Design, synthesis, and biological activity of novel, potent, and highly selective fused pyrimidine-2-carboxamide-4-one-based matrix metalloproteinase (MMP)-13 zinc-binding inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.009

Matrix metalloproteinase-13 (MMP-13), a member of the collagenase family of enzymes, has been implicated to play a key role in the pathology of osteoarthritis. Recently, we have reported the discovery of a series of quinazoline-2-carboxamide based non-zin

Full text of DOI:10.1016/j.bmc.2016.09.009

Accepted Manuscript
Design, synthesis, and biological activity of novel, potent, and highly selective
fused pyrimidine-2-carboxamide-4-one-based matrix metalloproteinase
(MMP)-13 zinc-binding inhibitors
Hiroshi Nara, Kenjiro Sato, Akira Kaieda, Hideyuki Oki, Haruhiko Kuno,
Takashi Santou, Naoyuki Kanzaki, Jun Terauchi, Osamu Uchikawa, Masakuni
Kori
PII:
S0968-0896(16)30696-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.009
BMC 13262
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 July 2016
3 September 2016
6 September 2016
Please cite this article as: Nara, H., Sato, K., Kaieda, A., Oki, H., Kuno, H., Santou, T., Kanzaki, N., Terauchi, J.,
Uchikawa, O., Kori, M., Design, synthesis, and biological activity of novel, potent, and highly selective fused
pyrimidine-2-carboxamide-4-one-based matrix metalloproteinase (MMP)-13 zinc-binding inhibitors, Bioorganic
& Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.009
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Design, synthesis, and biological activity of novel, potent, and highly selective fused
pyrimidine-2-carboxamide-4-one-based matrix metalloproteinase (MMP)-13 zinc-binding
inhibitors
Hiroshi Nara,* Kenjiro Sato, Akira Kaieda, Hideyuki Oki, 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
ABSTRACT
Matrix metalloproteinase-13 (MMP-13), a member of the collagenase family of enzymes, has been
implicated to play a key role in the pathology of osteoarthritis. Recently, we have reported the discovery of
a series of quinazoline-2-carboxamide based non-zinc-binding MMP-13 selective inhibitors, as exemplified
by compound 1. We then continued our research of a novel class of zinc-binding inhibitors to obtain
follow-up compounds with different physicochemical, pharmacokinetic, and biological activity profiles. In
order to design selective MMP-13 inhibitors, we adopted a strategy of connecting a zinc-binding group
with the quinazoline-2-carboxamide system, a unique S1' binder, by an appropriate linker. Among
synthesized compounds, a triazolone inhibitor 35 exhibited excellent potency (IC50 = 0.071 nM) and
selectivity (greater than 170-fold) over other MMPs (MMP-1, 2, 3, 7, 8, 9, 10, 12, and 14) and tumor
necrosis factor-α converting enzyme (TACE). In this article, the design, synthesis, and biological activity of
novel zinc-binding MMP-13 inhibitors are described.
1

1. Introduction
Osteoarthritis (OA) is a chronic disease characterized by destruction of articular cartilage upon aging and
progressive degradation due to mechanical stress. OA differs from systemic autoimmune diseases such as
rheumatoid arthritis. The most common clinical signs are joint swelling, instability, and restricted range of
motion. OA is at the top of arthralgia for elderly patients1-3 and is a major health concern for an
increasingly aging worldwide population.4 The pathogenesis of OA is incompletely understood, but
multiple genetic and environmental factors are thought to be involved in the development of OA. Genetic
studies have identified multiple gene variations associated with an increased risk of OA.5-8 Current OA
treatments are limited to symptomatic relief with NSAIDs, intra-articular injections of hyaluronic acid
conjugates, or surgical joint replacement. In addition, the COX-2 inhibitors cause an increase in the risk of
cardiovascular side effects,9 resulting in the withdrawal of Vioxx (rofecoxib) and Bextra (valdecoxib).
Thus, there is still an urgent unmet medical need to develop new and safer disease modifying
osteoarthritis drugs ("DMOADs") that reduce or reverse the cartilage destruction associated with OA.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent, calcium-containing endopeptidases,
many of which may play a role in OA. Preclinical testing of MMP inhibitors has shown that these inhibitors
are able to prevent the destruction of cartilage in some OA animal models.10 However, most clinical trials
of broad spectrum MMP inhibitors have been halted by dose-limiting toxicity (skin rash and
musculoskeletal side effects, which are characterized by joint stiffness, pain, inflammation, and tendinitis).
This side effect has been postulated to result from the inhibition of MMP-1, or MMP-14, or sheddases,
such as TACE.11-13 As a result, interest in more selective MMP-13 inhibitors has increased. MMP-13
(collagenase-3) is the most efficient collagenase which cleaves type II collagen14, 15 and is overexpressed in
OA cartilage.14, 16 Therefore, inhibition of this enzyme is one of the most promising approaches for the
treatment of cartilage degradation in arthritis.17-32
Recently, we described highly selective MMP-13 inhibitors, which do not bind the catalytic zinc ion
(Figure 1).33-36 The inhibitors possess a pyrimidin-4-one-2-carboxamide core, which binds effectively to
the primed regions (S1') of the catalytic active site36, 37, and the fused pyrimidine carboxamide systems
were successfully applied to other selective MMP-13 inhibitors.38-40 Among the previously synthesized
compounds in our laboratories, promising derivatives, quinazoline-2-carboxamide and
thienopyrimidine-2-carboxamide based MMP-13 inhibitors, as exemplified by 1 and 2, were identified,
which exhibit potent inhibition of MMP-13, but weak inhibitory activity toward other members of the
MMP family. This article describes the strategy employed to modify the previous series in order to obtain a
follow-up compound with improved physicochemical and pharmacokinetic properties.
Figure 1. Structure of fused pyrimidine-derived MMP-13 selective inhibitors 1 and 2.
2

Figure 2. Schematic structure of quinazoline-2-carboxamide-based zinc-binding MMP-13 inhibitors.
There are three major components to most MMP inhibitors as follows: (1) the zinc binding group (ZBG);
(2) side chains which could interact with amino acids around the catalytic zinc ion; (3) the
pocket-occupying functionality referred to as the P1' group, which is bound in the S1' pocket.41-47
Historically, most MMP inhibitors gained affinity through interacting with the catalytic zinc via a chelating
moiety and by positioning hydrogen bonding groups such as backbone NH of Leu185, backbone carbonyl
oxygen of Ala186, and carboxylate of the Glu202 residue near the catalytic zinc. However, one major
concern with this approach is the issue of selectivity, since many of the important residues in the catalytic
site are well-conserved among the MMP family. In contrast, conformational diversity is localized at the S1'
loop region, which forms the bottom half of the S1' subsite. MMPs such as MMP-2, MMP-3, MMP-8,
MMP-9, MMP-12, MMP-13, and MMP-14 have large S1' pockets and can accommodate more diverse
large P1' groups. Thus, targeting the large S1' loop region assists in identifying MMP-13-specific
inhibitors.19-28, 48-52 As mentioned above, we described the discovery of potent MMP-13 inhibitors which
form β-sheet type hydrogen bond interactions with both Thr245 and Thr247 of the S1' site. As a result, we
designed MMP-13 inhibitors by incorporating
a
ZBG via appropriate spacers into the
quinazoline-2-carboxamide scaffold that can be recognized by the enzyme and forms a complex with it
(Figure 2). In addition, spacers were chosen that had a potential to form hydrogen bonds with the backbone
amides near the catalytic zinc.
Drugs for the treatment of OA are required to reach the site of action by diffusing into the cartilage matrix.
However, a carboxyl function may lead to poor penetration through the negatively charged cartilage matrix
because the carboxyl acid would be ionized at physiological pH.53-55 Hence, efforts were focused on
incorporation of non-carboxylate moieties or intra-articular therapy.56
We now describe the design, synthesis, in vitro MMP inhibitory activity, and oral pharmacokinetic
parameters of the MMP-13 inhibitors possessing a non-carboxylate zinc-binding function attached to the
pyrimidin-4-one-2-carboxamide core, which binds effectively to the primed regions of the catalytic active
site of the enzyme.
2. Chemistry
The synthesis of the biphenyl sulfonamides 12 and 13 is shown in Scheme 1. Benzylamine 9 was
prepared by sulfonylation of H-(DL)-Val-OMe (5) with 4-bromophenylsulfonyl chloride (6), followed by
Suzuki coupling with (3-cyanophenyl)boronic acid and cyano group reduction. Substituted-benzylamine 9
was condensed with quinazoline-2-carboxylic acid ethyl ester 10 by heating in the absence of base to give
amide 11. The pyrimidine NH of the quinazoline 10 accelerates rapid aminolysis of the ester through
intramolecular acid catalysis.33, 34 This neighboring group effect explains the high reactivity of the ester
3

function. The fully functionalized biphenyl ester sulfonamide 11 was then hydrolyzed to the carboxylic acid
12, and converted into the hydroxamic acid 13.
Scheme 1. Synthesis of Biphenylsulfonamide Derivatives 12 and 13a
a Reagents and conditions: (a) Na2CO3, acetone, H2O, 0 °C–rt, 84%; (b) (3-cyanophenyl)boronic acid,
Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O, reflux, 97%; (c) H2, Raney Ni, NH3, THF, MeOH, H2O, rt, 76%;
(d) 10, EtOH, 90 °C, 86%; (e) NaOH, H2O, THF, MeOH, 80 °C, 83%; (f) TMSONH2, WSCD·HCl, HOBt,
DMF, rt, 71%.
Scheme 2. Synthesis of 3-Phenoxypropane-1-sulfonamide Derivatives 20 and 21a
a Reagents and conditions: (a) TEA, THF, 0 °C–rt, 81%; (b) 3-hydroxybenzonitrile, K2CO3, NaI, DMF,
100 °C, 52%; (c) H2, Raney Ni, NH3, THF, MeOH, H2O, rt, quant.; (d) 10, TEA, EtOH, 80 °C, 59%; (e)
NaOH, H2O, THF, MeOH, 80 °C, 97%; (f) TMSONH2, WSCD·HCl, HOBt, DMF, rt, 78%.
Synthetic pathway to 3-phenoxypropane-1-sulfonamides 20 and 21 is shown in Scheme 2. The
3-phenoxypropane-1-sulfonamides 20 and 21 were prepared from the commercially available ethyl
pipecolate (14). Sulfonylation with 3-chloropropane-1-sulfonyl chloride (15) afforded sulfonamide 16.
Alkylation of 3-hydroxybenzonitrile with the chloride 16 yielded ether 17, and the following nitrile
reduction of 17 gave benzylamine 18. Condensation of the resulting benzylamine 18 with
4

quinazoline-2-carboxylic acid ethyl ester 10, accomplished in the same manner as 11 in Scheme 1, and
basic hydrolysis of the ethyl ester 19 afforded the carboxylic acid 20, which was then subsequently
converted into the desired hydroxamic acid 21 by condensation with O-(trimethylsilyl)hydroxylamine as
described in Scheme 2.
Scheme 3. Synthesis of Reverse Hydroxamate 29a and Its Analogues 29b–ca
a Reagents and conditions: (a) (3-cyanophenyl)boronic acid, Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O,
reflux, 95%; (b) (1) n-BuLi, THF, –78 °C; (2) isobutyraldehyde, –78 °C, 65%; (c) (1) H2, Raney Ni, NH3,
THF, MeOH, H2O, rt; (2) HCl, AcOEt, MeOH, Et2O, rt, 80%; (d) 10, TEA, EtOH, 80 °C, 78%; (e) MsCl,
TEA, THF, 0 °C–rt, 76%; (f) TMSONH2, THF, rt, 65%; (g) HCO2H, Ac2O, THF, rt, 37%; (h) Ac2O, AcOH,
THF, rt, 79%; (i) (1) triphosgene, DIEA, THF, 0 °C–rt; (2) NH3, H2O, rt, 83%.
Scheme 4. Synthesis of Triazolone Derivatives 35 and 37a
a Reagents and conditions: (a) (4-bromophenyl)methanethiol, K2CO3, DMF, rt, 88%; (b) mCPBA, DMF,
rt, 92%; (c) (3-cyanophenyl)boronic acid, Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O, reflux, 60%; (d) H2,
Raney Ni, NH3, THF, MeOH, H2O, rt, 79%; (e) 10, TEA, EtOH, DMA, 90 °C, 55%; (f) 3635, TEA, EtOH,
DMA, 90 °C, 45%.
5

Reverse hydroxamates 29a–c were prepared according to refs.57, 58 as illustrated in Scheme 3. Biphenyl
formation of 22 using Suzuki conditions, followed by treatment of a lithium anion of methyl sulfone 23
with isobutyraldehyde gave alcohol adduct 24. Reduction of the cyano group of 24 by catalytic
hydrogenation afforded aminomethyl derivative 25, which yielded amide 26 on aminolysis of 10. To
prepare reverse hydroxamates 29a–c, the hydroxy sulfone 26 was first converted to 1,2-unsaturated sulfone
27 by formation of mesylate of 26 followed by elimination. Treatment of the 1,2-unsaturated sulfone 27
with O-(trimethylsilyl)hydroxylamine in THF gave 1,4-Michael adduct 28. Acylation or carbamoylation of
28 afforded N-formyl hydroxylamine 29a, N-acetyl hydroxylamine 29b, and N-carbamoyl hydroxylamine
29c.
Conversion of chloromethyltriazolone 3059 to sulfide 31 followed by oxidation led to sulfone 32 as shown
in Scheme 4. Triazolone derivatives 35 and 37 were prepared from the bromide 32 using the same synthetic
sequence as described in Schemes 1 and 3.
Installation of a variety of 5-membered heterocycles linked via a piperazine spacer (45a-e) was performed
using divergent synthetic strategy whereby various potential ZBGs could be introduced on a common
intermediate 43 at the last stage of the synthesis (Scheme 5). The common intermediate 43 was prepared
from commercially available 1-(3-bromophenyl)piperazine (38) in five steps.
Scheme 5. Synthesis of Phenylpiperazine Derivatives 45a–ea
a Reagents and conditions: (a) Boc2O, TEA, THF, rt, 64%; (b) Zn(CN)2, Pd(PPh3)4, DMF, 80 °C, 80%;
(c) H2, Raney Ni, NH3, THF, MeOH, H2O, rt, 100%; (d) 10, TEA, EtOH, 80 °C, 92%; (e) HCl, AcOEt,
MeOH, THF, H2O, rt–50 °C, 97%; (f) 44a–e, WSCD·HCl, HOBt, TEA, DMF, rt, 32–79%.
Scheme 6. Synthesis of Pyrimidine-2,4,6-trione Derivative 51a
6

a Reagents and conditions: (a) K2CO3, DMF, rt, 69%; (b) (1) H2, Raney Ni, NH3, THF, MeOH, H2O, rt;
(2) NaOH, H2O, 95%; (c) 10, TEA, EtOH, 80 °C, 67%; (d) 46, K2CO3, DMF, rt, 51%.
Scheme 7. Synthesis of 1,2,4-Triazole Derivative 54a
a Reagents and conditions: (a) DIEA, EtOH, microwave, 100 °C, 78%; (b) TFA, Et3SiH, CH3CN, rt, 96%.
Scheme 8. Synthesis of 3-Chlorophenyl Derivative 55a
a Reagents and conditions: 3-methoxybenzylamine , EtOH, Et3N, 80 °C, 62%.
(A)
(B)
Leu 185
Ala 186
7

Figure 3. Superposition of the X-ray structures of MMP-13 selective inhibitors 333-35 (cyan) and 424, 27
(magenta). (A) Surface representation of MMP-13 illustrating the binding cavity. The figure was made with
program PyMOL.60 (B) Schematic representation of the binding site of MMP-13 and the inhibitors 3 and 4.
An attempt to introduce a barbiturate functionality into the quinazoline-2-carboxamide via a biphenyl
linker was performed using 3-(4-hydroxyphenyl)benzonitrile (47) as a starting material (Scheme 6).
Alkylation of 47 with alkyl bromide 46 to ether 48 followed by reduction of the nitrile to primary amine
with hydrogen gas in the presence of Raney nickel and ammonia proceeded with an unexpected elimination
of the barbiturate functionality to afford 4-[3-(aminomethyl)phenyl]phenol (49) as a sodium salt. After
condensation of 49 with 10, the barbiturate functionality was reinstalled using 46 under basic condition to
provide pyrimidine-2,4,6-trione derivative 51.
As shown in Scheme 7, ester 10 was subjected to aminolysis by treatment with amine 5235 in EtOH to
afford compound 53, which was converted to 1,2,4-triazole 54 under acidic condition.
Reaction of ester 3635 and 3-methoxybenzylamine afforded 3-chlorophenyl derivative 55 (Scheme 8).
3. Results and Discussion
3-1. Drug Design Recently, we have described quinazoline-2-carboxamide potent inhibitors that occupy
the S1' pocket of MMP-13 without interacting with the catalytic zinc and the residues near the zinc.33 X-ray
crystallography data have confirmed the unique binding mode that is characterized by forming a β-sheet
type interaction with hydrogen bonding to the enzyme’s backbone spanning the S1' pocket. In order to
design potent MMP-13 inhibitors, we adopted a strategy of connecting ZBGs with our P1' quinazoline
system by an appropriate linker (Figure 2). Molecule alignment study between our
quinazoline-2-carboxamide 3 and biphenyl sulfonamide carboxylate MMP inhibitor 424, 27 discovered at
Wyeth was performed. For comparison of the ligands complexed in the binding sites of the crystal
structures, the binding sites were superimposed using PyMOL. The structure diagrams of the inhibitors are
shown in Figure 3. The biphenyl sulfonamide inhibitor 4 consists of an extra terminal valine moiety
complexing the catalytic zinc and a benzofuran moiety extending deep into the S1' pocket. Two hydrogen
bonds are formed between the inhibitor’s sulfone oxygen and NH of Leu185 and Ala186. Alignment of
compound 3 (cyan) with Wyeth's compound 4 (magenta) shows the proximity of the phenyl ring A of
quinazoline-2-carboxamide 3 to the phenyl ring B of biphenyl sulfonamide inhibitor 4. On the basis of the
above observations, the substituent on the phenyl ring A of quinazoline-2-carboxamide 3 should be
positioned in a meta-position to fill the hydrophobic S1' tunnel toward Leu185, Ala186, and the catalytic
zinc. Hence, we newly designed quinazoline-2-carboxamide derivatives with various ZBGs via spacers
(Figure 4). For a comprehensive schematic representation of the designed compounds to MMP-13, see
Figure 5. Spacers were chosen that had the potential to form hydrogen bonds with the backbone amides of
Leu185 and Ala186, as observed between these residues and the sulfone oxygen of the biphenyl
sulfonamide carboxylate inhibitor 4 (Figure 3). Thus, ethers, amides, sulfones, and sulfonamides were
selected as linker elements that contain at least one hydrogen bond acceptor atom. In addition, efforts were
focused on inhibitors based on a variety of non-carboxylate ZBGs including heterocyclic ZBGs.
8

Figure 4. Quinazoline-2-carboxamide based zinc-binding MMP-13 inhibitors.
Figure 5. Schematic representation of the putative binding mode of the designed compounds to MMP-13.
Hydrogen bonds are shown as dashed lines.
3-2. MMPs Activity IC50 values of quinazoline derivatives have been determined on ten recombinant
MMP subtypes, namely MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12,
MMP-13, MMP-14, and TACE. The IC50 values are displayed in Table 1. As expected, all compounds
showed improved MMP-13 inhibitory activities in comparison with the lead compound 3. Hydroxamic acid
derivative 13 gave a > 1,000-fold increased potency in MMP-13 (IC50 = 3.2 pM) to 3, but decreased
selectivity over most of the MMPs showing at least nanomolar potency. Hydroxamate 21, which has a
methylene linker, showed a 100-fold increase in potency when compared to the lead 3 with subtype
selectivity for MMP-1, 7, and 14, but the selectivity of this compound over MMP-2, 3, 8, 10, and 12 was
relatively low.
Carboxylate series 12 and 20 showed improved potency compared to that of the lead 3 but had no
improved MMP-13 inhibitory activity over the corresponding hydroxamic acid derivatives 13 and 21,
9

respectively, resulting in sub-nanomolar inhibitory activity. However, in contrast to the biphenyl linker
series 12, the methylene linker series 20 showed greater MMP-13 selectivity than that of 12 against all
enzymes tested.
Additionally, substituents present on the nitrogen atom of reverse hydroxamates 29a-c affected the
potency to some extent. An N-formyl derivative 29a was the most potent compound of the series, showing
about 100-fold more potent inhibition of MMP-13 than that of the lead compound 3 accompanied by an
increase of unintended activity toward other MMPs. The N-Acetyl derivative 29b demonstrated a more
attractive balance of potency and selectivity than that of the N-formyl derivative 29a showing a somewhat
(5- to 6-fold) less potent MMP-13 inhibitory activity than that of 29a, and the selectivity against MMP-2, 8,
and 10 was high (> 200-fold). Urea 29c showed a lower degree of enzyme specificity toward subtypes of
MMPs than that of N-acetyl derivative 29b.
Incorporation of a triazolone ZBG, through a biphenylmethylsulfone linker group, to the
pyrimidin-4-one-2-carboxamide core (35) resulted in about 100-fold improved inhibitory potency (IC50
=
71 pM) with substantial selectivity (> 170-fold) over all other enzymes tested.
On the contrary, incorporation of 5-membered heterocycles via a piperazine linker was not effective,
giving relatively weak (IC50 > 100 pM) inhibitors 45a–e. Introduction of a hydantoin (45a), a triazolone
(45b), and a pyrazolone (45c), resulted in a 10-fold improved MMP-13 inhibitory activity in comparison to
that of the other non-carbonyl analogues (imidazole 45d and tetrazole 45e). This result indicates that the
carbonyl oxygen on the rings forms favorable hydrogen bonds with the MMP-13 backbone atoms close to
the active site zinc or interacts with the zinc itself. Among the compounds 45a–c, triazolone 45b showed
the highest potency but relatively low selectivity towards MMP-2, 3, 8, and 10 (MMP-8/MMP-13 ratio 35).
As shown in Table 1, an improvement in both potency and selectivity was observed for the
biphenylmethylsulfone linker analogue 35, which maintained the same triazolone ZBG, as compared to the
piperazine linker analogue 45b. This result indicates that an appropriate fine-tuning of the linker moiety
would improve selectivity toward MMP-13.
Table 1. Inhibitory Activities Against MMPs of Quinazoline Derivatives: Effect of ZBG and Linker
Attached to Phenyl Ring (A-Ring) at 3-Position
10

a IC50 values were calculated as an average of three experiments and are represented as means ± standard
b
deviations. Results are means of two experiments. c IC50 values were calculated as an average of three
experiments.
Figure 6. Introduction of P1" group: 5-aryl-thienopyrimidines from quinazolines.
The MMP-13 inhibitory activity of the barbituric acid derivative 51 was not enough (IC50 = 230 pM),
11

however, this compound showed at least 240-fold selectivity for MMP-13 over other MMPs.
Previously described non-zinc-binding MMP-13 inhibitors by our group such as compound 1 took
advantage of both S1' and S1" site binding pockets to achieve potent inhibition and high selectivity.33 The
zinc-binding inhibitors with P1' in this report resulted in an increased MMP-13 potency but decreased
selectivity over the other MMP subtypes. The use of strong chelators such as hydroxamates or reverse
hydroxamates as ZBGs may preclude the development of selective MMP inhibitors. Accordingly, further
attempts were made to improve both potency and selectivity by interacting with S1" side pocket, which is
unique to MMP-13. In the course of the study of the MMP inhibitor without a ZBG aimed at the designing
new P1" in our laboratory, we have found that introduction of a small aryl group into the 5-position of the
thienopyrimidine-2-carboxamide structure resulted in the excellent potency/selectivity profile (Figure 6).35
Thus, the 5-arylthienopyrimidine derivative 55 with a benzene ring directly attached to the thiophene ring
not only enhanced the inhibitory activity but also improved the selectivity profile compared to the
quinazoline 3 (Table 2, 3 vs 55). Therefore, in a series of derivatives containing ZBGs, small aromatic
rings were P1" substituents of choice for the inhibition of MMP-13 (Table 2, 37 and 56). As a result,
improvement of potency has been achieved by the combination of a simple linker with a triazole group as a
35
ZBG (Table 2, 56).
However, in contrast to the triazole ZBG series 56, incorporation of the
5-arylthienopyrimidine-type P1" group into the triazolone 35 was not proved to be well tolerated (Table 2，
35 vs 37). Probably, coupled with the size of the triazolone ZBG group, introduction of a rigid biphenyl P1'
linker and a sulfonyl group resulted in a small shift of the pyrimidine-4-one-2-carboxamide core structure
toward the primed side of the binding site, which would result in a significant loss in potency.
Further discussion on this issue, however, would best await X-ray crystal structures of the compounds 35
and 37 bound to MMP-13. On the other hand, it is presumed that a highly flexible alkylene linker of 54 and
56 that also have ZBG enabled 5-arylthienopyrimidine structure to interact effectively with the S1' and S1"
pockets of MMP-13, which resulted in an improvement of potency and selectivity.
Table 2. Inhibitory Activities Against MMPs: Effect of Introduction of P1" Group
12

a IC50 values were calculated as an average of three experiments and are represented as means ± standard
b
deviations. IC50 values were calculated as an average of three experiments. c Results are average of two
experiments. c A sulfonehydroxamate MMP inhibitor RS-130,83061 was used as a reference for comparison.
Table 3. DMPK/tox Profiles of Representative Compounds
a Human/rat liver microsomal clearance. b 0.1 mg/kg iv, 1 mg/kg po. c ATP was measured as an indicator of
viable cells.
3-3. DMPK/tox Profile Table 3 shows DMPK/tox properties of the derivatives 35, 51, and 29b with
more than 100-fold selectivity against related MMPs. Despite possessing good properties (high stability in
rat and human liver microsomes, low cytochrome P450 (CYP) 3A4 inhibition, and low toxicity by ATP
measurement), an oral bioavailability of the triazolone compound 35 in rats was less than 5%, perhaps due
13

to low solubility and permeability (assessed by parallel artificial membrane permeability assay (PAMPA))
issues. Alternatively, the barbiturate derivative 51 demonstrated good PAMPA permeability, good stability
against liver microsomes, and acceptable oral bioavailability (F% > 30) in rats. However, 51 showed a very
low volume of distribution, which may reflect high plasma protein binding. N-Acetylhydroxylamine
derivative 29b displayed poor in vitro metabolic stability and oral bioavailability in rat, and showed potent
CYP3A4 inhibition and cytotoxic effect.
4. Conclusion
We have previously used a fused pyrimidine-2-carboxamide scaffold represented by compound 1 for an
initial validation of two strategies to obtain potency and selectivity for MMP-13 by utilizing (i) the unique
side pocket of S1' cavity, referred to as S1" pocket and (ii) interactions toward catalytic zinc through a
linker.35 With the goal of further expanding the chemical diversity, we next investigated the potential of
ZBGs. In addition to accessing unique structure space, we expected that the inclusion of non-carboxylic
acid-based ZBGs could impart useful biological properties such as improved pharmacokinetics or
permeability into cartilage matrix. In conclusion, by applying a structure-based approach, we have
discovered a new series of MMP-13 inhibitors represented by 35 with high potency against MMP-13
activity (IC50 = 0.071 nM) and high selectivity against other MMPs (> 170-fold over MMP-1, 2, 3, 7, 8, 9,
10, 12, 14, and TACE). The series is characterized by having a triazolone as the zinc binding moiety
connected via a biphenyl spacer with a sulfonyl linkage to the P1' group, quinazoline-2-carboxamide.
While analog 35 showed a slow rat liver microsomal turnover rate, its oral availability in rat PK studies was
found to be much lower than expected. The discrepancy of these results may be due to solubility and
permeability issues. By application of known SAR, P1" substituents could be placed on the quinazoline
core with a view to improving the enzyme inhibition activity, selectivity, physicochemical, and PK
properties of this class of compounds. In vivo animal efficacy will be the subject of a future publication.
5. Experimental Section
5-1. Chemistry
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 () relative to
tetramethylsilane (TMS:  0.00 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 mm x 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%
14

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 mmx
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. Elemental analyses were performed by Takeda Analytical Research Laboratories, Ltd.
Methyl N-[(4-Bromophenyl)sulfonyl]valinate (7). To a mixture of methyl valinate hydrochloride (5,
10.0 g, 59.7 mmol) and Na2CO3 (15.8 g, 149 mmol) in a mixed solvent of acetone (200 mL) and H2O (100
mL) was added 4-bromobenzenesulfonyl chloride (6, 12.7 g, 49.7 mmol) at 0 °C, and the mixture was
stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure, diluted with
ethyl acetate, and successively washed with 0.1 N hydrochloric acid, H2O and brine. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by crystallization
1
from ethyl acetate–hexane to give the title compound as a white powder (14.7 g, 42.0 mmol, 84%). H
NMR (300 MHz, DMSO-d6)  0.76–0.85 (6H, m), 1.84–1.98 (1H, m), 3.37 (3H, s), 3.57 (1H, d, J = 7.2
Hz), 7.63–7.71 (2H, m), 7.76–7.83 (2H, m), 8.39 (1H, s).
Methyl N-[(3’-Cyanobiphenyl-4-yl)sulfonyl]valinate (8). A mixture of compound 7 (5.00 g, 14.3
mmol), (3-cyanophenyl)boronic acid (2.52 g, 17.1 mmol), tetrakis(triphenylphosphine)palladium(0) (0.330
g, 0.286 mmol), 2 M aqueous Na2CO3 solution (8.57 mL, 17.1 mmol), ethanol (15 mL), and toluene (50
mL) was refluxed for 8 h. After completeness of reaction was checked by LC-MS, the reaction mixture was
diluted with ethyl acetate and washed with H2O and brine. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by crystallization from ethyl acetate–hexane
to give the title compound as a yellow powder (5.18 g, 13.9 mmol, 97%). 1H NMR (300 MHz, DMSO-d6) 
0.81 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.8 Hz), 1.86–1.98 (1H, m), 3.34 (3H, s), 3.55–3.64 (1H, m), 7.72
(1H, t, J = 7.8 Hz), 7.81–7.86 (2H, m), 7.91 (1H, d, J = 7.6 Hz), 7.94–8.00 (2H, m), 8.10 (1H, d, J = 8.0
Hz), 8.27 (1H, s), 8.32–8.40 (1H, m).
Methyl N-{[3’-(Aminomethyl)biphenyl-4-yl]sulfonyl}valinate (9). A mixture of compound 8 (5.00 g,
13.4 mmol), Raney Ni (5.0 g), 28% aqueous NH3 solution (50 mL), THF (50 mL), and methanol (50 mL)
was stirred at room temperature for 15 h under hydrogen atmosphere (1 atm). The catalyst was removed by
vacuum filtration through a PTFE membrane and the filtrate was concentrated under reduced pressure. The
residue was diluted with a mixed solvent of ethyl acetate and hexane, and the insoluble materials were
filtered off. The filtrate was washed with brine, and dried over Na2SO4. After removal of the solvent, the
residue was triturated with diethyl ether to give the title compound as a white powder (3.84 g, 10.2 mmol,
76%). 1H NMR (300 MHz, DMSO-d6)  0.80 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.8 Hz), 1.85–1.98 (1H,
m), 3.34 (3H, s), 3.58 (1H, d, J = 7.2 Hz), 3.80 (2H, s), 7.35–7.48 (2H, m), 7.56 (1H, d, J = 7.2 Hz), 7.71
(1H, s), 7.78–7.84 (2H, m), 7.84–7.91 (2H, m).
Methyl
N-{[3’-({[(4-Oxo-3,4-dihydroquinazolin-2-yl)carbonyl]amino}methyl)biphenyl-4-yl]sul-
fonyl}valinate (11). The mixture of compound 9 (0.300 g, 0.797 mmol) and ethyl 4-oxo-3,4-dihydro-
quinazoline-2-carboxylate62 (10, 0.145 g, 0.664 mmol) in ethanol (9 mL) was stirred at 90 °C for 5 h. The
mixture was concentrated under reduced pressure, diluted with ethyl acetate, washed with 0.1 N
hydrochloric acid and brine, and dried over Na2SO4. The filtrate was concentrated under reduced pressure
15

to give the title compound as a colorless gum (0.375 g, 0.684 mmol, 86%). 1H NMR (300 MHz, DMSO-d6)
 0.76–0.86 (6H, m), 1.84–1.97 (1H, m), 3.33 (3H, s), 3.57 (1H, d, J = 7.0 Hz), 4.58 (2H, d, J = 6.2 Hz),
7.38–7.52 (2H, m), 7.57–7.67 (2H, m), 7.71–7.94 (7H, m), 8.13–8.21 (1H, m), 8.32 (1H, s), 9.65 (1H, t, J =
6.2 Hz), 11.95 (1H, s).
N-{[3’-({[(4-Oxo-3,4-dihydroquinazolin-2-yl)carbonyl]amino}methyl)biphenyl-4-yl]sulfonyl}valine
(12). A mixture of compound 11 (250 mg, 0.456 mmol), 4 N aqueous sodium hydroxide solution (0.399
mL, 1.60 mmol), H2O (3 mL), THF (3 mL) and methanol (3 mL) was stirred at 80 °C for 15 h. The mixture
was concentrated under reduced pressure, diluted with H2O, acidified with 1 N hydrochloric acid (2.4 mL),
and partitioned between ethyl acetate and H2O. The organic layer was washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was purified by crystallization from methanol to give the
1
title compound as a white powder (202 mg, 0.456 mmol, 83%). mp 141–143 °C. H NMR (300 MHz,
DMSO-d6)  0.75–0.88 (6H, m), 1.88–2.01 (1H, m), 3.54 (1H, dd, J = 8.6 Hz, 5.9 Hz), 4.57 (2H, d, J = 6.4
Hz), 7.40–7.53 (2H, m), 7.58–7.67 (2H, m), 7.72–7.94 (7H, m), 8.06 (1H, d, J = 8.9 Hz), 8.15–8.21 (1H,
m), 9.65 (1H, t, J = 6.3 Hz), 12.27 (1H, s), 12.61 (1H, s). Anal. Calcd for C27H26N4O6S: C, 60.66; H, 4.90;
N, 10.48. Found: C, 60.57; H, 5.11; N, 10.48.
N-({4’-[({1-[(Hydroxyamino)carbonyl]-2-methylpropyl}amino)sulfonyl]biphenyl-3-yl}methyl)-4-ox
o-3,4-dihydroquinazoline-2-carboxamide (13). The mixture of compound 12 (80 mg, 0.15 mmol),
O-(trimethylsilyl)hydroxylamine (0.055 mL, 0.45 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiim-
ide hydrochloride (86 mg, 0.45 mmol), and 1-hydroxybenzotriazole (61 mg, 0.45 mmol) in DMF (5 mL)
was stirred at room temperature for 12 h. The mixture was diluted with ethyl acetate, successively washed
with 0.05 N hydrochloric acid, aqueous NaHCO3 solution, H2O and brine, and dried over Na2SO4. After
removal of the solvent, the residue was purified by crystallization from methanol–diethyl ether to give the
1
title compound as a white powder (58 mg, 0.11 mmol, 71%). mp 153–155 °C. H NMR (300 MHz,
DMSO-d6)  0.75 (6H, dd, J = 6.1 Hz, 3.4 Hz), 1.70–1.84 (1H, m), 3.28–3.39 (1H, m), 4.57 (2H, d, J = 6.1
Hz), 7.37–7.52 (2H, m), 7.57–7.67 (2H, m), 7.71–7.93 (7H, m), 7.98 (1H, d, J = 9.1 Hz), 8.17 (1H, d, J =
8.0 Hz), 8.79 (1H, s), 9.63 (1H, t, J = 6.6 Hz), 10.52 (1H, s), 12.26 (1H, bs). Anal. Calcd for C27H27N5O6S:
C, 59.00; H, 4.95; N, 12.74. Found: C, 58.72; H, 5.05; N, 12.56.
Ethyl 1-[(3-Chloropropyl)sulfonyl]piperidine-2-carboxylate (16). Ethyl piperidine-2-carboxylate (14,
4.19 g, 26.7 mmol) and triethylamine (3.72 mL, 26.7 mmol) were added to a mixture of 3-chloropropane-
1-sulfonyl chloride (15, 4.72 g, 26.7 mmol) in THF (100 mL) at 0 °C, and the mixture was stirred for 12 h
at room temperature. After the mixture was concentrated under reduced pressure, the residue was diluted
with ethyl acetate, successively washed with H2O, 0.1 N hydrochloric acid and brine, and dried over
Na2SO4. The filtrate was concentrated under reduced pressure to give the title compound as a yellow oil
1
(6.44 g, 21.6 mmol, 81%). H NMR (300 MHz, DMSO-d6)  1.13–1.27 (1H, m), 1.22 (3H, t, J = 7.0 Hz),
1.33–1.50 (1H, m), 1.59–1.72 (3H, m), 2.05–2.17 (3H, m), 3.06–3.27 (3H, m), 3.54–3.63 (1H, m),
3.70–3.77 (2H, m), 4.12–4.22 (2H, m), 4.52–4.59 (1H, m).
Ethyl 1-({3-[(3-Cyanophenyl)oxy]propyl}sulfonyl)piperidine-2-carboxylate (17). A mixture of
compound 16 (2.00 g, 6.72 mmol), 3-hydroxybenzonitrile (800 mg, 6.72 mmol), K2CO3 (928 mg, 6.72
mmol), and sodium iodide (101 mg, 0.672 mmol) in DMF (20 mL) was stirred at 100 °C for 15 h. The
16

mixture was diluted with ethyl acetate, successively washed with H2O, 0.5 N aqueous sodium hydroxide
solution and brine, and dried over Na2SO4. After removal of the solvent, the residue was triturated with
1
methanol to give the title compound as a pale yellow powder (1.34 g, 3.52 mmol, 52%). H NMR (300
MHz, DMSO-d6)  1.13–1.27 (1H, m), 1.21 (3H, t, J = 7.2 Hz), 1.30–1.47 (1H, m), 1.59–1.73 (3H, m),
2.04–2.16 (3H, m), 3.08–3.20 (1H, m), 3.21–3.31 (2H, m), 3.56–3.66 (1H, m), 4.10–4.21 (4H, m),
4.53–4.59 (1H, m), 7.27–7.33 (1H, m), 7.38–7.46 (2H, m), 7.50 (1H, t, J = 8.0 Hz).
Ethyl 1-[(3-{[3-(Aminomethyl)phenyl]oxy}propyl)sulfonyl]piperidine-2-carboxylate (18). A mixture
of compound 17 (1.20 g, 3.15 mmol), Raney Ni (1.2 g), 28% aqueous NH3 solution (12 mL), THF (24 mL),
and methanol (12 mL) was stirred at room temperature for 12 h under hydrogen atmosphere (1 atm). The
catalyst was removed by vacuum filtration through a PTFE membrane, and the filtrate was concentrated
under reduced pressure to give the title compound as a green oil (1.21 g, 3.15 mmol, quant.). 1H NMR (300
MHz, DMSO-d6)  1.13–1.28 (4H, m), 1.31–1.48 (1H, m), 1.59–1.75 (3H, m), 2.03–2.15 (3H, m),
3.07–3.38 (5H, m), 3.61 (1H, d, J = 13.0 Hz), 3.98–4.09 (2H, m), 4.11–4.22 (2H, m), 4.51–4.63 (1H, m),
6.69–6.79 (1H, m), 6.80–6.96 (2H, m), 7.22 (1H, bs).
Ethyl 1-[(3-{[3-({[(4-Oxo-3,4-dihydroquinazolin-2-yl)carbonyl]amino}methyl)phenyl]oxy}propyl)-
sulfonyl]piperidine-2-carboxylate (19). A mixture of compound 18 (600 mg, 1.56 mmol), compound 1062
(310 mg, 1.42 mmol), and triethylamine (0.297 mL, 2.13 mmol) in ethanol (20 mL) was stirred at 80 °C for
12 h. After the mixture was allowed to cool to room temperature, 2 N ethanolic HCl solution (1.5 mL) was
added to the mixture. The precipitated solid was collected and washed with ethanol to give the title
compound as a white powder (464 mg, 0.834 mmol, 59%). mp 84–86 °C. 1H NMR (300 MHz, DMSO-d6)
 1.08–1.25 (1H, m), 1.19 (3H, t, J = 7.2 Hz), 1.27–1.45 (1H, m), 1.55–1.71 (3H, m), 2.00–2.15 (3H, m),
3.05–3.18 (1H, m), 3.19–3.29 (2H, m), 3.54–3.63 (1H, m), 4.05 (2H, t, J = 6.2 Hz), 4.09–4.18 (2H, m),
4.45 (2H, d, J = 6.4 Hz), 4.51–4.60 (1H, m), 6.83 (1H, d, J = 8.3 Hz), 6.90–6.97 (2H, m), 7.25 (1H, t, J =
8.0 Hz), 7.61 (1H, t, J = 7.6 Hz), 7.74–7.82 (1H, m), 7.84–7.93 (1H, m), 8.17 (1H, d, J = 8.0 Hz), 9.54 (1H,
t, J = 6.2 Hz), 12.25 (1H, bs). Anal. Calcd for C27H32N4O7S: C, 58.26; H, 5.79; N, 10.07. Found: C, 57.97;
H, 5.63; N, 10.04.
1-[(3-{[3-({[(4-Oxo-3,4-dihydroquinazolin-2-yl)carbonyl]amino}methyl)phenyl]oxy}propyl)sulfonyl
]piperidine-2-carboxylic Acid (20). A mixture of compound 19 (360 mg, 0.647 mmol), 4 N aqueous
sodium hydroxide solution (0.566 mL, 2.26 mmol), H2O (4 mL), THF (4 mL), and methanol (4 mL) was
stirred at 80 °C for 4 h. The mixture was diluted with H2O, acidified with 1 N hydrochloric acid (3 mL),
and partitioned between ethyl acetate and H2O. The organic layer was washed with brine, and dried over
Na2SO4. After removal of the solvent, the residue was purified by crystallization from ethyl acetate–diethyl
ether to give the title compound as a white powder (332 mg, 0.628 mmol, 97%). mp 204–206 °C. 1H NMR
(300 MHz, DMSO-d6)  1.06–1.45 (2H, m), 1.54–1.70 (3H, m), 2.02–2.17 (3H, m), 3.08–3.42 (3H, m),
3.49–3.63 (1H, m), 3.97–4.11 (2H, m), 4.36–4.51 (3H, m), 6.83 (1H, d, J = 8.0 Hz), 6.87–6.97 (2H, m),
7.25 (1H, t, J = 8.1 Hz), 7.62 (1H, t, J = 7.6 Hz), 7.74–7.83 (1H, m), 7.89 (1H, t, J = 7.6 Hz), 8.18 (1H, d, J
= 8.0 Hz), 9.55 (1H, t, J = 5.7 Hz), 12.28 (1H, bs), 13.04 (1H, bs). Anal. Calcd for C25H28N4O7S: C, 56.81;
H, 5.34; N, 10.60. Found: C, 56.82; H, 5.58; N, 10.33.
N-[(3-{[3-({2-[(Hydroxyamino)carbonyl]piperidin-1-yl}sulfonyl)propyl]oxy}phenyl)methyl]-4-oxo-
17

3,4-dihydroquinazoline-2-carboxamide (21). The mixture of compound 20 (150 mg, 0.284 mmol),
O-(trimethylsilyl)hydroxylamine (0.104 mL, 0.851 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiim-
ide hydrochloride (163 mg, 0.851 mmol), and 1-hydroxybenzotriazole (115 mg, 0.851 mmol) in DMF (5
mL) was stirred at room temperature for 12 h. The mixture was diluted with ethyl acetate, successively
washed with 0.05 N hydrochloric acid, H2O and brine, and dried over Na2SO4. After removal of the solvent,
the residue was purified by crystallization from methanol–ethyl acetate–diethyl ether–hexane to give the
1
title compound as a white powder (120 mg, 0.221 mmol, 78%). mp 106–108 °C. H NMR (300 MHz,
DMSO-d6)  1.29–1.48 (2H, m), 1.53–1.72 (3H, m), 1.85–1.94 (1H, m), 2.04–2.17 (2H, m), 3.07–3.59 (4H,
m), 4.00–4.08 (2H, m), 4.27 (1H, d), 4.45 (2H, d, J = 6.4 Hz), 6.79–6.86 (1H, m), 6.90–6.97 (2H, m), 7.25
(1H, t, J = 8.1 Hz), 7.61 (1H, t, J = 7.6 Hz), 7.75–7.81 (1H, m), 7.84–7.92 (1H, m), 8.17 (1H, d, J = 8.0 Hz),
8.91 (1H, bs), 9.54 (1H, t, J = 6.4 Hz), 10.66 (1H, bs), 12.22 (1H, bs). Anal. Calcd for
C25H29N5O7S·0.3H2O: C, 54.69; H, 5.43; N, 12.76. Found: C, 54.98; H, 5.44; N, 12.47.
4’-(Methylsulfonyl)biphenyl-3-carbonitrile (23). A mixture of 1-bromo-4-(methylsulfonyl)benzene (22,
10.0 g, 42.5 mmol), (3-cyanophenyl)boronic acid (7.50 g, 51.0 mmol), tetrakis(triphenylphosphine)-
palladium(0) (983 mg, 0.851 mmol), 2 M aqueous Na2CO3 solution (25.5 mL, 51.0 mmol), ethanol (30 mL),
and toluene (100 mL) was refluxed for 8 h. The reaction mixture was diluted with ethyl acetate and washed
with H2O and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure.
The residue was triturated with diisopropyl ether to give the title compound as a yellow powder (10.4 g,
40.4 mmol, 95%). 1H NMR (300 MHz, DMSO-d6)  3.28 (3H, s), 7.73 (1H, t, J = 8.0 Hz), 7.89–7.95 (1H,
m), 7.99–8.06 (4H, m), 8.09–8.15 (1H, m), 8.29 (1H, t).
4’-[(2-Hydroxy-3-methylbutyl)sulfonyl]biphenyl-3-carbonitrile (24). To a solution of compound 23
(2.00 g, 7.77 mmol.) in THF (40 mL) was added dropwise n-butyl lithium (1.6 M in hexane, 5.34 mL, 8.55
mmol) at –78 °C. The mixture was stirred at –78 °C for 1 h followed by the addition of isobutyraldehyde
(1.06 mL, 11.7 mmol). After the resulting mixture was stirred at –78 °C for further 3 h, the reaction was
quenched with saturated aqueous NH4Cl solution. The mixture was extracted with ethyl acetate, washed
with brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel
column chromatography (25–50% ethyl acetate/hexane) to give the title compound as a pale yellow oil
(1.66 g, 5.04 mmol, 65%). 1H NMR (300 MHz, DMSO-d6)  0.78 (3H, d, J = 6.8 Hz), 0.82 (3H, d, J = 6.8
Hz), 1.57–1.70 (1H, m), 3.39 (2H, d, J = 6.1 Hz), 3.74–3.82 (1H, m), 4.84 (1H, d, J = 5.7 Hz), 7.73 (1H, t,
J = 8.0 Hz), 7.90–7.95 (1H, m), 7.98–8.05 (4H, m), 8.10–8.15 (1H, m), 8.29 (1H, t, J = 1.5 Hz).
1-{[3’-(Aminomethyl)biphenyl-4-yl]sulfonyl}-3-methylbutan-2-ol Hydrochloride (25). A mixture of
compound 24 (1.60 g, 4.86 mmol), Raney Ni (1.6 g), 28% aqueous NH3 solution (16 mL), THF (32 mL),
and methanol (16 mL) was stirred at room temperature for 12 h under hydrogen atmosphere (1 atm). After
the catalyst was removed by vacuum filtration through a PTFE membrane, the filtrate was concentrated
under reduced pressure and the residual oil was dissolved in a mixed solvent of ethyl acetate, diethyl ether,
and methanol. To the solution was added dropwise 4 N HCl in ethyl acetate (1.82 mL) at room temperature.
The precipitated solid was collected and washed with diethyl ether to give the title compound as a white
powder (1.44 g, 3.89 mmol, 80%). 1H NMR (300 MHz, DMSO-d6)  0.77 (3H, d, J = 6.8 Hz), 0.81 (3H, d,
J = 6.8 Hz), 1.58–1.71 (1H, m), 3.35–3.40 (2H, m), 3.73–3.82 (1H, m), 4.12 (2H, bs), 4.84 (1H, d, J = 5.7
18

Hz), 7.53–7.60 (2H, m), 7.76–7.81 (1H, m), 7.92–8.04 (5H, m), 8.47 (3H, bs).
N-({4’-[(2-Hydroxy-3-methylbutyl)sulfonyl]biphenyl-3-yl}methyl)-4-oxo-3,4-dihydroquinazoline-2-
carboxamide (26). A mixture of compound 25 (800 mg, 2.16 mmol), compound 1062 (429 mg, 1.97 mmol)
and triethylamine (0.411 mL, 2.95 mmol) in ethanol (21 mL) was stirred at 80 °C for 12 h. After the
mixture was allowed to cool to room temperature, 2 N ethanolic HCl solution (2.2 mL) was added to the
mixture. The precipitated solid was collected and washed with ethanol to give the title compound as a white
powder (774 mg, 1.53 mmol, 78%). mp 169–171 °C. 1H NMR (300 MHz, DMSO-d6)  0.76 (3H, d, J = 6.8
Hz), 0.81 (3H, d, J = 6.8 Hz), 1.56–1.69 (1H, m), 3.34–3.38 (2H, m), 3.72–3.81 (1H, m), 4.58 (2H, d, J =
6.4 Hz), 4.81 (1H, d, J = 5.8 Hz), 7.42–7.53 (2H, m), 7.57–7.68 (2H, m), 7.74–7.81 (2H, m), 7.85–7.92 (3H,
m), 7.95–8.01 (2H, m), 8.17 (1H, dd, J = 7.9, 1.3 Hz), 9.65 (1H, t, J = 6.4 Hz), 12.03 (1H, bs). Anal. Calcd
for C27H27N3O5S: C, 64.14; H, 5.38; N, 8.31. Found: C, 63.99; H, 5.36; N, 8.22.
N-[(4’-{[(1E)-3-Methylbut-1-en-1-yl]sulfonyl}biphenyl-3-yl)methyl]-4-oxo-3,4-dihydroquinazoline-
2-carboxamide (27). To a mixture of compound 26 (300 mg, 0.593 mmol) and triethylamine (0.579 mL,
4.15 mmol) in THF (6 mL) was added methanesulfonyl chloride (0.083 mL, 1.1 mmol) at 0 °C, and the
mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ethyl acetate,
washed with 0.1 N hydrochloric acid and brine, and dried over Na2SO4. After removal of the solvent, the
residue was purified by crystallization from ethyl acetate–diethyl ether to give the title compound as a pale
yellow powder (220 mg, 0.593 mmol, 76%). mp 213–214 °C. 1H NMR (300 MHz, DMSO-d6)  1.02 (6H,
d, J = 6.8 Hz), 2.44–2.59 (1H, m), 4.58 (2H, d, J = 6.1 Hz), 6.71–6.80 (1H, m), 6.86–6.96 (1H, m),
7.37–7.54 (2H, m), 7.55–7.68 (2H, m), 7.70–7.83 (2H, m), 7.82–7.98 (5H, m), 8.17 (1H, d, J = 7.6 Hz),
9.65 (1H, t, J = 5.9 Hz), 12.29 (1H, bs). Anal. Calcd for C27H25N3O4S·0.2H2O: C, 66.02; H, 5.21; N, 8.50.
Found: C, 65.85; H, 5.21; N, 8.44.
N-[(4’-{[2-(Hydroxyamino)-3-methylbutyl]sulfonyl}biphenyl-3-yl)methyl]-4-oxo-3,4-dihydroquinaz
oline-2-carboxamide (28). To a solution of compound 27 (180 mg, 0.369 mmol) in THF (4 mL) was
added O-(trimethylsilyl)hydroxylamine (0.271 mL, 2.22 mmol) at room temperature, and the mixture was
stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure and crystallized
from ethyl acetate–diethyl ether to give the title compound as a white powder (125 mg, 0.240 mmol, 65%).
1
mp 197–199 °C. H NMR (300 MHz, DMSO-d6)  0.76 (3H, d, J = 6.8 Hz), 0.80 (3H, d, J = 6.8 Hz),
1.87–2.03 (1H, m), 2.86–2.96 (1H, m), 3.25–3.43 (2H, m), 4.58 (2H, d, J = 6.2 Hz), 5.52 (1H, bs), 7.31 (1H,
s), 7.41–7.55 (2H, m), 7.57–7.70 (2H, m), 7.74–7.83 (2H, m), 7.84–8.05 (5H, m), 8.14–8.21 (1H, m), 9.66
(1H, t, J = 6.4 Hz), 12.26 (1H, bs). Anal. Calcd for C27H28N4O5S: C, 62.29; H, 5.42; N, 10.76. Found: C,
62.04; H, 5.50; N, 10.49.
N-{[4’-({2-[Formyl(hydroxy)amino]-3-methylbutyl}sulfonyl)biphenyl-3-yl]methyl}-4-oxo-3,4-dihyd
roquinazoline-2-carboxamide (29a). Acetic anhydride (0.054 mL, 0.58 mmol) was added to formic acid
(0.217 mL) at room temperature and the mixture was stirred at room temperature for 2 h. The above
solution was added to a mixture of compound 28 (200 mg, 0.384 mmol) and formic acid (2 mL) in THF (20
mL) at room temperature and the mixture was stirred at room temperature for 4 h. After the mixture was
concentrated under reduced pressure, the residue was purified by preparative HPLC and crystallization
from ethanol–ethyl acetate–diethyl ether to give the title compound as a pale yellow powder (77.0 mg,
19

0.140 mmol, 37%). mp 153–155 °C. 1H NMR (300 MHz, DMSO-d6)  0.72–0.84 (6H, m), 1.68–1.86 (1H,
m), 3.46–3.57 (1H, m), 3.60–3.77 (1.6H, m), 4.24 (0.4H, t, J = 8.2 Hz), 4.59 (2H, d, J = 6.2 Hz), 7.42–7.55
(2H, m), 7.58–7.71 (2H, m), 7.74–7.83 (2H, m), 7.85–8.04 (5.6H, m), 8.12–8.22 (1.4H, m), 9.55 (0.6H, s),
9.66 (1H, t, J = 6.2 Hz), 9.89 (0.4H, s), 12.28 (1H, bs). Anal. Calcd for C28H28N4O6S·1.3H2O: C, 58.79; H,
5.39; N, 9.79. Found: C, 58.58; H, 5.10; N 9.64.
N-{[4’-({2-[Acetyl(hydroxy)amino]-3-methylbutyl}sulfonyl)biphenyl-3-yl]methyl}-4-oxo-3,4-dihydr
oquinazoline-2-carboxamide (29b). Acetic anhydride (0.018 mL, 0.19 mmol) was added to a mixture of
compound 28 (100 mg, 0.192 mmol) and acetic acid (2 mL) in THF (20 mL) at room temperature and the
mixture was stirred at room temperature for 2 h. After the mixture was concentrated under reduced pressure,
the residue was purified by crystallization from ethanol–ethyl acetate–diethyl ether–hexane to give the title
1
compound as a white powder (85.0 mg, 0.152 mmol, 79%). mp 167–169 °C. H NMR (300 MHz,
DMSO-d6)  0.78 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.8 Hz), 1.87–2.00 (4H, m), 3.14–3.24 (1H, m),
3.31–3.42 (1H, m), 3.49–3.59 (1H, m), 4.58 (2H, d, J = 6.4 Hz), 7.42–7.54 (2H, m), 7.57–7.70 (2H, m),
7.74–7.82 (2H, m), 7.83–8.05 (6H, m), 8.18 (1H, d, J = 6.8 Hz), 9.65 (1H, t, J = 5.9 Hz), 12.27 (1H, bs).
Anal. Calcd for C29H30N4O6S: C, 61.91; H, 5.37; N, 9.96. Found: C, 61.68; H, 5.29; N, 9.94.
N-{[4’-({2-[(Aminocarbonyl)(hydroxy)amino]-3-methylbutyl}sulfonyl)biphenyl-3-yl]methyl}-4-oxo-
3,4-dihydroquinazoline-2-carboxamide (29c). A suspension of compound 28 (50 mg, 0.096 mmol) and
N,N-diethylisopropylamine (0.017 mL, 0.096 mmol) in THF (10 mL) was added to a mixture of
triphosgene (9.4 mg, 0.032 mmol) in THF (2 mL) at 0 °C. After the mixture was stirred at room
temperature for 2 h, 28% aqueous NH3 solution (0.065 mL, 0.96 mmol) was added at room temperature and
the resulting mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced
pressure and the residue was triturated with ethanol–H2O to give the title compound as a white powder
1
(44.7 mg, 0.0793 mmol, 83%). mp 193–195 °C. H NMR (300 MHz, DMSO-d6)  0.69–0.80 (6H, m),
1.66–1.81 (1H, m), 3.29–3.39 (1H, m), 3.62 (1H, dd, J = 15.3, 8.3 Hz), 4.26–4.36 (1H, m), 4.58 (2H, d, J =
6.2 Hz), 6.10 (2H, bs), 7.40–7.54 (2H, m), 7.57–7.71 (2H, m), 7.73–7.82 (2H, m), 7.85–8.04 (5H, m), 8.18
(1H, d, J = 8.3 Hz), 9.07 (1H, s), 9.65 (1H, bs), 12.28 (1H, bs). Anal. Calcd for C28H29N5O6S·0.2H2O: C,
59.29; H, 5.22; N, 12.35. Found: C, 59.50; H, 5.10; N, 12.08.
5-({[(4-Bromophenyl)methyl]thio}methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (31). A mixture of
5-(chloromethyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (30, 2.00 g, 15.0 mmol, synthesized by the method of
Cowden et al.59), (4-bromophenyl)methanethiol (3.04 g, 15.0 mmol), and K2CO3 (2.48 g, 18.0 mmol) in
DMF (40 mL) was stirred at room temperature for 12 h. After H2O was added to the reaction mixture, the
precipitated solid was collected and washed with H2O and diethyl ether to give the title compound as a
white powder (3.97 g, 13.2 mmol, 88%). 1H NMR (300 MHz, DMSO-d6)  3.36 (2H, s), 3.70 (2H, s), 7.28
(2H, d, J = 8.3 Hz), 7.52 (2H, d, J = 8.3 Hz), 11.29 (1H, bs), 11.39 (1H, bs).
5-({[(4-Bromophenyl)methyl]sulfonyl}methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (32). To a solution
of compound 31 (500 mg, 1.67 mmol) in DMF (5 mL) was added 3-chloroperoxybenzoic acid (903 mg,
3.67 mmol) at 0 °C, and the mixture was stirred at room temperature for 12 h. After aqueous NaHCO3
solution was added to the mixture, the precipitated solid was collected and washed with H2O and diethyl
1
ether to give the title compound as a white powder (508 mg, 1.53 mmol, 92%). H NMR (300 MHz,
20

DMSO-d6)  4.32 (2H, s), 4.60 (2H, s), 7.38 (2H, d, J = 8.3 Hz), 7.63 (2H, d, J = 8.3 Hz), 11.62 (1H, bs),
11.68 (1H, bs).
4’-({[(5-Oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]sulfonyl}methyl)biphenyl-3-carbonitrile (33).
A mixture of compound 32 (480 mg, 1.44 mmol), (3-cyanophenyl)boronic acid (255 mg, 1.73 mmol),
tetrakis(triphenylphosphine)palladium(0) (33 mg, 0.029 mmol), and 2 M aqueous Na2CO3 solution (1.73
mL, 3.47 mmol), ethanol (3 mL), and toluene (10 mL) was refluxed for 12 h. The mixture was diluted with
H2O and washed with ethyl acetate. The aqueous layer was acidified with 1 N hydrochloric acid and the
precipitated solid was collected. The solid was washed with H2O and diethyl ether to give the title
compound as a pale yellow solid (308 mg, 0.869 mmol, 60%). 1H NMR (300 MHz, DMSO-d6)  4.36 (2H,
s), 4.67 (2H, s), 7.56 (2H, d, J = 8.3 Hz), 7.69 (1H, t, J = 7.8 Hz), 7.79–7.89 (3H, m), 8.06 (1H, d, J = 8.0
Hz), 8.20 (1H, s), 11.65 (1H, bs), 11.70 (1H, bs).
5-[({[3’-(Aminomethyl)biphenyl-4-yl]methyl}sulfonyl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one
(34). A mixture of compound 33 (75 mg, 0.21 mmol), Raney Ni (200 mg), 28% aqueous NH3 solution (2.5
mL), THF (5 mL), and methanol (2.5 mL) was stirred at room temperature for 2 h under hydrogen
atmosphere (1 atm). The catalyst was removed by vacuum filtration through a PTFE membrane and the
filtrate was concentrated under reduced pressure. The residue was triturated with diethyl ether to give the
title compound as a pale blue powder (60 mg, 0.17 mmol, 79%). The pale blue powder was used for the
next reaction without purification. MS m/z 359 [M + H]+.
4-Oxo-N-{[4’-({[(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]sulfonyl}methyl)biphenyl-3-yl]met
hyl}-3,4-dihydroquinazoline-2-carboxamide (35). A mixture of compound 34 (60 mg, 0.17 mmol),
compound 1062 (24 mg, 0.11 mmol), triethylamine (0.389 mL, 1.12 mmol), ethanol (3 mL), and
N,N-dimethylacetamide (3 mL) was stirred at 90 °C for 24 h. The mixture was diluted with ethyl acetate,
washed with 0.1 N hydrochloric acid and brine, and dried over Na2SO4. After removal of the solvent, the
residue was crystallized from ethanol–diethyl ether to give the title compound as a pale yellow powder (32
1
mg, 0.061 mmol, 55%). mp 245–247 °C. H NMR (300 MHz, DMSO-d6)  4.34 (2H, s), 4.57 (2H, d, J =
6.4 Hz), 4.64 (2H, s), 7.35–7.48 (2H, m), 7.52 (2H, d, J = 8.1 Hz), 7.56–7.74 (5H, m), 7.75–7.82 (1H, m),
7.89 (1H, t, J = 7.4 Hz), 8.17 (1H, d, J = 7.9 Hz), 9.62 (1H, t, J = 5.6 Hz), 11.52–11.78 (2H, m),
12.02–12.37 (1H, m). Anal. Calcd for C26H22N6O5S·0.5H2O: C, 57.88; H, 4.30; N, 15.58. Found: C, 57.61;
H, 4.40; N, 15.43.
5-(3-Chlorophenyl)-4-oxo-N-{[4'-({[(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]sulfonyl}methy
l)biphenyl-3-yl]methyl}-3,4-dihydrothieno[2,3-d]pyrimidine-2-carboxamide (37).
A
mixture of
compound 34 (0.100 g, 0.279 mmol), compound 3635 (0.078 g, 0.23 mmol), and triethylamine (0.162 ml,
1.16 mmol) in a mixed solvent of ethanol (5 mL) and N,N-dimethylacetamide (5 mL) was stirred at 90 °C
for 24 h. After removal of the solvent, the residue was purified by preparative HPLC and crystallized from
methanol to give the title compound as a pale yellow powder (0.0671 g, 0.104 mmol, 45%). mp
1
276–278 °C. H NMR (300 MHz, DMSO-d6)  4.34 (2H, s), 4.55 (2H, d, J = 6.1 Hz), 4.64 (2H, s),
7.33–7.90 (13H, m), 9.77 (1H, t, J = 5.9 Hz), 11.63 (1H, bs), 11.69 (1H, bs), 12.47 (1H, bs). Anal. Calcd
for C30H23ClN6O5S2·0.7H2O: C, 54.62; H, 3.73; N, 12.74. Found: C, 54.78; H, 3.76; N, 12.45.
1,1-Dimethylethyl 4-(3-Bromophenyl)piperazine-1-carboxylate (39). To a solution of 1-(3-bromo-
21

phenyl)piperazine (38, 6.00 g, 27.4 mmol) and triethylamine (3.82 mL, 27.4 mmol) in THF (60 mL) was
added dropwise di-t-butyl dicarbonate (5.97 g, 27.4 mmol) at room temperature, and the mixture was
stirred at room temperature for 3 h. After the mixture was concentrated under reduced pressure, the residue
was diluted with ethyl acetate and H2O, and neutralized with 1 N hydrochloric acid. The organic layer was
separated, washed with brine, and dried over Na2SO4. After removal of the solvent, the residue was
1
crystallized from hexane to give the title compound as a white powder (5.39 g, 15.8 mmol, 64%). H NMR
(300 MHz, DMSO-d6)  1.42 (9H, s), 3.09–3.17 (4H, m), 3.39–3.47 (4H, m), 6.91–6.97 (2H, m), 7.09 (1H,
t, J = 2.1 Hz), 7.12–7.19 (1H, m).
1,1-Dimethylethyl 4-(3-Cyanophenyl)piperazine-1-carboxylate (40). A mixture of compound 39 (3.10
g, 9.09 mmol), zinc cyanide (587 mg, 5.00 mmol), and tetrakis(triphenylphosphine)palladium(0) (525 mg,
0.454 mmol) in DMF (30 mL) was heated at 80 °C on an oil bath for 12 h under nitrogen atmosphere. The
mixture was diluted with ethyl acetate, washed with H2O and brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel column chromatography (25–40% ethyl
acetate/hexane) and triturated with hexane to give the title compound as a white power (2.10 g, 7.31 mmol,
1
80%). H NMR (300 MHz, DMSO-d6)  1.42 (9H, s), 3.16–3.24 (4H, m), 3.40–3.48 (4H, m), 7.16–7.21
(1H, m), 7.25–7.31 (1H, m), 7.33–7.36 (1H, m), 7.36–7.43 (1H, m).
1,1-Dimethylethyl 4-[3-(Aminomethyl)phenyl]piperazine-1-carboxylate (41).
A
mixture of
compound 40 (2.00 g, 6.96 mmol), Raney Ni (2.0 g), 28% aqueous NH3 solution (20 mL), THF (20 mL)
and methanol (20 mL) was stirred at room temperature for 4 h under hydrogen atmosphere (1 atm). The
catalyst was removed by vacuum filtration through a PTFE membrane and the filtrate was concentrated
under reduced pressure. The residue was dissolved in ethyl acetate, dried over Na2SO4, and concentrated
1
under reduced pressure to give the title compound as a pale blue oil (2.03 g, 6.97 mmol, 100%). H NMR
(300 MHz, DMSO-d6)  1.42 (9H, s), 3.04–3.13 (4H, m), 3.17–3.51 (6H, m), 3.50–3.82 (2H, m), 6.71–6.80
(2H, m), 6.92 (1H, bs), 7.16 (1H, t, J = 7.2 Hz).
1,1-Dimethylethyl
4-[3-({[(4-Oxo-3,4-dihydroquinazolin-2-yl)carbonyl]amino}methyl)phenyl]-
piperazine-1-carboxylate (42). A mixture of compound 41 (2.00 g, 6.86 mmol), compound 1062 (1.25 g,
5.72 mmol), and triethylamine (0.797 mL, 5.72 mmol) in ethanol (30 mL) was stirred at 80 °C for 12 h.
After the mixture was allowed to cool to room temperature, 1 N hydrochloric acid (5.7 mL) was added. The
precipitated solid was collected and washed with ethanol to give the title compound as a white powder
(2.44 g, 5.26 mmol, 92%). mp 184–185 °C. 1H NMR (300 MHz, DMSO-d6)  1.41 (9H, s), 3.03–3.14 (4H,
m), 3.39–3.50 (4H, m), 4.43 (2H, d, J = 6.4 Hz), 6.76–6.88 (2H, m), 6.97 (1H, s), 7.18 (1H, t, J = 7.8 Hz),
7.56–7.66 (1H, m), 7.74–7.82 (1H, m), 7.84–7.94 (1H, m), 8.17 (1H, dd, J = 7.9, 1.1 Hz), 9.48 (1H, t, J =
6.4 Hz), 12.27 (1H, bs). Anal. Calcd for C25H29N5O4: C, 64.78; H, 6.31; N, 15.11. Found: C, 64.62; H, 6.30;
N, 15.12.
4-Oxo-N-[(3-piperazin-1-ylphenyl)methyl]-3,4-dihydroquinazoline-2-carboxamide
Dihydrochlo-
ride (43). To a mixture of compound 42 (2.40 g, 6.86 mmol), ethyl acetate (10 mL), THF (50 mL), and
methanol (40 mL) was added 4 N HCl in ethyl acetate (10.4 mL) at room temperature. The mixture was
stirred at room temperature for 4 h and then heated at 50 °C for 2 h. Diethyl ether (100 mL) was added to
the mixture and the precipitated solid was collected to give the title compound as a white powder (2.18 g,
22

1
5.00 mmol, 97%). mp 243–245 °C. H NMR (300 MHz, DMSO-d6)  3.14–3.26 (4H, m), 3.31–3.42 (4H,
m), 4.45 (2H, d, J = 6.4 Hz), 6.83–6.93 (2H, m), 7.00 (1H, bs), 7.22 (1H, t, J = 7.8 Hz), 7.62 (1H, t, J = 7.6
Hz), 7.75–7.83 (1H, m), 7.90 (1H, t, J = 7.6 Hz), 8.18 (1H, d, J = 8.0 Hz), 9.25–9.40 (2H, m), 9.52 (1H, t, J
= 6.4 Hz). Anal. Calcd for C20H21N5O2·2HCl·0.2H2O: C, 51.52; H, 4.14; N, 7.51. Found: C, 51.42; H, 4.11;
N, 7.51.
N-[(3-{4-[(2,5-Dioxoimidazolidin-4-yl)acetyl]piperazin-1-yl}phenyl)methyl]-4-oxo-3,4-dihydroquin
azoline-2-carboxamide (45a).
A
mixture of compound 43 (109 mg, 0.250 mmol),
(2,5-dioxoimidazolidin-4-yl)acetic acid (47 mg, 0.30 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (96 mg, 0.50 mmol), and 1-hydroxybenzotriazole (68 mg, 0.50 mmol) in DMF (5
mL) was stirred at room temperature for 1 h, and triethylamine (0.139 mL, 1.00 mmol) was added. The
reaction mixture was stirred at room temperature for further 12 h. After the reaction mixture was
concentrated under reduced pressure, the residue was purified by preparative HPLC and crystallization
from methanol–diethyl ether to give the title compound as a pale yellow powder (78 mg, 0.16 mmol, 62%).
mp 280–282 °C. 1H NMR (300 MHz, DMSO-d6)  2.69–2.91 (2H, m), 3.04–3.21 (4H, m), 3.58 (4H, d, J =
3.8 Hz), 4.21–4.29 (1H, m), 4.43 (2H, d, J = 6.1 Hz), 6.76–6.90 (2H, m), 6.98 (1H, bs), 7.19 (1H, t, J = 8.0
Hz), 7.62 (1H, t, J = 7.4 Hz), 7.72 (1H, bs), 7.76–7.83 (1H, m), 7.89 (1H, t, J = 7.0 Hz), 8.18 (1H, d, J = 8.3
Hz), 9.49 (1H, t, J = 6.1 Hz), 10.56 (1H, bs), 12.26 (1H, bs). Anal. Calcd for C25H25N7O5·0.5H2O: C,
58.59; H, 5.11; N, 19.13. Found: C, 58.58; H, 5.17; N, 19.04.
4-Oxo-N-[(3-{4-[(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)acetyl]piperazin-1-yl}phenyl)methyl]-3,4-
dihydroquinazoline-2-carboxamide (45b). A mixture of compound 43 (109 mg, 0.250 mmol),
(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)acetic acid63 (43 mg, 0.30 mmol), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride (96 mg, 0.50 mmol), and 1-hydroxybenzotriazole (68 mg, 0.50 mmol)
in DMF (5 mL) was stirred at room temperature for 1 h, and triethylamine (0.139 mL, 1.00 mmol) was
added. The reaction mixture was stirred at room temperature for further 12 h. After the reaction mixture
was concentrated under reduced pressure, the residue was purified by preparative HPLC, and crystallization
from methanol–diethyl ether to give the title compound as a pale yellow powder (77 mg, 0.16 mmol, 63%).
mp 311–313 °C. 1H NMR (300 MHz, DMSO-d6)  3.06–3.22 (4H, m), 3.55–3.69 (6H, m), 4.44 (2H, d, J =
6.4 Hz), 6.77–6.90 (2H, m), 6.98 (1H, bs), 7.19 (1H, t, J = 8.0 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.75–7.82 (1H,
m), 7.85–7.94 (1H, m), 8.18 (1H, d, J = 7.2 Hz), 9.49 (1H, t, J = 6.2 Hz), 11.14 (1H, bs), 11.18 (1H, bs),
12.27 (1H, bs). Anal. Calcd for C24H24N8O4·0.5H2O: C, 57.94; H, 5.06; N, 22.52. Found: C, 57.82; H, 5.01;
N, 22.43.
4-Oxo-N-[(3-{4-[(5-oxo-2,5-dihydro-1H-pyrazol-3-yl)acetyl]piperazin-1-yl}phenyl)methyl]-3,4-dihy
droquinazoline-2-carboxamide (45c). A mixture of compound 43 (109 mg, 0.250 mmol), compound 44c
(43 mg, 0.30 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (96 mg, 0.50 mmol),
and 1-hydroxybenzotriazole (68 mg, 0.50 mmol) in DMF (5 mL) was stirred at room temperature for 1 h,
and triethylamine (0.139 mL, 1.00 mmol) was added. The reaction mixture was stirred at room temperature
for further 12 h. Water (5 mL) was added to the mixture and the resulting mixture was stirred at 50 °C for 4
h. After the reaction mixture was concentrated under reduced pressure, the residue was purified by
preparative HPLC, and crystallization from methanol–diethyl ether to give the title compound as a pale
23

1
yellow powder (40 mg, 0.082 mmol, 33%). mp 183–185 °C. H NMR (300 MHz, DMSO-d6)  3.03–3.17
(4H, m), 3.53–3.72 (6H, m), 4.43 (2H, d, J = 6.2 Hz), 5.33 (1H, s), 6.77–6.90 (2H, m), 6.97 (1H, bs), 7.19
(1H, t, J = 7.9 Hz), 7.57–7.67 (1H, m), 7.74–7.82 (1H, m), 7.84–7.95 (1H, m), 8.18 (1H, dd, J = 7.9, 0.9
Hz), 9.49 (1H, t, J = 6.1 Hz), 12.26 (1H, bs). Anal. Calcd for C25H25N7O4·2H2O: C, 57.35; H, 5.58; N,
18.73. Found: C, 57.66; H, 5.27; N, 18.35.
N-({3-[4-(1H-Imidazol-4-ylacetyl)piperazin-1-yl]phenyl}methyl)-4-oxo-3,4-dihydroquinazoline-2-ca
rboxamide Trifluoroacetate (45d).
1H-imidazol-4-ylacetic acid
A
mixture of compound 43 (109 mg, 0.250 mmol),
hydrochloride (49 mg, 0.30 mmol),
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (96 mg, 0.50 mmol), and
1-hydroxybenzotriazole (68 mg, 0.50 mmol) in DMF (5 mL) was stirred at room temperature for 1 h, and
triethylamine (0.174 mL, 1.25 mmol) was added. The reaction mixture was stirred at room temperature for
further 12 h. After the reaction mixture was concentrated under reduced pressure, the residue was purified
by preparative HPLC, and crystallization from methanol–diethyl ether to give the title compound as a pale
1
yellow powder (93 mg, 0.196 mmol, 79%). mp 202–204 °C. H NMR (300 MHz, DMSO-d6)  3.08–3.24
(4H, m), 3.59–3.73 (4H, m), 3.96 (2H, s), 4.44 (2H, d, J = 6.4 Hz), 6.79–6.92 (2H, m), 6.99 (1H, bs), 7.21
(1H, t, J = 7.8 Hz), 7.46 (1H, s), 7.62 (1H, t, J = 7.4 Hz), 7.75–7.82 (1H, m), 7.90 (1H, t, J = 7.2 Hz), 8.18
(1H, d, J = 8.0 Hz), 9.00 (1H, s), 9.51 (1H, t, J = 5.9 Hz), 12.27 (1H, bs), 14.17 (2H, bs). Anal. Calcd for
C25H25N7O3·CF3CO2H·H2O: C, 53.73; H, 4.68; N, 16.25. Found: C, 53.60; H, 4.74; N, 16.12.
4-Oxo-N-({3-[4-(1H-tetrazol-5-ylacetyl)piperazin-1-yl]phenyl}methyl)-3,4-dihydroquinazoline-2-ca
rboxamide (45e). A mixture of compound 43 (109 mg, 0.250 mmol), 1H-tetrazol-5-ylacetic acid (38 mg,
0.30 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (96 mg, 0.50 mmol), and
1-hydroxybenzotriazole (68 mg, 0.50 mmol) in DMF (5 mL) was stirred at room temperature for 1 h, and
triethylamine (0.139 mL, 1.00 mmol) was added. The reaction mixture was stirred at room temperature for
further 12 h. After the reaction mixture was concentrated under reduced pressure, the residue was purified
by preparative HPLC, and crystallization from methanol–diethyl ether to give the title compound as a pale
1
yellow powder (38 mg, 0.080 mmol, 32%). mp 163–165 °C. H NMR (300 MHz, DMSO-d6)  3.07–3.24
(4H, m), 3.57–3.76 (4H, m), 4.27 (2H, s), 4.44 (2H, d, J = 6.4 Hz), 6.78–6.91 (2H, m), 6.99 (1H, bs), 7.20
(1H, t, J = 8.1 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.75–7.83 (1H, m), 7.89 (1H, t, J = 7.8 Hz), 8.18 (1H, d, J =
7.6 Hz), 9.49 (1H, t, J = 5.7 Hz), 12.26 (1H, bs). Anal. Calcd for C23H23N9O3·1.2H2O: C, 55.80; H, 5.17; N,
25.46. Found: C, 56.10; H, 4.87; N, 25.17.
(5-Oxo-2,5-dihydro-1H-pyrazol-3-yl)acetic Acid (44c). Hydrazine (4.38 g, 129 mmol) was added to a
solution of diethyl 3-oxopentanedioate (26.0 g, 129 mmol) in ethanol (250 mL) at room temperature and
the mixture was stirred at 80 °C for 3 h. After the mixture was concentrated under reduced pressure, the
residue
was
crystallized
from
diethyl
ether–hexane
to
give
ethyl
1
(5-oxo-2,5-dihydro-1H-pyrazol-3-yl)acetate as a white powder (9.79 g, 57.5 mmol, 45%). H NMR (300
MHz, DMSO-d6)  1.19 (3H, t, J = 7.0 Hz), 3.53 (2H, s), 4.08 (2H, q, J = 6.9 Hz), 5.34 (1H, s), 9.53 (1H,
bs), 11.39 (1H, bs).
A mixture of ethyl (5-oxo-2,5-dihydro-1H- pyrazol-3-yl)acetate (5.00 g, 29.4 mmol), 8 N aqueous
sodium hydroxide solution (11.0 mL, 88.0 mmol), H2O (20 mL), THF (20 mL), and methanol (20 mL) was
24

stirred at 80 °C for 3 h. After THF and methanol were removed by evaporation, the residue was acidified
with 6 N hydrochloric acid (15 mL). The precipitated solid was collected, washed with H2O, and dried in a
1
stream of air to give the title compound as a white powder (3.43 g, 24.1 mmol, 82%). H NMR (300 MHz,
DMSO-d6)  3.45 (2H, s), 5.33 (1H, s), 10.52–12.13 (3H, m).
4’-({5-[2-(Ethyloxy)ethyl]-2,4,6-trioxohexahydropyrimidin-5-yl}oxy)biphenyl-3-carbonitrile (48). A
mixture of 5-bromo-5-[2-(ethyloxy)ethyl]pyrimidine-2,4,6(1H,3H,5H)-trione20, 64 (46, 2.00 g, 7.17 mmol),
4’-hydroxybiphenyl-3-carbonitrile65 (47, 1.40 g, 7.17 mmol), and K2CO3 (3.47 g, 25.1 mmol) in DMF (40
mL) was stirred at room temperature for 4 h. The reaction mixture was diluted with ethyl acetate, washed
with H2O and brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by
crystallization from methanol–diethyl ether to give the title compound as a white powder (1.95 g, 4.96
mmol, 69%). 1H NMR (300 MHz, DMSO-d6)  1.08 (3H, t, J = 7.0 Hz), 2.44 (2H, t, J = 5.9 Hz), 3.30–3.39
(2H, m), 3.52 (2H, t, J = 5.9 Hz), 6.78–6.84 (2H, m), 7.60–7.70 (3H, m), 7.76–7.81 (1H, m), 7.92–7.98 (1H,
m), 8.06–8.10 (1H, m), 11.88 (2H, bs).
Sodium 3’-(Aminomethyl)biphenyl-4-olate (49). A mixture of compound 48 (1.50 g, 3.81 mmol),
Raney Ni (1.5 g), 28% aqueous NH3 solution (15 mL), THF (30 mL), and methanol (15 mL) was stirred at
room temperature for 12 h under hydrogen atmosphere (1 atm). The catalyst was removed by vacuum
filtration through a PTFE membrane and the filtrate was concentrated under reduced pressure. The residue
was diluted with H2O, and acidified with 1 N hydrochloric acid. The insoluble materials were filtered off
through a pad of Celite and then the filtrate was basified with 1 N aqueous sodium hydroxide solution. The
precipitated solid was collected and washed with H2O and ethanol to give the title compound as a pale
green powder (800 mg, 3.62 mmol, 95%). 1H NMR (300 MHz, DMSO-d6)  3.74 (2H, bs), 6.68–6.93 (2H,
m), 7.05–7.68 (6H, m).
N-[(4’-Hydroxybiphenyl-3-yl)methyl]-4-oxo-3,4-dihydroquinazoline-2-carboxamide (50). A mixture
of compound 49 (200 mg, 0.904 mmol), compound 1062 (132 mg, 0.603 mmol), and triethylamine (0.336
mL, 2.41 mmol) in ethanol (10 mL) was stirred at 80 °C for 12 h. After the mixture was allowed to cool to
room temperature, 2 N HCl in ethanol (2 mL) was added. The precipitated solid was collected and washed
with ethanol to give the title compound as a white powder (149 mg, 0.401 mmol, 67%). mp 244–246 °C.
1H NMR (300 MHz, DMSO-d6)  4.53 (2H, d, J = 6.1 Hz), 6.84 (2H, d, J = 8.3 Hz), 7.23–7.30 (1H, m),
7.37 (1H, t, J = 7.6 Hz), 7.42–7.50 (3H, m), 7.55–7.64 (2H, m), 7.73–7.80 (1H, m), 7.87 (1H, t, J = 7.6 Hz),
8.16 (1H, d, J = 8.0 Hz), 9.51–9.64 (2H, m), 12.01 (1H, bs). Anal. Calcd for C22H17N3O3·0.3H2O: C, 70.13;
H, 4.71; N, 11.15. Found: C, 69.96; H, 4.56; N, 11.24.
N-{[4’-({5-[2-(Ethyloxy)ethyl]-2,4,6-trioxohexahydropyrimidin-5-yl}oxy)biphenyl-3-yl]methyl}-4-o
xo-3,4-dihydroquinazoline-2-carboxamide (51). A mixture of compound 46 (75 mg, 0.27 mmol),
compound 50 (100 mg, 0.269 mmol), and K2CO3 (186 mg, 1.34 mmol) in DMF (5 mL) was stirred at room
temperature for 5 h. The reaction mixture was diluted with ethyl acetate, washed with H2O and brine, and
dried over Na2SO4. After removal of the solvent, the residue was purified by crystallization from ethyl
acetate–diisopropyl ether to give the title compound as a white powder (78 mg, 0.137 mmol, 51%). mp
163–165 °C. 1H NMR (300 MHz, DMSO-d6)  1.01–1.08 (3H, m), 2.37–2.47 (2H, m), 3.29–3.39 (2H, m),
3.44–3.55 (2H, m), 4.53 (2H, d, J = 6.1 Hz), 6.73–6.82 (2H, m), 7.26–7.66 (7H, m), 7.73–7.94 (2H, m),
25

8.14–8.21 (1H, m), 9.60 (1H, t, J = 6.1 Hz), 11.73–12.01 (2H, m), 12.10–12.39 (1H, m). Anal. Calcd for
C30H27N5O7·0.1iPr2O: C, 63.39; H, 4.94; N, 12.08. Found: C, 63.16; H, 5.01; N, 11.79.
4-Oxo-N-(3-{3-[(1-trityl-1H-1,2,4-triazol-3-yl)oxy]propoxy}benzyl)-3,4-dihydroquinazoline-2-carbo
xamide (53). A mixture of compound 5235 (0.300 g, 0.611 mmol.), compound 1062 (0.133 g, 0.611 mmol),
and DIEA (0.213 mL, 1.22 mmol) in ethanol (4.5 mL) was heated under microwave irradiation at 100 °C
for 1.5 h. After the reaction mixture was cooled to room temperature, the precipitated solid was collected
and washed with ethanol to give the title compound 53 as a white powder (0.315 g, 0.475 mmol, 78%). MS
1
m/z 663.2 [M + H]+. H NMR (300 MHz, DMSO-d6) δ 2.05–2.17 (2H, m), 4.06 (2H, t, J = 6.1 Hz), 4.29
(2H, t, J = 6.1 Hz), 4.44 (2H, d, J = 6.4 Hz), 6.78–6.85 (1H, m), 6.88–6.96 (2H, m), 7.02–7.11 (6H, m),
7.23 (1 H, t, J = 8.0 Hz), 7.30–7.42 (9H, m), 7.60 (1H, t, J = 7.6 Hz), 7.74–7.82 (2H, m), 7.87 (1H, t, J =
7.6 Hz), 8.17 (1H, d, J = 7.2 Hz), 9.51 (1H, t, J = 6.2 Hz), 11.86 (1H, bs).
4-Oxo-N-{3-[3-(1H-1,2,4-triazol-3-yloxy)propoxy]benzyl}-3,4-dihydroquinazoline-2-carboxamide
(54). TFA (1.27 mL) and triethylsilane (0.082 mL, 0.52 mmol) were added to a mixture of compound 53
(0.285 g, 0.430 mmol) in CH3CN (6 mL) at room temperature, and the mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated in vacuo. The residue was crystallized from
diethyl ether–ethanol to give the title compound as a white powder (0.173 g, 0.411 mmol, 96%). MS m/z
421.1 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 2.09–2.21 (2H, m), 4.08 (2H, t, J = 6.2 Hz), 4.29–4.40
(2H, m), 4.45 (2H, d, J = 6.4 Hz), 6.84 (1H, dd, J = 8.1, 2.1 Hz), 6.89–6.98 (2H, m), 7.24 (1H, t, J = 7.8
Hz), 7.61 (1H, t, J = 7.4 Hz), 7.74–7.82 (1H, m), 7.84–7.94 (1H, m), 8.12–8.26 (2H, m), 9.53 (1H, t, J = 6.1
Hz), 12.24 (1H, bs), 13.27 (1H, bs). mp 190–192 °C. Anal. Calcd for C21H20N6O4·0.2H2O: C, 59.48; H,
4.85; N, 19.82. Found: C, 59.40; H, 4.72; N, 19.80.
5-(3-Chlorophenyl)-N-(3-methoxybenzyl)-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-2-carboxamide
(55). A mixture of compound 36 (0.110 g, 0.346 mmol) and 3-methoxybenzylamine (0.066 mL, 0.518
mmol) , and triethylamine (0.096 mL, 0.691 mmol) in ethanol (2.0 mL) was stirred at 80 °C for 12 hr (clear
solution). After the reaction mixture was allowed to cool to rt, the mixture was concentrated in vacuo. The
residue was taken up in ethyl acetate, and washed with 1 N HCl twice and brine. The organic phase was
dried over Na2SO4, and concentrated in vacuo to give a foam which was crystallized from ethyl acetate to
1
give the title compound as a white powder (0.092 g, 0.216 mmol, 62%). mp 164 °C. H NMR (300 MHz,
DMSO-d6)  3.74 (3H, s), 4.44 (2H, d, J = 6.4 Hz), 6.79–6.86 (1H, m), 6.88–6.95 (2H, m), 7.25 (1H, t, J =
8.1 Hz), 7.40–7.54 (3H, m), 7.61 (1H, q, J = 1.6 Hz), 7.8 (1H, s), 9.69 (1H, t, J = 6.4 Hz), 12.46 (1H, s).
Anal. Calcd for C21H16ClN3O3S: C, 59.22; H, 3.79; N, 9.87. Found: C, 59.11; H, 3.87; N, 9.69.
5-2. MMPs and TACE enzyme inhibition assay. Human recombinant MMP precursors were purchased
from Genzyme-Techne (MMP-1, 2, 7, 8, 9, 10, 12, 13, and TACE) or Biogenesis (MMP-3). Human
recombinant GST-MMP-14 was prepared as described by Sato et al.66 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, 12, 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 assay
buffer containing enzymes and fluorescence peptide (Cy3-PLGLK(Cy5Q)AR-NH2 for MMPs and
26

Cy3-PLAQAV(Cy5Q-L-2,3-diaminopropionic acid)-RSSSR-NH2 for TACE, Amersham Biosciences) in the
presence of the 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).
6. Acknowledgement
The authors thank our supervisors: Drs. Shigenori Ohkawa, Yuji Ishihara, and Yoshinori Ikeura, for
valuable discussions and critical comments throughout this work. We thank Drs. Keiji Kamiyama and
Nobuo Cho for their careful reading of the manuscript. We also thank the members of drug metabolism &
pharmacokinetics research laboratories for pharmacokinetic and physicochemical studies, and the
members of the Takeda analytical research laboratories, Ltd. for elemental analyses.
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