney Ni, EtOH, 40 °C to reflux, 40−88% (for 6a, 6c, and 6m), 16% over 2 steps (for 6g); (h) ethyl chloroglyoxylate, Et3N, THF, 0 °C to rt, 79−100%; (i) NaOEt, EtOH, 0 °C to rt, 38−87% (for 8b and 8d−m); (j) p-TsOH, toluene, reflux, 22−27% (for 8a and 8c); (k) 3-methoxybenzylamine, DMF or EtOH, 80−90 °C, 28−92%.

anthranilic acid amides 6a, 6m, and 6c, respectively.15 Anthranilic acid amide 6g was prepared in the same manner using nitrobenzonitrile 5d that was obtained through a Sandmeyer cyanation sequence from 2-nitro-5-trifluoromethylphenylamine (4). N-Acylation of the anthranilic acid amides 6a−m with ethyl chloroglyoxylate, followed by treating the intermediates 7a−m with an appropriate acid or base, provided 4-oxo-3,4-dihydroquinazoline-2-carboxylic acid ethyl esters 8a− m. Incorporation of an amide moiety at the 2-position of 4-oxo3,4-dihydroquinazoline was achieved by aminolysis of 4-oxo3,4-dihydroquinazoline-2-carboxylic acid ethyl esters 8a−m with 3-methoxybenzylamine under heating in DMF or EtOH. The ester group at the 2-position is highly reactive toward

primary aliphatic amines to give the corresponding amide derivatives 9a−m. Common intermediates 8n and 8o were obtained from readily prepared anthranilic acid esters 15 in a single-step procedure (method B) employing ethyl cyanoformate and 1 M HCl/AcOH.16 Hydrolysis of the diester 10 by aqueous lithium hydroxide provided the corresponding monoacid 11, which was homologated via the Arndt−Eistert sequence to give the methyl ester 12 (Scheme 2).17 Catalytic reduction of the nitro group of 12 over palladium on carbon yielded the anthranilic acid ester 15a. Selective lithiation at the 2-position of 13 with 2 equiv of n-butyl lithium followed by nucleophilic addition to the ethyl chlorocarbonate afforded exclusively the 6-amino-2,3-difluorobenzoic acid ethyl ester 14 C



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Scheme 2. Synthesis of 4-Oxo-3,4-dihydroquinazoline-2-carboxamide Derivatives 9n−o (Method B)a

a

Reagents and conditions: (a) LiOH, H2O, THF, rt, 96%; (b) (1) (COCl)2, DMF, THF, rt; (trimethylsilyl)diazomethane, TEA, Et2O, THF, CH3CN, 0 °C; (2) MeOH, AgOBz, TEA, THF, rt, 67% over 2 steps; (c) H2, Pd on carbon, THF, MeOH, rt, 99%; (d) n-BuLi, ClCO2Et, THF, −78 °C; (e) HCl, AcOEt, rt, 70% over 2 steps; (f) NCCO2Et, HCl, AcOH, 80 °C, 76−82%; (g) 3-methoxybenzylamine, EtOH, 80 °C, 78−84%.

Scheme 3. Modification of Substituents at the C-6 Positiona

a

Reagents and conditions: (a) H2, Pd on carbon, THF, MeOH, rt, 100%; (b) EtI, Cs2CO3, DMF, THF, rt, 21%; (c) (1) N,N-dimethylthiocarbamoyl chloride, DABCO, DMF, rt, 96%; (2) N,N-diethylaniline, 210 °C, 82%; (3) KOH, MeOH, reflux, 95%; (d) MeI, Et3N, rt, 85%; (e) mCPBA, CHCl3, rt, 95%.

in fair yield.18 Removal of the Boc group from 14 yielded the free amine 15b. As described above in Scheme 3, the 6-benzyloxy derivative 9i was debenzylated by catalytic hydrogenation using palladium on carbon to give the phenol 16, which was then converted to the 6-ethoxy derivative 17 in 21% yield. This low yield was due to the competing alkylation of the pyrimidine ring. Alternatively, the above-described phenol 16 was converted to the O-arylthiocarbamate, which was then subjected to the thermal Newman−Kwart rearrangement19 to afford the S-

arylthiocarbamate. The intermediate was readily hydrolyzed to give the thiol 18, which subsequently alkylated with iodomethane to give 6-methylthio derivative 19a. Finally, sulfide oxidation transformed 19a to the sulfone 19b. Synthesis of inhibitors 20a and 20b was based on the versatile palladium-catalyzed coupling chemistry. Compounds 20a and 20b were synthesized from the common intermediate 9j, as shown in Scheme 4. To synthesize 5-substituted derivatives 21a−g, the 5-fluoro group of 9k was displaced via a nucleophilic aromatic D



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Scheme 4. Modification of Substituents at the C-6 Positiona

Reagents and conditions: (a) PhB(OH)2, Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O, reflux, 64% (for 20a); (b) Zn(CN)2, Pd(PPh3)4, DMF, 80 °C, 68% (for 20b).

a

Scheme 5. Nucleophilic Substitution Reaction of 5-Fluoro Derivatives 9k and 9oa

a

Reagents and conditions: (a) ROH or RSH, NaH, DMA, rt to 80 °C (for 21a−c and 21e−k); (b) RNH2, DMA, 80−100 °C (for 21d).

Scheme 6. Synthesis of 5-Substituted Derivatives 26a−da

Reagents and conditions: (a) 2-phenylethanol, NaH, THF, 90 °C, 97%; (b) (1) (COCl)2, DMF, THF, 0 °C to rt; (2) NaOH, H2O, THF, MeOH, rt, 90% over 2 steps; (c) Tf2O, pyridine, CH2Cl2, 0 °C to rt, 97%; (d) Zn(CN)2, Pd(PPh3)4, DMF, 150 °C under microwave, 82% (for 25a); (e) PhB(OH)2, Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O, reflux, 91% (for 25b); (f) PhOH, Pd2(dba)3, 2-(di-tert-butylphosphino)biphenyl, DIEA, toluene, 80 °C, 19% (for 25c); (g) (1) 3-phenyl-1-propyne, Pd(PPh3)4, CuI, DIEA, DMF, rt; (2) H2, Pd on carbon, THF, MeOH, rt, 21% over 2 steps (for 25d); (h) HCl, H2O, AcOH, 60 °C, 53−80%. a

E



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Scheme 7. Synthesis of Amino Linker Analogue 29a

a Reagents and conditions: (a) NBS, AIBN, CHCl3, reflux, 79%; (b) N-methylbenzylamine, pyridine, DMF, THF, 0 °C to rt, 59%; (c) 3methoxybenzylamine, EtOH, 80 °C, 8%.

Scheme 8. Synthesis of Ether Linker Analogue 34a

a Reagents and conditions: (a) SEMCl, NaH, DMF, rt, 77%; (b) NBS, AIBN, CHCl3, reflux, 40%; (c) benzyl alcohol, NaH, THF, 0 °C to rt; (d) TFA, CH2Cl2, 0 °C to rt, 30% over 2 steps; (e) 3-methoxybenzylamine, EtOH, 80 °C, 51%.

Scheme 9. Synthesis of Amide Linker Analogue 36a

a

Reagents and conditions: (a) NaOH, H2O, THF, MeOH, 80 °C, 87%; (b) WSCD·HCl, HOBt, DMAP, DMF, 50 °C, 81%.

substitution reaction with various nucleophiles (RONa, RSNa, RNH2) as shown in Scheme 5.20 Regioselective nucleophilic aromatic substitution of the 5-position on 5,6-difluoro derivative 9o could be carried out under milder conditions, providing the ethers 21h−k in moderate yields. Substitution by phenethyl alcohols led to the product of displacement in modest yield with competing elimination to styrene derivatives. Compounds 26a−d were prepared from a common intermediate 24, derived from the starting fluoride 9k. As shown in Scheme 6, a phenethyloxy substitution/β-elimination in one pot followed by DMF-aminal protection strategy was employed. Since the pyrimidine ring system and the side chain amide NH of 22 could participate in reactions that lead to side product formation, the synthesis required the generation of these two moieties in protected form before the desired triflation reaction could be carried out. Thus, a novel 5membered cyclic DMF-aminal was utilized as a protecting group to protect quinazolin-4-one-2-carboxamide 22 by forming an imidazolidine ring. This protecting group can be easily introduced under Vilsmeier conditions, is stable to bases

and nucleophiles, and can be removed efficiently by hydrochloric acid in acetic acid at 60 °C for 2−3 h. The obtained fully protected phenol 23 was then converted to the triflate 24. Palladium-catalyzed coupling of 24 with various nucleophiles afforded the coupling products 25a−d in moderate yields. Finally, the new protecting group was deprotected with hydrochloric acid in acetic acid to afford the desired 26a−d. In this way, effective protection of a pyrimidin-4-one-2carboxamide system has been demonstrated, and, as far as we know, no such cyclic DMF-aminal protecting group has been reported. Syntheses of 5-substituted 4-oxo-3,4-dihydroquinazoline with modified or replaced linker chain containing three atoms were accomplished via the corresponding 5-bromomethyl derivatives 27, 31, or 5-carboxymethyl derivative 35. As shown in Scheme 7, benzylic bromination of 5-methyl-4-oxo-3,4-dihydroquinazoline 8f was followed by reaction with secondary Nmethylbenzylamine to give 28, which was subsequently treated with primary 3-methoxybenzylamine to give compound 29. The synthesis of ether linker analogue 34 could not be carried F



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Scheme 10. Preparation of Salts of Compound 21ka

a

Reagents and conditions: NaHCO3 (1 equiv), DMSO, THF, MeOH, H2O, 80 °C; MeOH, reflux, 89%

Figure 3. Crystal structure of complex of 37 and MMP-13. (A) Surface representation of MMP-13 illustrating the binding cavity. The inhibitor is buried deep into the S1′ pocket. The figure was made with PyMOL.34 (B) Schematic representation of the binding mode of 37 and MMP-13. Hydrogen bonds are shown as dashed lines.

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out by the same method described above due to the formation of multiple unidentified byproducts. Thus, in Scheme 8, the pyrimidine nucleus of 8f was protected by suitable protecting group.21 Protection of a nitrogen at the 3-position of pyrimidine ring by SEM-Cl followed by benzylic bromination with NBS gave the bromide 31, which was reacted with benzyl alcohol to give 32. After removal of the SEM protecting group under acidic conditions, the active ester 33 was subjected to aminolysis by treatment with 3-methoxybenzylamine in EtOH to afford compound 34 in a similar manner as that for the preparation of 29 in Scheme 7. Another series of compounds that had an amide linker was synthesized as described in Scheme 9. Saponification of the ester function of 9n delivered the carboxylic acid 35, which was subjected to condensation with aniline, affording the amide linked compound 36. Sodium salts of compound 21k were prepared by treatment of 21k with 1 or 2 equiv of sodium hydrogen carbonate in an aqueous solvent as described in Scheme 10. In the initial preparation of sodium salts, we adopted a solvent system of THF/MeOH/H2O; however, this solvent system would not be amenable to being scaled up due to requirement of a large volume of solvent to dissolve carboxylic acid 21k. A practical and scalable procedure was investigated to prepare multikilogram batches of 38 (monosodium salt of 21k). The addition of a small amount of DMSO as a cosolvent was effective to dissolve 21k in a small volume of solvent. Thus, a mixed solvent system consisting of THF/MeOH/DMSO/H2O (8:2:1:2 v/v) was developed, which allowed a kilogram-scale preparation of 38.

RESULTS AND DISCUSSION

Compound 37 (Table 4) was identified by high-throughput screening as a novel MMP-13 selective inhibitor. Interestingly, the inhibitor did not possess an obvious zinc binding group, and this compound showed selectivity greater than 25-fold versus other MMP isoenzymes. An X-ray crystallographic study of compound 37 bound to MMP-13 revealed that the quinazoline ring is pointing toward the S1′ subsite (Figure 3). The structural basis for MMP-13 selective inhibition by lead compound 37 was analyzed by modeling studies based on X-ray crystallographic data of the 37−MMP-13 complex. From the results, the backbone NH of Met253, which is located in the deeper region of S1′ pocket, and its side pocket were selected as the first target for enhancing specific interactions with MMP13. Moreover, the 5- or 6-position of the quinazoline ring of compound 37 was predicted to be a suitable position to introduce substituents. Indeed, even introduction of a small methoxy substituent at the 7- or 8-position of the quinazoline ring resulted in a large decrease in activity in both compounds (9c and 9d). These results indicate that the spatial tolerance around the 7- or 8position of quinazoline ring has a narrow range in coordination to the enzyme. Therefore, the SAR studies were focused on the substitutions around the quinazoline core at the C-5 and C-6 positions. As shown in Table 1, substitution at the 6-position with a fluoro group (9e) retained inhibitory potency in comparison with that for unsubstituted lead compound 37. However, the methyl 9f or trifluoromethyl 9g derivatives showed a reduced inhibition of MMP-13. By contrast, substitution with a methoxy G



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that of the methylthio 19a or cyano 20b, which were isosteres for a methoxy group. In the C-6-substituted series of MMP-13 inhibitors, small substituents (fluoro, methoxy, ethoxy, methylthio, and cyano group), which are possible hydrogenbonding partners with the Met253 backbone, were optimal for activity in this region (IC50 = 1.8−11 nM). These findings led us to continue our investigation of the SAR of the series by focusing on the 5-position to improve the affinity for the enzyme through efficient interaction with the protein target. Unlike 6-subsituted inhibitors, 5-substituted inhibitors 9k, 9a, and 26a with small substituents did not lead to a significant improvement in potency, as shown in Table 2. Modeling/X-ray studies had indicated that, in the proposed orientation of the quinazoline-2-carboxamide derivative which interacts with MMP-13, the fluoro, methoxy, and cyano group at the 5position did not have sufficient size to reach the hydrophobic binding pocket of the enzyme. The specific MMP-13 inhibitors, initially reported by a research group at Warner−Lambert, were derived by introducing a hydrophobic substituent to extend the P1′ substituent such that it could deeply fill the hydrophobic side pocket of S1′, which is referred to as the S1″ pocket.22 In order to design potent MMP-13 inhibitors, we adopted a strategy of connecting fragments with our quinazoline system by an appropriate linker. On the basis of information on S1″ bound MMP inhibitors, we selected candidate fragments that could be in contact with the binding side pocket. The compounds prepared for P1″ substitution are listed in Table 2. Increasing the length of the linker between the quinazoline core and the P1″ phenyl group progressively increased MMP13 inhibitory activity (26b, 26c, 21a, 9m, 21b, and 21c). One of the most potent compounds, phenethyloxy 9m with the optimized linker length, was 17-fold more potent than the

Table 1. Inhibitory Activities against MMP-13 of 6-, 7-, and 8-Substitued Derivatives

compd

R1

R2

R3

IC50 (nM)a

37 9e 9f 9g 9b 17 9h 9i 19a 19b 20a 20b 9c 9d

H F Me CF3 OMe OEt OCF3 OBn SMe SO2Me Ph CN H H

H H H H H H H H H H H H OMe H

H H H H H H H H H H H H H OMe

12 ± 1.5 11 ± 1.6 26 ± 3.1 97 ± 13 4.0 ± 0.53 2.4 ± 0.16 27 ± 3.7 110 ± 17 1.8 ± 0.19 23 ± 0.77 9.8 ± 0.56 6.3 ± 0.53 2200 ± 960 >10 000

IC50 against MMP-13. Each value is the mean ± SD from triplicate assay in a single experiment.

a

group 9b or an ethoxy group 17 resulted in 3−5-fold improvement in MMP-13 inhibitory activity, whereas substitution with trifluoromethoxy (9h) and benzyloxy (9i) groups reduced potency compared with that of 9b. Unlike compound 9b, having a methoxy group, the methylsulfonyl 19b does not improve potency compared to

Table 2. Inhibitory Activities against MMP-13 of 5-Substitued Derivatives

a

IC50 against MMP-13. Each value is the mean ± SD from triplicate assay in a single experiment. H



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accessibility and promising biological activity, we focused on the synthesis and structure−activity relationships of a family of 6-fluoroquinazolines. For compounds 21i, 21j, and 21k, the hydrogen-donor or -acceptor functionality, which would avoid potential oxidative metabolism of the para position of the terminal phenyl ring, was oriented to the ε-amino group of Lys140 at the bottom of S1″ pocket. A fluoro substitution in the headgroup produced analogue 21i, which was somewhat less potent than 21h. On the other hand, replacement with an amino group 21j retained inhibitory activity but demonstrated higher clearance and shorter elimination half-life than 21h in rats. A carboxylate based heterocycle such as 21k (IC50 = 0.0039 nM) showed a 10-fold increase in potency as compared with that of the parent compound 21h. It was highly orally bioavailable (F% = 29, rat, 1 mg/kg in 0.5% methyl cellulose) and exhibited greater metabolic stability relative to that of the parent 21h. These properties made the carboxylate 21k a promising candidate for further preclinical evaluation toward the treatment of MMP-13 related diseases. A representative series of the above inhibitors was assessed for their selectivity profiles against other matrix metalloproteinase homologues including MMP-1, 2, 3, 7, 8, 9, 10, and 14 and TACE (Table 4). Compound 21k exhibited >180 000-fold selectivity for MMP-13 over MMP-8 and >41 000-fold selectivity for MMP-13 over MMP-10. The compound also displayed excellent MMP-13 selectivity (>1 000 000-fold) against MMP-1, 2, 3, 7, 9, and 14 and TACE. The 21k−MMP-13 complex structure, obtained by a soaking experiment, revealed that compound 21k binds to MMP-13 in the same binding mode as that of lead compound 37 and does not undergo coordination with the catalytic zinc. The quinazoline ring system of compound 21k fills the deep S1′ pocket of MMP-13 consisting of Thr245 and Thr247, the enzyme’s specificity pocket. As shown in Figure 4, the inhibitor 21k is stabilized at the S1′ site of MMP-13 by three possible hydrogen bonds: (a) the O4 carbonyl oxygen of the quinazoline ring and the backbone amide of Thr247, (b) the N3 amide hydrogen of the quinazoline ring and the carbonyl oxygen of Thr245, and (c) the exocyclic carbonyl oxygen at the 2-position of the quinazoline ring and the backbone amide of Thr245. This β-sheet type interaction confers the potent inhibitory activity of the quinazolin-4-one-2-carboxamide inhibitors with high selectivity toward MMP-13. The

unsubstituted lead compound 37 against MMP-13. Accordingly, further work was focused on increasing the inhibitory activity for the derivatives with three-atom linkers. Replacement of the oxygen atom of the linker in phenethyloxy 9m with other atoms furnished the amino, thio, and carbon analogues 21d, 21e, and 26d, respectively, which showed 4−14-fold reduced MMP-13 activity. Similarly, a series of compounds with varied arrangement of heteroatoms in the linker (34, 29, and 36), except the aniline amide 36, showed decreased activities compared to that of the parent compound 9m. The substitution of the aromatic phenyl ring in the P1″ position with a cyclohexyl group causes a slight decrease in MMP-13 inhibitory activity (21f vs 9m). Incorporation of a basic amine functionality in the cyclohexyl group also led to the essentially inactive piperidine (21g vs 9m). From these results, we examined detailed structure−activity relationship studies on one of the most potent phenethyloxy series 9m. Moreover, the introduction of a small fluoro group on the benzene ring at the 6-position in phenethyloxy 9m afforded a slight increase in MMP-13 inhibitory activity with a substantially lowered rat iv clearance (21h, Table 3). Therefore, because of both synthetic Table 3. Inhibitory Activities against MMP-13 and Rat Pharmacokinetic Parameters of 5-Phenethyloxy Derivatives

compd

R1

R2

IC50 (nM)a

CLb

metabolic stabilityc

9m 21h 21i 21j 21k

H H F NH2 CO2H

H F F F F

0.69 ± 0.058 0.040 ± 0.013 0.15 ± 0.013 0.030 ± 0.0018 0.0039 ± 0.0011

1.7 1.0 1.7 14 1.7

170 150 180 140 70

IC50 against MMP-13. Each value is the mean ± SD from triplicate assay in a single experiment. bRat iv clearance (L/h/kg). cRat hepatic microsomal metabolic stability (μL/min/mg). a

Table 4. Selectivity Profiles for Compounds 37, 21k, and 39 (RS-130,830)

IC50 (nM)a

a

compd

MMP-13

MMP-1

MMP-2

MMP-3

MMP-7

MMP-8

MMP-9

MMP-10

MMP-14

TACE

37 21k 39

12 0.0039 0.010

>10 000 >10 000 34

300 5300 0.029

>10 000 4000 0.30

>10 000 >10 000 210

1100 720 0.097

>10 000 >10 000 0.11

3400 160 0.54

>10 000 >10 000 1.1

>10 000 >10 000 14

Each value is the mean from triplicate assay in a single experiment. I



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Figure 4. Crystal structure of the complex of carboxylate 21k and MMP-13. (A) Surface representation of MMP-13 illustrating the binding cavity. The inhibitor is buried deep into the S1′ pocket and extends into an additional S1′ side pocket (S1″), which is unique to MMP-13. The figure was made with PyMOL.34 (B) The distances of hydrogen bonds and ionic interactions between 21k and MMP-13 are depicted as dashed lines.

Table 5. Pharmacokinetic Parameters of Sodium Salt 38a

intravenousb

oralc

species

dose (mg/kg)

Vd,ss (mL/kg)

CLtotal (mL/h/kg)

dose (mg/kg)

Tmax (h)

Cmax (ng/mL)

AUC (ng·h/mL)

rat guinea pig rabbit dog monkey human

1.0 1.0 0.1 1.0 1.0 NT

995 5387 1142 731 7025 NT

2077 1615 222 185 1256 NT

10 3.0 10 10 1.0 NT

0.25 0.50 1.8 1.7 2.8 NT

439 439 2293 3873 45 NT

1597 1413 15 677 31 810 456 NT

Fd (%) 23 74 35 58 45

(4.9f) (6.3f) (11g) (3.0f) (1.5f) NT

metabolic stabilitye 70 1.0 3.0 NDh 2.0 3.0

a

All experiments were performed using three male animals. NT = not tested. bCompounds were dosed in DMA/PEG400 or DMSO. cCompounds were dosed in 0.5% methyl cellulose. dBioavailabilities of 21k (free form) are given in parentheses. eHepatic microsomal metabolic stability (μL/ min/mg) of free acid of 38. fBioavailability at a dose of 3 mg/kg, po. gBioavailability at a dose of 1 mg/kg, po. hNo elimination of 38 was observed. ND = not determined.

phenethyloxy group of compound 21k is deeply buried in the S1″ specificity pocket, and the carboxylic group on the terminal phenyl ring provides an anchor to the protein through a salt bridge with the ε-amino group of Lys140. A fluoro group at the 6-position of the inhibitor is weakly interacting with the backbone amide nitrogen of Met253 with a slightly longer F−N bonding distance (3.3 Å). Furthermore, preliminary safety assessments with 21k by several in vitro assays indicated no obvious safety concerns (inhibition of the cytochrome P450 system, effect on hERG current, cytotoxicity, phototoxicity, and mutagenic side effects; data not shown). This compound proceeded into further preclinical studies for pharmacokinetic and safety evaluation. Unfortunately, compound 21k exhibited poor oral bioavailability in all tested species (rats, guinea pigs, rabbits, beagle dogs, and cynomolgus monkeys; Table 5). Given its high metabolic stability in liver microsomes (Table 5) and high permeability in Caco-2 membranes (apparent permeability

coefficient (Papp) = 47.7 nm/s), this low bioavailability is probably due to poor solubility. These findings led us to continue our investigation by focusing on the formation of salts of 21k to remediate the low bioavailability. Compound 21k has a carboxyl function and an amide NH of the pyrimidinone ring with relatively high acidity, which allows the formation of salts to improve solubility. We prepared several salt forms of compound 21k (monosodium, disodium, monopotassium, and dipotassium salts; not shown) and examined the nature of the salts, including aqueous solubility, hygroscopicity, crystallinity, presence of polymorphs, and reproducibility of the preparation. The storage stability tests under accelerated temperature and relative humidity conditions showed that the monosodium salt (38) had the favorable properties of being stable and nonhygroscopic, whereas the disodium, monopotassium, and dipotassium salts J



Article

whereas 38 showed a significant (P < 0.01) reduction of CTX-II release (69%), comparable to that of the broad spectrum hydroxamate-type MMP inhibitor 39 (RS-130,830) (87%). These data indicate that MMP-13 selective inhibition is sufficient to inhibit collagen degradation in the rat MIAinduced OA model. The non-zinc binding selective MMP-13 inhibitor 38 was judged to be the most promising candidate for further development due to its favorable potency, selectivity, bioavailability, and in vivo activity.

had hygroscopic characteristics and their crystal form changes when hydrate formation occurred (data not shown). Additionally, the monosodium salt 38 had a high degree of crystallinity (data not shown) and was efficiently recovered from the crystallization solvents with good reproducibility. On the basis of these data, the monosodium salt 38 was selected for further evaluation. The pharmacokinetics of 38 were assessed in rats, guinea pigs, rabbits, beagle dogs, and cynomolgus monkeys. Thus, 38 was found to have favorable oral bioavailability in all tested species (F% = 23, 74, 35, 58, and 45, respectively) with a moderate to high volume of distribution. The improvement of oral bioavailability is thought to be the result of enhanced solubility and/or dissolution rate of the compound. In guinea pig and monkey, 38 exhibited moderate plasma AUC with a relatively high volume of distribution. By contrast, in rabbit and dog, this compound showed high plasma AUC with a moderate volume of distribution. This compound exceptionally exhibited lower plasma AUC in spite of the moderate volume of distribution in rat as compared to those in other species. Carboxylate 21k showed moderate metabolic stability only in rat microsomes, likely resulting in the high total body clearance and low plasma AUC. On the other hand, the excellent microsomal stability of 21k in human microsomes indicates that this compound will show good pharmacokinetic profiles in human. In a preliminary 2 week toxicological study in rats, none of the symptoms associated with toxicity were observed up to a dose of 200 mg/ kg/day. These properties made 38 a promising candidate for further preclinical evaluation toward the treatment of MMP-13 related diseases. The rat model of monoiodoacetate (MIA) induced OA was used for assessing the effects of MMP-13 selective inhibitors on cartilage degradation. The injection of iodoacetate into the knees of rats results in chondrocyte necrosis and an increase in MMP activity.23−25 The cartilage marker C-telopeptide of type II collagen (CTX-II) is generated from cartilage collagen by MMP cleavage.26,27 Compounds were administrated once orally (10 mg/kg) at day 7 after MIA injection. Four hours after oral administration of the test compounds at a 10 mg/kg dose, the rats were sacrificed, and synovial fluid samples from the joints were collected for CTX-II measurement. After MIA injection, 18.6-fold increased levels of intra-articular CTX-II were observed (Figure 5) in the vehicle-treated animals,

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CONCLUSIONS We have developed an efficient method for the synthesis of quinazoline-2-carboxamide derivatives and synthesized a new class of MMP-13 selective inhibitors. On the basis of X-ray crystallography of the complex of lead compound 37 with MMP-13, we designed a series of potent MMP-13 selective inhibitors to exploit the primed side of the active site, which is involved in the interactions with the backbone NH of Met253 in the deeper region of the S1′ pocket, the additional S1′ side pocket, and the ε-amino group of Lys140. Introduction of the phenethyloxy group at the 5-position of the quinazoline ring as in 9m and 21h−k induces a notable increase in potency. Among them, carboxylic acid inhibitor 21k, which interacts with the ε-amino group of Lys140, exhibited excellent potency (IC50 = 0.0039 nM) and selectivity (greater than 41 000-fold) over that of other MMPs (MMP-1, 2, 3, 7, 8, 9, 10, and 14 and TACE). Compound 21k may avoid the musculoskeletal side effects observed during clinical trials with nonspecific MMP inhibitors. X-ray analysis of the complex of 21k with MMP-13 confirmed that the inhibitor is buried deep into the S1′ pocket by forming a β-sheet type interaction through hydrogen bonding to the enzyme’s backbone spanning the S1′ pocket, which extends into an additional S1′ side pocket (S1″ pocket) that is unique to MMP-13. Due to the insolubility of the carboxylic acid 21k in water (0.7 μg/mL, Britton−Robinson buffer (pH 7)), alkali metal salts were generated to increase oral bioavailability. The monosodium salt 38 exhibited favorable bioavailability and pharmacokinetic parameters in various species with desirable physicochemical properties. Compound 21k was evaluated in a rat monoiodoacetate (MIA) induced arthritis model and was found to suppress cartilage degradation in this model following oral administration. Additionally, no overt toxicity was found in a preliminary 2 week toxicological study in rats. These results warrant further preclinical evaluation of 38 as a promising candidate for the treatment of MMP-13 related diseases.

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EXPERIMENTAL SECTION

General Methods. Melting points were determined in open capillary tubes on a Büchi melting point apparatus B545 and are uncorrected. 1H NMR spectra were recorded on a Varian Gemini-200 (200 MHz), Varian Gemini-300 (300 MHz), or Bruker DPX-300 (300 MHz) spectrometer and are reported in parts per million (δ) relative to tetramethylsilane (TMS, δ 0.0 ppm). Data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, ddd = doublet of doublet of doublet, bs = broad singlet), and coupling constants (J, Hz). Unless otherwise specified, all solvents and reagents were obtained from commercial suppliers and used without further purification. Column chromatography was performed using Merck silica gel 60 (70−230 mesh). Thin-layer chromatography (TLC) was performed on Merck silica gel plates 60F254. LC-MS analysis was performed on a Shiseido CAPCELL PACK C-18 UG120 S-3 column (1.5 mmϕ × 35 mm) in a Waters Alliance 2795 or an Agilent 1100 LC

Figure 5. Protective effect of MMP inhibitors on cartilage degradation in the rat model of monoiodoacetate (MIA)-induced OA. Synovial Cterminal telopeptide of type II collagen (CTX-II) levels were measured at 4 h after intra-articular injection of MIA. Data are expressed as mean ± SEM (n = 6). ** denotes P < 0.01 versus vehicle group by two-way analysis of variance with Dunnett’s test. K



Article

from compound 8f (white powder, 68%). mp 175−177 °C. 1H NMR (200 MHz, DMSO-d6) δ 2.48 (3H, s), 3.74 (3H, s), 4.45 (2H, d, J = 6.6 Hz), 6.80−6.94 (3H, m), 7.25 (1H, t, J = 8.0 Hz), 7.65−7.75 (2H, m), 7.98 (1H, s), 9.50 (1H, t, J = 6.4 Hz), 12.16 (1H, bs). Anal. Calcd for C18H17N3O4·0.2H2O: C, 63.04; H, 5.11; N, 12.25. Found: C, 62.93; H, 5.11; N, 12.08. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-6-(trifluoromethyl)3,4-dihydroquinazoline-2-carboxamide (9g). Compound 9g was prepared from compound 8g (pale yellow powder, 61%). mp 186−187 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.73 (3H, s), 4.46 (2H, d, J = 6.3 Hz), 6.80−6.84 (1H, m), 6.90−6.93 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.95 (1H, dd, J = 8.1, 0.6 Hz), 8.18 (1H, dd, J = 8.4, 2.1 Hz), 8.38 (1H, d, J = 0.9 Hz), 9.63 (1H, t, J = 6.3 Hz), 12.71 (1H, bs). Anal. Calcd for C18H14F3N3O3: C, 57.30; H, 3.74; N, 11.14. Found: C, 57.22; H, 3.78; N, 11.22. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-6[(trifluoromethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (9h). Compound 9h was prepared from compound 8h (white powder, 62%). mp 156−159 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.46 (2H, d, J = 6.3 Hz), 6.83 (1H, dd, J = 8.1, 2.4 Hz), 6.90−6.94 (2H, m), 7.25 (1H, t, J = 8.1 Hz), 7.86−7.93 (2H, m), 8.00 (1H, s), 9.58 (1H, t, J = 6.3 Hz), 12.59 (1H, bs). Anal. Calcd for C18H14F3N3O4: C, 54.97; H, 3.59; N, 10.68. Found: C, 54.80; H, 3.53; N, 10.73. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-6-[(phenylmethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (9i). Compound 9i was prepared from compound 8i (white powder, 86%). mp 179−182 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.73 (3H, s), 4.44 (2H, d, J = 6.6 Hz), 5.27 (2H, s), 6.82 (1H, dd, J = 8.1, 2.4 Hz), 6.90−6.94 (2H, m), 7.24 (1H, t, J = 7.6 Hz), 7.32−7.42 (3H, m), 7.43−7.51 (1H, m), 7.54−7.58 (1H, m), 7.66 (1H, d, J = 2.4 Hz), 7.75 (1H, d, J = 8.7 Hz), 9.48 (1H, t, J = 6.4 Hz), 11.80−11.90 (1H, m). Anal. Calcd for C24H21N3O4: C, 69.39; H, 5.10; N, 10.11. Found: C, 69.09; H, 5.07; N, 10.21. 5-Fluoro-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (9k). Compound 9k was prepared from compound 8k (pale yellow powder, 89%). mp 159−161 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.44 (2H, d, J = 6.3 Hz), 6.80−6.84 (1H, m), 6.89−6.92 (2H, m), 7.21−7.27 (1H, m), 7.31− 7.38 (1H, m), 7.58 (1H, d, J = 8.1 Hz), 7.81−7.88 (1H, m), 9.54 (1H, t, J = 6.3 Hz), 12.28 (1H, bs). Anal. Calcd for C17H14FN3O3: C, 62.38; H, 4.31; N, 12.84. Found: C, 62.43; H, 4.38; N, 12.88. 5-Methyl-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (9l). Compound 9l was prepared from compound 8l (white powder, 78%). mp 150−152 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.79 (3H, s), 3.74 (3H, s), 4.44 (2H, d, J = 6.3 Hz), 6.82 (1H, dd, J = 8.1, 2.4 Hz), 6.89−6.93 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.35 (1H, d, J = 7.5 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.7 (1H, t, J = 7.8 Hz), 9.48 (1H, t, J = 6.3 Hz), 11.94 (1H, bs). Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C, 66.86; H, 5.37; N, 13.08. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[(2-phenylethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (9m). Compound 9m was prepared from compound 8m (pale yellow powder, 61%). mp 150−151 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.10 (2H, d, J = 6.6 Hz), 3.73 (3H, s), 4.25 (2H, t, J = 6.6 Hz), 4.43 (2H, d, J = 6.6 Hz), 6.82 (1H, dd, J = 8.1, 1.8 Hz), 6.89−6.92 (2H, m), 7.09 (1H, d, J = 8.1 Hz), 7.17−7.30 (5H, m), 7.46−7.49 (2H, m), 7.71 (1H, t, J = 8.1 Hz), 9.47 (1H, t, J = 6.6 Hz), 11.77 (1H, bs). Anal. Calcd for C25H23N3O4· 0.1H2O: C, 69.62; H, 5.42; N, 9.68. Found: C, 69.44; H, 5.40; N, 9.68. 6-(Ethyloxy)-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (17). A mixture of compound 16 (120 mg, 0.368 mmol), cesium carbonate (240 mg, 0.736 mmol), iodoethane (0.087 mL, 1.10 mmol), THF (3 mL), and DMF (1 mL) was stirred at room temperature for 4 h. The mixture was partitioned between ethyl acetate and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude materials were purified by preparative HPLC and recrystallization from ethanol to give the title compound as a white powder (27.0 mg, 0.0764 mmol, 21%). mp 163−165 °C. 1H NMR (300 MHz, DMSO-d6) δ 1.38 (3H, t, J = 6.9 Hz), 3.74 (3H, s), 4.17

system equipped with a Waters 2487 absorbance detector and a Micromass ZQ2000 mass spectrometer. Analytes were eluted using a linear gradient of water (0.05% TFA)/acetonitrile (0.04% TFA) from 90:10 to 0:100 over 4 min at a flow rate of 0.5 mL/min. UV detection was at 220 nm. Preparative HPLC was performed on a Shiseido CAPCELL PACK C-18 UG120 S-5 column (20 mmϕ × 50 mm), eluting at 25 mL/min with a gradient of water (0.1% TFA)/ acetonitrile (0.1% TFA). UV detection was at 220 nm. Compound purity for all tested compounds was determined by elemental analysis or HPLC analysis. Experimentally determined hydrogen, carbon, and nitrogen composition by elemental analysis was within ±0.4% of the expected value, implying a purity of ≥95%. Analytical HPLC was performed with Corona charged aerosol detector (CAD) on an Lcolumn 2 ODS (30 mm × 2.0 mm i.d., CERI, Japan) operated at 50 °C, eluting at 0.5 mL/min using a linear gradient. Mobile phases were as follows: A, 50 mmol/L ammonium acetate, water, and acetonitrile (1:8:1, v/v/v); B, 50 mmol/L ammonium acetate and acetonitrile (1:9, v/v). The ratio of mobile phase B was increased linearly from 5 to 95% over 3 min followed by 95% over the next 1 min. All experiments using animals were reviewed and approved by the Internal Animal Care and Use Committee of Takeda Pharmaceutical Research Division. Representative Procedure for the Synthesis of Compounds 9a−o: 5-(Methyloxy)-N-{[3-(methyloxy) phenyl]methyl}-4-oxo3,4-dihydroquinazoline-2-carboxamide (9a). A mixture of compound 8a (150 mg, 0.604 mmol) and 3-methoxybenzylamine (166 mg, 1.21 mmol) in DMF (4 mL) was stirred at 80 °C for 15 h. The mixture was concentrated under reduced pressure, and the residue was triturated with diisopropylether to give crude 9a. The crude product was recrystallized from ethanol−diisopropylether to give the title compound as a pale yellow powder (117 mg, 0.345 mmol, 57%). mp 188−190 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.73 (3H, s), 3.87 (3H, s), 4.42 (2H, d, J = 6.6 Hz), 6.79−6.83 (1H, m), 6.88−6.93 (2H, m), 7.10 (1H, d, J = 8.1 Hz), 7.23 (1H, t, J = 8.1 Hz), 7.28 (1H, d, J = 8.1 Hz), 7.74 (1H, t, J = 8.1 Hz), 9.45 (1H, t, J = 6.6 Hz), 11.75 (1H, bs). Anal. Calcd for C18H17N3O4·0.1H2O: C, 63.37; H, 5.08; N, 12.32. Found: C, 63.38; H, 4.90; N, 12.21. 6-(Methyloxy)-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4dihydroquinazoline-2-carboxamide (9b). Compound 9b was prepared from compound 8b (white powder, 28%). mp 174−176 °C. 1H NMR (200 MHz, DMSO-d6) δ 3.74 (3H, s), 3.91 (3H, s), 4.45 (2H, d, J = 6.6 Hz), 6.81−6.94 (3H, m), 7.25 (1H, t, J = 8.0 Hz), 7.49 (1H, dd, J = 8.8, 3.0 Hz), 7.56 (1H, d, J = 3.0 Hz), 7.74 (1H, d, J = 8.6 Hz), 9.42−9.52 (1H, m). Anal. Calcd for C18H17N3O4·0.2H2O: C, 63.04; H, 5.11; N, 12.25. Found: C, 62.93; H, 5.11; N, 12.08. 7-(Methyloxy)-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4dihydroquinazoline-2-carboxamide (9c). Compound 9c was prepared from compound 8c (white powder, 47%). mp 228−230 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 3.91 (3H, s), 4.45 (2H, d, J = 6.2 Hz), 6.81−6.94 (3H, m), 7.16−7.29 (3H, m), 8.05− 8.10 (1H, m), 9.50 (1H, t, J = 6.2 Hz), 12.12 (1H, bs). Anal. Calcd for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38. Found: C, 63.41; H, 5.02; N, 12.42. 8-(Methyloxy)-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4dihydroquinazoline-2-carboxamide (9d). Compound 9d was prepared from compound 8d (white powder, 71%). mp 238−239 °C. 1H NMR (200 MHz, DMSO-d6) δ 3.74 (3H, s), 3.94 (3H, s), 4.47 (2H, d, J = 6.4 Hz), 6.81−6.99 (3H, m), 7.21−7.29 (1H, m), 7.41− 7.58 (2H, m), 7.70−7.74 (1H, m), 9.21 (1H, t, J = 6.4 Hz), 12.26 (1H, bs). Anal. Calcd for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38. Found: C, 63.50; H, 5.14; N, 12.29. 6-Fluoro-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (9e). Compound 9e was prepared from compound 8e (pale yellow powder, 69%). mp 177−179 °C. 1H NMR (200 MHz, DMSO-d6) δ 3.74 (3H, s), 4.45 (2H, d, J = 6.4 Hz), 7.25 (1H, t, J = 8.0 Hz), 6.80−6.94 (3H, m), 7.74−7.89 (3H, m), 9.50−9.57 (1H, m). Anal. Calcd for C17H14FN3O3·0.2H2O: C, 61.70; H, 4.39; N, 12.70. Found: C, 61.44; H, 4.27; N, 2.71. 6-Methyl-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (9f). Compound 9f was prepared L



Article

Representative Procedure for the Synthesis of Compounds 21a−c, 21e−g, and 21k: N-{[3-(Methyloxy)-phenyl]methyl}-4oxo-5-[(phenylmethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (21a). To a solution of benzyl alcohol (99.0 mg, 0.917 mmol) in DMA (6 mL) was added sodium hydride (60% oil dispersion, 122 mg, 3.06 mmol), and the mixture was stirred at room temperature for 30 min. Compound 9k (200 mg, 0.611 mmol) was added to the mixture, and the resulting mixture was stirred at 80 °C for 1 h. The reaction mixture was acidified with 0.5 N hydrochloric acid to pH 3−4 and extracted with a mixed solvent of ethyl acetate and THF. The organic layer was washed with H2O and brine, dried over Na2SO4, and concentrated under reduced pressure to give crude 21a. The crude product was crystallized from ethanol to give the title compound as a pale yellow powder (207 mg, 0.498 mmol, 81%). mp 188−190 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.44 (2H, d, J = 6.6 Hz), 5.28 (2H, s), 6.80−6.84 (1H, m), 6.90−6.93 (2H, m), 7.18−7.33 (4H, m), 7.37−7.43 (2H, m), 7.75 (1H, t, J = 8.4 Hz), 7.61−7.63 (2H, m), 9.50 (1H, t, J = 6.3 Hz), 11.81 (1H, bs). Anal. Calcd for C24H21N3O4: C, 69.39; H, 5.10; N, 10.11. Found: C, 69.11; H, 4.97; N, 10.40. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[(3phenylpropyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (21b). Compound 21b was prepared from compound 9k and 3phenylpropan-1-ol (pale yellow powder, 44%). mp 165−167 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.01−2.11 (2H, m), 2.89 (2H, t, J = 7.5 Hz), 3.74 (3H, s), 4.05 (2H, t, J = 6 Hz), 4.44 (2H, d, J = 6.3 Hz), 6.80−6.84 (1H, m), 6.90−6.92 (2H, m), 7.06 (1H, d, J = 8.4 Hz), 7.13−7.29 (7H, m), 7.71 (1H, t, J = 8.1 Hz), 9.48 (1H, t, J = 6.3 Hz), 11.76 (1H, bs). Anal. Calcd for C26H25N3O4·0.1H2O: C, 70.13; H, 5.70; N, 9.44. Found: C, 69.91; H, 5.62; N, 9.69. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-({2[(phenylmethyl)oxy]ethyl}oxy)-3,4-dihydroquinazoline-2-carboxamide (21c). Compound 21c was prepared from compound 9k and 2-[(phenylmethyl)oxy]ethanol (pale yellow powder, 76%). mp 136−138 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.73 (3H, s), 3.84 (2H, t, J = 4.5 Hz), 4.26 (2H, t, J = 4.5 Hz), 4.43 (2H, d, J = 6.6 Hz), 4.69 (2H, s), 6.80−6.84 (1H, m), 6.89−6.92 (2H, m), 7.12 (1H, d, J = 8.4 Hz), 7.21−7.38 (7H, m), 7.72 (1H, t, J = 8.1 Hz), 9.47 (1H, t, J = 6.3 Hz), 11.76 (1H, bs). Anal. Calcd for C26H25N3O5: C, 67.96; H, 5.48; N, 9.14. Found: C, 67.76; H, 5.52; N, 9.22. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[(2-phenylethyl)thio]-3,4-dihydroquinazoline-2-carboxamide (21e). Compound 21e was prepared from compound 9k and 2-phenylethanethiol (pale yellow powder, 55%). mp 178−180 °C. 1H NMR (300 MHz, DMSOd6) δ 2.98 (2H, t, J = 7.5 Hz), 3.20 (2H, t, J = 7.5 Hz), 3.74 (3H, s), 4.44 (2H, d, J = 6.3 Hz), 6.81−6.84 (1H, m), 6.90−6.92 (2H, m), 7.22−7.34 (6H, m), 7.41−7.59 (2H, m), 7.75 (1H, t, J = 7.8 Hz), 9.50 (1H, t, J = 6.6 Hz), 12.17 (1H, bs). Anal. Calcd for C25H23N3O3S· 0.4H2O: C, 66.32; H, 5.30; N, 9.28. Found: C, 66.16; H, 5.10; N, 9.52. 5-[(2-Cyclohexylethyl)oxy]-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (21f). Compound 21f was prepared from compound 9k and 2-cyclohexylethanol (white powder, 55%). mp 130−132 °C. 1H NMR (300 MHz, DMSO-d6) δ 0.90−1.05 (2H, m), 1.13−1.30 (3H, m), 1.60− 1.78 (8H, m), 3.73 (3H, s), 4.10 (2H, t, J = 6.3 Hz), 4.43 (2H, d, J = 6.3 Hz), 6.80−6.83 (1H, m), 6.89−6.92 (2H, m), 7.10 (1H, d, J = 8.1 Hz), 7.21−7.27 (2H, m), 7.71 (1H, t, J = 8.1 Hz), 9.46 (1H, t, J = 6.3 Hz), 11.69 (1H, bs). Anal. Calcd for C25H29N3O4: C, 68.95; H, 6.71; N, 9.65. Found: C, 68.71; H, 6.71; N, 9.77. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[(2-piperidin-1ylethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (21g). Compound 21g was prepared from compound 9k and 2-piperidin-1ylethanol (pale yellow powder, 35%). mp 225−227 °C. 1H NMR (300 MHz, DMSO-d6) δ 1.45−1.79 (2H, m), 1.76−1.83 (4H, m), 3.05− 3.39 (2H, m), 3.48−3.75 (4H, m), 3.73 (3H, s), 4.43 (2H, d, J = 6.3 Hz), 4.49−4.55 (2H, m), 6.81−6.85 (1H, m), 6.88−6.92 (2H, m), 7.18−7.27 (2H, m), 7.39 (1H, d, J = 8.4 Hz), 7.81 (1H, t, J = 8.1 Hz), 9.52 (1H, t, J = 6.3 Hz), 11.90−12.10 (1H, m). Anal. Calcd for C24H28N4O4·1.5H2O: C, 62.19; H, 6.74; N, 12.09. Found: C, 61.92; H, 6.45; N, 11.85. 4-[2-({6-Fluoro-2-[({[3-(methyloxy)phenyl]methyl}amino)carbonyl]-4-oxo-3,4-dihydroquinazolin- 5-yl}oxy)ethyl]-

(2H, q, J = 6.9 Hz), 4.44 (2H, d, J = 6.3 Hz), 6.83 (1H, dd, J = 8.4, 2.4 Hz), 6.90−6.93 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.47 (1H, dd, J = 9.0, 3.0 Hz), 7.54 (1H, d, J = 3.0 Hz), 7.73 (1H, d, J = 9.3 Hz), 9.47 (1H, t, J = 6.3 Hz), 12.18 (1H, bs). Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N, 11.89. Found: C, 64.55; H, 5.52; N, 11.89. N-{[3-(Methyloxy)phenyl]methyl}-6-(methylthio)-4-oxo-3,4dihydroquinazoline-2-carboxamide (19a). A mixture of compound 18 (270 mg, 0.791 mmol), iodomethane (0.049 mL, 0.791 mmol), and triethylamine (0.110 mL, 0.791 mmol) in THF (5 mL) was stirred at room temperature for 1 h. The mixture was partitioned between ethyl acetate and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by recrystallization from ethanol to give the title compound as a pale yellow powder (240 mg, 0.675 mmol, 85%). mp 168−170 °C. 1H NMR (200 MHz, DMSO-d6) δ 2.60 (3H, s), 3.74 (3H, s), 4.45 (2H, d, J = 6.2 Hz), 6.81−6.94 (3H, m), 7.25 (1H, t, J = 8.0 Hz), 7.70 (1H, d, J = 8.8 Hz), 7.77 (1H, dd, J = 8.8, 2.2 Hz), 7.89 (1H, d, J = 1.8 Hz), 9.50 (1H, t, J = 6.2 Hz), 12.28 (1H, bs). Anal. Calcd for C18H17N3O3S: C, 60.83; H, 4.82; N, 11.82. Found: C, 60.65; H, 4.76; N, 11.98. N-{[3-(Methyloxy)phenyl]methyl}-6-(methylsulfonyl)-4-oxo3,4-dihydroquinazoline-2-carboxamide (19b). To a solution of compound 19a (60.0 mg, 0.169 mmol) in chloroform (2 mL) was added 3-chloroperoxybenzoic acid (84.0 mg, 338 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The mixture was partitioned between ethyl acetate and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by recrystallization from ethanol to give the title compound as a pale yellow powder (62.0 mg, 0.161 mmol, 95%). mp 186−188 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.33 (3H, s), 3.74 (3H, s), 4.46 (2H, d, J = 6.6 Hz), 6.80−6.84 (1H, m), 6.90−6.93 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.96 (1H, d, J = 8.4 Hz), 8.31 (1H, dd, J = 8.4, 2.1 Hz), 8.60 (1H, s), 9.63 (1H, t, J = 6.3 Hz), 12.77 (1H, bs). Anal. Calcd for C18H17N3O5S· 0.4H2O: C, 54.79; H, 4.55; N, 10.65. Found: C, 54.83; H, 4.36; N, 10.66. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-6-phenyl-3,4-dihydroquinazoline-2-carboxamide (20a). A mixture of compound 9j (300 mg, 0.689 mmol), phenylboronic acid (167 mg, 1.37 mmol), tetrakis(triphenylphosphine)palladium(0) (32.0 mg, 0.0277 mmol), and 2 N aqueous Na2CO3 solution (1.03 mL, 2.06 mmol) in a mixed solvent of ethanol (2 mL) and toluene (6 mL) was refluxed for 18 h under a nitrogen atmosphere. The reaction mixture was diluted with ethyl acetate, washed with H2O and brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by recrystallization from ethanol to give the title compound as a white powder (170 mg, 0.441 mmol, 64%). mp 202−204 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.46 (2H, d, J = 6.6 Hz), 6.81−6.85 (1H, m), 6.90−6.94 (2H, m), 7.25 (1H, t, J = 8.1 Hz), 7.40−7.45 (1H, m), 7.49−7.55 (2H, m), 7.78−7.88 (3H, m), 8.20 (1H, dd, J = 8.4, 2.4 Hz), 8.37 (1H, d, J = 2.1 Hz), 9.56 (1H, t, J = 6.3 Hz), 12.34 (1H, bs). Anal. Calcd for C23H19N3O3·0.2H2O: C, 71.01; H, 5.03; N, 10.80. Found: C, 71.12; H, 5.00; N, 10.51. 6-Cyano-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (20b). A mixture of compound 9j (1.00 g, 2.30 mmol), zinc cyanide (148 mg, 1.26 mmol), and tetrakis(triphenylphosphine)palladium(0) (132 mg, 0.115 mmol) in DMF (10 mL) was stirred at 80 °C for 3 h under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure, and the residue was suspended in ethyl acetate. The resulting precipitate was collected to give crude 20b as a white powder (537 mg). Two hundred milligrams of the crude product was recrystallized from ethanol to give the title compound as a white powder (193 mg, 0.577 mmol, 68%). mp 206−208 °C. 1H NMR (200 MHz, DMSO-d6) δ 3.74 (3H, s), 4.46 (2H, d, J = 6.6 Hz), 6.81−6.95 (3H, m), 7.25 (1H, t, J = 8.0 Hz), 7.89 (1H, d, J = 8.4 Hz), 8.23 (1H, dd, J = 8.6, 2.0 Hz), 8.55 (1H, d, J = 2.0 Hz), 9.63 (1H, t, J = 6.6 Hz). Anal. Calcd for C18H14N4O3·0.1H2O: C, 64.32; H, 4.26; N, 16.67. Found: C, 64.17; H, 4.23; N, 16.74. M



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and acetic acid (1 mL) was heated at 80 °C for 2 h. After completeness of reaction was checked by LC-MS, the reaction mixture was concentrated under reduced pressure, and the residue was crystallized from ethanol to give the title compound as a white powder (20 mg, 0.060 mmol, 80%). mp 185−187 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.46 (2H, d, J = 6.4 Hz), 6.79−6.87 (1H, m), 6.88− 6.97 (2H, m), 7.20−7.30 (1H, m), 7.92−8.13 (3H, m), 9.61 (1H, t, J = 6.1 Hz), 12.74 (1H, s). Anal. Calcd for C18H14N4O3: C, 64.66; H, 4.22; N, 16.76. Found: C, 64.39; H, 4.25; N, 17.04. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-phenyl-3,4-dihydroquinazoline-2-carboxamide (26b). Compound 26b was prepared from compound 25b (white powder, 53%). mp 196−198 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.46 (2H, d, J = 6.1 Hz), 6.80−6.87 (1H, m), 6.90−6.97 (2H, m), 7.21−7.40 (7H, m), 7.75−7.89 (2H, m), 9.55 (1H, t, J = 6.2 Hz), 12.00 (1H, bs). Anal. Calcd for C23H19N3O3: C, 71.67; H, 4.97; N, 10.90. Found: C, 71.38; H, 5.07; N, 10.72. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-(phenyloxy)-3,4dihydroquinazoline-2-carboxamide (26c). Compound 26c was prepared from compound 25c (white powder, 55%). Chromatographic purity (HPLC) 95.7%. mp 148−150 °C. 1H NMR (300 MHz, DMSOd6) δ 3.74 (3H, s), 4.45 (2H, d, J = 6.4 Hz), 6.80−6.86 (1H, m), 6.89− 6.96 (4H, m), 7.00−7.14 (2H, m), 7.25 (1H, t, J = 8.1 Hz), 7.31−7.40 (2H, m), 7.57 (1H, d, J = 8.0 Hz), 7.81 (1H, t, J = 8.1 Hz), 9.54 (1H, t, J = 6.2 Hz), 12.03 (1H, bs). Anal. Calcd for C23H19N3O4·0.25H2O: C, 68.05; H, 4.84; N, 10.35. Found: C, 67.77; H, 4.70; N, 10.51. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-(3-phenylpropyl)3,4-dihydroquinazoline-2-carboxamide (26d). Compound 26d was prepared from compound 25d (white powder, 56%). mp 132− 134 °C. 1H NMR (300 MHz, DMSO-d6) δ 1.80−1.94 (2H, m), 2.63− 2.71 (2H, m), 3.22−3.33 (2H, m), 3.73 (3H, s), 4.45 (2H, d, J = 6.4 Hz), 6.78−6.87 (1H, m), 6.88−6.95 (2H, m), 7.10−7.31 (6H, m), 7.34 (1H, d, J = 7.2 Hz), 7.57−7.66 (1H, m), 7.73 (1H, t, J = 7.8 Hz), 9.50 (1H, t, J = 6.1 Hz), 12.00 (1H, bs). Anal. Calcd for C26H25N3O3· 0.1H2O: C, 72.74; H, 5.92; N, 9.79. Found: C, 72.70; H, 5.85; N, 9.87. N-{[3-(Methyloxy)phenyl]methyl}-5-{[methyl(phenylmethyl)amino]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (29). Compound 29 was prepared from compound 28 with the same procedure as that described for 9a (white powder, 8%). Chromatographic purity (HPLC) 93.4%. mp 127−128 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.17 (3H, s), 3.64 (2H, s), 3.73 (3H, s), 4.24 (2H, s), 4.45 (2H, d, J = 5.7 Hz), 6.81−6.93 (3H, m), 7.21−7.38 (6H, m), 7.64−7.67 (1H, m), 7.82−7.90 (2H, m), 9.52 (1H, t, J = 5.7 Hz), 11.42 (1H, bs). N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-{[(phenylmethyl)oxy]methyl}-3,4-dihydroquinazoline-2-carboxamide (34). Compound 34 was prepared from compound 33 with the same procedure as that described for 9a (white powder, 51%). mp 160−161 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.73 (3H, s), 4.44 (2H, d, J = 6.3 Hz), 4.71 (2H, s), 5.19 (2H, s), 6.81−6.93 (3H, m), 7.21−7.44 (6H, m), 7.68 (1H, d, J = 7.8 Hz), 7.79−7.89 (2H, m), 9.53 (1H, t, J = 6.3 Hz), 12.10 (1H, bs). Anal. Calcd for C25H23N3O4·0.1H2O: C, 69.62; H, 5.42; N, 9.74. Found: C, 69.47; H, 5.39; N, 9.91. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[2-oxo-2(phenylamino)ethyl]-3,4-dihydroquinazoline-2-carboxamide (36). A mixture of compound 35 (120 mg, 0.327 mmol), aniline (0.0600 mL, 0.653 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (125 mg, 0.653 mmol), 1-hydroxybenzotriazole (88.0 mg, 0.653 mmol), and 4-dimethylaminopyridine (40.0 mg, 0.327 mmol) in DMF (3 mL) was stirred at 50 °C for 12 h. The reaction mixture was diluted with ethyl acetate and washed with H2O, 0.1 N hydrochloric acid, H2O, and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was triturated with ethanol to give the title compound as a pale yellow powder (117 mg, 0.264 mmol, 81%). mp 206−208 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.74 (3H, s), 4.33 (2H, s), 4.45 (2H, d, J = 6.4 Hz), 6.79−6.86 (1H, m), 6.89−6.95 (2H, m), 6.99 (1H, t, J = 7.4 Hz), 7.20−7.31 (3H, m), 7.43 (1H, d, J = 7.2 Hz), 7.57 (2H, d, J = 7.6 Hz), 7.67−7.73 (1H, m), 7.79 (1H, t, J = 7.8 Hz), 9.53 (1H, t, J = 6.2 Hz), 10.08 (1H, bs), 12.10 (1H, bs). Anal. Calcd for C25H22N4O4·0.1H2O: C, 67.59; H, 5.04; N, 12.61. Found: C, 67.54; H, 4.91; N, 12.82.

benzoic Acid (21k). Compound 21k was prepared from compound 9o and 4-(2-hydroxyethyl)benzoic acid synthesized by the method of Gilman et al.28 (pale yellow powder, 29%). mp 227−229 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.18 (2H, t, J = 6.9 Hz), 3.73 (3H, s), 4.32 (2H, t, J = 6.9 Hz), 4.44 (2H, d, J = 6.0 Hz), 6.81−6.84 (1H, m), 6.89−6.92 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.46 (2H, d, J = 8.4 Hz), 7.53 (1H, dd, J = 6.3, 4.8 Hz), 7.79 (1H, t, J = 9.6 Hz), 7.87 (2H, d, J = 8.1 Hz), 9.50 (1H, t, J = 6.3 Hz), 12.20 (1H, bs), 12.87 (1H, bs). Anal. Calcd for C26H22FN3O6·0.1H2O: C, 63.31; H, 4.54; N, 8.52. Found: C, 63.21; H, 4.52; N, 8.36. N-{[3-(Methyloxy)phenyl]methyl}-4-oxo-5-[(2-phenylethyl)amino]-3,4-dihydroquinazoline-2-carboxamide (21d). A mixture of compound 9k (100 mg, 0.309 mmol) and 2-phenylethanamine (280 mg, 2.32 mmol) in DMA (2 mL) was stirred at 80 °C for 72 h. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with 0.1 N hydrochloric acid and brine, dried over Na2SO4, and concentrated under reduced pressure to give crude 21d. The crude product was crystallized from ethanol to give the title compound as a yellow powder (68.0 mg, 159 mmol, 51%). mp 164−166 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.94 (2H, t, J = 7.2 Hz), 3.39−3.47 (2H, m), 3.73 (3H, s), 4.42 (2H, d, J = 6.3 Hz), 6.67 (1H, d, J = 8.4 Hz), 6.81−6.92 (4H, m), 7.21−7.32 (6H, m), 7.55 (1H, t, J = 8.1 Hz), 8.66−8.70 (1H, m), 9.40−9.45 (1H, m), 11.89 (1H, bs). Anal. Calcd for C25H24N4O3·0.1H2O: C, 69.78; H, 5.67; N, 13.02. Found: C, 69.55; H, 5.56; N, 13.19. Representative Procedure for the Synthesis of Compounds 21h−j: 6-Fluoro-N-{[3-(methyloxy) phenyl]methyl}-4-oxo-5-[(2phenylethyl)oxy]-3,4-dihydroquinazoline-2-carboxamide (21h). To a solution of compound 9o (100 mg, 0.290 mmol) in DMA (2 mL) was added sodium hydride (60% oil dispersion, 46.0 mg, 1.16 mmol). After the mixture was stirred at room temperature for 30 min, 2-phenylethanol (53.0 mg, 0.434 mmol) was added, and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was acidified with 0.5 N hydrochloric acid to pH 3−4 and extracted with ethyl acetate. The organic layer was washed with H2O and brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by preparative HPLC and crystallization from ethanol/diethyl ether to give the title compound as a pale yellow powder (45.0 mg, 0.101 mmol, 35%). mp 134−135 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.10 (2H, t, J = 7.2 Hz), 3.74 (3H, s), 4.28 (2H, t, J = 7.2 Hz), 4.44 (2H, d, J = 6.3 Hz), 6.81−6.84 (1H, m), 6.89−6.92 (2H, m), 7.20−7.33 (6H, m), 7.52 (1H, dd, J = 9.0, 4.5 Hz), 7.79 (1H, t, J = 9.6 Hz), 9.50 (1H, t, J = 5.7 Hz), 12.14 (1H, bs). Anal. Calcd for C25H22FN3O4·0.7H2O: C, 65.27; H, 5.13; N, 9.13. Found: C, 65.04; H, 5.09; N, 8.84. 6-Fluoro-5-{[2-(4-fluorophenyl)ethyl]oxy}-N-{[3-(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2-carboxamide (21i). Compound 21i was prepared from compound 9o and 2-(4fluorophenyl)ethanol (pale yellow powder, 32%). mp 146−148 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.09 (2H, t, J = 7.2 Hz), 3.73 (3H, s), 4.27 (2H, t, J = 7.2 Hz), 4.44 (2H, d, J = 6.3 Hz), 6.80−6.84 (1H, m), 6.88−6.92 (2H, m), 7.11 (2H, t, J = 8.7 Hz), 7.25 (1H, t, J = 8.1 Hz), 7.37 (2H, dd, J = 8.4, 5.7 Hz), 7.53 (1H, dd, J = 9.0, 4.8 Hz), 7.78 (1H, dd, J = 10.2, 9.0 Hz), 9.50 (1H, t, J = 6.3 Hz), 12.15 (1H, bs). Anal. Calcd for C25H21F2N3O4·0.6H2O: C, 63.05; H, 4.70; N, 8.82. Found: C, 62.85; H, 4.57; N, 8.96. 5-{[2-(4-Aminophenyl)ethyl]oxy}-6-fluoro-N-{[3(methyloxy)phenyl]methyl}-4-oxo-3,4-dihydroquinazoline-2carboxamide (21j). Compound 21j was prepared from compound 9o and 2-(4-aminophenyl)ethanol (beige powder, 54%). mp 129−131 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.91 (2H, t, J = 7.5 Hz), 3.73 (3H, s), 4.14 (2H, t, J = 7.8 Hz), 4.44 (2H, d, J = 6.3 Hz), 4.90 (2H, bs), 6.48 (2H, d, J = 8.4 Hz), 6.80−6.84 (1H, m), 6.90−6.94 (4H, m), 7.24 (1H, t, J = 7.8 Hz), 7.53 (1H, dd, J = 9.0, 4.8 Hz), 7.76−7.83 (1H, m), 9.51 (1H, t, J = 6.6 Hz), 12.12 (1H, bs). Anal. Calcd for C25H23FN4O4·0.1H2O: C, 64.67; H, 5.04; N, 12.07. Found: C, 64.61; H, 5.05; N, 12.20. Representative Procedure for the Synthesis of Compounds 26a−d: 5-Cyano-N-{[3-(methyloxy)phenyl]-methyl}-4-oxo-3,4dihydroquinazoline-2-carboxamide (26a). A mixture of compound 25a (29 mg, 0.074 mmol), 6 N hydrochloric acid (0.496 mL), N



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Sodium 4-[2-({6-Fluoro-2-[({[3-(methyloxy)phenyl]methyl}amino)carbonyl]-4-oxo-3,4-dihydroquinazolin-5-yl}oxy)ethyl]benzoate (38). To a solution of compound 21k (1.50 g, 3.05 mmol) in a mixed solvent of THF (30 mL), methanol (7.5 mL), and DMSO (3.75 mL) was added a solution of sodium hydrogen carbonate (256 mg, 3.05 mmol) in H2O (7.5 mL) at 80 °C, and the mixture was stirred at 80 °C for 30 min. After THF and methanol were removed by evaporation, methanol (30 mL) was added to the residual suspension. The resulting suspension was stirred at 80 °C for 30 min and allowed to cool to room temperature. The precipitated solid was collected and washed with methanol to give the title compound as a pale yellow powder (1.40 g, 2.73 mmol, 89%). mp 319−321 °C. 1H NMR (300 MHz, DMSO-d6) δ 3.10 (2H, t, J = 7.3 Hz), 3.73 (3H, s), 4.26 (2H, t, J = 7.3 Hz), 4.43 (2H, d, J = 6.2 Hz), 6.79−6.85 (1H, m), 6.88−6.93 (2H, m), 7.20−7.30 (3H, m), 7.45 (1H, dd, J = 9.0, 4.7 Hz), 7.63 (1H, t, J = 9.7 Hz), 7.79 (2H, d, J = 8.1 Hz), 9.33 (1H, t, J = 6.5 Hz), 12.51 (1H, s). Anal. Calcd for C26H21N3O6FNa: C, 60.82; H, 4.12; N, 8.18. Found: C, 60.62; H, 4.11; N, 8.38. MMPs and TACE Enzyme Inhibition Assay. Human recombinant MMP precursors were purchased from Genzyme-Techne (MMP1, 2, 7, 8, 9, 10, and 13 and TACE) or Biogenesis (MMP-3). Human recombinant GST-MMP-14 was prepared as described by Sato et al.29 The MMP assay buffer consisted of 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35. The pro-MMPs were activated by preincubation with 1 mM aminophenylmercuric acetate (APMA) in assay buffer at 37 °C for 2 h (MMP-1, 2, 7, 8, 10, and 13) or 18 h (MMP-3 and 9). The TACE assay buffer consisted of 25 mM Tris-HCl (pH 9.0), 2.5 μM ZnCl2, and 0.005% Brij-35. Enzyme inhibition assays were performed in an assay buffer containing enzymes and fluorescence peptide (Cy3-PLGLK(Cy5Q)AR-NH2 for MMPs, Cy3-PLAQAV(Cy5Q-L-2,3-diaminopropionic acid)-RSSSRNH2 for TACE, Amersham Biosciences) in the presence of various concentrations of inhibitors. Following incubation at 37 °C for 40 min, the reaction was terminated by addition of EDTA (pH 8.0). The increase in fluorescence was measured by Farcyte spectrofluorimeter (Amersham Bioscience, λem = 535 nm; λex = 595 nm). Enzyme activity (%) was determined according to the following equation: enzyme activity (%) = (X − C)/(T − C) × 100, where X is the fluorescence count with inhibitor, T is the fluorescence count without inhibitor, and C is the fluorescence count with EDTA. IC50 values of inhibitors were obtained with iterative fitting package (GraphPad Prism software). Crystallization and Structure Determination. The human MMP-13 catalytic domain was prepared as described previously. Crystals were grown by the hanging drop vapor diffusion method at 20 °C (the temperature was modified). Prior to crystallization, a solution containing 6−14 mg/mL MMP-13 catalytic domain, 5 μM Zn(OAc)2, 5 mM CaCl2, 50 mM NaCl, 20 mM Tris HCl buffer (pH 8.0), and 0.5 mM compound was prepared. Equal volumes (0.5 μL) of the protein solution and reservoir solution containing 8−16% w/v PEG8000, 1.0− 1.5 M ammonium formate, and 0.1 M Tris HCl (pH 8.5) buffer were mixed and equilibrated in the hanging drop against a reservoir solution. Crystals were dipped into a 1:1:1 mixture of protein solution, reservoir solution, and glycerol, soaked for a few minutes, and then treated by flash-cooling method. X-ray diffraction data were collected at SPring-8 BL32B2 and processed with the program CrystalClear (Rigaku/MSC, Inc.). Phase determination was solved by the molecular replacement method with the program MOLREP30 using the structure with PDB accession number 830C.31 This initial model excluded solvents and inhibitors. The refinement program REFMAC32 was used, and some rebuilding of parts of the molecule was performed with WinCoot.33 X-ray coordinates have been deposited with the RCSB Protein Data Bank (PDB) for 37 in complex with MMP-13 (3WV2) and 21k in complex with MMP-13 (3WV1). The statistic data and the refinement statistics are shown in Table 6. Chemically Induced OA. Monoiodoacetate (MIA, Wako Pure Chemical Industries LTD, Japan) was dissolved in saline at 20 mg/mL. Diethyl ether-anesthetized female Sprague−Dawley rats (12 weeks old, Charles River, Japan) were injected with 25 μL of 20 mg/mL MIA in the right knee using a sterile syringe and 27-gauge needle. Control animals were injected with an equivalent amount of saline as that used

Table 6. X-ray Crystallographic Data Collection and Refinement Statistics for Complexes of 37 and 21k with MMP-13 37 (3WV2) X-ray source wavelength (Å) space group unit cell dimensions resolution (Å) unique reflections redundancy completeness (%) I/σ(I) Rsyma reflections used RMS bonds (Å) RMS angles (deg) average B value (Å2) R-valueb Rfreeb

21k (3WV1)

Data Collection SPring-8 BL32B2 1.0 C2 a = 143.5 Å, b = 35.7 Å, c = 94.8 Å, α = 90.0°, β = 135.3°, γ = 90.0° 2.30 14 744

SPring-8 BL32B2 1.0 C2 a = 134.4 Å, b = 36.1 Å, c = 95.3 Å, α = 90.0°, β = 130.9°, γ = 90.0° 1.98 24 131

4.0 95.4 (97.2)

3.69 98.0 (80.5)

9.0 (3.6) 0.077 (0.201) Refinement 14 007 0.009 1.212

9.3 (3.3) 0.085 (0.321)

13.0

26.1

0.174 0.241

0.177 0.220

22 911 0.009 1.318

Rsym = ∑h∑j |⟨I(h)⟩ − I(h)j|/∑h∑j⟨I(h)⟩, where ⟨I(h)⟩ is the mean intensity of symmetry-related reflections. bR-value = ∑||Fobs| − | Fcalc||/∑|Fobs|. Rfree for 5% of reflections excluded from refinement. Values in parentheses are for the highest resolution shell. a

for the treatment group. On day 7, rats were administered 10 mg/kg 39 (RS-130,830) or 38 orally. Four hours after oral administration of MMP inhibitors, the rats were euthanized by CO2, and the joints were lavaged with saline (50 μL) and analyzed for CTX-II in synovial fluid by the Serum Pre-Clinical CartiLaps ELISA (3CAL4000, Nordic Bioscience Diagnostics, Denmark).

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures and compound characterization data for intermediate compounds. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB entries for 37 in complex with MMP-13 and for 21k in complex with MMP-13 are 3WV2 and 3WV1, respectively.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Hiroaki Omae and Yoko Tanaka for the preparation of the catalytic domain of human MMP-13 for X-ray structure analysis, Mika Murabayashi for hit identification by NMR measurements, Dr. Satoshi Endo and Terufumi Takagi for helpful discussions about drug design, and Akira Fujishima for supporting crystallographic study. We acknowledge Drs. Yoshinori Ikeura, Keiji Kamiyama, and Nobuo Cho for careful reading of the manuscript and valuable suggestions. We O



Article

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acknowledge Akira Kaieda for preparing key compounds. We also thank Drs. Shigenori Ohkawa and Yuji Ishihara, our supervisors. We also thank the members of drug metabolism & pharmacokinetics research laboratories, biomolecular research laboratories, and analytical development laboratories for pharmacokinetic and physicochemical studies, the members of drug safety research laboratories for toxicokinetic and toxicological studies, and the members of the Takeda analytical research laboratories, Ltd., for elemental analyses.

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ABBREVIATIONS USED CTX-II, C-telopeptide fragments of type II collagen; SEM, 2(trimethylsilyl)ethoxymethyl; PTFE, poly(tetrafluoroethylene); TEA, triethylamine; mCPBA, m-chloroperoxybenzoic acid; DIEA, N,N-diisopropylethylamine; dba, dibenzylideneacetone; WSCD, water-soluble carbodiimide; HOBt, 1-hydroxybenzotriazole

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