Structure-based drug design to overcome species differences in kallikrein 7 inhibition of 1,3,6-trisubstituted 1,4-diazepan-7-ones

Article abstract of DOI:10.1016/j.bmc.2018.05.044

A series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were prepared as kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Previously reported compounds 1–3 were potent human KLK7 inhibitors; however, they did not exhibit inhibitory activity against mouse KLK7. Comparison of the human and mouse KLK7 structures reveals the cause of this species differences; therefore, compounds that could inhibit both KLK7s were designed, synthesized, and evaluated. Through this structure-based drug design, compound 22g was identified as an inhibitor against human and mouse KLK7, and only one of the enantiomers, (–)–22g, exhibited potent inhibitory activity. Furthermore, the crystal structure of mouse KLK7 complexed with 22g enabled the elucidation of structure–activity relationships and justified 22g as a valuable compound to overcome the species differences.

Full text of DOI:10.1016/j.bmc.2018.05.044

Accepted Manuscript
Structure-based drug design to overcome species differences in kallikrein 7 in-
hibition of 1,3,6-trisubstituted 1,4-diazepan-7-ones
Hidenobu Murafuji, Hajime Sugawara, Megumi Goto, Yoshiaki Oyama, Hiroki
Sakai, Seiichi Imajo, Toshiyuki Tomoo, Tsuyoshi Muto
PII:
S0968-0896(18)30522-4
https://doi.org/10.1016/j.bmc.2018.05.044
BMC 14386
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 March 2018
23 May 2018
24 May 2018
Please cite this article as: Murafuji, H., Sugawara, H., Goto, M., Oyama, Y., Sakai, H., Imajo, S., Tomoo, T., Muto,
T., Structure-based drug design to overcome species differences in kallikrein 7 inhibition of 1,3,6-trisubstituted 1,4-
diazepan-7-ones, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.05.044
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Bioorganic & Medicinal Chemistry
Structure-based drug design to overcome species differences in kallikrein 7
inhibition of 1,3,6-trisubstituted 1,4-diazepan-7-ones
Hidenobu Murafujia, 1, , Hajime Sugawaraa, 1, Megumi Goto1, Yoshiaki Oyama2, Hiroki Sakai1, Seiichi
Imajo, Toshiyuki Tomoo3 and Tsuyoshi Muto1, 
Asubio Pharma Co., Ltd., 6-4-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were prepared as kallikrein 7 (KLK7,
stratum corneum chymotryptic enzyme) inhibitors. Previously reported compounds 1–3 were
potent human KLK7 inhibitors; however, they did not exhibit inhibitory activity against mouse
KLK7. Comparison of the human and mouse KLK7 structures reveals the cause of this species
differences; therefore, compounds that could inhibit both KLK7s were designed, synthesized,
and evaluated. Through this structure-based drug design, compound 22g was identified as an
inhibitor against human and mouse KLK7, and only one of the enantiomers, (–)-22g, exhibited
potent inhibitory activity. Furthermore, the crystal structure of mouse KLK7 complexed with
22g enabled the elucidation of structure-activity relationships and justified 22g as a valuable
compound to overcome the species differences.
Available online
Keywords:
1,4-Diazepan-7-one
Serine protease
KLK7 inhibitor
Species differences
Structure-based drug design
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding authors.
E-mail addresses: murafuji.hidenobu.bj@daiichisankyo.co.jp (H. Murafuji), muto.tsuyoshi.zu@daiichisankyo.co.jp (T. Muto)
aThese authors contributed equally to this work.
1Present address: Shinagawa R&D Center, Daiichi-Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
2Present address: Shinagawa R&D Center, Daiichi-Sankyo RD Novare Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
3Present address: Process Technology Research Laboratories, Daiichi-Sankyo Co., Ltd., 1-12-1 Shinomiya, Hiratsuka, Kanagawa 254-0014,
Japan.

increasing the risk of inflammation.9,12 Atopic dermatitis (AD) is
a common and multifactorial chronic allergic skin disease. Skin
barrier dysfunction has been implicated in the development of
AD,9, 12, 13 and increased serine protease activities are thought to
be associated with impaired skin barrier function.14 Therefore, the
restoration of this barrier function of the skin would promote AD
therapy.15-17 KLK7 is a chymotryptic serine protease abundantly
expressed in the skin,18 and its levels have been reported to
significantly increase in AD patients.19,20 Therefore, we focused
our attention on KLK7 inhibitors that would aid AD therapy by
restoring the barrier function of the skin.
1. Introduction
Tissue kallikreins (KLKs) constitute a family of 15 serine
proteases (KLK1–KLK15) that are associated with various
physiological and pathological processes.1,2 KLKs have recently
received attention as attractive drug targets.2,3 The expression of
various KLKs were identified in the skin,4 among which
kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme)
plays an important role in epidermal desquamation by degrading
corneodesmosome proteins.5-7 The desquamation of corneocytes
is an essential process that occurs in normal skin8 upon
degradation of corneodesmosomes by skin-specific proteases.9
Desquamation is regulated by an appropriate balance of various
proteases, including KLK7 and their intrinsic inhibitors such as
lympho-epithelial Kazal-type-related inhibitor (LEKTI), which is
encoded by serine protease inhibitor Kazal-type 5 (SPINK5), to
maintain skin barrier function.10,11 The stratum corneum (SC),
which is composed of corneocytes held together by
In previous publications, we reported the discovery and
structure-activity relationship study of a 1,3,6-trisubstituted 1,4-
diazepan-7-one scaffold as novel human KLK7 inhibitors,21 and
synthesized highly potent and selective human KLK7
inhibitors.22 However, these compounds showed significantly
decreased potency for mouse KLK7 (Table 1). In order to use
these inhibitors as tools to elucidate the pathophysiological role
of KLK7, it was necessary to break through this species
differences by structural modification.
corneodesmosomes, is responsible for this barrier function.9,12
defective epidermal barrier increases moisture loss, and
facilitates the penetration of irritants and allergens, thereby
A
Table 1. Species differences between recombinant human and mouse KLK7 inhibitory activity.
mouse KLK7
inhibition ratio
human KLK7
IC50 (µM)
Compound
Structure
IC50 (µM)
@ 30µM (%)
1.9
>30
11
1
0.52
>30
>30
−1.2
2
3
0.058
32
In order to clarify the cause of the species differences in
inhibitory activity of our scaffold, we have determined the crystal
structure of mouse KLK7. Based on the structural comparison
between mouse and human KLK7, we identified the compound
substructure that interfered with the binding to mouse KLK7.
Moreover, using the crystal structures of mouse and human
KLK7 we obtained compounds that overcame the species
differences.
2. Results and discussions
2.1. Crystal structure of mouse apo-KLK7

Figure 1. Crystal structure of mouse apo-KLK7 (PDB ID: 5ZFH) and human apo-KLK7 (PDB ID: 3BSQ23). (A) Superimposition of mouse and human KLK7
structures. These two structures were overlapped by motion-tree analysis.24 For human structure, the coordinates of the C chain, which has the smallest
magnitude of the top two nodes in the asymmetric unit, were chosen. The residues that belong to two minor clusters in the mouse structure are indicated in green.
Mouse and human structures are colored in grey and orange, respectively. (B) Sequence alignment of mouse and human KLK7. The residues located within 5 Å
from compound 1 are boxed in blue. Residues 190 and 218, which are not conserved between mouse and human KLK7, are shown within the magenta frame.
Sequence alignment was calculated with ClustalW.25
Figure 2. Structural comparison around compound 1. Surfaces of mouse apo and human complex KLK7 are represented by grey and pink meshes, respectively.
Carbon atoms are shown in grey and green in mouse apo and human complex, respectively. Nitrogen and oxygen atoms are colored in blue and red, respectively.
(A) Surface structure around active site in mouse KLK7 (PDB ID: 5ZFH). The key residue Thr190 and the surrounding water molecule to determine the shape of
the binding pocket are also shown. (B) Surface structure around the binding site of 1 in human complex (PDB ID: 5Y9L21). The corresponding residue Ala190 is
also shown. Other residues were indicated in previous report.21 (C) Superimposition of the surface structures of mouse and human KLK7.
We have determined the crystal structure of mouse KLK7 at
1.93 Å resolution (PDB ID: 5ZFH). The crystal contains one
monomer in the asymmetric unit, and the main-chain atoms of all
residues are ordered. The structures of mouse and human KLK7
are very similar (Fig. 1A). Both are composed of two six-
stranded β-barrel structures and six disulfide bonds. The
sequence identity between mouse and human KLK7 is 76% (Fig.
1B), and the value of root mean square deviation (r.m.s.d.) of Cα
atoms between mouse and human (PDB ID: 3BSQ23) is 0.55 Å,
which was calculated for all residues except ten Cα atoms in two
minor clusters by motion-tree analysis24 (Figs. S1 and S2).
Comparing the mouse and human KLK7 structures, two small
regions were found to be slightly different: the C-terminal region
(residues 242–245), and residues 73–80. The binding site of
compound 1 is far from these regions (9.6 Å of nearest atoms
between Ile73 and 1) as observed in the crystal structure of
human KLK7 in complex with compound 1 previously reported
by us.21 Since the structures of mouse apo and human complexes
with 1 are also very similar (r.m.s.d = 0.43 Å), the complex
structure of human KLK7 with 1 was used for the structural

comparisons around the binding site of 1. The superimposition of
mouse apo and human KLK7 complexed with 1 (PDB ID:
5Y9L21) revealed that the bottom of the S1 pocket of the mouse
structure narrows around the side chain of Thr190 and a water
molecule (Fig. 2). Thr190 in mouse KLK7 is replaced by alanine
in human KLK7 (Fig. 1B). Furthermore, the water molecule in
mouse KLK7 is captured by the oxygen atom of Thr190 side
chain and the oxygen and nitrogen atoms of Val227 main chain
with hydrogen-bonding contacts (hydrogen-bonding distances =
2.6, 3.1, and 3.3 Å, respectively). The temperature factor of the
water oxygen atom is 34.1 Å2, which is almost the same as the
average value for water molecules (33.8 Å2). The composite omit
electron density map26 contoured at 1σ clearly shows this water
molecule (Fig. S3). The above two results support the existence
of a water molecule at this position. By filling the bottom of the
S1 pocket, the interaction of 1 appears to be hindered in mouse
KLK7. The same interference of interaction might occur in
mouse KLK7 for more potent human KLK7 inhibitors 2 and 3
since these compounds bear the same benzyl substituent. To
improve the inhibition activity in mouse KLK7 we have modified
the 5-chloro-2-methoxy benzyl group of our scaffold and
removed the chlorine atom at the meta position of 1.
2.2. Chemistry
Considering its potency against human KLK7 and ease of
synthesis, the benzyl group of compound 2 was modified. Thus,
compound 8, the dechloro derivative of 2, was prepared as shown
in Scheme 1. Dechlorination of 421 was achieved via
hydrogenolysis by Pd-C catalyst. Then, compound 5 was
condensed with 3-(1-methyl-1H-pyrazol-4-yl)aniline to yield
amide derivative 6, which was converted to thioamide 7. This
thioamidation proceeded at the more reactive and less sterically
hindered position. Finally, dimethylamidrazone 8 was obtained
by the treatment of 7 with N,N-dimethylhydrazine in the presence
of silver acetate.
Scheme 1. Reagents and conditions: (a) H2, 5% Pd-C, Et3N, MeOH, AcOEt, rt; (b) 3-(1-methyl-1H-pyrazol-4-yl)aniline, EDCI·HCl, CH2Cl2, rt; (c) Lawesson’s
reagent, THF, 50 °C; (d) Me2NNH2, AgOAc, pyridine, rt.
Compound 14, a key intermediate for the modification at the
6-position of the 1,4-diazepane core, was prepared as shown in
Scheme 2. Di-tert-butoxycarbonyl (Boc) derivative 10 was
synthesized by substitution of commercially available ethyl 2-
(bromomethyl)acrylate 9 with di-tert-butyl iminodicarboxylate
under basic conditions. The ethyl ester of 10 was hydrolyzed, and
the resulting carboxylic acid 11 was coupled with glycine ethyl
ester hydrochloride to yield 12. After Boc deprotection,
introduction of 2,4,6-trimethoxybenzyl (TMB) group to the
resulting amino group by reductive alkylation gave 13. Ester
hydrolysis of 13, followed by lactamization afforded exo-olefin
compound 14.
Scheme 2. Reagents and conditions: (a) di-t-butyl iminodicarboxylate, K2CO3, MeCN, rt; (b) 2 mol/L NaOH aq., MeOH, rt; (c) glycine ethyl ester hydrochloride,
HOBt, EDCI·HCl, Et3N, CH2Cl2, rt; (d) MeSO3H, EtOH, 40 °C, then Et3N, 2,4,6-(MeO)3PhCHO, NaBH(OAc)3, EtOH, 0–40 °C; (e) 4 mol/L NaOH aq. MeOH,
rt; (f) HOBt, EDCI·HCl, MeCN, rt.

The synthesis of compounds 22a–n (racemic compounds)
containing the modified 6-benzyl group on the 1,4-diazepane
core is shown in Scheme 3. Compound 14 was alkylated with
tert-butyl bromoacetate to afford 15. Then, compounds 18 were
synthesized by the rhodium-catalyzed 1,4-addition reaction27 of
aryl boronic acids to Michael acceptor 15. Alternatively, 18 were
prepared by the 1,4-addition of bis(pinacolato)diboron to
compound 15, and the obtained pinacolboronate 16 was
subsequently converted to potassium trifluroroborate 17. Suzuki-
Miyaura cross-coupling of 17 with aryl halides gave the
corresponding 18. The obtained racemic compounds 18a–n were
treated with Belleau’s reagent28 to prepare thioamides 19a–n,
which were converted to amidorazones 20a–n by reacting with
N,N-dimethylhydrazine in the presence of silver acetate. After the
simultaneous removal of tert-butyl and TMB groups under acidic
conditions, the condensation of 3-(1-methyl-1H-pyrazol-4-
yl)aniline with the resulting carboxylic acid derivatives 21a–n
afforded 22a–n. Optically active compounds (+)-22g and (–)-22g
(not shown in the scheme) were obtained by chromatographic
separation using chiral HPLC column (CHIRALPAK ID).
Scheme 3. Reagents and conditions: (a) BrCH2CO2t-Bu, 60% NaH, NaI, DMF, 0 °C; (b) bis(pinacolato)diboron, CuCl, 1,2-bis(diphenylphosphino)benzene, t-
BuONa, MeOH, THF, rt; (c) KHF2, MeCN, H2O, 0 °C; (d) ArI, or ArBr, Pd(OAc)2, 2-dicyclohexylphosphino-2',6'-di-i-propoxy-1,1'-biphenyl, Cs2CO3, toluene,
H2O, 80–85 °C; (e) boronic acid derivatives, [Rh(cod)OH]2, dioxane, H2O, 80–100 °C; (f) Belleau’s reagent, THF, rt–50 °C; (g) Me2NNH2, AgOAc, pyridine, 0
°C; (h) anisole, TFA, rt–60 °C; (i) 3-(1-methyl-1H-pyrazol-4-yl)aniline, EDCI·HCl, CH2Cl2, rt; (j) 3-(1-methyl-1H-pyrazol-4-yl)aniline, 1-propanephosphonic
acid anhydride, (i-Pr)2NEt, AcOEt, CH2Cl2, rt.
Compound 22o was synthesized by an alternative approach
shown in Scheme 4. Compound 18o was prepared analogously to
the described method in Scheme 3. Simultaneous removal of
TMB and tert-butyl groups under acidic conditions gave
compound 23, which was condensed with 3-(1-methyl-1H-
pyrazol-4-yl)aniline to furnish 24. The selective thioamidation of
compound 24 was accomplished with Lawesson’s reagent to give
compound 25, which was converted to compound 22o by
treatment with N,N-dimethylhydrazine and silver acetate.
Scheme 4. Reagents and conditions: (a) 4-chloro-2-methoxyphenylboronic acid, [Rh(cod)OH]2, dioxane, H2O, 80 °C; (b) anisole, TFA, rt; (c) 3-(1-methyl-
1H-pyrazol-4-yl)aniline, EDCI·HCl, CH2Cl2, rt; (d) Lawesson’s reagent, THF, 50 °C; (e) Me2NNH2, AgOAc, MeOH, THF, rt.

2.3. Structure-activity relationships for human and mouse KLK7
investigations of the substituent effect were conducted by using
easily prepared racemic compounds. The replacement of the
methoxy group of 8 by a cyano group decreased the potency of
compound 22a, instead, the replacement by a chloro group (22b)
resulted in retention of potency for human KLK7 inhibition and
increased potency for mouse KLK7 inhibition. Moving the
chloro substituent of 2 to para (22o) or ortho (22c) positions
decreased the inhibitory activities against human KLK7.
However, it is worth emphasizing that compound 22c showed
moderate but equipotent inhibitory activity against both human
and mouse KLK7. The comparison between ortho substituted
22b and di-ortho substituted 22c suggests that the di-ortho
substitution of the benzyl group is effective to overcome the
species differences.
The series of benzyl derivatives synthesized as above were
evaluated for the inhibitory activity against human and mouse
KLK7 (Table 2). As already mentioned, compound 2 showed
potent inhibitory activity against human KLK7, but no inhibitory
activity against mouse KLK7 was observed even at 30 μM
concentration. The inhibition ratio against mouse KLK7 at 30
μM of dechloro derivative 8 was improved compared to 2,
although its inhibitory potency against human KLK7 slightly
decreased. This result suggests that the problem with species
differences in the inhibitory activity could be solved by the
modification of the substituents on the benzyl group as already
indicated by the crystal structure of mouse KLK7. Further
Table 2. Investigation of the benzyl substituents for inhibitory activity against recombinant human and mouse KLK7.
mouse KLK7
human KLK7
Compound
R1
R2
R3
R4
6-Config.
inhibition ratio
IC50 (µM)
0.52
2.4
IC50 (µM)
@ 30 µM (%)
2
OMe
OMe
CN
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
H
R
>30
−1.2
41
8
R
>30
>30
21
22a
22b
22o
22c
racemate
racemate
racemate
racemate
17
22
Cl
2.5
59
OMe
OMe
7.5
>30
14
−4.1
67
11
To optimize the effects of the di-ortho substitution on the
benzyl group, various combinations of di-ortho substituents were
evaluated (Table 3). Compared to 22c, compound 22d bearing
methoxy and fluoro groups exhibited slightly higher inhibitory
activity for human KLK7, but also showed large differences in
the inhibitory activity between human and mouse KLK7. On the
other hand, compound 22e containing methoxy and methyl
groups displayed similar potency to 22c against both KLK7s.
Compounds combining a chloro substituent with another group
(22f–i) all, except for larger nitrile substituted derivative 22i,
showed improved potency against both human and mouse KLK7.
The fluoro substituted derivative 22f exhibited species
differences between human and mouse KLK7 inhibition, instead,
chloro (22g) or methyl (22h) derivatives showed similar
inhibitory potency for both KLK7s. In particular, 22g was more
than tenfold more potent than 22c against both human and mouse
KLK7. A similar tendency was observed in the case of the series
containing a methyl group (22e, 22h, and 22j–22l). Fluoro
substituted benzyl analogs (22d, 22f, 22j, 22m, and 22n) showed
potent human KLK7 inhibition activity, but species differences in
the inhibitory activity were observed in all cases. In summary,
benzyl di-ortho substitution was a useful strategy for overcoming
the species differences in inhibitory activity, and di-chloro analog
22g was the preferred compound not only for its inhibitory
activity but also for the absence of species differences.

Table 3. Optimization of the substituents for the inhibitory activity against recombinant human and mouse KLK7.
human KLK7
IC50 (µM)
mouse KLK7
IC50 (µM)
Compounda
R1
R2
22c
OMe
OMe
OMe
Cl
Cl
F
11
6.8
13
14
>30
19
22d
22e
Me
F
22f
0.94
0.79
1.5
11
6.0
1.2
1.6
7.8
7.7
3.8
7.1
>30
>30
22g
Cl
Cl
22h
Cl
Me
CN
F
22i
Cl
22j
Me
Me
Me
F
2.3
5.2
11
22k
Me
CN
F
22l
22m
6.9
2.7
22n
F
CF3
aAll compounds were racemate.
previous report, the configuration of the 6-position of the 1,4-
diazepane core affected the activity of KLK7 inhibition; the R-
enantiomer was more potent than the S-enantiomer.21 Therefore,
we assumed that more potent enantiomer (–)-22g has R
configuration.
Table 4. Inhibitory activity against recombinant human and
mouse KLK7 of the chiral compounds (+)-22g and (–)-22g.
human KLK7
mouse KLK7
Compound
IC50 (µM)
IC50 (µM)
(+)-22g
>30
16
2.4. Complex structure analysis
(–)-22g
0.38
0.70
In order to visualize the binding mode of 22g, in particular
around the benzyl group, the crystal structure of mouse KLK7
with (±)-22g was solved (Fig. 3A). While 22g was racemate, the
crystal structure analysis was carried out assuming that the ligand
was (–)-22g, which would have R configuration based on its
potent inhibitory activity as described our previous report.21
Next, we prepared chiral compounds (+)-22g and (–)-22g and
evaluated their human and mouse KLK7 inhibitory activity to
determine their differences in KLK7 inhibitions (Table 4).
Compound (–)-22g showed more potent inhibitory activity
against both human and mouse KLK7 than (+)-22g. In our

Figure 3. (A) Complex structure of 22g around the binding site of mouse KLK7 (PDB ID: 5ZFI). The surface of the protease is represented by the gray mesh and
22g is shown in ball-and-stick form (C: pink, O: red, N: blue, Cl: dark green). The chloro substituent interacts with the subpocket at the S1 site composed by the
main chain of Ser195 and side chain of Val213. (B) Complex structure of human KLK7 with 1 (PDB ID: 5Y9L) around the binding site viewed from the same
perspective as (A).
mode in this paper. On the other hand, the benzyl group of 22g
moves to a shallower position in the S1 pocket of mouse KLK7
(this binding pose is defined as type A binding mode in this
paper) than in that of human KLK7. In mouse KLK7, one of the
chloro substituents of 22g is located at the subpocket composed
of the main chain of Ser195 and side chain of Val213, and makes
van der Waals contacts with the side chains of Thr190 and
Val213 (Fig. 3A). In addition, one of the meta site carbon atoms
of the benzyl group in 22g is positioned within van der Waals
distance to the side chain oxygen atom of Thr190, and the other
meta site carbon atom interacts with the side chain of Cys220 and
main chain oxygen atom of Thr217. The other chlorine atom is
positioned within van der Waals distances to the side chains of
Asn192 and Tyr218. These two residues also form van der Waals
contacts with the methoxy group of 1 in human KLK7; Tyr218 is
replaced by Phe218 in human KLK7.
Considering Figs. 3B and 4, one of the ortho chlorine atoms
on the benzyl group might induce steric hindrance in the case of
type B binding mode in human KLK7. Therefore, the position of
the benzyl group of 22g in human KLK7 might also change from
that of 1, and the ortho chlorine atom could make van der Waals
contacts with Val213. The residues that interact with the two
chlorine atoms in 22g, except for Thr190 and Tyr218, are
conserved between mouse and human KLK7. Furthermore, in
mouse KLK7, compound 22g interacts with the Cβ atom of
Thr190, which is equivalent to the interaction to the methyl
group of Ala190 in human KLK7, and also with the phenyl ring
of Tyr218, which corresponds to the phenyl ring of Phe218 in
human KLK7. To summarize, our results indicate that human
KLK7 interacts with 22g in the same manner as with mouse
KLK7. In fact, the inhibitory activities against mouse and human
KLK7 are similar (Table 3).
Figure 4. Superimposition of the crystal structure complexes of mouse KLK7
with 22g (PDB ID: 5ZFI) and of human KLK7 with 1 (PDB ID: 5Y9L). The
surface of KLK7 is represented as mesh (gray for mouse and pink for human)
and the inhibitors are shown in ball-and stick model (C: pink for 22g, and
green for 1, O: red, N: blue, Cl: dark green).
Both, 22g in mouse KLK7 and 1 in human KLK721 interact
with a common binding site; however, the positions of the benzyl
group located at the S1 site are different from each other (Figs. 3
and 4). The benzyl group of 1 inserts deeply into the S1 pocket of
human KLK7, and this binding pose is defined as type B binding
The above binding mode information enables the elucidation
of structure-activity relationships. In mouse KLK7, the benzyl
group of compound 8 and 22c would locate in the shallower

position in the S1 pocket similarly to 22g (type A binding mode),
and would not be able to form type B binding mode because of
the steric hindrance that arises from the Thr190 residue and water
molecule. The methoxy group of 8 might be positioned toward
Asn192 and Tyr218, because the methoxy group would be too
large to fit in the subpocket composed of Ser195 and Val213.
Therefore, it is speculated that there is no interaction with the
subpocket, which causes the inhibitory activity of 8 against
mouse KLK7 to decrease. Compound 22c, in which the hydrogen
atom at the ortho position on the benzyl group of 8 is substituted
with a chlorine atom, exhibited increased inhibitory potency
against mouse KLK7 because the chlorine atom could interact
with the subpocket. On the other hand, the potency of 22c against
human KLK7 was decreased compared with that of 8 because the
binding modes of 8 and 22c to human KLK7 would be different.
In human KLK7, the binding mode of 8 might be type B where
the methoxy group would interact with Asn192 and Phe218 as
observed for 1. However, the binding mode of 22c in human
KLK7 would be type A because the two large substituents at both
ortho sites prevent the benzyl group from penetrating into the S1
pocket deeply, allowing the chlorine atom to interact with the
conserved subpocket as described above. The binding mode of
22c would be the same in mouse and human, thereby 22c
exhibited similar inhibitory activity against mouse and human
KLK7. In type A binding mode, the methoxy group of 22c is too
large to interact with Asn192 and Phe218. Therefore, 22g, in
which the methoxy group of 22c is replaced by a chlorine atom,
showed ten times more potent inhibitory activity than 22c against
both of human and mouse KLK7. Similarly, other compounds
with functional groups at both ortho sites (22e, 22g, 22h, 22i,
22k and 22l) are predicted to exhibit the same binding mode in
mouse and human KLK7, and these compounds showed
equipotent inhibitory activity against both KLK7s (Table 3 and
Fig. 5). In contrast, compounds that displayed different human
and mouse KLK7 inhibitory activity (represented by triangles in
the Fig. 5) would present different binding modes in each KLK7.
These compounds can penetrate deeply into the S1 site of human
KLK7 (Type B binding mode) because the ortho-substituents of
the benzyl group are small (hydrogen or fluorine atom).
Exceptionally, compounds 22f, 22j, and 22b would be able to
present both, type A and B, binding modes in human KLK7
because they would not only be able to penetrate deeply into the
S1 site but also interact with the subpocket via the methyl or
chloro substituents on the benzyl group. Therefore, these
compounds exhibited more potent inhibitory activity against
human KLK7 than against mouse KLK7.
Figure 5. Comparison between human and mouse KLK7 inhibitory activities. R1 and R2 are shown in parentheses. R1 were reassigned by the substituent that
would be positioned in the subpocket in mouse KLK7. Circles represent type A binding mode and triangles indicate other binding mode in human KLK7.
Inhibitory activity is displayed as pIC50 value (−logIC50). In the case of >30 μM of IC50 value, pIC50 was calculated with IC50 as 30 μM.

scaled with HKL2000.29 The crystal structure was solved by the
molecular replacement method using the human KLK7 structure
(PDB ID: 5Y9L).21 A molecular-replacement calculation was
performed using AMoRe.30 Model fitting was carried out using
Coot31 and refined with Refmac-5.32 Secondary structure
assignments and 3D structural figures were prepared by Maestro
ver. 11.2.33
3. Conclusion
The structural difference between human and mouse KLK7
revealed the cause of the species differences of 1,3,6-
trisubstituted 1,4-diazepan-7-one derivatives. To overcome the
difference in inhibitory activity between human and mouse
KLK7, the substituents on the benzyl group of the 1,4-diazepane
scaffold were modified according to structure-based drug design
studies. The series of di-ortho substituted benzyl derivatives
eliminated the species differences, and ultimately, di-ortho
chlorosubstituted compound 22g was identified as a potent
human and mouse KLK7 inhibitor. Moreover, only one of the
optically active isomers of 22g, (–)-22g, exhibited highly potent
inhibitory activity against both KLK7s. The elucidation of the
crystal structure of mouse KLK7 complexed with 22g and human
KLK7 complexed with 1 reveals the differences in the binding
modes and justifies the structure-activity relationships found in
human and mouse KLK7, respectively. We found that the
binding position of the benzyl group in the S1 site was important
for overcoming the species differences and prepared a series of
di-ortho substituted benzyl compounds that would form the type
A binding mode in both KLK7s, caused by similar van der Waals
contacts between the substituents on the benzyl group and the
residues of both KLK7s. Furthermore, the interaction to the
subpocket in the S1 site was essential for the highly potent
inhibitory activity.
4.2. Chemistry
4.2.1. General
Proton nuclear magnetic resonance spectra (1H NMR) and was
recorded on Brucker ARX-400 or Brucker Avance III (400 MHz)
spectrometer in the indicated solvent. Chemical shifts (δ) are
reported in parts per million relative to the internal standard
tetramethylsilane. High-resolution mass spectra (HRMS) and fast
atom bombardment (FAB) mass spectra were recorded on JEOL
JMS-700 mass spectrometer. Electro-spray ionization (ESI) mass
spectra were recorded on Agilent G1956A or Agilent G6120B
MSD spectrometer system. Optical rotation were measured by
JASCO P-1020. Chemical reagents and solvents were purchased
from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical
Industries, Kanto Kagaku, Nacalai Tesque or other commercial
suppliers and were used without purification. Chromatographic
purification was performed using Merck Silica Gel 60 (230-400
mesh) or Purif-Pack® SI 30 μm, Purif-Pack® NH 60 μm supplied
by Shoko Scientific.
4. Experimental
4.2.2. (R)-2-(6-(2-Methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl)acetic acid (5)
4.1. Crystal structure determinations of mouse KLK7
Expression, refolding, and purification of mouse KLK7 were
performed as previously reported for human KLK7.21 Briefly,
cDNA encoding mouse KLK7 with DDDDK, enterokinase
cutting site at the N-terminal side, was inserted into the pQE80
vector (Qiagen). BL21 cells transformed by the plasmid were
grown in 2 x YT medium at 37 °C. When A660 in the medium
reached 0.9, isopropyl β-D-1-thiogalactopyranoside was added to
reach a final concentration of 1 mM. After culturing for 5 h, the
cells were harvested.
To a solution of 4 (1.0 g, 2.93 mmol) in MeOH (15 ml) and
AcOEt (15 ml), Et3N (0.50 ml, 3.6 mmol) and 5% palladium
carbon (0.15 g) were added. The mixture was stirred under H2
atmosphere at rt for 24 h. Then the catalyst was filtered off, and
the filtrate was concentrated in vacuo. The residue was diluted
with 0.1 M HCl aq., and the product was extracted with CHCl3.
The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo to give the title compound as a white solid
1
(335mg, 37%). H NMR (400MHz, CD3OD) δ 7.24 - 7.15 (m,
2H), 6.95 (dd, J = 0.6, 8.1 Hz, 1H), 6.87 (dt, J = 1.1, 7.4 Hz, 1H),
4.68 (d, J = 17.4 Hz, 1H), 4.35 (d, J = 17.6 Hz, 1H), 4.07 (d, J =
17.4 Hz, 1H), 3.85 (s, 3H), 3.74 (d, J = 17.6 Hz, 1H), 3.62 - 3.52
(m, 1H), 3.25 - 3.19 (m, 2H), 3.13 (dd, J = 5.0, 14.1 Hz, 1H),
2.67 (dd, J = 9.2, 14.0 Hz, 1H); MS (ESI): 307 (M+H)+.
KLK7 was purified using HisTrap HP column (GE healthcare)
with 8 M urea. Refolding was performed using the step-wise
dialysis method. The eluted KLK7 solution (0.4 mg/ml) in the
dialysis
membrane
(2000
MWCO,
Slide-a-Lyzer™,
ThermoFisher Scientific) was incubated in a 100-fold volume of
50 mM Tris/HCl (pH 8.0), 100 mM NaCl, and 10 mM
dithiothreitol overnight at 25 °C. It was dialyzed at 4 °C by
lowering the urea concentration from 4 M to 0 M in buffer
conditions (50 mM Tris/HCl (pH 8.0), 100 mM NaCl, 2 mM
CaCl2, 0.4 M L-Arg, 5 mM reduced glutathione, and 0.5 mM
oxidized glutathione).
4.2.3. (R)-2-(6-(2-Methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-yl)-
N-(3-(1-methyl-1H-pyrazol-4-yl)phenyl)acetamide (6)
To a solution of 5 (120 mg, 0.39 mmol) and 3-(1-methyl-1H-
pyrazol-4-yl)aniline (67 mg, 0.39 mmol) in CH2Cl2 (10 ml),
EDCI·HCl (113 mg, 0.59 mmol) was added. The mixture was
stirred at rt overnight and then poured into sat. NH4Cl aq. The
product was extracted with AcOEt, and the organic layer was
successively washed with water, brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (CHCl3/MeOH = 99/1 to
90/10) to give the title compound as a yellow solid (161 mg,
89%). 1H NMR (400MHz, CDCl3) δ 7.93 (br. s, 1H), 7.75 (d, J =
0.6 Hz, 1H), 7.68 (br. s, 1H), 7.63 (s, 1H), 7.33 - 7.20 (m, 4H),
7.19 - 7.15 (m, 1H), 6.92 - 6.85 (m, 2H), 5.70 (br. s, 1H), 4.61 (d,
J = 17.3 Hz, 1H), 4.38 (d, J = 15.2 Hz, 1H), 4.13 (d, J = 15.1 Hz,
1H), 3.95 (d, J = 17.3 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H), 3.58 -
3.46 (m, 1H), 3.36 - 3.22 (m, 3H), 2.75 (dd, J = 9.0, 13.9 Hz,
1H); MS (ESI): 462 (M+H)+.
After the cleavage of the His-tag of KLK7 by enterokinase of
weight ratio of 1/100 (Sigma), refolded KLK7 was purified using
a HiTrap heparin HP column (GE healthcare).
Crystallization of KLK7 (10 mg/ml) was performed by the
sitting-drop vapor diffusion method. Crystals of apo KLK7 were
obtained by the crystallization condition of 20% w/v PEG 8000,
23% w/v PEG 400, 0.1 M MgCl2, and 0.1 M Tris/HCl (pH 8.0).
Crystals of the complex with 22g were obtained by soaking into a
crystallization solution a saturated solution of compound for 1.5
h. Crystals were flash-frozen in a cold nitrogen stream around
100ꢀK, and X-ray diffraction data were collected using an R-
AXIS IV++imaging-plate area detector mounted on a Rigaku
MicroMax-007 rotating-anode source with CuKα radiation
(λꢀ=ꢀ1.5418ꢀÅ, 40ꢀkV, 20ꢀmA). All data were processed and
4.2.4. (R)-2-(6-(2-Methoxybenzyl)-7-oxo-3-thioxo-1,4-diazepan-
1-yl)-N-(3-(1-methyl-1H-pyrazol-4-yl)phenyl)acetamide (7)

To a solution of 6 (160 mg, 0.347 mmol) in THF (5.0 ml) was
added Lawesson’s reagent (67 mg, 0.166 mmol), and the mixture
was stirred at 50 °C for 1 h. Then, water was added to the
reaction mixture, and the product was extracted with AcOEt. The
organic layer was washed with brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
NH silica gel column chromatography (CHCl3/MeOH = 99/1 to
90/10) to give the title compound as a pale yellow solid (90 mg,
the resultant mixture were added 2,4,6-trimethoxybenzaldehyde
(52.7 g, 0.268 mol), NaBH(OAc)3 (113.3 g, 0.535 mol) at 0 °C.
After stirring at 40 °C for 1.5 h, the mixture was concentrated in
vacuo. The residue was diluted with CHCl3 and water, and to the
resulting mixture was added sat. NaHCO3 aq. slowly. The
product was extracted and the organic layer was washed with sat.
NaHCO3 aq. and brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt = 100/0 to 50/50) to
give the title compound as a pale yellow solid (50.4 g, 4steps
1
54%). H NMR (400MHz, CDCl3) δ 7.96 (br. s, 1H), 7.90 (br. s,
1H), 7.75 (d, J = 0.5 Hz, 1H), 7.68 (br. s, 1H), 7.63 (s, 1H), 7.34 -
7.20 (m, 4H), 7.18 - 7.14 (m, 1H), 6.92 - 6.86 (m, 2H), 4.88 (d, J
= 17.3 Hz, 1H), 4.56 (d, J = 17.1 Hz, 1H), 4.44 (d, J = 15.3 Hz,
1H), 4.13 (d, J = 15.4 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.62 -
3.51 (m, 1H), 3.50 - 3.31 (m, 2H), 3.27 (dd, J = 5.1, 13.7 Hz,
1H), 2.78 (dd, J = 9.0, 13.7 Hz, 1H); MS (ESI): 478 (M+H)+.
1
52%). H-NMR (400MHz, CDCl3) δ 9.96 (t, J = 5.3 Hz, 1H) ,
6.15 (d, J = 2.0 Hz, 1H), 6.13 (s, 2H), 5.34 - 5.37 (m, 1H), 4.19
(q, J = 7.1 Hz, 2H), 4.09 (d, J = 5.3 Hz, 2H), 3.82 (s, 3H), 3.79
(s, 6H), 3.79 (s, 2H), 3.42 (br. s, 2H), 1.27 (t, J = 7.1 Hz, 3H).
4.2.7. 6-Methylene-1-(2,4,6-trimethoxybenzyl)-1,4-diazepane-
4.2.5. (R)-2-(3-(2,2-Dimethylhydrazono)-6-(2-methoxybenzyl)-7-
oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (8)
2,5-dione (14)
To a solution of 13 (50.0 g, 0.136 mol) in MeOH (164 ml)
was added 4 M NaOH aq. (40.5 ml), and the mixture was stirred
at rt for 14 h. After cooling to 0 °C, the mixture was acidified
(pH = 4.9) with sat. KHSO4 aq. and concentrated in vacuo. The
residue was diluted with MeCN, and the solvent was
concentrated in vacuo, and the same procedure was repeated 4
times. Then, MeCN (1000 ml) was added to the residue, and to
the resultant mixture were added HOBt (18.4 g, 0.137 mol),
EDCI·HCl (26.1 g, 0.136 mol), and the mixture was stirred at rt
for 2 days. The mixture was concentrated in vacuo, and the
residue was diluted with CHCl3 and water. After slow addition of
sat. NaHCO3 aq., the product was extracted. The organic layer
was washed with brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (CHCl3/MeOH = 96/4 to 95/5) to give
the title compound as a pale yellow solid (28.0 g, 64%). 1H-NMR
(400MHz, DMSO-d6) δ 8.02 (t, J = 5.2 Hz, 1H), 6.20 (s, 2H),
5.84 (d, J = 2.4 Hz, 1H), 4.87 (m, 1H), 4.43 (s, 2H), 3.98 (s, 2H),
3.85 (d, J = 5.2 Hz, 2H), 3.78 (s, 3H), 3.72 (s, 6H); MS: 321
(M+H)+.
To a solution of 7 (45 mg, 0.094 mmol) and N,N-
dimethylhydrazine (0.012 ml, 0.16 mmol) in pyridine (2.0 ml)
was added AgOAc (58.2 mg, 0.35 mmol), and the reaction
mixture was stirred at rt for 2 h. Then, water was added to the
reaction mixture, and the product was extracted with AcOEt. The
organic layer was washed with brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
NH silica gel column chromatography (CHCl3/MeOH = 98/2 to
90/10) to give the title compound as a pale yellow solid (28.7 mg,
61%). [α]24 = −64° (c 0.38, CHCl3); 1H NMR (400MHz, CDCl3) δ
D
8.25 (br. s, 1H), 7.73 (d, J = 0.8 Hz, 1H), 7.67 (br. s, 1H), 7.60 (s,
1H), 7.29 - 7.15 (m, 5H), 6.91 - 6.85 (m, 2H), 6.27 (br. s, 1H),
4.55 (d, J = 16.1 Hz, 1H), 4.35 (d, J = 15.4 Hz, 1H), 4.18 (d, J =
15.4 Hz, 1H), 3.93 (s, 3H), 3.84 (s, 3H), 3.83 (d, J = 16.1 Hz,
1H), 3.53 - 3.36 (m, 2H), 3.28 - 3.16 (m, 2H), 2.78 (dd, J = 8.9,
13.8 Hz, 1H), 2.32 (s, 6H); MS (ESI): 504 (M+H)+; HRMS
+
(FAB) Calcd for C27H34N7O3 : 504.2718; Found: 504.2707.
4.2.6. Ethyl 2-(2-(((2,4,6-
trimethoxybenzyl)amino)methyl)acrylamido)acetate (13)
4.2.8. tert-Butyl 2-(6-methylene-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (15)
To a solution of ethyl 2-(bromomethyl)acrylate 9 (104.4 g,
0.541 mol) in acetonitrile (900 ml) were added di-t-butyl
iminodicarboxylate (122.4g 0.564 mol) and K2CO3 (156.9 g, 1.14
mol). The mixture was stirred at rt for 2 days and concentrated in
vacuo. The residue was diluted with AcOEt and water and the
product was extracted. The organic layer was washed with water,
dried over anhydrous Na2SO4 and concentrated in vacuo to give
the crude 10 (182.7 g). To a solution of crude 10 (182.0 g) in
MeOH (1000 ml) was added 2 M NaOH aq. (552 ml) slowly, and
the mixture was stirred at rt for 1.5 h. The mixture was
concentrated in vacuo, and the residue was diluted with CHCl3
and water. After slow addition of sat. KHSO4 aq., the product
was extracted with CHCl3. The organic layer was washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo to
give the crude 11 (171.8 g). To a solution of crude 11 (171.0 g)
in CH2Cl2 (1200 ml) were added glycine ethyl ester
hydrochloride (94.4 g, 0.676 mol), HOBt (94.4 g, 0.698 mol),
EDCI·HCl (131.9 g, 0.688 mol), and Et3N (94.2 ml, 0.676 mol),
the reaction was stirred at rt for 4 h. To the mixture were added
water and sat. NaHCO3 aq. and the product was extracted with
CHCl3. The organic layer was successively washed with water,
KHSO4 aq., NaHCO3 aq., and brine, dried over anhydrous
Na2SO4 and concentrated in vacuo to give the crude 12 (237.5 g).
To a solution of crude 12 (118.0 g) in EtOH (1015 ml) was added
methanesulfonic acid (60 ml, 0.924 mol), and the mixture was
stirred at 40 °C for 17 h. After cooling to 0 °C, to the mixture
were added EtOH (252 ml), and Et3N (129 ml, 0.926 mol). To
To a suspension of 14 (10 g, 31.2 mmol) and NaI (6.08 g, 40.6
mmol) in DMF (70 ml), t-butyl bromoacetate (6.00 ml, 40.6
mmol) and 60% NaH (1.50 g, 37.5 mmol) were added at 0 °C,
and the reaction mixture was stirred for 1 h at the same
temperature. After t-butyl bromoacetate (1.00 ml, 6.77 mmol)
and 60% NaH (0.25 g, 6.25 mmol) were added, the reaction
mixture was stirred at 0 °C for 1 h. Then, sat. NH4Cl aq. was
added to the reaction mixture, and the product was extracted with
AcOEt. The organic layer was successively washed with water,
brine, dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane/AcOEt = 50/50 to 20/80) to give the title compound as a
1
white solid (12.8 g, 94%). H NMR (400MHz, CDCl3) δ 6.10 -
6.07 (m, 1H), 6.08 (s, 2H), 5.01 - 4.98 (m, 1H), 4.65 (s, 2H), 4.27
(s, 2H), 4.19 (s, 2H), 4.01 (s, 2H), 3.81 (s, 3H), 3.76 (s, 6H), 1.47
(s, 9H); MS (ESI): 435 (M+H)+.
4.2.9. tert-Butyl 2-(3,7-dioxo-6-((4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)methyl)-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (16)
A mixture of CuCl (10.3 mg, 0.104 mmol), NaOt-Bu (30 mg,
0.311mmol) and 1,2-bis(diphenylphosphino)benzene (46 mg,
0.104 mmol) in THF (3 ml) was stirred at rt for 30 min. To the
resultant mixture, a solution of bis(pinacolato)diboron (964 mg,
3.80 mmol) in THF (6 ml) was added. After stirring for 15 min at
rt, compound 15 (1.50 g, 3.45 mmol) and MeOH (0.28 ml, 6.90

mmol) were added, and the mixture was stirred at rt for 18 h.
Then, the reaction mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt = 20/80 to 0/100) to
The reaction was carried out according to the procedure for
18a with 2-chloro-6-methoxyphenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a pale
1
yellow solid (134 mg, 98%). H NMR (400MHz, CDCl3) δ 7.10
1
give the title compound as a white solid (1.89 g, 97%). H NMR
(t, J = 8.2 Hz, 1H), 6.90 (dd, J = 1.0, 8.2 Hz, 1H), 6.68 (dd, J =
0.8, 8.2 Hz, 1H), 5.94 (s, 2H), 4.62 (d, J = 13.7 Hz, 1H), 4.43 (d,
J = 13.8 Hz, 1H), 4.24 (d, J = 17.3 Hz, 1H), 4.21 (s, 2H), 4.11 (d,
J = 17.3 Hz, 1H), 3.82 (s, 3H), 3.72 (s, 3H), 3.62 (s, 6H), 3.31
(dd, J = 12.2, 14.4 Hz, 1H), 3.16 (dd, J = 3.6, 13.4 Hz, 1H), 3.08
(tt, J = 3.8, 11.5 Hz, 1H), 2.93 (dd, J = 10.9, 13.4 Hz, 1H), 2.82
(dd, J = 4.0, 14.4 Hz, 1H), 1.48 (s, 9H); MS (ESI): 577 (M+H)+.
(400MHz, CDCl3) δ 6.10 (s, 2H), 4.68 (d, J = 13.7 Hz, 1H), 4.62
(d, J = 13.7 Hz, 1H), 4.44 (d, J = 16.9 Hz, 1H), 4.24 (d, J = 17.3
Hz, 1H), 3.96 (d, J = 16.8 Hz, 1H), 3.95 (d, J = 17.4 Hz, 1H),
3.81 (s, 3H), 3.77 (s, 6H), 3.25 - 3.05 (m, 3H), 1.44 (s, 9H), 1.19
(s, 6H), 1.17 (s, 6H), 1.04 - 0.94 (m, 1H), 0.82 - 0.74 (m, 1H);
MS (ESI): 563 (M+H)+.
4.2.10. Potassium ((4-(2-(tert-butoxy)-2-oxoethyl)-2,5-dioxo-1-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-6-
yl)methyl)trifluoroborate (17)
4.2.14. tert-Butyl 2-(6-(2-fluoro-6-methoxybenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18d)
The reaction was carried out according to the procedure for
18a with 2-fluoro-6-methoxybebzebeboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a white
To a solution of 16 (1.85 g, 3.29 mmol) in MeCN (30 ml) was
added a solution of KHF2 (0.771 g, 9.87 mmol) in H2O (10 ml) at
0 °C. After stirring at 0 °C for 2h, solvent was concentrated in
vacuo. The residue was diluted with acetone, the insolubles were
removed by filtration and filtrate was concentrated in vacuo. The
residue was triturated with diethylether, and the precipitate was
collected by filtration to give the title compound as a white solid
(1.74 g, 98%). 1H NMR (400MHz, DMSO-d6) δ 6.17 (s, 2H),
4.48 (d, J = 13.4 Hz, 1H), 4.34 (d, J = 13.4 Hz, 1H), 4.09 (d, J =
16.3 Hz, 1H), 4.02 (d, J = 16.9 Hz, 1H), 3.98 (d, J = 16.3 Hz,
1H), 3.88 (d, J = 16.9 Hz, 1H), 3.76 (s, 3H), 3.69 (s, 6H), 3.33 -
3.24 (m, 1H), 2.86 (dd, J = 11.9, 14.3 Hz, 1H), 2.63 - 2.53 (m,
1H), 1.39 (s, 9H), 0.41 - 0.28 (m, 1H), -0.04 - -0.18 (m, 1H); MS
(FAB): 503 (M-K)−.
1
solid (112 mg, 87%). H NMR (400MHz, CDCl3) δ 7.13 (dt, J =
6.7, 8.3 Hz, 1H), 6.63 - 6.55 (m, 2H), 5.97 (s, 2H), 4.57 (d, J =
13.7 Hz, 1H), 4.50 (d, J = 13.7 Hz, 1H), 4.26 (d, J = 16.6 Hz,
1H), 4.19 (d, J = 17.3 Hz, 1H), 4.14 (d, J = 16.7 Hz, 1H), 4.14 (d,
J = 17.4 Hz, 1H), 3.82 (s, 3H), 3.74 (s, 3H), 3.63 (s, 6H), 3.29 -
3.19 (m, 1H), 3.11 - 2.98 (m, 2H), 2.88 (dd, J = 3.7, 14.2 Hz,
1H), 2.71 (dd, J = 11.4, 13.9 Hz, 1H), 1.47 (s, 9H); MS (ESI):
561 (M+H)+.
4.2.15. tert-Butyl 2-(6-(2-methoxy-6-methylbenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18e)
A mixture of 17 (200 mg, 0.369 mmol), 2-bromo-1-methoxy-
3-methylbenzene (148 mg, 0.737 mmol), Pd(OAc)2 (8.3 mg,
0.037 mmol), 2-dicyclohexylphosphino-2',6'-di-iso-propoxy-1,1'-
biphenyl (34.4 mg, 0.074 mmol), and Cs2CO3 (360 mg, 1.11
mmol) in toluene (7.5 ml) and water (1.5 ml) was stirred at 80 °C
for 25 h. Then, water was added to the reaction mixture, and the
product was extracted with AcOEt. The organic layer was
successively washed with water and brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (hexane/AcOEt = 40/60 to
10/90) to give the title compound as a white solid (43 mg, 21%).
1H NMR (400MHz, CDCl3) δ 7.06 (t, J = 7.9 Hz, 1H), 6.70 (d, J
= 7.5 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.93 (s, 2H), 4.62 (d, J =
13.7 Hz, 1H), 4.39 (d, J = 13.7 Hz, 1H), 4.33 (d, J = 17.3 Hz,
1H), 4.30 (d, J = 16.6 Hz, 1H), 4.11 (d, J = 16.4 Hz, 1H), 4.05 (d,
J = 17.3 Hz, 1H), 3.82 (s, 3H), 3.72 (s, 3H), 3.58 (s, 6H), 3.36 -
3.25 (m, 1H), 3.03 (dd, J = 3.1, 13.4 Hz, 1H), 2.92 - 2.71 (m,
3H), 2.07 (s, 3H), 1.48 (s, 9H); MS (ESI): 557 (M+H)+.
4.2.11. tert-Butyl 2-(6-(2-cyanobenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18a)
To a solution of 15 (260 mg, 0.598 mmol) in dioxane/H2O
(10/1) (3 ml) were added 2-cyanophenylboronic acid (172 mg,
1.17 mmol) and hydroxy(1,5-cyclooctadiene)rhodium (I) dimer
(27.3 mg, 0.060 mmol), and the mixture was stirred at 80 °C for
2h. The reaction mixture was diluted with AcOEt, and
successively washed with sat. KHSO4 aq., sat. NaHCO3 aq., and
brine. The organic layer was dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (eluent A: hexane/AcOEt (1/1), eluent
B: MeOH; A/B = 95/5 to 90/10) to give the title compound as a
pale yellow solid (321 mg, quant.). 1H NMR (400MHz, CDCl3) δ
7.55 (dd, J = 1.1, 7.8 Hz, 1H), 7.45 (dt, J = 1.4, 7.7 Hz, 1H), 7.29
(dt, J = 1.1, 7.5 Hz, 1H), 7.22 - 7.18 (m, 1H), 6.00 (s, 2H), 4.72
(d, J = 13.7 Hz, 1H), 4.44 (d, J = 13.7 Hz, 1H), 4.28 (d, J = 17.3
Hz, 1H), 4.20 (d, J = 16.6 Hz, 1H), 4.14 (d, J = 16.6 Hz, 1H),
4.04 (d, J = 17.3 Hz, 1H), 3.83 (s, 3H), 3.69 (s, 6H), 3.40 - 3.27
(m, 2H), 3.09 - 2.92 (m, 2H), 2.86 (dd, J = 8.4, 14.2 Hz, 1H),
1.46 (s, 9H); MS (ESI): 538 (M+H)+.
4.2.16. tert-Butyl 2-(6-(2-chloro-6-fluorobenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18f)
The reaction was carried out according to the procedure for
18a with 2-chloro-6-fluorobenzeneboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a white
solid (47 mg, 36%). 1H NMR (400MHz, CDCl3) δ 7.17 - 7.08 (m,
2H), 6.92 - 6.86 (m, 1H), 5.95 (s, 2H), 4.66 (d, J = 13.7 Hz, 1H),
4.45 (d, J = 13.7 Hz, 1H), 4.25 (d, J = 16.7 Hz, 1H), 4.24 (d, J =
17.3 Hz, 1H), 4.18 (d, J = 16.8 Hz, 1H), 4.09 (d, J = 17.3 Hz,
1H), 3.82 (s, 3H), 3.65 (s, 6H), 3.33 (dd, J = 12.5, 14.0 Hz, 1H),
3.26 - 3.18 (m, 1H), 3.09 - 2.99 (m, 1H), 2.93 - 2.79 (m, 2H),
1.47 (s, 9H); MS (ESI): 565 (M+H)+.
4.2.12. tert-Butyl 2-(6-(2-chlorobenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18b)
The reaction was carried out according to the procedure for
18a with 2-chlorophenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a pale
yellow solid (114 mg, 91%). 1H NMR (400MHz, CDCl3) δ 7.30 -
7.27 (m, 1H), 7.18 - 7.09 (m, 2H), 7.06 - 7.02 (m, 1H), 5.99 (s,
2H), 4.65 (d, J = 13.7 Hz, 1H), 4.47 (d, J = 13.8 Hz, 1H), 4.25 (d,
J = 17.3 Hz, 1H), 4.21 (d, J = 16.7 Hz, 1H), 4.16 (d, J = 16.7 Hz,
1H), 4.07 (d, J = 17.3 Hz, 1H), 3.83 (s, 3H), 3.66 (s, 6H), 3.33 -
3.21 (m, 2H), 3.09 - 2.97 (m, 2H), 2.69 (dd, J = 9.3, 14.1 Hz,
1H), 1.46 (s, 9H); MS (ESI): 547 (M+H)+.
4.2.17. tert-Butyl 2-(6-(2,6-dichlorobenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18g)
The reaction was carried out according to the procedure for
18a with 2,6-dichlorophenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a white
4.2.13. tert-Butyl 2-(6-(2-chloro-6-methoxybenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18c)
1
solid (116 mg, 87%). H NMR (400MHz, CDCl3) δ 7.21 (d, J =

8.7 Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H), 7.08 (dd, J = 7.4, 8.7 Hz,
1H), 5.93 (s, 2H), 4.70 (d, J = 13.7 Hz, 1H), 4.41 (d, J = 13.8 Hz,
1H), 4.26 (d, J = 17.3 Hz, 1H), 4.23 (s, 2H), 4.09 (d, J = 17.3 Hz,
1H), 3.82 (s, 3H), 3.65 (s, 6H), 3.41 (dd, J = 11.9, 14.4 Hz, 1H),
3.33 (dd, J = 3.5, 13.7 Hz, 1H), 3.19 - 3.10 (m, 1H), 3.04 (dd, J =
11.0, 13.7 Hz, 1H), 2.88 (dd, J = 3.7, 14.4 Hz, 1H), 1.48 (s, 9H);
MS (ESI): 581 (M+H)+.
methoxy-3-methylbenzene to give the title compound as a pale
yellow solid (25.2 mg, 12%). H NMR (400MHz, CDCl3) δ 7.41
1
(dd, J = 1.0, 7.7 Hz, 1H), 7.30 (br. d, J = 7.7 Hz, 1H), 7.21 (t, J =
7.7 Hz, 1H), 5.88 (s, 2H), 4.81 (d, J = 13.7 Hz, 1H), 4.46 (d, J =
16.2 Hz, 1H), 4.41 (d, J = 17.3 Hz, 1H), 4.27 (d, J = 13.7 Hz,
1H), 3.99 (d, J = 16.1 Hz, 1H), 3.96 (d, J = 17.2 Hz, 1H), 3.81 (s,
3H), 3.60 (s, 6H), 3.58 (dd, J = 12.2, 14.7 Hz, 1H), 3.39 (dd, J =
3.8, 14.3 Hz, 1H), 2.94 - 2.81 (m, 2H), 2.65 (tt, J = 4.1, 11.5 Hz,
1H), 2.17 (s, 3H), 1.48 (s, 9H); MS (ESI): 552 (M+H)+.
4.2.18. tert-Butyl 2-(6-(2-chloro-6-methylbenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18h)
4.2.23. tert-Butyl 2-(6-(2,6-difluorobenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18m)
The reaction was carried out according to the procedure for
18e with 3-chloro-2-iodotoluene instead of 2-bromo-1-methoxy-
3-methylbenzene to give the title compound as a white solid (28
The reaction was carried out according to the procedure for
18a with 2,6-difluorophenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a white
solid (50 mg, 20%). 1H NMR (400MHz, CDCl3) δ 7.20 - 7.11 (m,
1H), 6.83 - 6.75 (m, 2H), 5.98 (s, 2H), 4.62 (d, J = 13.7 Hz, 1H),
4.51 (d, J = 13.6 Hz, 1H), 4.28 (d, J = 16.8 Hz, 1H), 4.19 (d, J =
17.3 Hz, 1H), 4.12 (d, J = 17.3 Hz, 1H), 4.12 (d, J = 16.8 Hz,
1H), 3.82 (s, 3H), 3.66 (s, 6H), 3.31 - 3.22 (m, 1H), 3.17 - 3.09
(m, 1H), 3.07 - 2.97 (m, 1H), 2.97 - 2.90 (m, 1H), 2.69 (dd, J =
10.7, 14.2 Hz, 1H), 1.47 (s, 9H); MS (ESI): 549 (M+H)+.
1
mg, 14%). H NMR (400MHz, CDCl3) δ 7.15 (dd, J = 0.9, 7.9
Hz, 1H), 7.03 (t, J = 7.8 Hz, 1H), 6.99 - 6.96 (m, 1H), 5.93 (s,
2H), 4.70 (d, J = 13.7 Hz, 1H), 4.36 (d, J = 13.8 Hz, 1H), 4.34 (d,
J = 17.3 Hz, 1H), 4.32 (d, J = 16.4 Hz, 1H), 4.12 (d, J = 16.4 Hz,
1H), 4.03 (d, J = 17.2 Hz, 1H), 3.82 (s, 3H), 3.61 (s, 6H), 3.48 -
3.37 (m, 1H), 3.26 - 3.15 (m, 1H), 2.95 - 2.83 (m, 3H), 2.13 (s,
3H), 1.48 (s, 9H); MS (ESI): 561 (M+H)+.
4.2.19. tert-Butyl 2-(6-(2-chloro-6-cyanobenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18i)
4.2.24. tert-Butyl 2-(6-(2-fluoro-6-(trifluoromethyl)benzyl)-3,7-
dioxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18n)
The reaction was carried out according to the procedure for
18e with 2-bromo-3-chlorobenzonitrile instead of 2-bromo-1-
methoxy-3-methylbenzene to give the title compound as a pale
The reaction was carried out according to the procedure for
18a with 2-fluoro-6-(trifluoromethyl)phenylboronic acid instead
of 2-cyanophenylboronic acid to give the title compound as a
white solid (35 mg, 21%). 1H NMR (400MHz, CDCl3) δ 7.40 (br.
d, J = 7.7 Hz, 1H), 7.35 - 7.28 (m, 1H), 7.14 (br. t, J = 8.8 Hz,
1H), 5.93 (s, 2H), 4.67 (d, J = 13.8 Hz, 1H), 4.42 (d, J = 13.7 Hz,
1H), 4.29 (d, J = 16.8 Hz, 1H), 4.19 (d, J = 17.3 Hz, 1H), 4.14 (d,
J = 16.9 Hz, 1H), 4.11 (d, J = 17.3 Hz, 1H), 3.81 (s, 3H), 3.63 (s,
6H), 3.33 - 3.23 (m, 2H), 3.12 (tt, J = 3.7, 11.3 Hz, 1H), 2.91 -
2.81 (m, 2H), 1.48 (s, 9H); MS (ESI): 599 (M+H)+.
1
yellow solid (16.3 mg, 8%). H NMR (400MHz, CDCl3) δ 7.51
(dd, J = 1.3, 8.1 Hz, 1H), 7.46 (dd, J = 1.3, 7.7 Hz, 1H), 7.29 -
7.23 (m, 1H), 5.93 (s, 2H), 4.78 (d, J = 13.7 Hz, 1H), 4.38 (d, J =
13.7 Hz, 1H), 4.26 (d, J = 16.6 Hz, 1H), 4.26 (d, J = 17.3 Hz,
1H), 4.19 (d, J = 16.6 Hz, 1H), 4.07 (d, J = 17.3 Hz, 1H), 3.83 (s,
3H), 3.67 (s, 6H), 3.54 - 3.43 (m, 2H), 3.13 - 3.04 (m, 1H), 2.99 -
2.88 (m, 2H), 1.47 (s, 9H); MS (ESI): 572 (M+H)+.
4.2.20. tert-Butyl 2-(6-(2-fluoro-6-methylbenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18j)
4.2.25. tert-Butyl 2-(6-(2-chlorobenzyl)-7-oxo-3-thioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19b)
The reaction was carried out according to the procedure for
18e with 2-bromo-1-fluoro-3-methylbenzene instead of 2-bromo-
1-methoxy-3-methylbenzene to give the title compound as a
white solid (37 mg, 18%). 1H NMR (400MHz, CDCl3) δ 7.07 (dt,
J = 5.8, 7.9 Hz, 1H), 6.87 (br. d, J = 7.5 Hz, 1H), 6.81 (br. t, J =
9.2 Hz, 1H), 5.93 (s, 2H), 4.69 (d, J = 13.6 Hz, 1H), 4.37 (d, J =
13.6 Hz, 1H), 4.35 (d, J = 17.3 Hz, 1H), 4.34 (d, J = 16.4 Hz,
1H), 4.08 (d, J = 16.3 Hz, 1H), 4.02 (d, J = 17.3 Hz, 1H), 3.81 (s,
3H), 3.60 (s, 6H), 3.41 - 3.32 (m, 1H), 3.14 - 3.03 (m, 1H), 2.89
(dd, J = 3.3, 14.5 Hz, 1H), 2.77 - 2.66 (m, 2H), 2.12 (s, 3H), 1.47
(s, 9H); MS (ESI): 545 (M+H)+.
To a solution of 18b (106 mg, 0.194 mmol) in THF (2 ml) was
added Belleau’s reagent (56.3 mg, 0.107 mmol), and the mixture
was stirred at rt for 2 h. Then, the solvent was concentrated in
vacuo, and the residue was purified by silica gel column
chromatography (hexane/AcOEt = 80/20 to 60/40) to give the
1
title compound as white solid (96 mg, 88%). H NMR (400MHz,
CDCl3) δ 7.28 - 7.23 (m, 1H), 7.17 - 7.08 (m, 2H), 6.96 (dd, J =
2.0, 7.3 Hz, 1H), 5.96 (s, 2H), 5.34 (d, J = 14.2 Hz, 1H), 4.90 (d,
J = 16.1 Hz, 1H), 4.67 (d, J = 14.2 Hz, 1H), 4.66 (d, J = 17.3 Hz,
1H), 4.40 (d, J = 16.1 Hz, 1H), 3.90 (d, J = 17.3 Hz, 1H), 3.84 (s,
3H), 3.63 (s, 6H), 3.66 - 3.53 (m, 1H), 3.33 - 3.24 (m, 1H), 3.14
(dd, J = 4.9, 15.1 Hz, 1H), 2.78 - 2.65 (m, 2H), 1.48 (s, 9H); MS
(ESI): 563 (M+H)+.
4.2.21. tert-Butyl 2-(6-(2,6-dimethylbenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18k)
The reaction was carried out according to the procedure for
18a with 2,6-dimethylphenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound as a white
4.2.26. tert-Butyl 2-(6-(2-methoxy-6-methylbenzyl)-7-oxo-3-
thioxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate
(19e)
1
solid (111 mg, 89%). H NMR (400MHz, CDCl3) δ 7.00 (dd, J =
6.5, 8.3 Hz, 1H), 6.95 - 6.91 (m, 2H), 5.93 (s, 2H), 4.67 (d, J =
13.7 Hz, 1H), 4.36 (d, J = 17.3 Hz, 1H), 4.35 (d, J = 13.7 Hz,
1H), 4.34 (d, J = 16.3 Hz, 1H), 4.09 (d, J = 16.6 Hz, 1H), 4.04 (d,
J = 17.3 Hz, 1H), 3.83 (s, 3H), 3.58 (s, 6H), 3.35 (dd, J = 12.0,
14.1 Hz, 1H), 3.15 (dd, J = 2.6, 13.9 Hz, 1H), 2.87 (dd, J = 3.8,
14.4 Hz, 1H), 2.80 - 2.71 (m, 1H), 2.67 (dd, J = 11.5, 13.8 Hz,
1H), 2.13 (s, 6H), 1.48 (s, 9H); MS (ESI): 541 (M+H)+.
The reaction was carried out according to the procedure for
19b with 18e instead of 18b to give the title compound as a white
solid (38 mg, 90%). 1H NMR (400MHz, CDCl3) δ 7.06 (t, J = 7.9
Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 5.88
(s, 2H), 5.37 (d, J = 14.1 Hz, 1H), 5.02 (d, J = 15.9 Hz, 1H), 4.72
(d, J = 17.3 Hz, 1H), 4.57 (d, J = 14.1 Hz, 1H), 4.39 (d, J = 15.9
Hz, 1H), 3.87 (d, J = 17.3 Hz, 1H), 3.82 (s, 3H), 3.73 - 3.64 (m,
1H), 3.70 (s, 3H), 3.55 (s, 6H), 3.06 - 2.94 (m, 2H), 2.85 (dd, J =
11.5, 13.7 Hz, 1H), 2.51 - 2.40 (m, 1H), 2.05 (s, 3H), 1.48 (s,
9H); MS (ESI): 573 (M+H)+.
4.2.22. tert-Butyl 2-(6-(2-cyano-6-methylbenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18l)
The reaction was carried out according to the procedure for
18e with 2-iodo-3-methylbenzonitrile instead of 2-bromo-1-

4.2.27. tert-Butyl 2-(6-(2-chloro-6-fluorobenzyl)-7-oxo-3-thioxo-
4.2.32. tert-Butyl 2-(6-(2,6-difluorobenzyl)-7-oxo-3-thioxo-4-
4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19f)
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19m)
The reaction was carried out according to the procedure for
19b with 18f instead of 18b to give the title compound as a white
solid (33 mg, 80%). 1H NMR (400MHz, CDCl3) δ 7.16 - 7.06 (m,
2H), 6.87 (ddd, J = 1.5, 7.9, 9.2 Hz, 1H), 5.90 (s, 2H), 5.33 (d, J
= 14.2 Hz, 1H), 4.98 (d, J = 16.2 Hz, 1H), 4.65 (d, J = 14.2 Hz,
1H), 4.64 (d, J = 17.3 Hz, 1H), 4.45 (d, J = 16.2 Hz, 1H), 3.90 (d,
J = 17.4 Hz, 1H), 3.83 (s, 3H), 3.74 - 3.65 (m, 1H), 3.63 (s, 6H),
3.32 (td, J = 3.2, 13.4 Hz, 1H), 3.05 (dd, J = 4.9, 15.2 Hz, 1H),
2.84 - 2.76 (m, 1H), 2.76 - 2.66 (m, 1H), 1.48 (s, 9H); MS (ESI):
581 (M+H)+.
The reaction was carried out according to the procedure for
19b with 18m instead of 18b to give the title compound as a
1
white solid (45 mg, 87%). H NMR (400MHz, CDCl3) δ 7.20 -
7.11 (m, 1H), 6.81 - 6.73 (m, 2H), 5.93 (s, 2H), 5.29 (d, J = 14.2
Hz, 1H), 4.93 (d, J = 16.1 Hz, 1H), 4.72 (d, J = 14.2 Hz, 1H),
4.60 (d, J = 17.4 Hz, 1H), 4.46 (d, J = 16.2 Hz, 1H), 3.93 (d, J =
17.3 Hz, 1H), 3.83 (s, 3H), 3.67 - 3.56 (m, 1H), 3.63 (s, 6H), 3.24
- 3.19 (m, 1H), 3.08 (dd, J = 4.3, 14.8 Hz, 1H), 2.71 - 2.62 (m,
2H), 1.48 (s, 9H); MS (ESI): 565 (M+H)+.
4.2.33. tert-Butyl 2-(6-(2-fluoro-6-(trifluoromethyl)benzyl)-7-
oxo-3-thioxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-
yl)acetate (19n)
4.2.28. tert-Butyl 2-(6-(2,6-dichlorobenzyl)-7-oxo-3-thioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19g)
The reaction was carried out according to the procedure for
The reaction was carried out according to the procedure for
19b with 18n instead of 18b to give the title compound as a
white solid (31 mg, 94%). 1H NMR (400MHz, CDCl3) δ 7.39 (br.
d, J = 7.8 Hz, 1H), 7.35 - 7.28 (m, 1H), 7.13 (br. t, J = 8.7 Hz,
1H), 5.89 (s, 2H), 5.34 (d, J = 14.2 Hz, 1H), 4.96 (d, J = 16.3 Hz,
1H), 4.65 (d, J = 14.3 Hz, 1H), 4.60 (d, J = 17.3 Hz, 1H), 4.48 (d,
J = 16.3 Hz, 1H), 3.92 (d, J = 17.4 Hz, 1H), 3.82 (s, 3H), 3.69 -
3.58 (m, 1H), 3.61 (s, 6H), 3.45 - 3.34 (m, 1H), 3.06 (dd, J = 3.8,
15.4 Hz, 1H), 2.84 - 2.72 (m, 2H), 1.48 (s, 9H); MS (ESI): 615
(M+H)+.
19b with 18g instead of 18b to give the title compound as a white
1
solid (110 mg, 97%). H NMR (400MHz, CDCl3) δ 7.19 (d, J =
8.8 Hz, 1H), 7.19 (d, J = 7.3 Hz, 1H), 7.08 (dd, J = 7.3, 8.8 Hz,
1H), 5.89 (s, 2H), 5.36 (d, J = 14.3 Hz, 1H), 5.02 (d, J = 16.2 Hz,
1H), 4.65 (d, J = 17.3 Hz, 1H), 4.60 (d, J = 14.2 Hz, 1H), 4.46 (d,
J = 16.1 Hz, 1H), 3.89 (d, J = 17.3 Hz, 1H), 3.85 - 3.76 (m, 1H),
3.83 (s, 3H), 3.63 (s, 6H), 3.45 (dd, J = 4.1, 13.9 Hz, 1H), 3.06
(dd, J = 5.0, 15.1 Hz, 1H), 2.99 (dd, J = 11.2, 13.9 Hz, 1H), 2.87
- 2.77 (m, 1H), 1.48 (s, 9H); MS (ESI): 597 (M+H)+.
4.2.29. tert-Butyl 2-(6-(2-chloro-6-methylbenzyl)-7-oxo-3-thioxo-
4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19h)
4.2.34. tert-Butyl 2-(6-(2-cyanobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20a)
The reaction was carried out according to the procedure for
19b with 18h instead of 18b to give the title compound as a
white solid (24 mg, 86%). H NMR (400MHz, CDCl3) δ 7.14 -
To a solution of 18a (325 mg, 0.605 mmol) in THF (3 ml) was
added Belleau’s reagent (320 mg, 0.605 mmol), and the mixture
was stirred at rt for 1 h. Then, the reaction mixture was diluted
with AcOEt, and was successively washed with sat. NaHCO3 aq.,
water, and brine. The organic layer was dried over anhydrous
Na2SO4, and concentrated in vacuo to give crude 19a as a pale
yellow foam (409 mg). To a solution of crude 19a (409 mg) in
Pyridine (5 ml) were added N,N-dimethylhydrazine (115 μl, 1.49
mmol) and AgOAc (340 mg, 2.04 mmol) at 0 °C, and the mixture
was stirred for 30 min. The reaction mixture was diluted with
AcOEt and filtered, then filtrate was concentrated in vacuo. The
residue was diluted with AcOEt and insolubles were filtered off.
The filtrate was concentrated, and the residue was purified by NH
silica gel column chromatography (hexane/AcOEt = 70/30 to
35/65) to give the title compound as a pale yellow solid (299 mg,
2 steps 85%). 1H NMR (400MHz, CDCl3) δ 7.53 (dd, J = 1.1, 7.7
Hz, 1H), 7.42 (dt, J = 1.4, 7.7 Hz, 1H), 7.29 - 7.23 (m, 1H), 7.16
(br. d, J = 7.5 Hz, 1H), 5.99 (s, 2H), 4.78 (d, J = 15.7 Hz, 1H),
4.64 (d, J = 13.3 Hz, 1H), 4.48 (d, J = 15.7 Hz, 1H), 4.29 (d, J =
17.1 Hz, 1H), 4.28 (d, J = 13.4 Hz, 1H), 3.92 (d, J = 17.1 Hz,
1H), 3.82 (s, 3H), 3.65 (s, 6H), 3.29 - 3.18 (m, 2H), 2.95 (dd, J =
6.0, 14.7 Hz, 1H), 2.86 (dd, J = 8.3, 14.2 Hz, 1H), 2.75 - 2.65 (m,
1H), 2.41 (s, 6H), 1.48 (s, 9H); MS (ESI): 580 (M+H)+.
1
7.10 (m, 1H), 7.03 (t, J = 7.7 Hz, 1H), 6.99 - 6.95 (m, 1H), 5.88
(s, 2H), 5.42 (d, J = 14.2 Hz, 1H), 5.04 (d, J = 16.1 Hz, 1H), 4.71
(d, J = 17.4 Hz, 1H), 4.55 (d, J = 14.2 Hz, 1H), 4.40 (d, J = 16.1
Hz, 1H), 3.86 (d, J = 17.3 Hz, 1H), 3.86 - 3.77 (m, 1H), 3.82 (s,
3H), 3.59 (s, 6H), 3.25 (dd, J = 3.9, 14.2 Hz, 1H), 3.09 (dd, J =
5.3, 15.1 Hz, 1H), 2.91 (dd, J = 11.3, 14.2 Hz, 1H), 2.58 - 2.48
(m, 1H), 2.12 (s, 3H), 1.48 (s, 9H); MS (ESI): 577 (M+H)+.
4.2.30. tert-Butyl 2-(6-(2-fluoro-6-methylbenzyl)-7-oxo-3-thioxo-
4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19j)
The reaction was carried out according to the procedure for
19b with 18j instead of 18b to give the title compound as a white
1
solid (29 mg, 78%). H NMR (400MHz, CDCl3) δ 7.07 (dt, J =
5.9, 7.8 Hz, 1H), 6.86 (br. d, J = 7.4 Hz, 1H), 6.79 (br. t, J = 8.9
Hz, 1H), 5.87 (s, 2H), 5.42 (d, J = 14.1 Hz, 1H), 5.02 (d, J = 15.9
Hz, 1H), 4.72 (d, J = 17.3 Hz, 1H), 4.56 (d, J = 14.1 Hz, 1H),
4.37 (d, J = 16.1 Hz, 1H), 3.86 (d, J = 17.3 Hz, 1H), 3.81 (s, 3H),
3.77 - 3.67 (m, 1H), 3.57 (s, 6H), 3.15 - 3.01 (m, 2H), 2.79 - 2.70
(m, 1H), 2.42 - 2.32 (m, 1H), 2.09 (s, 3H), 1.48 (s, 9H); MS
(ESI): 561 (M+H)+.
4.2.31. tert-Butyl 2-(6-(2,6-dimethylbenzyl)-7-oxo-3-thioxo-4-
4.2.35. tert-Butyl 2-(6-(2-chlorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20b)
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (19k)
The reaction was carried out according to the procedure for
19b with 18k instead of 18b, and THF and CH2Cl2 was used as a
solvent to give the title compound as a white solid (105 mg,
To a solution of 19b (92 mg, 0.163 mmol) in pyridine (2 ml)
were added AgOAc (82 mg, 0.49 mmol) and N,N-
dimethylhydrazine (25 μl, 0.33 mmol) at 0 °C, and the mixture
was stirred for 3h. The reaction mixture was diluted with AcOEt
and insolubles were filtered off. Then, filtrate was concentrated
in vacuo, and the residue was purified by NH silica gel column
chromatography (hexane/AcOEt = 85/15 to 60/40) to give the
title compound as a white solid (83 mg, 86%). 1H NMR
(400MHz, CDCl3) δ 7.29 - 7.23 (m, 1H), 7.14 - 7.05 (m, 2H),
1
92%). H NMR (400MHz, CDCl3) δ 7.02 - 6.97 (m, 1H), 6.93 -
6.89 (m, 2H), 5.88 (s, 2H), 5.42 (d, J = 14.1 Hz, 1H), 5.02 (d, J =
16.2 Hz, 1H), 4.73 (d, J = 17.4 Hz, 1H), 4.53 (d, J = 14.1 Hz,
1H), 4.40 (d, J = 16.1 Hz, 1H), 3.87 (d, J = 17.3 Hz, 1H), 3.83 (s,
3H), 3.73 - 3.64 (m, 1H), 3.56 (s, 6H), 3.20 (dd, J = 3.5, 14.2 Hz,
1H), 3.04 (dd, J = 5.4, 14.9 Hz, 1H), 2.67 (dd, J = 11.4, 14.3 Hz,
1H), 2.52 - 2.41 (m, 1H), 2.11 (s, 6H), 1.48 (s, 9H); MS (ESI):
557 (M+H)+.

7.02 - 6.96 (m, 1H), 5.98 (s, 2H), 4.75 (d, J = 15.8 Hz, 1H), 4.60
(d, J = 13.3 Hz, 1H), 4.55 (d, J = 15.8 Hz, 1H), 4.28 (d, J = 13.3
Hz, 1H), 4.26 (d, J = 17.1 Hz, 1H), 3.95 (d, J = 17.1 Hz, 1H),
3.82 (s, 3H), 3.62 (s, 6H), 3.24 - 3.15 (m, 2H), 2.89 (dd, J = 5.8,
14.6 Hz, 1H), 2.79 - 2.64 (m, 2H), 2.41 (s, 6H), 1.48 (s, 9H); MS
(ESI): 589 (M+H)+.
The reaction was carried out according to the procedure for
20b with 19g instead of 19b to give the title compound as a pale
1
yellow solid (92 mg, 88%). H NMR (400MHz, CDCl3) δ 7.18
(d, J = 8.5 Hz, 1H), 7.18 (d, J = 7.4 Hz, 1H), 7.05 (dd, J = 7.4,
8.5 Hz, 1H), 5.91 (s, 2H), 4.83 (d, J = 15.7 Hz, 1H), 4.68 (d, J =
13.3 Hz, 1H), 4.61 (d, J = 15.7 Hz, 1H), 4.32 (d, J = 17.1 Hz,
1H), 4.20 (d, J = 13.2 Hz, 1H), 3.93 (d, J = 17.1 Hz, 1H), 3.81 (s,
3H), 3.62 (s, 6H), 3.43 (dd, J = 13.1, 15.9 Hz, 1H), 3.35 (dd, J =
4.4, 13.8 Hz, 1H), 2.98 (dd, J = 10.2, 13.8 Hz, 1H), 2.85 - 2.75
(m, 2H), 2.43 (s, 6H), 1.49 (s, 9H); MS (ESI): 623 (M+H)+.
4.2.36. tert-Butyl 2-(6-(2-chloro-6-methoxybenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20c)
The reaction was carried out according to the procedure for
4.2.41. tert-Butyl 2-(6-(2-chloro-6-methylbenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20h)
20a with 18c instead of 18a to give the title compound as a pale
1
yellow solid (83.2 mg, 2 steps 62%). H NMR (400MHz, CDCl3)
δ 7.06 (t, J = 8.2 Hz, 1H), 6.88 (dd, J = 1.0, 8.2 Hz, 1H), 6.65
(dd, J = 0.7, 8.2 Hz, 1H), 5.91 (s, 2H), 4.85 (d, J = 15.7 Hz, 1H),
4.65 (d, J = 13.3 Hz, 1H), 4.55 (d, J = 15.7 Hz, 1H), 4.36 (d, J =
17.1 Hz, 1H), 4.18 (d, J = 13.2 Hz, 1H), 3.89 (d, J = 17.1 Hz,
1H), 3.80 (s, 3H), 3.70 (s, 3H), 3.58 (s, 6H), 3.40 - 3.29 (m, 1H),
3.22 - 3.13 (m, 1H), 2.89 (dd, J = 10.7, 13.4 Hz, 1H), 2.76 - 2.64
(m, 2H), 2.41 (s, 6H), 1.49 (s, 9H); MS (ESI): 619 (M+H)+.
The reaction was carried out according to the procedure for
20b with 19h instead of 19b to give the title compound as a
1
white solid (17 mg, 71%). H NMR (400MHz, CDCl3) δ 7.12
(dd, J = 1.0, 7.8 Hz, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.97 - 6.93 (m,
1H), 5.89 (s, 2H), 4.92 (d, J = 15.7 Hz, 1H), 4.73 (d, J = 13.3 Hz,
1H), 4.52 (d, J = 15.6 Hz, 1H), 4.41 (d, J = 17.1 Hz, 1H), 4.16 (d,
J = 13.3 Hz, 1H), 3.84 (d, J = 17.1 Hz, 1H), 3.80 (s, 3H), 3.56 (s,
6H), 3.45 (dd, J = 12.0, 14.9 Hz, 1H), 3.17 (dd, J = 4.1, 14.1 Hz,
1H), 2.87 (dd, J = 10.7, 14.1 Hz, 1H), 2.80 (dd, J = 5.6, 14.9 Hz,
1H), 2.59 - 2.48 (m, 1H), 2.42 (s, 6H), 2.13 (s, 3H), 1.49 (s, 9H);
MS (ESI): 603 (M+H)+.
4.2.37. tert-Butyl 2-(3-(2,2-dimethylhydrazono)-6-(2-fluoro-6-
methoxybenzyl)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-
1-yl)acetate (20d)
The reaction was carried out according to the procedure for
20a with 18d instead of 18a to give the title compound as a pale
yellow solid (73.3 mg, 2 steps 63%). 1H NMR (400MHz, CDCl3)
δ 7.09 (dt, J=6.7, 8.3 Hz, 1H), 6.61 - 6.51 (m, 2H), 5.94 (s, 2H),
4.77 (d, J = 15.8 Hz, 1H), 4.59 (d, J = 13.2 Hz, 1H), 4.58 (d, J =
15.8 Hz, 1H), 4.27 (d, J = 17.1 Hz, 1H), 4.24 (d, J = 13.3 Hz,
1H), 3.95 (d, J = 17.1 Hz, 1H), 3.80 (s, 3H), 3.71 (s, 3H), 3.59 (s,
6H), 3.28 - 3.18 (m, 1H), 3.10 - 2.99 (m, 1H), 2.80 - 2.62 (m,
3H), 2.41 (s, 6H), 1.49 (s, 9H); MS (ESI): 603 (M+H)+.
4.2.42. tert-Butyl 2-(6-(2-chloro-6-cyanobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20i)
The reaction was carried out according to the procedure for
20a with 18i instead of 18a to give the title compound as a
1
colorless oil (15.5 mg, 2 steps 69%). H NMR (400MHz, CDCl3)
δ 7.48 (dd, J = 1.3, 8.0 Hz, 1H), 7.44 (dd, J = 1.3, 7.8 Hz, 1H),
7.22 (t, J = 7.9 Hz, 1H), 5.92 (s, 2H), 4.81 (d, J = 15.4 Hz, 1H),
4.75 (d, J = 13.3 Hz, 1H), 4.64 (d, J = 15.4 Hz, 1H), 4.27 (d, J =
17.1 Hz, 1H), 4.20 (d, J = 13.3 Hz, 1H), 3.99 (d, J = 17.2 Hz,
1H), 3.82 (s, 3H), 3.64 (s, 6H), 3.50 - 3.41 (m, 2H), 2.98 - 2.85
(m, 2H), 2.83 - 2.73 (m, 1H), 2.43 (s, 6H), 1.48 (s, 9H); MS
(ESI): 614 (M+H)+.
4.2.38. tert-Butyl 2-(3-(2,2-dimethylhydrazono)-6-(2-methoxy-6-
methylbenzyl)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-
yl)acetate (20e)
The reaction was carried out according to the procedure for
20b with 19e instead of 19b to give the title compound as a pale
yellow solid (34 mg, 88%). 1H NMR (400MHz, CDCl3) δ 7.03 (t,
J = 7.9 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.61 (d, J = 8.0 Hz,
1H), 5.89 (s, 2H), 4.92 (d, J = 15.7 Hz, 1H), 4.69 (d, J = 13.2 Hz,
1H), 4.49 (d, J = 15.6 Hz, 1H), 4.45 (d, J = 17.1 Hz, 1H), 4.15 (d,
J = 13.2 Hz, 1H), 3.82 (d, J = 17.1 Hz, 1H), 3.80 (s, 3H), 3.69 (s,
3H), 3.52 (s, 6H), 3.35 (dd, J = 12.3, 14.9 Hz, 1H), 2.96 (dd, J =
4.0, 13.6 Hz, 1H), 2.81 (dd, J = 11.2, 13.7 Hz, 1H), 2.70 (dd, J =
6.0, 15.1 Hz, 1H), 2.48 - 2.35 (m, 1H), 2.42 (s, 6H), 2.09 (s, 3H),
1.49 (s, 9H); MS (ESI): 599 (M+H)+.
4.2.43. tert-Butyl 2-(3-(2,2-dimethylhydrazono)-6-(2-fluoro-6-
methylbenzyl)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-
yl)acetate (20j)
The reaction was carried out according to the procedure for
20b with 19j instead of 19b to give the title compound as a pale
1
yellow solid (20 mg, 71%). H NMR (400MHz, CDCl3) δ 7.03
(dt, J = 6.0, 7.8 Hz, 1H), 6.85 (br. d, J = 7.4 Hz, 1H), 6.78 (br. t,
J = 8.8 Hz, 1H), 5.89 (s, 2H), 4.92 (d, J = 15.7 Hz, 1H), 4.72 (d, J
= 13.3 Hz, 1H), 4.49 (d, J = 15.6 Hz, 1H), 4.42 (d, J = 17.1 Hz,
1H), 4.16 (d, J = 13.3 Hz, 1H), 3.83 (d, J = 17.1 Hz, 1H), 3.80 (s,
3H), 3.54 (s, 6H), 3.40 - 3.31 (m, 1H), 3.02 (td, J = 3.4, 13.9 Hz,
1H), 2.83 - 2.66 (m, 2H), 2.47 - 2.31 (m, 1H), 2.42 (s, 6H), 2.11
(s, 3H), 1.49 (s, 9H); MS (ESI): 587 (M+H)+.
4.2.39. tert-Butyl 2-(6-(2-chloro-6-fluorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20f)
The reaction was carried out according to the procedure for
20b with 19f instead of 19b to give the title compound as a white
solid (25 mg, 83%). 1H NMR (400MHz, CDCl3) δ 7.13 - 7.05 (m,
2H), 6.89 - 6.82 (m, 1H), 5.93 (s, 2H), 4.78 (d, J = 15.8 Hz, 1H),
4.63 (d, J = 13.2 Hz, 1H), 4.62 (d, J = 15.7 Hz, 1H), 4.28 (d, J =
17.2 Hz, 1H), 4.24 (d, J = 13.3 Hz, 1H), 3.95 (d, J = 17.1 Hz,
1H), 3.81 (s, 3H), 3.61 (s, 6H), 3.31 (dd, J = 11.0, 14.1 Hz, 1H),
3.21 (td, J = 3.3, 12.9 Hz, 1H), 2.85 - 2.68 (m, 3H), 2.42 (s, 6H),
1.49 (s, 9H); MS (ESI): 607 (M+H)+.
4.2.44. tert-Butyl 2-(6-(2,6-dimethylbenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20k)
The reaction was carried out according to the procedure for
20b with 19k instead of 19b to give the title compound as a
1
white solid (97.7 mg, 89%). H NMR (400MHz, CDCl3) δ 6.97
(dd, J = 6.3, 8.4 Hz, 1H), 6.93 - 6.88 (m, 2H), 5.88 (s, 2H), 4.95
(d, J = 15.6 Hz, 1H), 4.74 (d, J = 13.6 Hz, 1H), 4.48 (d, J = 15.8
Hz, 1H), 4.46 (d, J = 17.1 Hz, 1H), 4.13 (d, J = 13.2 Hz, 1H),
3.81 (s, 3H), 3.81 (d, J = 17.2 Hz, 1H), 3.52 (s, 6H), 3.40 - 3.31
(m, 1H), 3.16 - 3.10 (m, 1H), 2.77 (dd, J = 5.6, 14.7 Hz, 1H),
4.2.40. tert-Butyl 2-(6-(2,6-dichlorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20g)

2.63 (dd, J = 11.0, 14.1 Hz, 1H), 2.42 (s, 6H), 2.45 - 2.38 (m,
1H), 4.56 (d, J = 16.3 Hz, 1H), 4.30 (d, J = 15.6 Hz, 1H), 4.19 (d,
J = 15.6 Hz, 1H), 3.94 (s, 3H), 3.84 (d, J = 16.4 Hz, 1H), 3.64 -
3.47 (m, 3H), 3.42 - 3.33 (m, 1H), 2.99 (dd, J = 6.3, 14.1 Hz,
1H), 2.31 (s, 6H); MS (ESI): 499 (M+H)+; HRMS (FAB) Calcd
1H), 2.13 (s, 6H), 1.49 (s, 9H); MS (ESI): 583 (M+H)+.
4.2.45. tert-Butyl 2-(6-(2-cyano-6-methylbenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20l)
+
for C27H31N8O2 : 499.2564; Found: 499.2543.
4.2.49. 2-(6-(2-Chlorobenzyl)-3-(2,2-dimethylhydrazono)-7-oxo-
1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (22b)
The reaction was carried out according to the procedure for
20a with 18l instead of 18a to give the title compound as a
1
colorless oil (30.3 mg, 2 steps 73%). H NMR (400MHz, CDCl3)
δ 7.38 (dd, J = 0.8, 7.6 Hz, 1H), 7.27 (br. d, J = 7.7 Hz, 1H), 7.18
(t, J = 7.7 Hz, 1H), 5.85 (s, 2H), 5.00 (d, J = 15.4 Hz, 1H), 4.80
(d, J = 13.3 Hz, 1H), 4.46 (d, J = 15.4 Hz, 1H), 4.45 (d, J = 17.1
Hz, 1H), 4.11 (d, J = 13.2 Hz, 1H), 3.81 (d, J = 16.8 Hz, 1H),
3.80 (s, 3H), 3.55 (s, 6H), 3.60 - 3.52 (m, 1H), 3.34 (dd, J = 4.0,
14.1 Hz, 1H), 2.89 - 2.77 (m, 2H), 2.43 (s, 6H), 2.40 - 2.31 (m,
1H), 2.16 (s, 3H), 1.49 (s, 9H); MS (ESI): 594 (M+H)+.
Anisole (0.143 ml, 1.31 mmol) and TFA (1.5 ml, 19.5 mmol)
were added to 20b (77 mg, 0.131 mmol), and the reaction
mixture was stirred at 60 °C for 5 h. Then, the mixture was
azeotropically concentrated with toluene, and the residue was
triturated with Et2O. The supernatant liquid was removed by
decantation, and the residue was collected to give the 21b as a
white solid (55 mg). To a suspension of 21b in CH2Cl2 (2 ml)
were added 3-(1-methyl-1H-pyrazol-4-yl)aniline (47.3 mg , 0.273
mmol), EDCI·HCl (52.4 mg, 0.273 mmol), and the mixture was
stirred at rt for 1 h. To the reaction mixture was added sat.
NaHCO3 aq., and the product was extracted with AcOEt. The
organic layer was successively washed with water and brine,
dried over anhydrous Na2SO4 and concentrated in vacuo. The
residue was purified by NH silica gel column chromatography
(eluent A: hexane/AcOEt (1/1), eluent B: MeOH, A/B = 98/2 to
92/8) to give the title compound as a white solid (54 mg, 2 steps
81%). 1H NMR (400MHz, CDCl3) δ 8.02 (br. s, 1H), 7.74 (d, J =
0.8 Hz, 1H), 7.65 (br. s, 1H), 7.61 (s, 1H), 7.40 - 7.30 (m, 2H),
7.30 - 7.15 (m, 5H), 6.26 (br. s, 1H), 4.57 (d, J = 16.3 Hz, 1H),
4.31 (d, J = 15.4 Hz, 1H), 4.20 (d, J = 15.6 Hz, 1H), 3.94 (s, 3H),
3.83 (d, J = 16.3 Hz, 1H), 3.66 - 3.53 (m, 1H), 3.49 - 3.27 (m,
3H), 2.88 (dd, J = 7.7, 14.0 Hz, 1H), 2.31 (s, 6H); MS (ESI): 508
4.2.46. tert-Butyl 2-(6-(2,6-difluorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20m)
The reaction was carried out according to the procedure for
20b with 19m instead of 19b to give the title compound as a
1
white solid (40 mg, 85%). H NMR (400MHz, CDCl3) δ 7.16 -
7.07 (m, 1H), 6.80 - 6.72 (m, 2H), 5.96 (s, 2H), 4.70 (d, J = 15.8
Hz, 1H), 4.65 (d, J = 15.9 Hz, 1H), 4.57 (d, J = 13.3 Hz, 1H),
4.29 (d, J = 13.2 Hz, 1H), 4.20 (d, J = 17.1 Hz, 1H), 4.01 (d, J =
17.1 Hz, 1H), 3.81 (s, 3H), 3.63 (s, 6H), 3.27 - 3.17 (m, 1H), 3.14
- 3.06 (m, 1H), 2.84 (dd, J = 5.4, 13.6 Hz, 1H), 2.77 - 2.61 (m,
2H), 2.41 (s, 6H), 1.48 (s, 9H); MS (ESI): 591 (M+H)+.
4.2.47. tert-Butyl 2-(3-(2,2-dimethylhydrazono)-6-(2-fluoro-6-
(trifluoromethyl)benzyl)-7-oxo-4-(2,4,6-trimethoxybenzyl)-1,4-
diazepan-1-yl)acetate (20n)
+
(M+H)+; HRMS (FAB) Calcd for C26H31ClN7O2 : 508.2222;
Found: 508.2222.
The reaction was carried out according to the procedure for
20b with 19n instead of 19b to give the title compound as a
white solid (26 mg, 83%). 1H NMR (400MHz, CDCl3) δ 7.37 (br.
d, J = 7.9 Hz, 1H), 7.31 - 7.23 (m, 1H), 7.11 (br. t, J = 9.2 Hz,
1H), 5.93 (s, 2H), 4.72 (d, J = 15.8 Hz, 1H), 4.67 (d, J = 15.9 Hz,
1H), 4.66 (d, J = 13.3 Hz, 1H), 4.22 (d, J = 13.2 Hz, 1H), 4.22 (d,
J = 17.2 Hz, 1H), 4.00 (d, J = 17.1 Hz, 1H), 3.80 (s, 3H), 3.61 (s,
6H), 3.33 - 3.21 (m, 2H), 2.88 - 2.74 (m, 3H), 2.42 (s, 6H), 1.48
(s, 9H); MS (ESI): 641 (M+H)+.
4.2.50. 2-(6-(2-Chloro-6-methoxybenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22c)
The reaction was carried out according to the procedure for
22a with 20c instead of 20a to give the title compound as a white
1
solid (14.8 mg, 2 steps 21%). H NMR (400MHz, CDCl3) δ 8.16
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.68 (br. s, 1H), 7.62 (s, 1H),
7.30 - 7.27 (m, 2H), 7.24 - 7.19 (m, 1H), 7.16 (t, J = 8.2 Hz, 1H),
7.01 (dd, J = 1.1, 8.1 Hz, 1H), 6.79 (dd, J = 0.8, 8.3 Hz, 1H),
6.31 (br. s, 1H), 4.55 (d, J = 15.9 Hz, 1H), 4.34 (d, J = 15.4 Hz,
1H), 4.23 (d, J = 15.4 Hz, 1H), 3.93 (s, 3H), 3.90 (br. d, J = 15.9
Hz, 1H), 3.84 (s, 3H), 3.55 - 3.45 (m, 1H), 3.35 - 3.25 (m, 3H),
3.16 (dd, J = 10.5, 13.8 Hz, 1H), 2.31 (s, 6H); MS (ESI): 538
4.2.48. 2-(6-(2-Cyanobenzyl)-3-(2,2-dimethylhydrazono)-7-oxo-
1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (22a)
Anisole (0.26 ml, 2.38 mmol) and TFA (3 ml, 38.9 mmol)
were added to 20a (274 mg, 0.473 mmol), and the mixture was
stirred at rt for 5 h and at 40 °C for 20 h. Then, the mixture was
azeotropically concentrated with toluene 3 times, and the residue
was triturated with Et2O. The appeared solid was collected by
filtration to give 21a as a white solid (214 mg). To a mixture of
21a in CH2Cl2 (2 ml) and AcOEt (2 ml) were added N,N-
diisopropylethylamine (0.33 ml, 1.89 mmol), 3-(1-methyl-1H-
pyrazol-4-yl)aniline (81 mg, 0.468 mmol), and 1.7 mol/l 1-
propanephosphonic acid cyclic anhydride in AcOEt (0.83 ml,
1.41 mmol), and the reaction mixture was stirred at rt for 15 min.
Then, the reaction mixture was diluted with AcOEt, and
successively washed with sat. NaHCO3 aq., water, and brine. The
organic layer was dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by NH silica gel
column chromatography (eluent A: hexane/EtOAc (1/1), eluent
B: MeOH, A/B = 95/5 to 88/12) to give the title compound as a
+
(M+H)+; HRMS (FAB) Calcd for C27H33ClN7O3 : 538.2328;
Found: 538.2343.
4.2.51. 2-(3-(2,2-Dimethylhydrazono)-6-(2-fluoro-6-
methoxybenzyl)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-
pyrazol-4-yl)phenyl)acetamide (22d)
The reaction was carried out according to the procedure for
22a with 20d instead of 20a to give the title compound as a white
1
solid (21.8 mg, 2 steps 36%). H NMR (400MHz, CDCl3) δ 8.17
(br. s, 1H), 7.74 (d, J = 0.6 Hz, 1H), 7.68 (br. s, 1H), 7.62 (s, 1H),
7.32 - 7.24 (m, 2H), 7.24 - 7.13 (m, 2H), 6.73 - 6.65 (m, 2H),
6.27 (br. s, 1H), 4.55 (d, J = 16.1 Hz, 1H), 4.35 (d, J = 15.4 Hz,
1H), 4.20 (d, J = 15.4 Hz, 1H), 3.93 (s, 3H), 3.87 (d, J = 16.1 Hz,
1H), 3.85 (s, 3H), 3.48 - 3.39 (m, 1H), 3.39 - 3.31 (m, 1H), 3.30 -
3.15 (m, 2H), 2.94 (dd, J = 10.5, 13.7 Hz, 1H), 2.31 (s, 6H); MS
(ESI): 522 (M+H)+; HRMS (FAB) Calcd for C27H33FN7O3 :
+
1
522.2623; Found: 522.2592.
white solid (224 mg, 2 steps 95%). H NMR (400MHz, CDCl3) δ
7.96 (br. s, 1H), 7.73 (d, J = 0.8 Hz, 1H), 7.67 - 7.62 (m, 2H),
7.61 (s, 1H), 7.53 - 7.44 (m, 2H), 7.35 - 7.17 (m, 4H), 6.30 (br. s,

27
1
4.2.52. 2-(3-(2,2-Dimethylhydrazono)-6-(2-methoxy-6-
methylbenzyl)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-
pyrazol-4-yl)phenyl)acetamide (22e)
(–)-22g: white solid. [α]D = −34° (c 0.50, CHCl3); H NMR
(400MHz, CDCl3) δ 8.26 (br. s, 1H), 7.73 (d, J=0.8 Hz, 1H), 7.66
(br. s, 1H), 7.60 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.28 - 7.23 (m,
2H), 7.22 - 7.17 (m, 1H), 7.14 (dd, J = 7.7, 8.4 Hz, 1H), 6.31 (br.
s, 1H), 4.56 (d, J = 16.2 Hz, 1H), 4.36 (d, J = 15.6 Hz, 1H), 4.22
(d, J = 15.6 Hz, 1H), 3.92 (s, 3H), 3.90 (d, J = 16.2 Hz, 1H), 3.68
- 3.59 (m, 1H), 3.50 - 3.40 (m, 2H), 3.36 (td, J = 3.9, 13.1 Hz,
1H), 3.27 (dd, J = 10.0, 14.3 Hz, 1H), 2.31 (s, 6H); MS (ESI):
The reaction was carried out according to the procedure for
22b with 20e instead of 20b to give the title compound as a white
1
solid (18 mg, 2 steps 66%). H NMR (400MHz, CDCl3) δ 8.15
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.69 (br. s, 1H), 7.62 (s, 1H),
7.31 - 7.24 (m, 2H), 7.23 - 7.18 (m, 1H), 7.13 (t, J = 7.9 Hz, 1H),
6.82 (d, J = 7.5 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.40 (br. s,
1H), 4.55 (d, J = 15.8 Hz, 1H), 4.36 (d, J = 15.4 Hz, 1H), 4.21 (d,
J = 15.4 Hz, 1H), 3.93 (s, 3H), 3.88 (d, J = 15.6 Hz, 1H), 3.84 (s,
3H), 3.43 - 3.27 (m, 2H), 3.27 - 3.10 (m, 2H), 3.01 (dd, J = 10.2,
13.9 Hz, 1H), 2.38 (s, 3H), 2.34 (s, 6H); MS (ESI): 518 (M+H)+;
542 (M+H)+; HRMS (FAB) Calcd for C26H30Cl2N7O2 : 542.1833;
+
Found: 542.1842.
4.2.56. 2-(6-(2-Chloro-6-methylbenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22h)
+
HRMS (FAB) Calcd for C28H36N7O3 : 518.2874; Found:
The reaction was carried out according to the procedure for
22b with 20h instead of 20b to give the title compound as a
white solid (9 mg, 2 steps 63%). H NMR (400MHz, CDCl3) δ
8.02 (br. s, 1H), 7.74 (d, J = 0.6 Hz, 1H), 7.67 (br. s, 1H), 7.61 (s,
1H), 7.32 - 7.18 (m, 4H), 7.11 - 7.06 (m, 2H), 6.31 (br. s, 1H),
4.54 (d, J = 16.1 Hz, 1H), 4.32 (d, J = 15.6 Hz, 1H), 4.24 (d, J =
15.6 Hz, 1H), 3.94 (s, 3H), 3.91 (d, J = 16.1 Hz, 1H), 3.59 - 3.49
(m, 1H), 3.45 - 3.31 (m, 3H), 3.08 (dd, J = 9.5, 14.4 Hz, 1H),
2.41 (s, 3H), 2.30 (s, 6H); MS (ESI): 522 (M+H)+; HRMS (FAB)
518.2870.
1
4.2.53. 2-(6-(2-Chloro-6-fluorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22f)
The reaction was carried out according to the procedure for
22b with 20f instead of 20b to give the title compound as a white
1
solid (12 mg, 2 steps 64%). H NMR (400MHz, CDCl3) δ 8.03
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.68 (br. s, 1H), 7.62 (s, 1H),
7.32 - 7.25 (m, 2H), 7.23 - 7.15 (m, 3H), 7.03 - 6.96 (m, 1H),
6.29 (br. s, 1H), 4.55 (d, J = 16.2 Hz, 1H), 4.32 (d, J = 15.4 Hz,
1H), 4.24 (d, J = 15.6 Hz, 1H), 3.94 (s, 3H), 3.91 (d, J = 16.2 Hz,
1H), 3.59 - 3.48 (m, 1H), 3.40 - 3.32 (m, 3H), 3.11 - 3.03 (m,
1H), 2.30 (s, 6H); MS (ESI): 526 (M+H)+; HRMS (FAB) Calcd
+
Calcd for C27H33ClN7O2 : 522.2379; Found: 522.2352.
4.2.57. 2-(6-(2-Chloro-6-cyanobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22i)
The reaction was carried out according to the procedure for
22a with 20i instead of 20a to give the title compound as a white
solid (10.2 mg, 2 steps 83%). H NMR (400MHz, CDCl3) δ 8.00
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.65 (br. s, 1H), 7.62 (s, 1H),
7.63 - 7.60 (m, 1H), 7.58 (dd, J = 1.3, 7.8 Hz, 1H), 7.31 (t, J =
8.0 Hz, 1H), 7.29 - 7.23 (m, 2H), 7.23 - 7.18 (m, 1H), 6.36 (br. s,
1H), 4.54 (d, J = 16.3 Hz, 1H), 4.35 (d, J = 15.6 Hz, 1H), 4.21 (d,
J = 15.6 Hz, 1H), 3.94 (s, 3H), 3.94 (d, J = 16.2 Hz, 1H), 3.71 -
3.57 (m, 2H), 3.55 - 3.40 (m, 2H), 3.18 (dd, J = 8.0, 13.9 Hz,
1H), 2.31 (s, 6H); MS (ESI): 533 (M+H)+; HRMS (FAB) Calcd
+
for C26H30ClFN7O2 : 526.2128; Found: 526.2095.
1
4.2.54. 2-(6-(2,6-Dichlorobenzyl)-3-(2,2-dimethylhydrazono)-7-
oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (22g)
The reaction was carried out according to the procedure for
22b with 20g instead of 20b to give the title compound as a white
1
solid (30 mg, 2 steps 88%). H NMR (400MHz, CDCl3) δ 8.10
(br. s, 1H), 7.74 (d, J = 0.6 Hz, 1H), 7.67 (br. s, 1H), 7.62 (s, 1H),
7.32 (d, J = 7.9 Hz, 2H), 7.29 - 7.25 (m, 2H), 7.23 - 7.18 (m, 1H),
7.14 (dd, J = 7.7, 8.4 Hz, 1H), 6.31 (br. s, 1H), 4.55 (d, J = 16.2
Hz, 1H), 4.33 (d, J = 15.4 Hz, 1H), 4.24 (d, J = 15.4 Hz, 1H),
3.93 (s, 3H), 3.92 (d, J = 16.2 Hz, 1H), 3.68 - 3.59 (m, 1H), 3.51
- 3.41 (m, 2H), 3.36 (td, J = 3.9, 13.1 Hz, 1H), 3.28 (dd, J = 10.0,
14.3 Hz, 1H), 2.30 (s, 6H); MS (ESI): 542 (M+H)+; HRMS
+
for C27H30ClN8O2 : 533.2175; Found: 533.2194.
4.2.58. 2-(3-(2,2-Dimethylhydrazono)-6-(2-fluoro-6-
methylbenzyl)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-
pyrazol-4-yl)phenyl)acetamide (22j)
The reaction was carried out according to the procedure for
22b with 20j instead of 20b to give the title compound as a white
solid (10 mg, 2 steps 63%). H NMR (400MHz, CDCl3) δ 8.00
+
(FAB) Calcd for C26H30Cl2N7O2 : 542.1833; Found: 542.1823.
1
4.2.55. (+)-2-(6-(2,6-Dichlorobenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide ((+)-22g) and (–)-2-(6-(2,6-
Dichlorobenzyl)-3-(2,2-dimethylhydrazono)-7-oxo-1,4-diazepan-
1-yl)-N-(3-(1-methyl-1H-pyrazol-4-yl)phenyl)acetamide ((–)-22g)
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.68 (br. s, 1H), 7.62 (s, 1H),
7.32 - 7.18 (m, 3H), 7.12 (dt, J = 5.8, 7.9 Hz, 1H), 6.98 (br. d, J =
7.7 Hz, 1H), 6.89 (br. t, J = 9.0 Hz, 1H), 6.30 (br. s, 1H), 4.54 (d,
J = 16.1 Hz, 1H), 4.33 (d, J = 15.6 Hz, 1H), 4.22 (d, J = 15.6 Hz,
1H), 3.94 (s, 3H), 3.90 (d, J = 16.1 Hz, 1H), 3.44 - 3.27 (m, 3H),
3.25 - 3.18 (m, 1H), 2.94 (dd, J = 8.6, 13.9 Hz, 1H), 2.39 (s, 3H),
2.31 (s, 6H); MS (ESI): 506 (M+H)+; HRMS (FAB) Calcd for
The compound 22g was separated using CHIRALPAK ID
(Daicel
Chemical
Industries)
(movement
phase:
hexane/EtOH/Et2NH = 50/50/0.1) to give the (+)-22g and (–)-
22g.
+
C27H33FN7O2 : 506.2674; Found: 506.2695.
4.2.59. 2-(6-(2,6-Dimethylbenzyl)-3-(2,2-dimethylhydrazono)-7-
oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (22k)
27
1
(+)-22g: white solid. [α]D = +34° (c 0.50, CHCl3); H NMR
(400MHz, CDCl3) δ 8.30 (br. s, 1H), 7.73 (d, J = 0.8 Hz, 1H),
7.66 (br. s, 1H), 7.59 (s, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.28 - 7.22
(m, 2H), 7.22 - 7.16 (m, 1H), 7.14 (dd, J = 7.7, 8.4 Hz, 1H), 6.31
(br. s, 1H), 4.57 (d, J = 16.2 Hz, 1H), 4.37 (d, J = 15.6 Hz, 1H),
4.22 (d, J = 15.4 Hz, 1H), 3.92 (s, 3H), 3.89 (d, J = 16.2 Hz, 1H),
3.68 - 3.59 (m, 1H), 3.50 - 3.40 (m, 2H), 3.36 (td, J = 4.0, 13.2
Hz, 1H), 3.27 (dd, J = 10.2, 14.3 Hz, 1H), 2.31 (s, 6H); MS
The reaction was carried out according to the procedure for
22b with 20k instead of 20b to give the title compound as a
white solid (82.3 mg, 2 steps 98%). 1H NMR (400MHz, CDCl3) δ
8.49 (br. s, 1H), 7.72 (d, J = 0.8 Hz, 1H), 7.67 (br. s, 1H), 7.57 (s,
1H), 7.26 - 7.21 (m, 2H), 7.20 - 7.14 (m, 1H), 7.08 - 7.00 (m,
3H), 6.27 (br. s, 1H), 4.49 (d, J = 16.3 Hz, 1H), 4.36 (d, J = 15.7
Hz, 1H), 4.27 (d, J = 15.7 Hz, 1H), 3.93 (d, J = 16.3 Hz, 1H),
3.91 (s, 3H), 3.43 - 3.22 (m, 4H), 2.96 - 2.87 (m, 1H), 2.33 (s,
(ESI): 542 (M+H)+; HRMS (FAB) Calcd for C26H30Cl2N7O2 :
542.1833; Found: 542.1862.
+

6H), 2.30 (s, 6H); MS (ESI): 502 (M+H)+; HRMS (FAB) Calcd
for C28H36N7O2 : 502.2925; Found: 502.2945.
(d, J = 0.6 Hz, 1H), 7.68 (br. s, 1H), 7.61 (s, 1H), 7.31 - 7.25 (m,
1H), 7.24 - 7.18 (m, 2H), 7.09 (d, J = 7.9 Hz, 1H), 6.89 - 6.84 (m,
2H), 5.83 (br. s., 1H), 4.60 (d, J = 17.4 Hz, 1H), 4.35 (d, J = 15.3
Hz, 1H), 4.15 (d, J = 15.3 Hz, 1H), 3.94 (d, J = 17.1 Hz, 1H),
3.93 (s, 3H), 3.83 (s, 3H), 3.53 - 3.44 (m, 1H), 3.34 - 3.27 (m,
2H), 3.19 (dd, J = 5.6, 13.9 Hz, 1H), 2.70 (dd, J = 8.3, 14.0 Hz,
1H).
+
4.2.60. 2-(6-(2-Cyano-6-methylbenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22l)
The reaction was carried out according to the procedure for
22a with 20l instead of 20a to give the title compound as a white
solid (22.6 mg, 2 steps 90%). H NMR (400MHz, CDCl3) δ 7.97
4.2.65. 2-(6-(4-Chloro-2-methoxybenzyl)-7-oxo-3-thioxo-1,4-
diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (25)
1
(br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.67 (br. s, 1H), 7.62 (s, 1H),
7.51 (br. d, J = 7.7 Hz, 1H), 7.41 (br. d, J = 7.3 Hz, 1H), 7.30 -
7.23 (m, 3H), 7.23 - 7.18 (m, 1H), 6.38 (br. s, 1H), 4.54 (d, J =
16.2 Hz, 1H), 4.36 (d, J = 15.7 Hz, 1H), 4.21 (d, J = 15.7 Hz,
1H), 3.94 (s, 3H), 3.95 (d, J = 16.1 Hz, 1H), 3.56 - 3.43 (m, 4H),
3.10 - 3.02 (m, 1H), 2.44 (s, 3H), 2.32 (s, 6H); MS (ESI): 513
The reaction was carried out according to the procedure for
19b with 24 instead of 18b and Lawesson’s reagent instead of
Belleau’s reagent to give the title compound (184 mg, quant.). 1H
NMR (400MHz, CDCl3) δ 7.97 (br. s, 1H), 7.86 (br. s, 1H), 7.75
(d, J = 0.4 Hz, 1H), 7.68 (br. s, 1H), 7.62 (s, 1H), 7.32 - 7.20 (m,
3H), 7.09 (d, J = 8.0 Hz, 1H), 6.89 - 6.85 (m, 2H), 4.87 (d, J =
17.3 Hz, 1H), 4.56 (d, J = 17.4 Hz, 1H), 4.41 (d, J = 15.2 Hz,
1H), 4.14 (d, J = 15.3 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.59 -
3.49 (m, 1H), 3.47 - 3.32 (m, 2H), 3.20 (dd, J = 5.5, 13.7 Hz,
1H), 2.72 (dd, J = 8.5, 13.9 Hz, 1H); MS (ESI): 512 (M+H)+.
+
(M+H)+; HRMS (FAB) Calcd for C28H33N8O2 : 513.2721;
Found: 513.2725.
4.2.61. 2-(6-(2,6-Difluorobenzyl)-3-(2,2-dimethylhydrazono)-7-
oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (22m)
The reaction was carried out according to the procedure for
22b with 20m instead of 20b to give the title compound as a
white solid (34 mg, 2 steps quant.). 1H NMR (400MHz, CDCl3) δ
8.29 (br. s, 1H), 7.73 (d, J = 0.5 Hz, 1H), 7.66 (br. s, 1H), 7.59 (s,
1H), 7.29 - 7.15 (m, 4H), 6.93 - 6.84 (m, 2H), 6.30 (br. s, 1H),
4.57 (d, J = 16.2 Hz, 1H), 4.36 (d, J = 15.6 Hz, 1H), 4.21 (d, J =
15.6 Hz, 1H), 3.92 (s, 3H), 3.86 (d, J = 16.3 Hz, 1H), 3.53 - 3.43
(m, 1H), 3.42 - 3.21 (m, 3H), 2.91 (dd, J = 9.6, 14.2 Hz, 1H),
2.31 (s, 6H); MS (ESI): 510 (M+H)+; HRMS (FAB) Calcd for
4.2.66. 2-(6-(4-Chloro-2-methoxybenzyl)-3-(2,2-
dimethylhydrazono)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-methyl-
1H-pyrazol-4-yl)phenyl)acetamide (22o)
The reaction was carried out according to the procedure for
20b with 25 instead of 19b and pyridine instead of MeOH/THF
(1/10) to give the title compound as white solid (13.7 mg, 43%).
1H NMR (400MHz, CDCl3) δ 8.06 (br. s., 1H), 7.74 (d, J = 0.6
Hz, 1H), 7.69 (br. s, 1H), 7.61 (s, 1H), 7.31 - 7.25 (m, 1H), 7.23 -
7.18 (m, 2H), 7.13 (d, J = 7.9 Hz, 1H), 6.89 - 6.84 (m, 2H), 6.27
(br. s, 1H), 4.54 (d, J = 16.1 Hz, 1H), 4.32 (d, J = 15.4 Hz, 1H),
4.18 (d, J = 15.4 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H), 3.84 (d, J =
16.4 Hz, 1H), 3.49 - 3.35 (m, 2H), 3.25 - 3.15 (m, 2H), 2.74 (dd,
J = 8.3, 13.7 Hz, 1H), 2.31 (s, 6H); MS (ESI): 538 (M+H)+;
+
C26H30F2N7O2 : 510.2424; Found: 510.2391.
4.2.62. 2-(3-(2,2-Dimethylhydrazono)-6-(2-fluoro-6-
(trifluoromethyl)benzyl)-7-oxo-1,4-diazepan-1-yl)-N-(3-(1-
methyl-1H-pyrazol-4-yl)phenyl)acetamide (22n)
+
HRMS (FAB) Calcd for C27H33ClN7O3 : 538.2328; Found:
The reaction was carried out according to the procedure for
538.2343.
22b with 20n instead of 20b to give the title compound as a
1
white solid (16 mg, 2 steps 78%). H NMR (400MHz, CDCl3) δ
4.3. Enzymatic assay
8.03 (br. s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.67 (br. s, 1H), 7.62 (s,
1H), 7.50 (br. d, J = 7.9 Hz, 1H), 7.41 - 7.33 (m, 1H), 7.30 - 7.19
(m, 4H), 6.28 (br. s, 1H), 4.54 (d, J = 16.2 Hz, 1H), 4.33 (d, J =
15.4 Hz, 1H), 4.22 (d, J = 15.4 Hz, 1H), 3.94 (s, 3H), 3.90 (d, J =
16.4 Hz, 1H), 3.60 - 3.50 (m, 1H), 3.47 - 3.38 (m, 1H), 3.38 -
3.32 (m, 2H), 3.08 (dd, J = 9.6, 14.7 Hz, 1H), 2.30 (s, 6H); MS
Recombinant human KLK7 (R&D systems) was activated
with thermolysin for 2 h at 37 °C. Recombinant mouse KLK7 for
crystallization before activation was activated with enterokinase
for 30 min at 37 °C. Test compounds were incubated with
activated enzyme (1 µg/mL) in reaction buffer (50 mM Tris-HCl
(pH 8.5), 0.15 M NaCl, 0.1% BSA) for 10 min at room
temperature, and 10 µM of substrate (MOCAc-Arg-Pro-Lys-Pro-
Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2, Peptide Institute) was
added. After 30 min of incubation at room temperature, the
reaction was stopped by adding 30% acetic acid, and
fluorescence (Ex. 340/30 nm, Em. 400/30 nm) was measured.
+
(ESI): 560 (M+H)+; HRMS (FAB) Calcd for C27H30F4N7O2 :
560.2392; Found: 560.2365.
4.2.63. tert-Butyl 2-(6-(4-chloro-2-methoxybenzyl)-3,7-dioxo-4-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-1-yl)acetate (18o)
The reaction was carried out according to the procedure for
18a with 4-chloro-2-methoxyphenylboronic acid instead of 2-
cyanophenylboronic acid to give the title compound (270 mg,
quant.). 1H NMR (400MHz, CDCl3) δ 6.83 (d, J = 8.0 Hz, 1H),
6.78 (dd, J = 1.9, 7.9 Hz, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.00 (s,
2H), 4.64 (d, J = 13.7 Hz, 1H), 4.42 (d, J = 13.7 Hz, 1H), 4.26 (d,
J = 17.3 Hz, 1H), 4.20 (d, J = 16.7 Hz, 1H), 4.15 (d, J = 16.6 Hz,
1H), 4.06 (d, J = 17.3 Hz, 1H), 3.84 (s, 3H), 3.73 (s, 3H), 3.65 (s,
6H), 3.23 - 3.08 (m, 2H), 2.97 - 2.87 (m, 2H), 2.50 (dd, J = 9.8,
13.8 Hz, 1H), 1.46 (s, 9H).
Supplementary Material
Supplementary data associated with this article can be found,
in the online version at:
References and notes
1. Yousef GM, Diamandis EP. The new human tissue kallikrein gene
family: structure, function, and association to disease. Endoc Rev.
2001;22:184–204.
2. Prassas I, Eissa A, Poda G, Diamandis EP. Unleashing the
therapeutic potential of human kallikrein-related serine proteases.
Nat Rev Drug discovery. 2015;14:183–202.
3. Sotiropoulou G, Pampalakis G. Targeting the kallikrein-related
peptidases for drug development. Trends Pharmacol Sci.
2012;33:623–634.
4.2.64. 2-(6-(4-Chloro-2-methoxybenzyl)-3,7-dioxo-1,4-
diazepan-1-yl)-N-(3-(1-methyl-1H-pyrazol-4-
yl)phenyl)acetamide (24)
The reaction was carried out according to the procedure for
22b with 18o instead of 20b to give the title compound (190 mg,
2 steps 83%).1H NMR (400MHz, CDCl3) δ 7.95 (br. s, 1H), 7.74

4. Komatsu N, Takata M, Otsuki N, et al. Expression and
localization of tissue kallikrein mRNAs in human epidermis and
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Structure-based drug design to overcome
species differences in kallikrein 7 inhibition
of 1,3,6-trisubstituted 1,4-diazepan-7-ones
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Hidenobu Murafuji*, Hajime Sugawara, Megumi Goto, Yoshiaki Oyama, Hiroki Sakai, Seiichi Imajo,
Toshiyuki Tomoo and Tsuyoshi Muto*
Asubio Pharma Co., Ltd., 6-4-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan