Discovery of novel, highly potent, and selective matrix metalloproteinase (MMP)-13 inhibitors with a 1,2,4-triazol-3-yl moiety as a zinc binding group using a structure-based design approach

Article abstract of DOI:10.1021/acs.jmedchem.6b01007

On the basis of a superposition study of X-ray crystal structures of complexes of quinazoline derivative 1 and triazole derivative 2 with matrix metalloproteinase (MMP)-13 catalytic domain, a novel series of fused pyrimidine compounds which possess a 1,2,4-triazol-3-yl group as a zinc binding group (ZBG) was designed. Among the herein described and evaluated compounds, 31f exhibited excellent potency for MMP-13 (IC₅₀ = 0.036 nM) and selectivities (greater than 1,500-fold) over other MMPs (MMP-1, -2, -3, -7, -8, -9, -10, and -14) and tumor necrosis factor-α converting enzyme (TACE). Furthermore, the inhibitor was shown to protect bovine nasal cartilage explants against degradation induced by interleukin-1 and oncostatin M. In this article, we report the discovery of extremely potent, highly selective, and orally bioavailable fused pyrimidine derivatives that possess a 1,2,4-triazol-3-yl group as a novel ZBG for selective MMP-13 inhibition.

Full text of DOI:10.1021/acs.jmedchem.6b01007

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Article
Discovery of Novel, Highly Potent, and Selective Matrix Metalloproteinase
(MMP)-13 Inhibitors with a 1,2,4-Triazol-3-yl Moiety as a Zinc
Binding Group Using a Structure-Based Design Approach
Hiroshi Nara, Akira Kaieda, Kenjiro Sato, Takako Naito, Hideyuki Mototani, Hideyuki Oki, Yoshio Yamamoto,
Haruhiko Kuno, Takashi Santou, Naoyuki Kanzaki, Jun Terauchi, Osamu Uchikawa, and Masakuni Kori
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01007 • Publication Date (Web): 14 Dec 2016
Downloaded from http://pubs.acs.org on December 14, 2016
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Discovery of Novel, Highly Potent, and
Selective Matrix Metalloproteinase (MMP)ꢀ13
Inhibitors with a 1,2,4ꢀTriazolꢀ3ꢀyl Moiety as a
Zinc Binding Group Using a StructureꢀBased
Design Approach
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Hiroshi Nara,* Akira Kaieda, 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, 26ꢀ1,
MuraokaꢀHigashi 2ꢀchome, Fujisawa, Kanagawa 251ꢀ8555, Japan
Abstract
On the basis of a superposition study of Xꢀray crystal structures of complexes of
quinazoline derivative 1 and triazole derivative 2 with matrix metalloproteinase
(MMP)ꢀ13 catalytic domain, a novel series of fused pyrimidine compounds which
possess a 1,2,4ꢀtriazolꢀ3ꢀyl group as a zinc binding group (ZBG) was designed. Among
the herein described and evaluated compounds, 31f exhibited excellent potency for
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MMPꢀ13 (IC = 0.036 nM) and selectivities (greater than 1,500ꢀfold) over other MMPs
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(MMPꢀ1, 2, 3, 7, 8, 9, 10, and 14) and tumor necrosis factorꢀα converting enzyme
(TACE). Furthermore, the inhibitor was shown to protect bovine nasal cartilage
explants against degradation induced by interleukinꢀ1 and oncostatin M. In this article,
we report the discovery of extremely potent, highly selective, and orally bioavailable
fused pyrimidine derivatives that possess a 1,2,4ꢀtriazolꢀ3ꢀyl group as a novel ZBG for
selective MMPꢀ13 inhibition.
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INTRODUCTION
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Osteoarthritis (OA) is one of the most common forms of arthritis affecting more than 30
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million patients worldwide. The principal morphological characteristic of OA is
progressive cartilage damage that leads to pain and reduced mobility in affected joints.
The recommended
pharmaceutical therapies in the guidelines from the American College of Rheumatology
are
2ꢀ5
limited to alleviation of pain and inhibition of inflammation. The therapies are oral
treatment with acetaminophen, nonꢀsteroidal antiꢀinflammatory drugs (NSAIDs), or
cyclooxygenaseꢀ2 (COXꢀ2) inhibitors and intraꢀarticular injections of hyaluronic acid or
corticosteroids. In addition, because some COXꢀ2 selective inhibitors (rofecoxib and
6, 7
valdecoxib) were withdrawn from the market in 2004 and 2005,
there is a significant
unmet medical need for safe oral diseaseꢀmodifying osteoarthritis drugs that may be
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, 9
able to prevent, slow down, or reverse any advanced cartilage destruction.
The matrix metalloproteinases (MMPs) are a family of structurally related
zincꢀdependent endopeptidases that degrade varied extracellular matrix. Among MMPs,
10, 11
MMPꢀ13 (collagenaseꢀ3) specifically catalyzes the hydrolysis of type II collagen,
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the main structural component of the cartilage matrix, while the protein is resistant to
most proteases. MMPꢀ13 is expressed at higher levels by OA chondrocytes than by
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normal chondrocytes. In addition, regulated expression of human MMPꢀ13 in joint
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cartilages induces OA in genetically modified mice. Further, an MMP inhibitor that
preferentially inhibits MMPꢀ13 has been shown to block the degradation of explanted
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human OA cartilage. These findings suggest that MMPꢀ13 plays a crucial role in the
destruction of articular cartilage in OA.
In preclinical testing, MMP inhibitors have inhibited the destruction of cartilage in some
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animal models of OA. However, most clinical trials of broadꢀspectrum MMP
inhibitors have been discontinued due to concerns of doseꢀlimiting toxicity (skin rash
and musculoskeletal side effects (MSS) characterized by joint stiffness and pain).
Although a number of hypotheses have been proposed for the cause of MSS, including a
nonꢀselective inhibition of other metalloproteinases or a combined inhibition of a series
of critical MMPs, the pharmacological basis for such joint side effects remains
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unknown.
Therefore, considerable interest has been directed toward potent
inhibitors of MMPꢀ13 with a high degree of selectivity over other MMPs, which may
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avoid undesirable side effects.
Most MMP inhibitors generally incorporate a P1'
fragment that can be accommodated in the S1' subsite of the enzyme active site and a
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functional group capable of binding the catalytic zinc ion. While a number of P1'
fragment allows modulation of potency and selectivity against various MMPs, only a
small set of zinc binding groups (ZBGs) have been identified and employed in the
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27, 30ꢀ32
design of selective MMP inhibitors.
Highꢀthroughput screening for MMPꢀ13 inhibition led to a moderately potent (IC = 12
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3, 25, 33
nM) quinazolineꢀ2ꢀcarboxamide 1
triazole 2, which were successfully coꢀcrystallized with MMPꢀ13 catalytic domain
Figure 1). These observations allowed the design of a hybrid molecule that combined
and a weakly potent (IC = 1,900 nM)
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the quinazoline 1 with the triazole ZBG 2 as shown in Figure 2.
We have previously used the fused pyrimidineꢀ2ꢀcarboxamideꢀ4ꢀone scaffold
represented by compound 3 for initial validation of a strategy to obtain inhibitory
potency and selectivity for MMPꢀ13 by utilizing (i) a hydrophobic interaction with the
S1' and its side pocket (S1") as clearly revealed by Xꢀray analysis and (ii) a hydrogen
bonding interaction with the εꢀamino group of Lys140 at the bottom of the S1" pocket
2
3, 25, 33, 34
(
Figure 3).
Furthermore, the fused pyrimidine system has been successfully
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applied by several groups.
To further increase our repertoire of selective MMPꢀ13
inhibitors, we next investigated the potential of substituents on the aromatic ring of the
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Nꢀbenzyl group of 1, thereby potentially addressing the catalytic zinc as shown in
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Figure 2 (C). Beside our early patent application, a similar combination approach was
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recently reported by Fischer et al.
In this article, we report the design, synthesis, and biological activity of novel fused
pyrimidine derivatives which possess a 1,2,4ꢀtriazolꢀ3ꢀyl group as a ZBG with potent
MMPꢀ13 inhibitory activities, excellent selectivities, and good oral bioavailabilities.
CHEMISTRY
Scheme 1 and Scheme 2 depict the synthesis of benzylamine derivatives used in
Scheme 4 and Scheme 5. As shown in Scheme 1, benzylamine with an aliphatic side
chain bearing a carboxylic ester functionality 6 was synthesized by catalytic
hydrogenation of the cyano group of benzonitrile 5 prepared by alkylation of
3ꢀcyanophenol 4 with ethyl 4ꢀbromobutanoate. Another series of compounds that has a
thioether linker was also synthesized. Benzylamines 9a–c were obtained in the
following manner. Alkylation of 3ꢀcyanophenol (4) with the corresponding
1ꢀbromoalkyl chloride afforded alkyl chlorides 7a–c. Substitution reaction of the
chlorides 7a–c with triazolylthiols afforded 8a, 8b, and 8d. Benzonitrile 8a underwent
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Raney nickel catalyzed hydrogenation to give the benzylamine derivative 9a.
Triphenylmethyl protection followed by reduction with lithium aluminum hydride of
each cyano group of benzonitriles 8b and 8d led to the benzylamines 9b and 9c,
respectively. Benzylamine analogues 15a and 15b with diether linkers were synthesized
by the following method. Commercially available nitrotriazole 10 was protected in a
regioselective manner with the sterically demanding triphenylmethyl group to give
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3ꢀnitroꢀ1ꢀ(triphenylmethyl)ꢀ1Hꢀ1,2,4ꢀtriazole (11). Alkylation of commercially
available 3ꢀcyanophenol (4) with ethyl bromoacetate gave ethyl ester 12. Subsequent
chemoselective reduction of the ester group with sodium borohydride afforded
benzonitrile 13a. Oneꢀcarbon homologue of 13a, benzonitrile 13b, was obtained
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directly from 3ꢀcyanophenol 4 by alkylation with 3ꢀbromopropanol. Precursors of
benzylamines 15a and 15b, benzonitriles 14a and 14b, were synthesized by aromatic
nucleophilic substitution reaction of nitrotriazole 11 with hydroxyalkyl substituted
benzonitriles 13a or 13b, respectively. Lithium aluminum hydride reduction of the
cyano group of benzonitriles 14a and 14b led to the desired benzylamine derivatives
15a and 15b.
Carboxylic acid derivatives 20a–c were prepared as shown in Scheme 3.
Thiosemicarbazide 16 was reacted with ethyl 3ꢀchloroꢀ3ꢀoxopropanoate or ethyl
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succinyl chloride followed by cyclization with sodium ethoxide to give triazoleꢀ3ꢀthiol
18a and 18b, respectively. Desulfurization of 18a using Raney Ni provided ester 19c in
a moderate yield (54%), while desulfurization of 18b with sodium nitrite in nitric acid
and concomitant hydrolysis of the ester group led to carboxylic acid 19d in excellent
yield (99%). Reesterification of 19d and triphenylmethyl protection gave ester 19f.
Alkaline hydrolysis of esters 19b, 19c, and 19f afforded the desired carboxylic acids
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20a–c, respectively.
The synthetic routes for 4ꢀoxoꢀ3,4ꢀdihydroquinazolineꢀ2ꢀcarboxamide derivatives 22a–
c and 23 via ethyl 4ꢀoxoꢀ3,4ꢀdihydroquinazolineꢀ2ꢀcarboxylate (21) are shown in
Scheme 4. Taking advantage of the high reactivity of the ester group at the 2ꢀposition of
the quinazoline 21, amide formations at the 2ꢀposition of 4ꢀoxoꢀ3,4ꢀdihydroquinazoline
were achieved by aminolysis of the ethyl 4ꢀoxoꢀ3,4ꢀdihydroquinazolineꢀ2ꢀcarboxylate
(21) with the corresponding benzylamines 6 and 9a (see Scheme 1 for preparation) to
23, 25, 40
give the compounds 22a and 23, respectively.
The ethyl ester 22a was
hydrolyzed, and the resulting carboxylic acid 22b was treated with oxalyl chloride
followed by hydroxylamine to afford the desired hydroxamic acid 22c.
The synthesis of 4ꢀoxoꢀ3,4ꢀdihydrothienopyrimidineꢀ2ꢀcarboxamide derivatives is
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demonstrated in Scheme 5. The thienopyrimidine derivatives 25a–g were synthesized in
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the same manner as described for the quinazoline derivatives in Scheme 4.
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Accordingly, aminolysis of the commercially available ethyl
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ꢀmethylꢀ4ꢀoxoꢀ3,4ꢀdihydrothieno[2,3ꢀd]pyrimidineꢀ2ꢀcarboxylate (24) with
corresponding benzylamines, either commercially available or synthesized (9a–c and
5a) as described in Scheme 1 and Scheme 2, and if required, followed by the removal
of the triphenylmethyl groups under an acidic condition gave the amide derivatives 26a,
6b, and 25a–d. Deprotection of the Boc group of the amide 26b afforded amine 26c.
Condensation of the resulting amines 26a and 26c with corresponding carboxylic acids
0c and 20a (see Scheme 3 for preparation), followed by removal of the
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triphenylmethyl group afforded the amideꢀlinked derivatives 25g and 25e, respectively.
Similarly, condensation of 26c with 20b (see Scheme 3 for preparation) led to 25f. In
the triphenylmethyl deprotection step for 25b–e and 25g, cation scavengers are essential
for achieving consistently high yields of the triazoles, with triethylsilane proving to be
superior to anisole in terms of reaction reproducibility.
The synthesis of 5ꢀsubstituted 4ꢀoxoꢀ3,4ꢀdihydrothienopyrimidineꢀ2ꢀcarboxamide
derivatives 31a–q is described in Scheme 6. Ethyl 2ꢀaminothiophenecarboxylates 29c,
29e, 29f, 29h, 29i, 29k, and 29l were prepared from the corresponding ketones 27a–g in
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oneꢀpot Gewald reaction. Thus, Knoevenagel adducts of the ketones 27a–g and ethyl
cyanoacetate 28 were prepared and subsequently treated with elemental sulfur and
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morpholine in toluene. Ester intermediates 30a–m were obtained by reaction of
thiophenes 29a–m with ethyl cyanoformate in 1 M hydrogen chloride in acetic acid
solution. In the same fashion as for the synthesis of 5ꢀmethylthienopyrimidine
derivatives shown in Scheme 5, aminolysis of the reactive ester group at the 2ꢀposition
of the key intermediates 30a–m with benzylamines 15a and 15b (see Scheme 2 for
preparation) followed by deprotection of the triphenylmethyl group led to the
corresponding amide derivatives 31a–q.
RESULTS AND DISCUSSION
23, 25
A lead quinazoline derivative 1
and a triazole derivative 2 were identified by high
throughput screening as MMPꢀ13 inhibitors (Figure 1). Interestingly, the quinazoline 1
did not possess an obvious ZBG, such as a hydroxamic acid or a carboxylic acid, and
displayed only 25ꢀfold selectivity over other MMP isoenzymes (Table 4).
An Xꢀray crystallographic study of the quinazoline 1 bound to MMPꢀ13 revealed that
no interaction with the catalytic zinc was observed and that the quinazoline ring
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occupies the deep S1' subsite
(Figure 4 (A) and 4 (B)). On the other hand, the
1,2,4ꢀtriazolꢀ3ꢀyl moiety of the triazole 2 coordinates in a monodentate fashion to the
catalytic zinc (II) center. Furthermore, unlike the quinazoline 1, the
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phenylaminothiazole moiety of 2 only partially fills the available space within the
MMPꢀ13 S1' pocket (Figure 4 (C) and (D)). On the basis of a superposition of the
crystal structures, the distance between the phenyl ring on the side chain and the ZBG
was estimated to be about 4 Å (Figure 5). As shown in Table 1, analogues were
designed and synthesized by connection of the methoxy moiety of the benzyl unit of the
quinazoline 1 with various ZBGs by a simple linker element.
Compounds 22a and 22b having an ester or a carboxylic acid group via a
nꢀpropyleneoxy linker at the 3ꢀposition of the leftꢀhand phenyl group showed a decrease
in MMPꢀ13 inhibitory activity in comparison to the original lead quinazoline 1.
Conversion of the carboxylic acid to hydroxamic acid, known as a potent ZBG, resulted
in a slight increase in potency (22c vs 1). On the other hand, quinazoline/triazole hybrid
compound 23 that is linked to the triazolyl group through a fourꢀatom linker showed a
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8ꢀfold increase in inhibitory activity against MMPꢀ13 (23 vs 1).
We have previously shown that the fused pyrimidine core markedly influences the
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MMPꢀ13 inhibitory potency, and thus, the quinazoline scaffold was replaced with a
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previously identified thieno[2,3ꢀd]pyrimidine core 25a–g (Table 2). The scaffold
gave analogues that were potent compounds (25a, 25b, and 25d) in the MMPꢀ13
inhibitory assay, however, an increased CYP3A4 inhibition was observed for the
compounds with sulfurꢀcontaining linkers (25a–c). Investigation of the length of the
linker between the benzylamide at the 2ꢀposition of the pyrimidine and the
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1,2,4ꢀtriazolꢀ3ꢀyl group revealed that the compound with a fiveꢀatom linker exhibited
comparable activity (25b vs 25a) to that of a fourꢀatom linker, but a sixꢀatom linker
derivative showed reduced activity (25c vs 25a). Since the inhibition of the CYP3A4
enzyme is well known to potentially induce drug–drug interactions, elimination of such
undesired property was clearly necessary. Fortuitously, the undesirable CYP3A4
inhibition was decreased in the oxygen linker analogue 25d. A series of compounds
with more rigid linkers (25e–g) showed decreased potency compared to that of
compound 25a. Subsequent rat pharmacokinetic studies revealed that thienopyrimidine
25d with an ethylenedioxy linker was one of the most potent inhibitors, had the least
risk of CYP3A4 inhibition among a series of fused pyrimidine derivatives, and showed
high oral exposure (AUC = 10855 ng•h/mL) in rats (F% = 18). Further optimization to
improve the pharmacokinetic profile and attenuation of offꢀtarget CYP3A4 inhibition
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while maintaining high potency was attempted at 5ꢀposition of the thienopyrimidine
core.
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In the course of another study of nonꢀzinc binding inhibitors having the
thienopyrimidine core, it was found that the 5ꢀposition of the core can accommodate a
2
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wide range of substituents without loss of potency. In Table 3, MMPꢀ13 inhibitory
activities, CYP3A4 inhibitory activities, and pharmacokinetic parameters of
5ꢀsubstituted thienopyrimidine derivatives are summarized. Introduction of an isopropyl
group at the 5ꢀposition maintained a potent MMPꢀ13 inhibitory activity (31a), and
substitution of the isopropyl group with a phenyl group showed a 3ꢀfold increased
MMPꢀ13 inhibitory activity (31d). Replacement of the phenyl group with thiophenes
maintained potent MMPꢀ13 inhibitory activities (31b and 31c), however, increased
unwanted CYP3A4 inhibitory activities. Introduction of an additional substituent at the
para position of the phenyl ring tended to reduce MMPꢀ13 inhibitory activities (31g, 31j,
and 31m). On the other hand, substitution at the meta position exhibited a tendency to
retain potency even with rather bulky substituent (31f, 31i, and 31l). Substitution by a
fluoro group (31e) or a chloro group (31h) at the ortho position of the phenyl ring also
retained MMPꢀ13 inhibitory activities. However, a methoxy group at the orthoꢀposition
of the benzene ring (31k) significantly reduced the activity. The reduction of the activity
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observed for 31k compared to 31l could be attributed to a steric repulsion between the
relatively large oꢀmethoxy group of 31k and the wall of the S1' subsite. Fiveꢀatom
linker compounds, such as 31n, 31o, 31p, and 31q gave comparable MMPꢀ13 inhibitory
activities compared to those of the corresponding fourꢀatom linker series 31e, 31f, 31h,
and 31i, but the fiveꢀatom series showed potent CYP3A4 inhibition. It is noteworthy
that in contrast to the triazole ZBG series exemplified by 31i and 31q, incorporation of
the mꢀchloroꢀsubstituted 5ꢀaryl group into other triazolone ZBG series was not proved
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to be well tolerated in terms of MMPꢀ13 inhibitory activity. Among the 5ꢀsubstituted
thienopyrimidine derivatives, mꢀfluorophenyl 31f showed the best combination of
CYP3A4 inhibition risk and oral exposure at a dose of 1 mg/kg in rats and mice (F% =
33 and 38, respectively). The overall properties of 31f made it an attractive candidate
for further preclinical evaluation towards the treatment of MMPꢀ13 related diseases.
Compound 31f, which exhibited a highly potent MMPꢀ13 inhibitory activity and a
promising DMPK profile, was assessed for its selectivity profile against other matrix
metalloproteinase homologues including MMPꢀ1, 2, 3, 7, 8, 9, 10, 14, and TACE as
shown in Table 4. Compound 31f exhibited 5,000ꢀfold selectivity for MMPꢀ13 over
MMPꢀ2, >1,500ꢀfold selectivity over MMPꢀ10, and >27,000ꢀfold selectivity over
MMPꢀ1, 3, 7, 8, 9, 14, and TACE. Consequently, compound 31f is one of the most
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selective and potent MMPꢀ13 inhibitors containing a ZBG.
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As exemplified by the crystal structure of the triazole inhibitor 3 bound to MMPꢀ13,
obtained by soaking experiment, the 1,2,4ꢀtriazolꢀ3ꢀyl group of 23 coordinates to the
catalytic zinc ion of MMPꢀ13 shown in Figure 6. The quinazoline ring system of the
hybrid inhibitor 23 fills the deep S1' pocket, the enzyme’s specificity pocket, of
MMPꢀ13 consisting of Thr245 and Thr247 in the same fashion as the binding mode of
the lead quinazoline 1 (Figure 4 (B)). Although the fourꢀatom linker between the
triazole ring and the phenyl group has no obvious interaction with MMPꢀ13, the linker
should play an important role in the orientation of the triazolyl group to form the metal
binding. The triazolyl group of 23 is coordinated to the zinc ion in a monodentate
fashion as that of the lead triazole 2 (Figure 4 (D)). The inhibitor 23 is stabilized at the
S1' site of MMPꢀ13 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
quinazoline ring and the carbonyl oxygen of Thr245, and (c) the exocyclic carbonyl
oxygen at the 2ꢀposition of the quinazoline ring and the backbone amide of Thr245. The
observed βꢀsheet type interaction confers the potent inhibitory activity of the
quinazolinꢀ4ꢀoneꢀ2ꢀcarboxamide inhibitors.
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Compound 31f was further evaluated for its ability to block the release of collagen from
cartilage. To examine the chondroprotective effect of the MMPꢀ13 selective inhibitor, a
bovine nasal cartilage (BNC) assay was performed. The BNC assay is an established
and widely used procedure that allows evaluation of cartilage degradation by
upregulating proteolytic enzymes such as the collagenases MMPꢀ1 and MMPꢀ13 in
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vitro.
The chondrocyteꢀmediated degradation of cartilage was studied using bovine
nasal cartilage slices cultured for up to 14 days in the presence or absence of MMP
inhibitors. Collagen release, measured quantitatively as hydroxyproline, was stimulated
by proꢀinflammatory cytokines interleukinꢀ1 (ILꢀ1) and oncostatin M (OSM).
As shown in Table 5, the protection of cartilage degradation by compound 31f in this
assay was concentration dependent and statistically significant at 1 µM (70.8% of
5
0
inhibition), whereas at the same concentration of 1 µM, 32 (RS-130,830 ), which
inhibits MMPs broadly, showed complete inhibition. This difference of inhibitory
activity profile between 31f and 32 in this assay may be explained by high isoform
selectivity of 31f for MMPꢀ13 over other collagenolytic enzymes in the
chondroprotective efficacy and/or a difference in the degree of tissue permeability of the
two inhibitors.
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CONCLUSIONS
On the basis of the superposition of Xꢀray crystal structures of the complexes of
quinazoline derivative 1 and triazole derivative 2 with MMPꢀ13 catalytic domain, we
have designed and identified a new class of MMPꢀ13 selective inhibitors that possesses
a 1,2,4ꢀtriazolꢀ3ꢀyl group as a ZBG, and developed an efficient synthetic method to
prepare quinazolineꢀ2ꢀcarboxamide and thienopyrimidineꢀ2ꢀcarboxamide derivatives.
Quinazoline and its isosteric thienopyrimidine derivatives that were linked via the
2ꢀposition with the 1,2,4ꢀtriazolꢀ3ꢀyl group by a 4ꢀatom linker induced a considerable
increase in potency. Particularly, diether linked 1,2,4ꢀtriazolꢀ3ꢀyl derivatives retained
potent inhibition and reduced undesirable inhibition of CYP3A4. Xꢀray analysis of the
complex, as exemplified by 23 with the MMPꢀ13 catalytic domain, confirmed that the
1,2,4ꢀtriazolꢀ3ꢀyl group directly interacts with the catalytic zinc ion and that the
quinazoline core is buried deeply into the S1' pocket by forming a βꢀsheet type
interaction with hydrogen bonding to the enzyme’s backbone spanning the S1' pocket.
Among a series of quinazoline and thienopyrimidine derivatives investigated,
5ꢀ(3ꢀfluorophenyl)ꢀ4ꢀoxoꢀNꢀ{3ꢀ[2ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ3ꢀylꢀoxy)ethoxy]benzyl}ꢀ3,4ꢀdihyd
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rothieno[2,3ꢀd]pyrimidineꢀ2ꢀcarboxamide (31f) exhibited excellent potency (IC =
50
0.036 nM) and selectivity (greater than 1,500ꢀfold) over other MMPs (MMPꢀ1, 2, 3, 7,
8, 9, 10, and 14) and TACE, and demonstrated favorable pharmacokinetic parameters,
making it an attractive candidate for further studies. To evaluate compound 31f for its
potential utility in the treatment of collagenase related disease and disorders, BNC assay
was employed. The selective MMPꢀ13 inhibitor 31f was effective at preventing the
ILꢀ1/OSM induced in vitro degradation of BNC (70.8% inhibition of cartilage
degradation at 1 µM). Taken together with the fact that MMPꢀ13 is indicated as the
primary collagenase in the human OA cartilage, selective MMPꢀ13 inhibitors may be a
potential treatment of OA while avoiding the toxicity associated with inhibition of
MMPs other than MMPꢀ13.
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EXPERIMENTAL SECTION
General Methods. Melting points were determined in open capillary tubes on a Büchi
1
melting point apparatus B545 and are uncorrected. H NMR spectra were recorded on a
Bruker DPXꢀ300 (300 MHz) or a Bruker Avance 400 (400 MHz) spectrometer and are
reported in parts per million (δ) relative to tetramethylsilane (TMS: δ 0.00 ppm). Data
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are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet, tt =
triplet of triplet, 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). Thinꢀlayer chromatography (TLC) was performed on Merck
silica gel plates 60F254. Preparative HPLC was performed on a Shiseido CAPCELL
PACK Cꢀ18 UG120 Sꢀ5 column (20 mmΦ × 50 mm), eluting at a flow rate of 25
mL/min with a linear gradient of water (0.1% TFA)/acetonitrile (0.1% TFA) from 90:10
to 0:100 over 10 min. UV detection was at 220 nm.
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The purity of all compounds used in biological studies was determined to be ≥ 95% by
elemental analysis or HPLC analysis. Elemental analyses were performed by Takeda
Analytical Research Laboratories, Ltd. Experimentally determined hydrogen, carbon,
and nitrogen composition by elemental analysis was within ± 0.4% of the expected
value, implying a purity of ≥ 95%. Liquid chromatography−mass spectrometry
(LC/MS) analysis was performed using one of the following two conditions: (a)
Lꢀcolumn 2 ODS (50 mm × 3.0 mm I.D., 3 ꢁm particle size, CERI, Japan) in a
Shimadzu LCꢀ20AD equipped with a Shimadzu LCMSꢀ2020 eluting with 5 mM
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AcONH in ultrapure water/acetonitrile = 90/10 (mobile phase A) and 5 mM AcONH
4
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in ultrapure water/acetonitrile = 10/90 (mobile phase B), using the following elution
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gradient of 5% B to 90% B over 0.9 min followed by 90% B isocratic over 1.1 min at a
flow rate of 1.5 mL/min (UV detection at 220 or 254 nm); (b) Shiseido CAPCELL
PACK Cꢀ18 UG120 Sꢀ3 column (1.5 mmΦ × 35 mm) in a Waters Alliance 2795 or an
Agilent 1100 LC 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.
Ethyl 4-[(3-cyanophenyl)oxy]butanoate (5). A solution of 4ꢀbromobutanoic acid (25.0
g, 150 mmol), DMF (5 drops), and oxalyl chloride (17.0 mL, 195 mmol) in
dichloromethane (250 mL) was stirred at room temperature for 5 h. The reaction
mixture was concentrated under reduced pressure, dichloromethane (200 mL) and
ethanol (9.0 mL, 170 mmol) were added to the residue, and the mixture was stirred at
room temperature for 15 h. The reaction mixture was washed with saturated aqueous
NaHCO solution and brine, and the organic layer was dried over Na SO , and
3
2
4
concentrated. The residue was purified by silica gel column chromatography (0–5%
ethyl acetate/hexane) to give ethyl 4ꢀbromobutanoate as a colorless oil (24.5 g, 84%). A
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suspension of 3ꢀhydroxybenzonitrile (4) (5.00 g, 42.0 mmol) and 60% sodium hydride
(oil dispersion, 2.01 g, 50.3 mmol) in DMF (200 mL) was stirred at room temperature
for 30 min, and ethyl 4ꢀbromobutanoate (9.82 g, 50.3 mmol) was added to the mixture.
The mixture was stirred at room temperature for 15 h and concentrated under reduced
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pressure. The residue was extracted with ethyl acetate and saturated aqueous NH Cl
4
solution. The organic layer was washed with saturated aqueous NH Cl solution and
4
brine, dried over Na SO , and concentrated. The obtained residue was purified by silica
2
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gel column chromatography (0–10% ethyl acetate/hexane) to give the title compound as
1
a colorless oil (10.2 g, quant.). H NMR (300 MHz, CDCl
3
)
δ
1.27 (3H, t, J = 7.2 Hz),
2.13 (2H, tt, J = 6.7, 6.7 Hz), 2.52 (2H, t, J = 7.2 Hz), 4.03 (2H, t, J = 6.1 Hz), 4.16 (2H,
q, J = 7.2 Hz), 7.08–7.16 (2H, m), 7.21–7.26 (1H, m), 7.32–7.40 (1H, m).
Ethyl 4-{[3-(Aminomethyl)phenyl]oxy}butanoate Hydrochloride (6). A suspension
of compound 5 (8.50 g, 36.4 mmol), 10% palladium on carbon (containing 50% water)
(12.8 g), and formic acid (98 mL) in methanol (80 mL) was stirred at room temperature
for 5 h under hydrogen atmosphere at 1 atm. The catalyst was filtered off through a
Celite pad, and the filtrate was concentrated under reduced pressure. To the residue was
added 4 N hydrogen chloride in ethyl acetate solution (15.0 mL, 60.0 mmol). The
mixture was stirred at room temperature for 1 h and concentrated under reduced
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pressure. The residue was crystallized from toluene, and the crude crystals were
recrystallized from diethyl ether to give the title compound as a white powder (7.79 g,
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78% for 2 steps). H NMR (300 MHz, DMSOꢀd
6
)
δ
1.18 (3H, t, J = 7.2 Hz), 1.98 (2H, tt,
J = 6.8, 6.8 Hz), 2.46 (2H, t, J = 7.3 Hz), 3.95–4.03 (4H, m), 4.07 (2H, q, J = 7.1 Hz),
.92 (1H, dd, J = 8.2, 2.2 Hz), 7.04 (1H, d, J = 7.3 Hz), 7.12 (1H, s), 7.31 (1H, t, J = 7.9
Hz), 8.26 (3H, s).
6
3-[(2-Chloroethyl)oxy]benzonitrile (7a). A suspension of 3ꢀhydroxybenzonitrile (4)
(5.00 g, 42.0 mmol), 1ꢀbromoꢀ2ꢀchloroethane (9.00 g, 62.8 mmol), and potassium
hydroxide (2.50 g, 44.6 mmol) in ethanol (100 mL) was stirred at 90 °C for 24 h. After
cooling to room temperature, the reaction mixture was concentrated under reduced
pressure. To the residue were added diethyl ether and water. The organic layer was
washed with brine, dried over Na SO , and concentrated. The residual oil was purified
2
4
by silica gel column chromatography (2–50% ethyl acetate/hexane) to give the title
1
compound as a colorless oil (1.39 g, 18%). H NMR (300 MHz, CDCl
3
)
δ
3.83 (2H, t, J
=
5.7 Hz), 4.25 (2H, t, J = 5.7 Hz), 7.14–7.19 (2H, m), 7.29 (1H, dt, J = 7.6, 1.3 Hz),
7.37–7.43 (1H, m).
3-[(3-Chloropropyl)oxy]benzonitrile (7b). A suspension of 1ꢀbromoꢀ3ꢀchloropropane
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(13.9 g, 88.1 mmol) and 60% sodium hydride (oil dispersion, 3.02 g, 126 mmol) in
ethanol (50 mL) was stirred at room temperature for 30 min. To the reaction mixture
was added 3ꢀhydroxybenzonitrile (4) (10.0 g, 83.9 mmol) at 0 °C, and the mixture was
stirred at 60 °C for 15 h and concentrated under reduced pressure. To the residue was
added ethyl acetate and water. The organic layer was washed with brine, dried over
Na SO , and concentrated. The residual oil was purified by silica gel column
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chromatography (5–15% ethyl acetate/hexane) to give the title compound as a colorless
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oil (14.2 g, 86%). H NMR (300 MHz, CDCl
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δ
2.26 (2H, tt, J = 6.0, 6.0 Hz), 3.75 (2H,
t, J = 6.2 Hz), 4.14 (2H, t, J = 5.8 Hz), 7.11–7.18 (2H, m), 7.23–7.28 (1H, m), 7.34–7.41
1H, m).
(
3-[(4-Chlorobutyl)oxy]benzonitrile (7c). Compound 7c was prepared from
3ꢀhydroxybenzonitrile (4) and 1ꢀbromoꢀ4ꢀchlorobutane with a similar procedure as
1
described for compound 7b (colorless oil, 95%). H NMR (300 MHz, CDCl
3
)
δ
1.94–
2.02 (4H, m), 3.60–3.66 (2H, m), 4.02 (2H, t, J = 5.4 Hz), 7.08–7.15 (2H, m), 7.21–7.27
(1H, m), 7.33–7.41 (1H, m).
3
-{[2-(1H-1,2,4-Triazol-3-ylthio)ethyl]oxy}benzonitrile (8a). A solution of compound
a (11.3 g, 62.3 mmol), 1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol (6.00 g, 59.3 mmol), and triethylamine
7
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(
8.40 mL, 62.3 mmol) in ethanol (50 mL) was stirred at 80 °C for 15 h. After cooling to
room temperature, the reaction mixture was concentrated under reduced pressure. To the
residue were added ethyl acetate and water. The organic layer was washed with brine,
0
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0
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dried over Na SO , and concentrated to give the title compound as a white powder (14.4
2
4
1
6
g, 98%). H NMR (300 MHz, DMSOꢀd ) δ 3.48 (2H, t, J = 6.7 Hz), 4.32 (2H, t, J = 6.6
Hz), 7.29–7.36 (1H, m), 7.38–7.43 (1H, m), 7.44–7.54 (2H, m), 8.46 (1H, s), 14.09 (1H,
s).
3
-{[3-(1H-1,2,4-Triazol-3-ylthio)propyl]oxy}benzonitrile (8b). Compound 8b was
prepared from compound 7b and 1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol with a similar procedure as
1
described for compound 8a (white powder, quant.). H NMR (300 MHz, DMSOꢀd
6
)
δ
2.11 (2H, tt, J = 6.8, 6.6 Hz), 3.22 (2H, t, J = 7.1 Hz), 4.14 (2H, t, J = 6.2 Hz), 7.26–
7.53 (4H, m), 8.42 (1H, s), 14.03 (1H, s).
3-[(3-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]thio}propyl)oxy]benzonitrile (8c).
A solution of compound 8b (4.00 g, 15.4 mmol), triphenylmethyl chloride (6.43 g, 23.0
mmol), and triethylamine (2.33 g, 23.0 mmol) in THF (50 mL) was stirred at room
temperature for 48 h and concentrated under reduced pressure. To the residue were
added ethyl acetate and water. The organic layer was washed with brine, dried over
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Na SO , and concentrated. The obtained oil was purified by silica gel column
2
4
chromatography (5–40% ethyl acetate/hexane) to give the title compound as a white
10
11
12
13
14
15
16
17
18
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47
48
49
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59
60
1
powder (3.52 g, 46%). H NMR (300 MHz, DMSOꢀd
6
)
δ
2.00–2.09 (2H, m), 3.16 (2H, t,
J = 7.1 Hz), 4.02–4.09 (2H, m), 7.01–7.11 (6H, m), 7.19–7.49 (13H, m), 8.13 (1H, s).
3
-{[4-(1H-1,2,4-Triazol-3-ylthio)butyl]oxy}benzonitrile (8d). Compound 8d was
prepared from compound 7c and 1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol with a similar procedure as
1
described for compound 8a (white powder, quant.). H NMR (300 MHz, CDCl
3
)
δ
1
.87–2.02 (5H, m), 3.20–3.31 (2H, m), 3.96–4.05 (2H, m), 7.06–7.16 (2H, m), 7.20–
7
.27 (1H, m), 7.32–7.40 (1H, m), 8.13 (1H, s).
3
-[(4-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]thio}butyl)oxy]benzonitrile (8e).
Compound 8e was prepared from compound 8d with a similar procedure as described
1
for compound 8c (white powder, 78%). H NMR (300 MHz, CDCl
3
)
δ
1.81–1.89 (4H,
m), 3.07–3.16 (2H, m), 3.83–3.90 (2H, m), 7.01–7.06 (2H, m), 7.11–7.17 (6H, m),
7
.19–7.24 (1H, m), 7.28–7.36 (10H, m), 7.87 (1H, s).
1
-(3-{[2-(1H-1,2,4-Triazol-3-ylthio)ethyl]oxy}phenyl)methanamine (9a). A solution
of compound 8a (9.00 g, 36.5 mmol) and Raneyꢀnickel (5.00 g) in 5 N ammonia in
methanol solution (300 mL) was stirred at room temperature under hydrogen
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atmosphere at 1 atm for 15 h. The catalyst was filtered off through a Celite pad, and the
filtrate was concentrated. The obtained residue was crystallized from toluene to give the
title compound as a pale blue powder (6.45 g, 71%). The compound was used without
further purification.
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1-{3-[(3-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]thio}propyl)oxy]phenyl}metha
namine (9b). Compound 9b was prepared from compound 8c with a similar procedure
1
as described for compound 15a (pale yellow oil, 95%). H NMR (300 MHz, DMSOꢀd )
6
δ
1.94–2.08 (2H, m), 3.16 (2H, t, J = 7.1 Hz), 3.62–3.67 (2H, m), 3.97 (2H, t, J = 6.1
Hz), 6.60–6.74 (1H, m), 6.82–6.92 (2H, m), 6.98–7.11 (6H, m), 7.12–7.45 (12H, m),
.14 (1H, s).
8
1-{3-[(4-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]thio}butyl)oxy]phenyl}methan
amine (9c). Compound 9c was prepared from compound 8e with a similar procedure as
1
described for compound 15a (yellow oil, 86%). H NMR (300 MHz, CDCl
3
)
δ
1.79–
1.89 (4H, m), 2.35 (2H, s), 3.09–3.16 (2H, m), 3.81 (2H, s), 3.85–3.93 (2H, m), 6.71
(1H, dd, J = 8.1, 2.3 Hz), 6.81 (1H, s), 6.87 (1H, d, J = 7.0 Hz), 7.08–7.37 (16H, m),
7.86 (1H, s).
3-Nitro-1-(triphenylmethyl)-1H-1,2,4-triazole (11). A solution of
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ꢀnitroꢀ1Hꢀ1,2,4ꢀtriazole (10) (1.00 g, 8.77 mmol), triphenylmethyl chloride (4.89 g,
17.5 mmol), and N,Nꢀdiisopropylethylamine (3.05 mL, 17.5 mmol) in THF (50 mL) was
stirred at room temperature for 15 h. The mixture was concentrated under reduced
pressure, and the residue was extracted with ethyl acetate and water. The organic layer
was washed with brine, dried over Na SO , and concentrated. The residue was purified
10
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4
by silica gel column chromatography (15–30% ethyl acetate/hexane) to give the title
1
compound as a white powder (2.90 g, 93%). H NMR (300 MHz, CDCl
3
)
δ
7.08–7.17
(6H, m), 7.33–7.47 (9H, m), 8.04 (1H, s).
Ethyl [(3-Cyanophenyl)oxy]acetate (12). Ethyl bromoacetate (14.7 g, 88.1 mmol) was
added dropwise to a suspension of potassium carbonate (12.8 g, 92.3 mmol) and
3ꢀhydroxybenzonitrile (4) (10.0 g, 83.9 mmol) in THF (50 mL) at room temperature,
and the mixture was stirred at 50 °C for 24 h. The mixture was extracted with ethyl
acetate and saturated aqueous NH Cl solution, and the organic layer was washed with
4
brine, dried over Na SO , and concentrated to give the title compound as a pale yellow
2
4
1
powder (17.5 g, 99%). H NMR (300 MHz, CDCl
3
) δ 1.31 (3H, t, J = 7.2 Hz), 4.29 (2H,
q, J = 7.1 Hz), 4.65 (2H, s), 7.13–7.18 (2H, m), 7.28–7.32 (1H, m), 7.36–7.44 (1H, m).
3
-[(2-Hydroxyethyl)oxy]benzonitrile (13a). A suspension of compound 12 (8.00 g,
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39.0 mmol) and sodium borohydride (1.47 g, 39.0 mmol) in ethanol (120 mL) was
stirred at 50 °C for 15 h, and the mixture was concentrated under reduced pressure. The
residue was extracted with ethyl acetate and water, and the organic layer was washed
0
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with brine, dried over Na SO , and concentrated to give the title compound as a white
2
4
1
powder (5.85 g, 92%). H NMR (300 MHz, CDCl
3
)
δ
1.67 (1H, bs), 3.97–4.02 (2H, m),
4.08–4.13 (2H, m), 7.13–7.20 (2H, m), 7.24–7.30 (1H, m), 7.35–7.43 (1H, m).
3-[(2-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]oxy}ethyl)oxy]benzonitrile (14a).
A solution of compound 13a (19.2 g, 118 mmol) in THF (100 mL) was added dropwise
to a suspension of compound 11 (40.0 g, 112 mmol) and 60% sodium hydride (oil
dispersion, 6.06 g, 152 mmol) in THF (300 mL), and the mixture was stirred at room
temperature for 12 h. To the mixture cooled to 0 °C were added water and ethyl acetate.
The organic layer was washed with brine, dried over Na SO , and concentrated. The
2
4
residue was purified by silica gel column chromatography (0–30% ethyl acetate/hexane)
1
to give the title compound as a white powder (25.0 g, 46%). H NMR (300 MHz,
3
CDCl ) δ 4.23–4.29 (2H, m), 4.56–4.63 (2H, m), 7.07–7.41 (19H, m), 7.67 (1H, s).
3-[(3-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]oxy}propyl)oxy]benzonitrile
39
(
14b). A solution of compound 13b (4.97 g, 28.1 mmol) in THF (50 mL) was added
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dropwise to a mixture of compound 11 (10.0 g, 28.1 mmol) and 60% sodium hydride
(oil dispersion, 2.25 g, 56.1 mmol) in THF (150 mL) at room temperature and the
resulting mixture was stirred at room temperature for 15 h. The reaction mixture was
10
11
12
13
14
15
16
17
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diluted with ethyl acetate and washed with H O and brine. The organic layer was dried
2
over Na SO and concentrated under reduced pressure to give the title compound as a
2
4
pale yellow amorphous (14.0 g). The crude 14b was used for the next reaction without
1
further purification. H NMR (300 MHz, DMSOꢀd
6
)
δ
2.08–2.19 (2H, m), 4.14 (2H, t, J
=
6.2 Hz), 4.30 (2H, t, J = 6.2 Hz), 7.04–7.12 (6H, m), 7.24–7.30 (1H, m), 7.34–7.43
(11H, m), 7.47 (1H, t, J = 7.9 Hz), 7.82 (1H, s).
1-{3-[(2-{[1-(Triphenylmethyl)-1H-1,2,4-triazol-3-yl]oxy}ethyl)oxy]phenyl}methan
amine (15a). To a suspension of lithium aluminum hydride (3.21 g, 84.6 mmol) in THF
(30 mL) was added dropwise a solution of compound 14a (4.00 g, 8.46 mmol) in THF
(20 mL) at room temperature, and the mixture was stirred at room temperature for 4 h.
2 4 2
To the mixture cooled to 0 °C was added Na SO •10H O (10.9 g, 33.9 mmol). The
mixture was stirred at room temperature for 30 min. The insoluble material was filtered
off through a Celite pad. The filtrate was dried over Na SO and concentrated. The
2
4
residual solid was washed with diethylether and dried to give the title compound as a
1
white powder (2.35 g, 58%). H NMR (300 MHz, DMSOꢀd
6
)
δ
1.58–2.00 (2H, m), 3.66
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