Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4- diazepane-2,5-diones as human chymase inhibitors using structure-based drug design

Article abstract of DOI:10.1016/j.bmc.2013.04.079

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5- diones were explored as human chymase inhibitors using structure-based drug design according to the X-ray cocrystal structure of chymase and compound 1. The optimization focused on

Full text of DOI:10.1016/j.bmc.2013.04.079

Accepted Manuscript
Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diaze‐
pane-2,5-diones as human chymase inhibitors using structure-based drug design
Taisaku Tanaka, Hajime Sugawara, Hiroshi Maruoka, Seiichi Imajo, Tsuyoshi
Muto
PII:
S0968-0896(13)00418-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.04.079
BMC 10819
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 March 2013
26 April 2013
27 April 2013
Please cite this article as: Tanaka, T., Sugawara, H., Maruoka, H., Imajo, S., Muto, T., Discovery of novel series of
6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as human chymase inhibitors using structure-based
drug design, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.04.079
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-
diones as human chymase inhibitors using structure-based drug design
Taisaku Tanaka*, Hajime Sugawara, Hiroshi Maruoka, Seiichi Imajo and Tsuyoshi Muto*
Asubio Pharma Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones were explored
as human chymase inhibitors using structure-based drug design according to the X-ray cocrystal
structure of chymase and compound 1. The optimization focused on the prime site led to the
attainment of compounds that showed potent inhibitory activity, and among them, 18R shows a
novel interaction mode.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Chymase inbibitor
Atopic Dermatitis
Amino acid derivatives
Heteroaromatic derivatives
1. Introduction
different physical property, was explored using a structure-based
drug design approach. Heteroaromatic derivatives and amino acid
Chymase is a chymotrypsin-like serine protease stored in mast
cell granules1 that hydrolyzes
variety of physiological
derivatives were designed by referring to the cocrystal structure
of chymase and compound 1 which is SUN13834 series
compound (Figure 1). In this paper, the approach used in the
design, in addition to the structure activity relationships, is
described. In the course of our study, various derivatives were
synthesized that showed potent inhibitory activity, comparable to
a
substrates, including angiotensin I.2 It is known to play a role in
the pathogenesis of atopic dermatitis; for example, it has been
reported that an intradermal injection of chymase to mouse skin
elicits edema as well as inflammatory cell accumulation,3,4 and a
correlation between a particular single nucleotide polymorphism
of the chymase gene and incidence of atopic dermatitis has been
observed.5–7 In a previous paper,8,9 we reported on novel, orally
active human chymase inhibitors with a 6-benzyl substituted 4-
aminocarbonyl-1,4-diazepane-2,5-dione
scaffold,
which
demonstrated selectivity against other serine proteases. Oral
administration of the representative compound SUN13834
reduced skin inflammation in NC/Nga mice10 that spontaneously
develop dermatitis under conventional conditions,11,12 in addition
to significantly decreasing the amount of scratching induced by
DNFB challenge in the mouse model.13 SUN13834 also showed
promising results when tested on humans with atopic
dermatitis.14
SUN13834.
In order to increase the chances of successfully launching our
chymase inhibitor with lower dosage and/or different
pharmacokinetic profile onto the market, a new class of the
compound, which showed enhanced inhibitory activity and/or
Figure1. Approach to obtaining new inhibitors
*Corresponding author. Tel.: +81-78-306-5307; fax: +81-78-306-5971; e-mail: tanaka.taisaku.fu@asubio.co.jp (T.Tanaka), Tel.: +81-
78-306-5338; fax: +81-78-306-5971; e-mail: muto.tsuyoshi.ba@asubio.co.jp (T.Muto)

2. Chemistry
hydrochloride, followed by deprotection of the Boc group,
provided amine 8. Introduction of a 2,4,6-trimethoxybenzyl
(TMB) group to 8 by reductive alkylationand subsequent
hydrolysis of the ester moiety, gave amino acid 10.
Lactamization of 10, followed by catalytic hydrogenation,
afforded the seven-membered lactam rac-12. (R)-1215 was
prepared by optical resolution of rac-12 using a chiral HPLC
column (CHIRALCEL OD). Following this, rac-12 and (R)-12
were converted to the carbamate rac-13 and (R)-13, respectively,
by sequential treatment with n-butyllithium and 2-
chlorophenylchloroformate.
Rac-13 (racemic mixture) and (R)-13 (R form at 6-position of
1,4-diazepane core; the configuration was selected by referring to
the structure of compound 1), which were used as a common
intermediate for both derivatives, were prepared from 5-chloro-2-
methoxybenzaldehyde (2), as shown in Scheme 1. The Baylis–
Hillman reaction of 2 with methyl acrylate, followed by
acetylation, gave adduct 4. SN2′-type substitution of the acetoxy
group of 4 by sodium azide, and subsequent reduction of the
azide group and hydrolysis of the methyl ester, led to β-amino
acid 6, which was then converted to N-Boc derivative 7 using
standard methods. Condensation of 7 with glycine ethyl ester
Scheme 1.Reagents and conditions: (a) methyl acrylate, DABCO, La(OTf)3, N(CH2CH2OH)2, rt; (b) AcCl, Et3N, CH2Cl2, 0C; (c) NaN3, DMSO, rt; (d) PPh3,
H2O, THF, rt; (e) aq NaOH, MeOH, rt; (f) (Boc)2O, aq NaOH, THF, 0C; (g) glycine ethyl ester HCl, EDCI HCl, HOBt, Et3N, CH2Cl2, rt; (h) 4M HCl/EtOAc, rt;
(i) 2,4,6-(MeO)3PhCHO, NaBH(OAc)3, THF, rt; (j) aq NaOH, MeOH, rt; (k) EDCI HCl, HOBt, Et3N, DMF, rt; (l) H2, 2%Pt(sulfide).C, THF, rt; (m) n-BuLi,
THF, -78°C (forrac-13) or -20C (for(R)-13), then 2-ClPhOCOCl, -78C (forrac-13) or -20C (for(R)-13); (n) optical resolution using chiral HPLC
(CHIRALCEL OD). *THF solvate
As shown in Scheme 2, the heteroaromatic derivatives listed in
Table1 were prepared from optically active carbamate (R)-13 as a
common intermediate. Coupling reactions of (R)-13 with the
corresponding amines furnished compounds 15A–F and H,
which were deprotected to yield heteroaromatic derivatives 16A–
F and H. Compound 16G was synthesized in a similar manner
from deprotected carbamate (R)-14.
coupling reaction of rac-13 with tert-butyl 2-aminobutylate,
followed by deprotection of the TMB group, provided rac-17.
Amino acid derivatives 18A–R were furnished by condensation
of rac-17 with the corresponding amines and/or deprotection.
The optically active compounds (R)-15 and (R)-16R, which were
used for cocrystallization with human chymase for X-ray
structure analysis, were synthesized in a similar manner from
(R)-13 via the deprotected carbamate (R)-14.
As shown in Scheme 3, the amino acid derivatives listed in Table
2 and 3 were prepared from rac-13 as a common intermediate. A

Scheme 2. Reagents and conditions: (a) amines, Et3N, DMAP, DMF, 0C; (b) DDQ, CH2Cl2, H2O, rt; (c) 1 M HCl/AcOH, rt (d) 1 M HCl/AcOH, anisole, rt; (e)
Zn, AcOH, rt; (f) TFA, anisole, rt.; (g) 42, DIPEA, DMAP, DMF, 0C.
Scheme 3. Reagents and conditions: (a) (R)-tert-butyl 2- aminobutanoate, Et3N, DMF, 0C; (b) TFA, CH2Cl2, rt; (c) TFA, anisole, rt; (d) (R)-tert-butyl 2-
aminobutanoate, DIPEA, DMAP, DMF, 0C; (e) 4 M HCl/EtOAc, rt; (f) amines n-propyl phosphoric acid anhydride, Et3N, CH2Cl2, rt; (g) 1 M HCl/AcOH, rt.
Amines for preparation of 16A–C were synthesized from the
corresponding aromatic halide 19a–c, as shown in Scheme 4.
tert-Butyl esterification of 19a–c with O-tert-butyl N,N′-
diisopropylisourea, followed by palladium-catalyzed cyanation,
provided aromatic cyanide 21a–c. The cyanide group was then
converted into an ethyl carbonyl moiety. Asymmetric reduction16
of the carbonyl group, followed by the Mitsunobu reaction with
phthalimide, provided 24a–c. After hydrazinolysis of the
phthalimide moiety, tartrate salts 25a–c17 were formed from the
obtained amines.
manipulation (protection of carboxyl group and enantioselective
addition of Et2Zn18 to the aldehyde). The desired amine 3117 was
then synthesized in a similar manner to Scheme 4 from 28,
shown in Scheme 5.
As demonstrated in Scheme 6, amines for the preparation of
16E and 16F were synthesized from 3-carboxyl furan (32a) and
3-carboxyl thiophene (32b), respectively. Friedel-Crafts reaction
using indium(III) triflate as a Lewis acid,19 followed by tert-butyl
esterification with O-tert-butyl N,N′-diisopropylisourea, provided
aryl ethyl ketones 33a,b. The desired amines 36a,b17 were then
synthesized in a similar manner to Scheme 4 from the obtained
ketones shown in Scheme 6.
As shown in Scheme 5, the amine for preparation of 16D was
synthesized from 5-formyl-2-furancarboxylic acid (26), which
was converted to secondary alcohol 28 by sequential

Scheme 4. Reagents and conditions: (a) O-tert- butyl N,N′-diidopropylisourea, CH2Cl2, reflux; (b) Zn(CN)2,Pd2(dba)3, dppf,Zn,AcNMe2, 120C; (c) CuI,
EtMgBr, THF, 0C; (d) (-)-Ipc2BCl, THF, -20C; (e) DEAD, PPh3,THF, phthalimide, rt; (f) NH2NH2.H2O, MeOH, 60C; (g) D-tartaric acid, EtOH, EtOAc.
Scheme 5. Reagents and conditions: (a) O-tert- butyl N,N′-diidopropylisourea, CH2Cl2, reflux; (b) (1S,2R)-2-(dibutylamino)-1-phenylpropan-1-ol, Et2Zn, hexane,
toluene, 0C; (c) DEAD, PPh3, THF, phthalimide, rt; (d) NH2NH2H2O, MeOH, 60C; (e) D-tartaric acid, EtOH, EtOAc.

Scheme 6. Reagents and conditions: (a) In(OTf)3,LiClO4, (EtCO)2O,MeNO2, 50°C; (b) O-tert- butyl N,N′-diidopropylisourea, CH2Cl2, reflux; (c) (-)-Ipc2BCl,
THF, -20C; (d )DEAD, PPh3, THF, phthalimide, rt; (e) NH2NH2H2O, MeOH, 60C; (f) D-tartaric acid, EtOH, EtOAc.
The amine for the preparation of 16G was synthesized from (R)-
2-(tert-butoxycarbonylamino)butyric acid (37), as shown in
Scheme 7. Condensation of 37 with (L)-serine methyl ester
hydrochloride, followed by cyclization using DAST20 and
dehydrogenation using bromotrichloromethane, provided 39.
After hydrolysis of the methyl ester moiety, obtained carboxylic
acid 40 was condensed with trichloroethanol to obtain 41.
Deprotection of the Boc group provided desired amine 42.
The amine used the for preparation of 16H was synthesized from
(R)-1-amino-1-thioxobutane-2-yl carbamic acid tert-butyl ester
(43), as shown in Scheme 8. Condensation of 43 with ethyl
bromopyruvate, followed by dehydroxylation using TFAA,
provided 45. The desired amine was then synthesized in a similar
manner to Scheme 7 from 45, as shown in Scheme 8.
Scheme 7. Reagents and conditions: (a) (L)-serine methyl ester HCl, EDCI HCl, HOBt, DIPEA, CH2Cl2, rt; (b) DAST, CH2Cl2, -20C, then
bromotrichloromethane, DBU, 0C; (c) aq. NaOH, MeOH, rt; (d) 2,2,2-trichloroethanol, EDCI HCl, 4-DMAP, CH2Cl2, rt; (e) 4M HCl/EtOAc, rt.
Scheme 8. Reagents and conditions: (a) ethyl bromopyruvate, KHCO3, DME, rt; (b)TFAA, pyridine, DME, 0C; (c) aq. NaOH, EtOH, rt; (d) 2,2,2-
trichloroethanol, EDCI HCl, 4-DMAP, CH2Cl2, rt; (e) 4M HCl/EtOAc, rt.

3. Results and discussion
It was first considered that conversion to a heteroaromatic ring
from a benzene ring in the prime site would lead to an additional
interaction of the heteroatom with the enzyme, for example, with
the carbonyl oxygen of Phe41. This would increase the affinity of
the inhibitor for the chymase. Table 1 shows the inhibitory
activities of the heteroaromatic derivatives thus designed.
The X-ray crystal structure of compound 1 within the structure
of human chymase is shown in Figure 2. In this configuration,
the benzyl unit and the ethyl unit are placed in S1 site and S1′ site,
respectively. There is a hydrogen bond between the amide
nitrogen of the seven-membered ring and the carbonyl oxygen of
Ser214.The anthranilic acid unit is located in the prime site. The
aromatic ring interacts hydrophobically with the cleft, and the
carboxyl group and the amino group interact with Arg143 by
ionic interaction and with the carbonyl oxygen of Phe41 by
hydrogen-bonding, respectively. This ionic interaction could be
responsible for the increase in affinity to human chymase that we
previously reported.9 However, the electron density of the
carboxyl group is unclear, as shown in Figure 3. This was
thought to be due to its flexibility within the prime site. We
therefore considered that there was still room for improvement in
the interaction of human chymase with compound 1, especially in
the prime site. In order to achieve this, we focused on
modification of that particular site, and designed heteroaromatic
derivatives and amino acid derivatives based on the X-ray crystal
structure.
Compounds 16C, 16D, and 16F showed potent activity, whereas,
the other derivatives were less effective. It was speculated that
the heteroaromatic rings did not interact directly with the
chymase in the manner that was predicted, but rather fixed the
configuration of the carboxyl group, leading to increased activity
due to an interaction with Lys40 and/or Arg143 in the S2′ site.
Figure 2. X-ray crystal structure of compound 1in the active site of human
chymase.
Figure 3.Electron density map of compound 1 on the crystal with human
chymase(contoured at 1 sigma).

Table 1. Inhibitory activity of heteroaromatic derivatives against recombinant human chymase.
It was next considered that an effective interaction with Lys40
would provide increased affinity compared with an interaction
with Arg143, as in compound 1.9 Therefore, we investigated
amino acid derivative rac-17 which also might show
improvement of pharmacokinetics profile due to smaller size.
Figure 4 shows the chymase inhibitory activity of rac-17 and the
X-ray cocrystal structure of (R)-17 (R form at 6-position of the
1,4-diazepane core) with human chymase. In the S1 and S1′ sites,
the benzyl unit and the ethyl unit show similar modes of
interaction to 1. From a standpoint of stereochemistry effect, R
form at 6-position of 1,4-diazepane core was more efficient than
S form for our inhibitor because (R)-17 showed more potent
inhibitory activity than rac-17. Referring to X-ray crystal
structure shown in Figure 4, it was supposed that if the benzyl
unit of S form derivative made favorable interaction with S1 site,
1,4-diazepane and prime site could not interact with enzyme well.
Furthermore, it was speculated that the carboxyl group interacted
with the amino group of Lys40; however, it was considered that
the configuration was not sufficient and that the interaction did
not compensate for the large desolvation penalty, and so, did not
contribute to an increase in affinity to human chymase.
It was therefore considered that increasing the enzyme–inhibitor
affinity by enhancing the interaction between Lys40 and the
amino acid unit was not a useful strategy. A different approach
was subsequently investigated that involved extension from the
carboxyl group by amide bond formation. This was hypothesized
to cause an increase in affinity owing to hydrophobic and/or ionic
interaction with the S2′ site, as previously described for
compound 1. Therefore, 18A–H were designed and synthesized,
and the inhibitory activities of the compounds are summarized
in Table 2. 18A–H showed moderate activity; however, no
further increase in activity was demonstrated. These results
indicated that both the hydrophobic and ionic interactions were
needed to achieve potent activity.
Figure 4. Inhibitory activity against recombinant human chymase of rac-17 (left); X-ray crystal structure of (R)-17 in the active site of human chymase (right).

Table 2. Inhibitory activity of amino acid derivatives against recombinant human chymase.
The introduction of a carboxyl group on the phenyl group of
18E was next attempted, with the intention of enhancing
interaction with the S2′site. Additionally, introduction of an
amino group to the phenyl group was also undertaken, similar
to another interaction of compound 1 with the S2′ site. As
shown in Table 3, 18I–M, which were all designed according
the aforementioned strategies, showed improvements in
activity, with 18I being the only exception. With the aim of
achieving a further enhancement in activity, structures that
combined each of the important interactions that had been
identified were designed (18N–Q). These compounds all
exhibited potent activity; in particular, 18P showed
comparable activity to 1.
In a separate approach, replacemant of the carboxyl group
with a tetrazolyl group, a known bioisostere, was carried out,
producing 18R, which showed comparable activity to 1.
According to the X-ray crystal structure shown in Figure 5, in
the case of (R)-18R (R form of 18R at 6-position of 1,4-
diazepane core), the tetrazolyl group is not placed in a cleft of
the prime site, making contact with the hydrophobic side
chain of Lys40 and the methyl group of Thr36. The potent
activity of 18R was assumed to be due to a different type of
interaction in the prime site compared to compounds 1 and
18P.

Table 3. Inhibitory activity of amino acid derivatives against recombinant human chymase.
Figure 5. X-ray crystal structure of (R)-18R in the active site of human chymase.
Several potent activity inhibitors were obtained overall, and
furthermore, they showed selectivity against other serine
proteases, including bovine α-chymotrypsin, human cathepsin G,
and human elastase, as shown in Table 4.

Table 4. IC50 (M) values of inhibition of human chymase and other serine
proteinases for 1, 16C, 16D, 18J, 18L, and 18R.
hydrogensulfate aqueous solution, distilled water, saturated
sodium hydrogencarbonate aqueous solution, and saturated saline,
dried over with anhydrous sodium sulfate, then concentrated. The
residue was dried in vacuo to obtain the title compound (11.8 g).
1H NMR (CDCl3) δ: 7.21-7.26 (m, 2H), 6.99 (s, 1H), 6.81 (d, J =
8.53 Hz, 1H), 6.41 (s, 1H), 5.62-5.66 (m, 1H), 3.81 (s, 3H), 3.75
(s, 3H), 2.11 (s, 3H); MS (ESI): 298 (M+H)+
5.1.4. (2E)-2-(Aminomethyl)-3-(5-chloro-2-
methoxyphenyl)-2-propenoic acid (6)
To the compound 4 (11.8 g) in dimethylsulfoxide (70 ml)
solution, sodium azide (3.9 g) was added and the mixture was
stirred at room temperature for 30 minutes. Distilled water was
added to the reaction solution, and the mixture was extracted
with diethylether. The organic layer was successively washed
with distilled water and saturated saline, dried over with
anhydrous sodium sulfate, and concentrated. The residue was
dried in vacuo to obtain the compound 5 (10.1 g). To the
compound 5 (10 g) in tetrahydrofuran (70 ml) solution,
triphenylphosphine (9.4 g) and distilled water (1 ml) were added
and the mixture was stirred at room temperature for 15 hours.
Next, tetrahydrofuran was distilled off in vacuo, methanol (70
ml) and 2N sodium hydroxide aqueous solution (35 ml) were
added to the remaining mixture, and the mixture was stirred at
room temperature for 2 hours. Next, the methanol was distilled
off in vacuo and the remaining aqueous layer was washed with
ethyl acetate. Further, the aqueous layer was neutralized by
hydrochloric acid, then precipitate was collected by filtration,
was washed with diethylether, and was dried in vacuo to obtain
4. Conclusion
A novel set of human chymase inhibitors were explored, which
included a heteroaromatic moiety and an amino acid moiety, and
their structure activity relationships and interaction modes were
investigated using X-ray crystal structure analysis. Several active
and selective inhibitors were obtained, with (R)-18R exhibiting
potent inhibitory activity comparable to SUN13834, which has
previously shown efficacy in treating humans with atopic
dermatitis, and in addition, demonstrating a new mode of
interaction.
5. Experimental section
5.1. Chemistry
5.1.1. Genaral
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 (d) are
reported in parts per million relative to the internal standard
tetramethylsilane. Electro-spray ionization (ESI) mass spectra
were recorded on Agilent G1956A MSD spectrometer system.
Other chemical reagents and solvents were purchased from
Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries,
Kanto Kagaku or Nacalai tesque and used without purification.
Flash column chromatography was performed using Merck Silica
Gel 60 (230-400 mesh) or Purif-Pack_ SI 30um supplied by
Shoko Scientific. Preparative TLC was performed using Merck
PTLC Glass Plates Si 60 20 x 20cm, 0.5mm or 1mm thick.
1
the compound 6 (6.3 g). H NMR (Acetic acid-d4) : 8.07 (1 H,
s), 7.41 (1H, dd, J = 2.6, 8.8 Hz), 7.34 (1H, d, J = 2.6 Hz), 7.03
(1H, d, J = 8.8 Hz), 4.07 (2H, s); MS (ESI): 242 (M+H)+
5.1.5. (2E)-2-{[(tert-
Butoxycarbonyl)amino]methyl}-3-(5-chloro-2-
methoxyphenyl)-2-propenoic acid (7)
To the compound 6 (15 g) in tetrahydrofuran (300 ml)
suspension, 2N sodium hydroxide aqueous solution (70 ml) and
di-tert-butyldicarbonate (15 g) were added and the mixture was
stirred at room temperature for 1 hour. Next, tetrahydrofuran was
distilled off in vacuo, the obtained aqueous mixture was acidified
by adding saturated potassium hydrogensulfate aqueous solution,
then the mixture was extracted with ethyl acetate. The organic
layer was washed with saturated saline and dried over with
anhydrous sodium sulfate and concentrated. The residue was
recrystallized by ethyl acetate/hexane to obtain the title
5.1.2. Methyl2-[(5-chloro-2-
methoxyphenyl)(hydroxy)methyl]propenoate (3)
A reaction mixture of 5-chloro-2-methoxybenzaldehyde (7 g),
methyl acrylate (6 ml), 1,4-diazabicyclo[2.2.2]octane (4.6 g),
lanthanum trifluoromethanesulfonate (1.2 g), and diethanol
amine (2.7 ml) was stirred at room temperature for 60 hours.
Distilled water and saturated potassium hydrogensulfate aqueous
solution were added to the reaction solution, and the mixture was
extracted with ethyl acetate. The organic layer was successively
washed with distilled water and saturated saline, dried over with
anhydrous sodium sulfate, then concentrated. The residue was
1
compound (19.8 g). H NMR (CDCl3) 7.93 (0.5H, br), 7.78
(0.5H, br), 7.42 (0.5H, br), 7.30 (1H, dd, J = 8.8, 2.4 Hz), 7.19
(0.5H, br), 6.84 (1H, d, J= 8.8 Hz), 6.76 (0.5H, br), 5.12 (0.5H,
br), 4.14 (2H, br), 3.84 (3H, s), 1.55-1.15 (9H, m); MS: 364
(M+Na)+
5.1.6. [((2E)-3-(5-Chloro-2-methoxyphenyl)-2-
{[(2,4,6-trimethoxybenzyl)amino]methyl}-2-
propenyl)amino]ethyl acetate hydrochloride (9)
1
dried in vacuo to obtain the title compound (11.1 g). H NMR
(CDCl3) 7.37 (1H, d, J = 2.9 Hz), 7.22 (1H, dd, J = 8.7, 2.9
Hz), 6.8 (1H, d, J = 8.7 Hz), 6.31 (1H, m), 5.83 (1H, d, J = 5.8
Hz), 5.69 (1H, m), 3.81 (3H, s), 3.77 (3H, s); MS (ESI): 239 (M-
OH)+
To the compound 7 (123 g) in methylene chloride (400 ml)
solution, glycine methyl ester hydrochloride (51 g), 1-
hydroxybenzotriazole (49 g), triethylamine (53 ml), and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (76 g)
were added under ice cooling and the mixture was stirred at room
temperature for 2 hours. The reaction solution was diluted with
distilled water, then the precipitate was filtered out and the
filtrate was extracted with ethyl acetate. The organic layer was
successively washed with saturated potassium hydrogensulfate
aqueous solution, distilled water, saturated sodium
hydrogencarbonate aqueous solution, and saturated saline, dried
over with anhydrous sodium sulfate, then concentrated. The
5.1.3. Methyl2-[(5-chloro-2-
methoxyphenyl)(acetoxy)methyl]propenoate (4)
To the compound 3 (11 g) in methylene chloride (100 ml)
solution, pyridine (3.5 ml) and acetylchloride (3.1 ml) were
added under ice cooling and the mixture was stirred at that
temperature for 1 hour. Distilled water was added to the reaction
solution, methylene chloride was distilled off in vacuo, and the
remaining aqueous layer was extracted with ethyl acetate. The
organic layer was successively washed with saturated potassium

residue was recrystallized by hexane/ethyl acetate. A mixed
solution of the obtained compound (131.5 g) and a 4M hydrogen
chloride/ethyl acetate solution (350 ml) was stirred at room
temperature for 20 minutes. The reaction solution was diluted
with diethylether, then the precipitate was collected by filtration
to obtain the compound 8 (109.3 g). To the compound 8 (55.8 g)
was filtered out and the filtrate was concentrated. The residue
was purified by silica gel column chromatography
(chloroform/ethyl acetate/methanol=7/1/0.5) to obtain the title
1
compound (13.1 g). H NMR (CDCl3) : 7.14 (1H, dd, J = 8.7,
2.5 Hz), 6.88 (1H, d, J = 2.5 Hz), 6.69 (1H, d, J = 8.7 Hz), 5.98
(2H, s), 5.94 (1H, br), 4.8 (1H, d, J = 13.7 Hz), 4.30-4.20 (2H, m),
3.83 (3H, s), 3.78-3.69 (4H, m), 3.63 (6H, s), 3.38 (1H, dd, J =
15.4, 12.2 Hz), 3.11 (1H, dd, J = 13.0, 3.2 Hz), 2.94 (1H, dd, J =
15.4, 4.9 Hz), 2.5 (1H, dd, J = 13.0, 10.7 Hz); MS (ESI): 461
(M+H)+
in
tetrahydrofuran
(800
ml)
solution,
2,4,6-
trimethoxybenzaldehyde (30.5 g) was added and the mixture was
stirred at room temperature for 20 minutes. Next, sodium
triacetoxy borohydride (50 g) was added to the reaction solution
and the mixture was stirred at room temperature for 1 hour.
Distilled water was added to the reaction solution, then
tetrahydrofuran was distilled off in vacuo. The remaining
aqueous layer was basified by a sodium hydroxide aqueous
solution, then the mixture was extracted with ethyl acetate. The
organic layer was washed with saturated saline, dried over with
anhydrous sodium sulfate, and partially distilled off in vacuo. A
4M hydrogen chloride/ethyl acetate solution was added to the
remaining solution and the mixture was stirred under ice cooling
for 30 minutes. The precipitate was collected by filtration to
5.1.9. (6R)-6-(5-Chloro-2-methoxybenzyl)-1-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-2,5-dione
tetrahydrofuran solvate((R)-12)
The compound rac-12 was separated using CHIRALCEL OD-
H
(Daicel
Chemical
Industries) (movement phase:
100/0.1) .The obtained
acetonitrile/trifluoroacetic acid
=
compound was diluted with tetrahydrofuran, and the resultant
suspension was stirred at 60 C until the solution became clear.
Then, the solution was gradually cooled to room temperature,
and the appeared precipitate was collected by filtration to
1
obtain the title compound (75.7 g). H NMR (DMSO-d6) : 9.12
1
obtained the title compound which contained 82% free form. H
(1H, br), 8.58 (1H, br), 7.59 (1H, s), 7.48 (1H, d, J = 8.9 Hz),
7.32 (1H, s), 7.13 (1H, d, J = 8.9 Hz), 6.23 (2H, s), 4.13 (2H, q, J
= 7.1 Hz), 3.96 (2H, d, J = 5.7 Hz), 3.84-3.70 (16H, m), 1.21 (3H,
t, J = 7.1 Hz); MS (ESI): 507 (M+H)+
NMR (CDCl3) : 7.14 (1H, dd, J = 2.4, 8.5 Hz), 6.88 (1H, d, J =
2.4 Hz), 6.70 (1H, d, J=8.5 Hz), 5.98 (2H, s), 4.81 (1H, d, J =
13.4 Hz), 4.27 - 4.20 (2H, m), 3.83 (3H, s), 3.77 - 3.73 (4H, m),
3.72 (3H, s), 3.63 (6H, s), 3.39 (1H, dd, J = 12.4, 15.2 Hz), 3.12
(1H, dd, J=3.4, 13.2 Hz), 2.94 (1H, dd, J = 4.7, 15.2 Hz), 2.54 -
2.38 (2H, m), 1.89 - 1.82 (4H, m); MS (ESI): 461 (M+H)+
5.1.7. (6E)-6-(5-Chloro-2-methoxybenzylidene)-1-
(2,4,6-trimethoxybenzyl)-1,4-diazepan-2,5-dione
(11)
5.1.10. 2-Chlorophenyl 6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-carboxylate
(rac-13)
To the compound 9 (148.5 g) in methanol (300 ml) solution,
2N sodium hydroxide aqueous solution (300 ml) was added and
the mixture was stirred at room temperature for 2 hours. The
reaction solution was neutralized by 6M hydrochloric acid (100
ml), then the methanol was distilled off in vacuo. Crystal nuclei
were added to the remaining solution and the mixture was stirred
under ice cooling for 30 minutes. The precipitate was collected
by filtration to obtain the compound 10 (143.1 g). A solution of
the compound 10 (53 g) and 1-hydroxybenzotriazole (14 g) in
N,N-dimethylformamide (1000 ml) was added dropwise to a
solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (24 g) and triethylamine (17 ml) in N,N-
dimethylformamide (500 ml) over 2 hours. After dropping, the
insoluble compound was filtered out, then the filtrate was
concentrated. Ethyl acetate and 1N hydrochloric acid were added
to the residue and the mixture was stirred for 30 minutes. The
insoluble compound was collected by filtration and washed with
distilled water and ethyl acetate. Ethyl acetate and 1N sodium
hydroxide aqueous solution were added to the obtained solid and
stirred for 30 minutes. The insoluble compound was collected by
filtration and washed with distilled water and ethyl acetate. A
mixed solvent of tetrahydrofuran/methanol=1/1 was added to the
obtained solid and the mixture was stirred under heating and
reflux for 30 minutes. The solution was allowed to cool to room
temperature, then the mixed solution was filtered by sellite and
the filtrate was concentrated. The residue was recrystallized by
ethyl acetate to obtain the title compound (22.1 g). 1H NMR
(CDCl3) : 7.6 (1H, s), 7.29-7.19 (1H, m), 6.76-6.69 (2H, m),
6.05 (1H, br), 5.75 (2H, s), 4.52 (2H, s), 4.23 (2H, s), 4.05 (2H, d,
J = 6.1 Hz), 3.80-3.74 (6H, m), 3.52 (6H, s)
To the compound rac-12 (4.07 g) in tetrahydrofuran (200 ml)
solution, a 1.59M hexane solution of n-butyllithium (6 ml) was
added at -78C and the mixture was stirred at that temperature for
20 minutes. Next, 2-chlorophenyl chlorocarbonate (1.4 ml) was
added to the reaction solution at -78C and the mixture was
stirred at that temperature for 20 minutes. The reaction solution
was diluted with saturated potassium hydrogensulfate aqueous
solution and distilled water, and the mixture was extracted with
ethyl acetate. The organic layer was successively washed with
saturated saline and distilled water, dried over with anhydrous
sodium sulfate, then concentrated. The residue was purified by
silica gel column chromatography (hexane/ethyl acetate/methanol
1
= 1/2/0 to 3/3/1) to obtain the title compound (4.21 g). H NMR
(CDCl3) : 7.44-7.40 (1H, m), 7.33-7.12 (4H, m), 6.93 (1H, d, J
= 2.6 Hz), 6.73 (1H, d, J = 8.8 Hz), 6.05 (2H, s), 5.06 (1H, d, J =
17.6 Hz), 4.83 (1H, d, J = 13.7 Hz), 4.44 (1H, d, J = 17.6 Hz),
4.34 (1H, d, J = 13.7 Hz), 3.83 (3H, s), 3.77 (3H, s), 3.7 (6H, s),
3.57-3.45 (1H, m), 3.29-3.14 (2H, m), 3.07 (1H, dd, J = 14.1, 3.8
Hz), 2.38 (1H, dd, J = 14.1, 9.8 Hz); MS (ESI): 617 (M+H)+
5.1.11. (6R)- 2-Chlorophenyl 6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-4-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-1-carboxylate
((R)-13)
To the compound (R)-12 (8.2g: net weight of free form) in
dimethoxyethane (200 ml) solution cooled at -20C, n-BuLi (12.2
ml)(1.60M hexane solution) was added , and the mixture was
stirred at that temperature for a few minutes. Then, 2-
chlorophenyl chloroformate (2.7 ml) was added to the mixture at
-20C, and the resultant mixture was subsequently stirred at that
temperature for 1 hour. Then, saturated ammonium chloride
aqueous solution was added to the reaction solution, the resultant
solution was extracted with ethyl acetate. Then, the organic layer
was successively washed with saturated saline, dried over with
5.1.8. 6-(5-Chloro-2-methoxybenzyl)-1-(2,4,6-
trimethoxybenzyl)-1,4-diazepan-2,5-dione (rac-
12)
To the compound 11 (16.3 g) in tetrahydrofuran (600 ml)
solution, 2% platinum carbon (sulfur poisoned catalyst) (7.3 g)
was added and the mixture was stirred under hydrogen
atmosphere at room temperature for 60 hours. Next, the catalyst

anhydrous sodium sulfate, then concentrated. The residue was
purified by silica gel column chromatography (hexane/ethyl
acetate = 1/2, then ethyl acetate/methanol = 10/1) to obtain the
title compound (1.29 g). 1H NMR (CDCl3) : 7.46 - 7.42 (1H, m),
7.32 - 7.25 (2H, m), 7.25 - 7.17 (2H, m), 6.95 (1H, d, J = 2.4 Hz),
6.76 (1H, d, J = 8.9 Hz), 6.07 (2H, s), 5.08 (1H, d, J = 17.9 Hz),
4.84 (1H, d, J = 13.8 Hz), 4.46 (1H, d, J = 17.0 Hz), 4.36 (1H, d,
J = 13.8 Hz), 3.85 (3H, s), 3.79 (3H, d, J = 5.3 Hz), 3.72 (6H, s),
3.55 - 3.46 (1H, m), 3.28 - 3.16 (2H, m), 3.12 - 3.05 (1H, m, J =
3.7 Hz), 2.40 (1H, dd, J = 9.7, 13.8 Hz)
procedure as in 5.1.13 to obtain the title compound. Yield:
34.3%; 1H NMR (DMSO-d6) : 9.75 (1H, d, J = 7.6 Hz), 8.75
(1H, d, J = 4.9 Hz), 7.81 (1H, brs), 7.75-7.68 (2H, m), 7.34 (1H,
d, J = 2.7 Hz), 7.27 (1H, dd, J = 8.8, 2.7 Hz), 7.01 (1H, d, J = 8.8
Hz), 4.96 (1H, dd, J = 14.1, 7.0 Hz), 4.77 (1H, d, J = 17.2 Hz),
4.50 (1H, d, J = 17.2 Hz), 3.94-3.80 (1H, m), 3.79 (3H, s), 3.17
(1H, t, J = 13.0 Hz), 3.07-2.97 (2H, m), 2.72-2.64 (1H, m), 1.92-
1.80 (2H, m), 0.80 (3H, t, J = 7.4 Hz); MS (ESI): 489 (M+H)+
5.1.15. 5-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]nicotinic acid (16C)
5.1.12. (6R)- 2-Chlorophenyl 6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
carboxylate ((R)-14)
To the compound (R)-13 (500 mg) in N,N-dimethylformamide
(2.5 ml) solution, the compound 25c (313 mg) , triethylamine
(113 l) and 4-dimethylaminopyridine (99 mg) were added under
ice cooling and the mixture was stirred at that temperature for 20
hours. Then, distilled water and ethyl acetate were added to the
reaction mixture, the organic layer and the aqueous layer were
separated. Then, the organic layer was successively washed with
saturated saline, dried over with anhydrous sodium sulfate, then
concentrated. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 1/1 to 1/2) to obtain the
compound 15C (518 mg). To the compound 15C (516 mg), 1M
hydrogen chloride/acetic acid solution (5 ml) was added , the
mixture was stirred at room temperature for 14 hours. Then, the
mixture was concentrated, the residue was purified by silica gel
column chromatography (ethyl acetate/acetic acid/methanol =
20/1, then ethyl acetate/acetic acid/methanol = 20/1/2) to obtain
Anisole (2.3 ml) and trifluoroacetic acid (10 ml) were added
to the compound (R)-13 (1.29 g), and the mixture was stirred at
room temperature for 2 hours. Then, the reaction mixture was
concentrated. The residue was purified by silica gel column
chromatography (hexane/ethyl = 1/1 to 1/99). The obtained solid
was suspended with diethyl ether, and subsequently collected by
filtration to obtaine the title compound (595 mg). 1H NMR
(CDCl3) : 7.91 (1H, d, J = 4.5 Hz), 7.63 - 7.60 (1H, m), 7.45 -
7.41 (2H, m), 7.39 - 7.32 (2H, m), 7.28 (1H, dd, J = 2.6, 8.7 Hz),
7.03 (1H, d, J = 8.9 Hz), 4.83 (1H, d, J = 17.5 Hz), 4.50 (1H, d, J
= 17.5 Hz), 3.89 - 3.83 (1H, m), 3.81 (3H, s), 3.30 - 3.22 (1H, m),
3.13 - 2.96 (2H, m), 2.63 (1H, dd, J = 8.9, 14.2 Hz); MS (ESI):
438 (M+H)+
5.1.13. 6-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]nicotinic acid (16A)
1
the title compound (118 mg). H NMR (DMSO-d6) : 9.48 (1H,
d, J = 7.1 Hz), 8.93 (1H, d, J = 1.8 Hz), 8.71 (1H, brs), 8.17 (1H,
s), 7.67 (1H, brd, J = 3.8 Hz), 7.34 (1H, d, J = 2.7 Hz), 7.27 (1H,
dd, J = 8.7, 2.7 Hz), 7.01 (1H, d, J = 8.7 Hz), 4.79 (1H, dd, J =
14.2, 7.0 Hz), 4.69 (1H, d, J = 17.1 Hz), 4.49 (1H, d, J = 17.1
Hz), 3.92-3.80 (1H, m), 3.79 (3H, s), 3.17 (1H, t, J = 12.9 Hz),
3.07-2.97 (2H, m), 2.78-2.65 (1H, m), 1.95-1.78 (2H, m), 0.87
(3H, t, J = 7.3 Hz); MS (ESI): 489 (M+H)+; Melting point: 138-
140C
To the compound (R)-13 (509m g) in N,N-dimethylformamide
(2.5 ml) solution, the compound 25a (319 mg) , triethylamine
(0.23 ml) and 4-dimethylaminopyridine (101 mg) were added
under ice cooling and the mixture was stirred at that temperature
for 16 hours. Then, saturated ammonium chloride aqueous
solution and ethylacetate were added to the reaction mixture, the
organic layer and the aqueous layer were separated. Then, the
organic layer was successively washed with saturated saline,
dried over with anhydrous sodium sulfate, then concentrated. The
residue was purified by silica gel column chromatography
(hexane/ethyl acetate = 1/3) to obtain the compound 15A (0.58 g).
To the compound 15A (0.57 g) in dichloromethane (11 ml)
solution, water (1.1 ml) and DDQ (0.36 g) were added , and the
mixture was stirred at room temperature for 20 hours. Then, the
mixture was filtrated through NH-silica gel (ethyl acetate), and
the filtrate was concentrated. The residue was purified by silica
gel column chromatography (hexane/ethyl =1/2 to 0/1). To the
obtained compound (221mg), 1M HCl/AcOH solution (2.2ml)
was added, and the mixture was stirred at room temperature for 4
hours. Then, the mixture was concentrated, and acetone and ethyl
acetate were added to the residue. The appeared precipitate was
5.1.16. 5-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]-2-furan carboxylic
acid (16D)
To the compound rac-13 (2.8 g) in N,N-dimethylformamide
(14 ml) solution, the compound 31 (1.7 g) , triethylamine (0.63
ml) and 4-dimethylaminopyridine (0.55 g) were added under ice
cooling and the mixture was stirred at that temperature for 16
hours. Then, distilled water and ethyl acetate were added to the
reaction mixture, the organic layer and the aqueous layer were
separated. Then, the organic layer was successively washed with
saturated saline, dried over with anhydrous sodium sulfate, then
concentrated. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate =1/2) to obtain the
compound 15D (1.02 g). To the compound 15D (1.02 g), 1M
hydrogen chloride/acetic acid solution (10 ml) was added , the
mixture was stirred at room temperature for 14 hours. Then, the
mixture was concentrated, the residue was purified by silica gel
1
collected by filtration to obtain the title compound (108 mg). H
NMR (DMSO-d6) : 9.75(1H, d, J = 7.5 Hz), 9.04 (1H, d, J =
2.1Hz), 8.25 (1H, dd, J = 8.1, 2.1 Hz), 7.70 (1H, brd, J = 3.9 Hz),
7.53 (1H, d, J = 8.1 Hz), 7.34 (1H, d, J = 2.7 Hz), 7.27 (1H, dd, J
= 8.7, 2.7 Hz), 7.01 (1H, d, J = 8.7 Hz), 4.94 (1H, dd, J = 14.1,
7.0 Hz), 4.77 (1H, d, J = 17.3 Hz), 4.51 (1H, d, J = 17.3 Hz),
3.94-3.82 (1H, m), 3.80 (3H, s), 3.17 (1H, t, J = 12.5 Hz), 3.08-
2.98 (2H, m), 2.72-2.64 (1H, m), 1.92-1.80 (2H, m), 0.80 (3H, t,
J = 7.4 Hz); MS (ESI): 489 (M+H)+
column
chromatography
(chloroform/ethyl
acetate/methanol/acetic acid = 4/4/1/0.1) to obtain the title
1
compound (284 mg). H NMR (DMSO-d6) : 9.35 (1H, d, J =
7.9 Hz), 7.69-7.60 (1H, br), 7.30 (1H, s), 7.24 (1H, d, J = 8.7 Hz),
7.11 (1H, d, J = 3.4 Hz), 6.97 (1H, d, J = 8.7 Hz), 6.46 (1H, d, J
= 3.4 Hz), 4.85 (1H, dd, J = 14.4, 7.1 Hz), 4.74 (1H, d, J = 17.3
Hz), 4.50 (1H, d, J = 17.3 Hz), 3.89-3.78 (1H, m), 3.76 (3H, s),
3.17-2.88 (3H, m), 2.65-2.57 (1H, m), 1.89-1.77 (2H, m), 0.84
(3H, t, J = 7.3 Hz); MS (ESI): 478 (M+H)+
5.1.14. 2-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]isonicotinic acid (16B)
Instead of the starting material compound of 5.1.13, that is,
the compound 25a, the compound 25b was used for the similar

5.1.17. 5-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]-3-furan carboxylic
acid (16E)
were added under ice cooling and the mixture was stirred at that
temperature for 3 days. Then, distilled water was added to the
reaction solution, the resultant mixture was extracted with ethyl
acetate. The organic layer was successively washed with
saturated potassium hydrogensulfate aqueous solution, saturated
sodium hydrogencarbonate aqueous solution and saturated saline,
and dried over with anhydrous sodium sulfate, then concentrated.
The residue was purified by silica gel column chromatography
(hexane/ethyl acetate = 1/1 to 0/100) to obtain the compound
15H (46 mg). 1M hydrogen chloride acetic acid solution (2 ml)
and anisole (89 ml) were added to the compound 15H, the
mixture was stirred at room temperature for 40 hours. Then, the
mixture was concentrated, the residue was purified by silica gel
column chromatography (chloroform/ethyl acetate/methanol =
15/15/1). To the obtained compound (42 mg) in acetic acid (0.8
ml) solution, zinc (80 mg) was added, and the mixture was stirred
at room temperature for 25 hours. Then, insoluble compound was
removed by filtration, and the filtrate was subsequently
concentrated. The residue was purified by preparative TLC
(chloroform/ethyl acetate/methanol = 10/10/1) to obtain the title
Instead of starting material compound of 5.1.13, that is, the
compound 25a, the compound 36a was used for the similar
procedure as in 5.1.13. to obtain the title compound. Yield:
40.6%; 1H NMR (DMSO-d6) : 12.71-12.59 (1H, br), 9.36 (1H,
d, J = 8.1 Hz), 8.20 (1H, brs), 7.69 (1H, brd, J = 3.9 Hz), 7.33
(1H, d, J = 2.6 Hz), 7.27 (1H, dd, J = 8.8, 2.6 Hz), 7.01 (1H, d, J
= 8.8 Hz), 6.54 (1H, s), 4.85 (1H, dd, J = 14.7, 7.1 Hz), 4.78 (1H,
d, J = 17.1 Hz), 4.52 (1H, d, J = 17.1 Hz), 3.91-3.81 (1H, m),
3.79 (3H, s) 3.15 (1H, t, J = 12.5 Hz), 3.07-2.92 (2H, m), 2.68-
2.57 (1H, m), 1.89-1.75 (2H, m), 0.86 (3H, t, J = 7.3 Hz); MS
(ESI): 478 (M+H)+
5.1.18. 5-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]-3-thiophencarboxylic
acid (16F)
Instead of starting material compound of 5.1.13, that is, the
compound 25a, the compound 36b was used for the similar
procedure as in 5.1.13. to obtain the title compound. Yield:
1
compound (14 mg). H NMR (CDCl3) : 9.62 - 9.53 (1H, m),
7.71 (1H, brs), 7.35 (1H, brs), 7.28 (1H, dd, J = 2.4, 8.5 Hz), 7.01
(1H, d, J = 8.5 Hz), 5.19 - 5.04 (1H, m), 4.80 - 4.67 (1H, m), 4.67
- 4.50 (1H, m), 3.94 - 3.84 (1H, m), 3.80 (3H, s), 3.48 - 3.37 (1H,
m), 3.23 - 3.15 (1H, m), 3.03 - 2.95 (2H, m), 2.74 - 2.62 (1H, m),
2.08 - 1.91 (2H, m), 0.94 - 0.89 (3H, m); MS (ESI): 495 (M+H)+
1
44.3%; H NMR (DMSO-d6) : 12.80-12.65 (1H, br), 9.42 (1H,
d, J = 7.8 Hz), 8.09 (1H, brs), 7.69 (1H, brd, J = 3.5 Hz), 7.33
(1H, d, J = 2.6 Hz), 7.30-7.24 (2H, m), 7.01 (1H, d, J = 8.8 Hz),
4.97 (1H, dd, J = 14.3, 7.1 Hz), 4.77 (1H, d, J = 17.1 Hz), 4.53
(1H, d, J = 17.1 Hz), 3.93-3.84 (1H, m), 3.79 (3H, s) 3.15 (1H, t,
J = 12.5 Hz), 3.07-2.94 (2H, m), 2.68-2.58 (1H, m), 1.94-1.85
(2H, m), 0.89 (3H, t, J = 7.3 Hz); MS (ESI): 494 (M+H)+
5.1.21. (2R)-2-({[6-(5-Chloro-2-methoxybenzyl)-
3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoic acid (rac-17)
To the compound rac-13 (3 g) in N,N-dimethylformamide (75
ml) solution, (R)-tert-butyl 2-aminobutanoate acetate (0.70 g) and
triethylamine (0.45 ml) were added under ice cooling and the
mixture was stirred at that temperature for 3 hours. Then,
distilled water was added to the reaction mixture, and the
resultant mixture was extracted with a mixed solution of
Hexane/EtOAc ( 1/1 ). Then, the organic layer was successively
washed with saturated saline, dried over with anhydrous sodium
sulfate, then concentrated. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 1/1 to 1/99) to
obtain tert-Butyl (2R)-2-({[6-(5-chloro-2-methoxybenzyl)-3,7-
dioxo-1,4-diazepan-1-yl]carbonyl}amino)butanoate (1.20 g). To
the compound (1.20 g) in CH2Cl2 cooled at 0C, TFA (15 ml)
was added, and the reaction mixture was stirred at room
temperature for 7.5 hours. Then, the mixture was concentrated,
and the residue was purified with Florisil column to give a
reddish brown oil. The oil was diluted with ethyl acetate, and the
solution was washed with distilled water and saturated sodium
5.1.19. 2-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]-4-oxazolcarboxylic
acid (16G)
To the compound 14 (173 mg) in N,N-dimethylformamide
(0.4 ml) solution cooled at 0C, the compound 42 (134 mg) , N,
N-diisopropylethylamine (69 l) and 4-dimethylaminopyridine
(37 mg) were added under ice cooling and the mixture was stirred
at that temperature for 15 hours. Then, distilled water was added
to the reaction solution, the resultant mixture was extracted with
ethyl acetate. The organic layer was successively washed with
saturated potassium hydrogensulfate aqueous solution, saturated
sodium hydrogencarbonate aqueous solution and saturated saline,
and dried over with anhydrous sodium sulfate, then concentrated.
The residue was purified by silica gel column chromatography
(chloroform/ethyl acetate = 1/1) to obtain the compound 15G
(188 mg). To the compound 15G (97 mg) in acetic acid (2 ml)
solution, zinc (200 mg) was added , and the mixture was stirred
at room temperature for 3 hours. Then, insoluble compound was
removed by filtration, and the filtrate was subsequently
concentrated. The residue was purified by preparative TLC
(chloroform/ethyl acetate/methanol/acetic acid = 5/5/1/0.1) to
obtain the title compound (7 mg). 1H NMR (CDCl3) : 9.53 (1 H,
d, J = 6.9 Hz), 7.69 (1H, s), 7.34 (1H, s), 7.27 (1H, d, J = 8.5 Hz),
7.01 (1H, d, J = 8.5 Hz), 4.99 - 4.92 (1H, m), 4.76 (1H, d, J =
15.8 Hz), 4.56 (1H, d, J = 17.0 Hz), 3.92 - 3.84 (1H, m), 3.79
(4H, s), 3.50 - 3.36 (1H, m), 3.25 - 3.10 (1H, m), 3.04 - 2.92 (2H,
m), 2.69 - 2.60 (1H, m), 1.99 - 1.80 (2H, m), 0.90 - 0.81 (3H, m);
MS (ESI): 479 (M+H)+
hydrogencarbonate
aqueous
solution.
The
sodium
hydrogencarbonate aqueous layer was acidified with HCl aq., and
the resultant solution was extracted with ethyl acetate. The
organic layer was washed with saturated saline, dried over with
anhydrous sodium sulfate, then concentrated to obtain the title
1
compound (638 mg). H NMR (CDCl3) : 9.45 (0.5H, d, J = 6.7
Hz), 9.44 (0.5H, d, J = 6.5 Hz), 7.24-7.20 (1H, m), 7.19-7.10
(0.5H, m), 7.15 (0.5H, d, J = 2.4 Hz), 7.11 (0.5H, d, J = 2.5 Hz),
6.95 (0.5H, br), 6.817 (0.5H, d, J = 8.8 Hz), 6.813 (0.5H, d, J =
8.7 Hz), 5.38-5.29 (1H, m), 4.48-4.41 (1H, m), 4.23-4.15 (1H, m),
3.84 (3H, s), 3.78-3.65 (1H, m), 3.40-3.33 (2H, m), 3.27-3.15
(1H, m), 2.69-2.58 (1H, m), 2.05-1.80 (2H, m), 1.09-1.00 (3H,
m) ); A NMR spectrum of the compound rac-17 was assigned as
mixture of two diastereomers. If each signal of correspond
protons was separated, the number of proton was assigned as 0.5.
NMR spectra of following diastereomers was assigned in the
same manner.; MS (ESI): 411 (M+H)+
5.1.20. 2-[(1R)-1-({[(6R)-6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)propyl]-4-thiazolcarboxylic
acid (16H)
To the compound (R)-13 (163 mg) in N,N-dimethylformamide
(5 ml) solution cooled at 0C, the compound 48 (93
mg) ,triethylamine (37 l) and 4-dimethylaminopyridine (32 mg)

5.1.22. (2R)-2-({[(6R)-6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoic acid ((R)-17)
solution, saturated saline, saturated sodium hydrogencarbonate
aqueous solution, and saturated saline, dried over with anhydrous
sodium sulfate, then concentrated. The precipitate was washed
with diethylether/hexane and collected by filtration to obtain {[2-
({[6-(5-chloro-2-methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}acetic acid tert-butyl ester
(41.6 mg). A mixed solution of the compound (33 mg) and 1N
hydrogen chloride/acetic acid solution (2 ml) was stirred at room
temperature for 5 hours. The reaction solution was concentrated,
then the residue was diluted with ethyl acetate. The insoluble
compound was filtered out, then the filtrate was concentrated.
The residue was recrystallized by hexane/ethyl acetate to obtain
To the compound (R)-14 (60 mg) in N,N-dimethylformamide
(0.14 ml) solution cooled at 0C, (R)-tert-butyl 2-aminobutanoate
acetate (27 mg), 4-dimethylaminopyridine (17 mg) and N, N-
diisopropylethylamine (0.024 ml) were added under ice cooling
and the mixture was stirred at that temperature for 15 hours. Then,
distilled water was added to the reaction mixture, and the
resultant mixture was extracted with ethyl acetate. Then, the
organic layer was successively washed with saturated potassium
hydrogensulfate
aqueous
solution,
saturated
sodium
1
the title compound (18 mg). H NMR (CDCl3) : 9.42 (0.5H, J =
hydrogencarbonate aqueous solution and saturated saline, dried
over with anhydrous sodium sulfate, then concentrated. The
residue was purified by silica gel column chromatography
(chloroform/ethyl acetate = 1/1) to obtain tert –Buthyl (2R)-2-
({[(6R)-6-(5-chloro-2-methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanate (51 mg). 4M hydrogen chloride
ethyl acetate solution (2 ml) was added to the compound (50 mg),
and the mixture was stirred at room temperature for 4 hours.
Then, the mixture was concentrated. The residue was purified by
d, 7.4 Hz), 9.39 (0.5H, d, J = 6.9 Hz), 7.25-7.05 (3.5H, m), 6.80
(1H, d, J = 8.9 Hz), 6.36 (0.5H, br), 5.28-5.16 (1H, m), 4.41-3.90
(5H, m), 3.83 (3H, s), 3.78-3.68 (1H, m), 3.38-3.30 (2H, m),
3.22-3.14 (1H, m), 3.67-3.55 (1H, m), 2.00-1.90 (1H, m), 1.85-
1.74 (1H, m), 1.05-0.99 (3H, m); MS (ESI): 469 (M+H)+
5.1.25. (2S)-2-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}propanoic
acid (18C)
silica
gel
column
chromatography
(chloroform/ethyl
acetate/methanol/acetic acid = 5/5/1/0.1) to obtain the title
compound (21 mg). 1H NMR (DMSO-d6) : 9.39 (2H, d, J = 6.5
Hz), 7.69 (1H, br), 7.34 (2H, br), 7.27 (2H, d, J = 9.3 Hz), 7.01
(2H, d, J = 8.9 Hz), 4.81 (1H, d, J = 17.0 Hz), 4.54 (1H, d, J =
17.5 Hz), 4.24 - 4.18 (1H, m), 3.89 (1H, br), 3.80 (3H, s), 3.14
(1H, br), 3.09 - 2.93 (2H, m), 2.70 - 2.58 (1H, m), 1.88 - 1.64
(2H, m), 0.86 (3H, t, J = 7.1 Hz); MS (ESI): 412 (M+H)+
Instead of the starting material compound of 5.1.16., that is,
the glycine tert-butyl ester hydrochloride, L-alanine tert-butyl
ester hydrochloride was used for the similar procedure as in
1
5.1.16. to obtain the title compound. Yield: 80.6%; H NMR
(DMSO-d6) : 12.79-12.19 (1H, br), 9.40 (0.5H, d, J = 7.5 Hz),
9.35 (0.5H, d, J = 7.7 Hz), 8.45 (0.5H, d, J = 8.0 Hz), 8.43 (0.5H,
d, J = 8.0 Hz), 7.64 (1H, br), 7.307 (0.5H, s), 7.301 (0.5H, s),
7.24 (0.5H, d, J = 8.8 Hz), 7.23 (0.5H, d, J = 8.8 Hz), 6.97 (1H, d,
J = 8.8 Hz), 4.78 (0.5H, d, J = 17.0 Hz), 4.76 (0.5H, d, J = 17.0
Hz), 4.48 (1H, d, J = 17.0 Hz), 4.35-4.28 (1H, m), 4.25-4.15 (1H,
m), 3.89-3.80 (1H, m), 3.76 (3H, s), 3.15-3.08 (1H, m), 3.03-2.91
(2H, m), 2.65-2.58 (1H, m), 1.72-1.57 (2H, m), 1.23 (3H, d, J =
7.3 Hz), 0.82-0.75 (3H, m); MS (ESI): 483 (M+H)+
5.1.23. 6-(5-Chloro-2-methoxybenzyl)-N-{(1R)-1-
[(methylamino)carbonyl]propyl}-3,7-dioxo-1,4-
diazepan-1-carboxamide (18A)
To the compound rac-17 (54 mg) in methylene chloride (3 ml)
solution, methylamine (30% ethyl acetate solution) (30 μl),
triethylamine (0.27 ml), and n-propyl phosphoric acid anhydride
(25% ethyl acetate solution) (0.39 ml) were added and the
mixture was stirred at room temperature for 4 hours. The reaction
solution was concentrated, then ethyl acetate was added to the
residue. The obtained solution was successively washed with
distilled water, saturated potassium hydrogensulfate aqueous
solution, saturated saline, saturated sodium hydrogencarbonate
aqueous solution, and saturated saline, dried over with anhydrous
sodium sulfate, then concentrated. The precipitate was washed
with diethylether/hexane and collected by filtration to obtain the
5.1.26. (2R)-2-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}propanoic
acid (18D)
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, D-alanine tert-butyl
ester hydrochloride was used for the similar procedure as in
1
5.1.24. to obtain the title compound. Yield: 77.5%; H NMR
(DMSO-d6) : 12.75-12.21 (1H, br), 9.37 (0.5H, d, J = 7.4 Hz),
9.33 (0.5H, d, J = 7.7 Hz), 8.41 (0.5H, d, J = 7.6 Hz), 8.39 (0.5H,
d, J = 8.0 Hz), 7.64 (1H, br), 7.30 (0.5H, s), 7.29 (0.5H, s), 7.24
(0.5H, d, J = 8.8 Hz), 7.23 (0.5H, d, J = 8.8 Hz), 6.97 (1H, d, J =
8.8 Hz), 4.77 (0.5H, d, J = 17.1 Hz), 4.76 (0.5H, d, J = 17.1 Hz),
4.48 (1H, d, J = 17.1 Hz), 4.35-4.26 (1H, m), 4.23-4.13 (1H, m),
3.89-3.80 (1H, m), 3.76 (3H, s), 3.16-3.05 (1H, m), 3.04-2.91
(2H, m), 2.67-2.57 (1H, m), 1.75-1.55 (2H, m), 1.25 (1.5H, d, J =
7.4 Hz), 1.24 (1.5H, d, J = 7.3 Hz), 0.88-0.81 (3H, m); MS (ESI):
483 (M+H)+
1
title compound (34 mg). H NMR (CDCl3) : 9.40 (0.5H, d, J =
7.2 Hz), 9.35 (0.5H, d, J = 7.2 Hz), 7.24-7.20 (1H, m), 7.14
(0.5H, d, J = 2.6 Hz), 7.11 (0.5H, d, J = 2.5 Hz), 6.81 (1H, d, J =
8.8 Hz), 6.06 (1H, br), 5.77 (1H, br), 5.35 (0.5H, d, J = 17.7 Hz),
5.32 (0.5H, d, J = 17.7 Hz), 4.28-4.18 (1H, m), 4.14 (1H, d, J =
17.7 Hz), 3.83 (3H, s), 3.76-3.67 (1H, m), 3.38-3.30 (2H, m),
3.25-3.18 (1H, m), 2.83 (1.5H, s), 2.82 (1.5H, s), 2.66-2.57 (1H,
m), 2.00-1.90 (1H, m), 1.83-1.73 (1H, m), 1.03-0.94 (3H, m); MS
(ESI): 425 (M+H)+
5.1.24. {[2-({[6-(5-Chloro-2-methoxybenzyl)-3,7-
dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}acetic acid
(18B)
To the compound rac-17 (50 mg) in methylene chloride (3 ml)
solution, glycine tert-butyl ester hydrochloride (41 mg),
triethylamine (0.29 ml), and n-propyl phosphoric acid anhydride
(25% ethyl acetate solution) (0.37 ml) were added and the
mixture was stirred at room temperature for 3 hours. The reaction
solution was concentrated, then ethyl acetate was added to the
residue. The obtained solution was successively washed with
distilled water, saturated potassium hydrogensulfate aqueous
5.1.27. N-[(1R)-1-(Anilinocarbonyl)propyl]-6-(5-
chloro-2-methoxybenzyl)-3,7-dioxo-1,4-diazepan-
1-carboxamide (18E)
Instead of the starting material compound of 5.1.23., that is,
methylamine, aniline was used for the similar procedure as in
1
5.1.23. to obtain the title compound. Yield: 41.5%; H NMR
(CDCl3) : 9.51 (0.5H, d, J = 7.3 Hz), 9.43 (0.5H, d, J = 6.9 Hz),
8.20 (0.5H, s), 8.15 (0.5H, s), 7.53 (2H, d, J = 8.0 Hz), 7.34-7.29
(2H, m), 7.21 (1H, dd, J = 8.7, 2.6 Hz), 7.15-7.00 (2H, m), 6.81
(0.5H, d, J = 8.7 Hz), 6.80 (0.5H, d, J = 8.7 Hz), 5.85-5.78 (1H,
m), 5.36 (0.5H, d, J = 17.4 Hz), 5.33 (0.5H, d, J = 17.4 Hz), 4.41-

4.33 (1H, m), 4.16 (0.5H, d, J = 17.4 Hz), 4.15 (0.5H, d, J = 17.4
Hz), 3.84 (1.5H, s), 3.83 (1.5H, s), 3.77-3.65 (1H, m), 3.37-3.30
(2H, m), 3.25-3.16 (1H, m), 2.65-2.55 (1H, m), 2.10-1.99 (1H,
m), 1.91-1.79 (1H, m), 1.10-1.00 (3H, m); MS (ESI): 487
(M+H)+
(1H, d, J = 7.5 Hz), 7.70-7.60 (1H, m), 7.57 (1H, t, J = 7.5 Hz),
7.35-7.31 (1H, m), 7.28-7.22 (1H, m), 7.15 (1H, t, J = 7.5 Hz),
6.99 (1H, d, J = 8.8 Hz), 4.77 (0.5H, d, J = 17.3 Hz), 4.73 (0.5H,
d, J = 17.3 Hz), 4.53 (1H, d, J = 17.3 Hz), 4.31-4.25 (1H, m),
3.95-3.84 (1H, m), 3.77 (3H, s), 3.15-3.06 (1H, m), 3.05-2.91
(2H, m), 2.69-2.61 (1H, m), 1.94-1.72 (2H, m), 0.95-0.89 (3H,
m); MS (ESI): 531 (M+H)+
5.1.28. N-{(1R)-1-[(tert-
Butoxyamino)carbonyl]propyl}-6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
carboxamide (18F)
5.1.32. 3-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}benzoic acid
(18J)
Instead of the starting material compound of 5.1.23., that is,
methylamine, 2-aminopyridine was used for the similar
procedure as in 5.1.23. to obtain the title compound.
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 3-aminobenzoic acid
tert-butyl ester was used for the similar procedure as in 5.1.24. to
obtain the title compound. Yield: 77.3%; 1H NMR (DMSO-d6) :
13.10-12.47 (1H, br), 10.35 (0.5H, s), 10.33 (0.5H, s), 9.48 (0.5H,
d, J = 7.2 Hz), 9.43 (0.5H, d, J = 7.3 Hz), 8.22-8.20 (1H, br),
7.78 (1H, d, 7.9), 7.65 (1H, s), 7.61 (1H, d, J = 7.9 Hz), 7.41 (1H,
t, J = 7.9 Hz), 7.31 (1H, s), 7.24 (0.5H, d, 8.8Hz), 7.23 (0.5H, d,
J = 8.8 Hz), 6.98 (1H, d, J = 8.8 Hz), 4.78 (0.5H, d, J = 17.2 Hz),
4.77 (0.5H, d, J = 17.2 Hz), 4.51 (1H, d, J = 17.2 Hz), 4.49-4.39
(1H, m), 3.90-3.81 (1H, m), 3.77 (3H, s), 3.18-3.07 (1H, m),
3.05-2.91 (2H, m), 2.69-2.60 (1H, m), 1.89-1.67 (2H, m), 0.92-
0.80 (3H, m); MS (ESI): 531 (M+H)+
1
Yield:33.2%; H NMR (CDCl3) : 9.34 (0.5H, d, J = 7.4 Hz),
9.28 (0.5H, d, J = 7.4 Hz), 8.42 (1H, s), 7.19 (1H, dd, J = 8.8, 2.5
Hz), 7.12 (0.5H, d, J = 2.5 Hz), 7.09 (0.5H, d, J = 2.5 Hz), 6.794
(0.5H, d, J = 8.8 Hz), 6.790 (0.5H, d, J = 8.8 Hz), 5.69 (1H, br),
5.31 (0.5H, d, J = 17.6 Hz), 5.28 (0.5H, d, J = 17.6 Hz), 4.15-
4.01 (1H, m), 4.12 (1H, d, J = 17.6 Hz), 3.82 (1.5H, s), 3.81
(1.5H, s), 3.74-3.62 (1H, m), 3.55-3.25 (2H, m), 3.23-3.12 (1H,
m), 2.63-2.53 (1H, m), 2.02-1.91 (1H, m), 1.83-1.72 (1H, m),
1.25 (9H, s), 1.01-0.95 (3H, m); MS (ESI): 483 (M+H)+
5.1.29. 6-(5-Chloro-2-methoxybenzyl)-3,7-dioxo-
N-{(1R)-1-[(3-pyridylamino)carbonyl]propyl}-
1,4-diazepan-1-carboxamide (18G)
5.1.33. 4-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}benzoic acid
(18K)
Instead of the starting material compound of 5.1.23., that is,
methylamine, 3-aminopyridine was used for the similar
procedure as in 5.1.23. to obtain the title compound. Yield:
1
41.5%; H NMR (CDCl3) : 9.51 (0.5H, d, J = 7.2 Hz), 9.42
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 4-aminobenzoic acid
tert-butyl ester was used for the similar procedure as in 5.1.24. to
obtain the title compound. Yield: 50.8%; 1H NMR (DMSO-d6) :
12.88-12.35 (1H, br), 10.47 (0.5H, s), 10.45 (0.5H, s), 9.47 (0.5H,
d, J = 7.2 Hz), 9.43 (0.5H, d, J = 7.3 Hz), 7.87 (2H, d, J = 8.6
Hz), 7.68 (2H, d, J = 8.6 Hz), 7.67 (1H, br), 7.31 (1H, s), 7.248
(0.5H, d, J = 8.8 Hz), 7.242 (0.5H, d, J = 8.8 Hz), 6.98 (1H, d, J
= 8.8 Hz), 4.77 (1H, d, J = 17.1 Hz), 4.51 (1H, d, J = 17.1 Hz),
4.48-4.40 (1H, m), 3.92-3.81 (1H, m), 3.77 (3H, s), 3.18-3.08
(1H, m), 3.05-2.92 (2H, m), 2.65-2.59 (1H, m), 1.85-1.65 (2H,
m), 0.90-0.85 (3H, m); MS (ESI): 531 (M+H)+
(0.5H, d, J = 6.8 Hz), 8.63-8.58 (2H, m), 8.31 (0.5H, s), 8.30
(0.5H, s), 8.15-8.10 (1H, m), 7.24-7.18 (2H, m), 7.13 (0.5H, d, J
= 2.6 Hz), 7.10 (0.5H, d, J = 2.6 Hz), 6.795 (0.5H, d, J = 8.8 Hz),
6.791 (0.5H, d, J = 8.7 Hz), 5.88 (1H, br), 5.32 (0.5H, d, J = 17.5
Hz), 5.29 (0.5H, d, J = 17.5 Hz), 4.44-4.32 (1H, m), 4.17 (1H, d,
J = 17.5 Hz), 3.82 (1.5H, s), 3.81 (1.5H, s), 3.75-3.65 (1H, m),
3.35-3.26 (2H, m), 3.22-3.15 (1H, m), 2.65-2.55 (1H, m), 2.10-
1.98 (1H, m), 1.90-1.77 (1H, m), 1.08-0.99 (3H, m); MS (ESI):
488 (M+H)+
5.1.30. 6-(5-Chloro-2-methoxybenzyl)-3,7-dioxo-
N-{(1R)-1-[(4-pyridylamino)carbonyl]propyl}-
1,4-diazepan-1-carboxamide (18H)
5.1.34. N-{(1R)-1-[(3-
Instead of the starting material compound of 5.1.23., that is,
methylamine, 4-aminopyridine was used for the similar
procedure as in 5.1.23. to obtain the title compound. Yield:
Aminoanilino)carbonyl]propyl}-6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
carboxamide (18L)
1
40.9%; H NMR (CDCl3) : 9.50 (0.5H, d, J = 7.1 Hz), 9.41
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 1-tert-butoxycarbonyl-
1,3-phenylenediamine was used for the similar procedure as in
(0.5H, d, J = 6.8 Hz), 8.79 (0.5H, s), 8.76 (0.5H, s), 8.46 (2H, d,
J = 6.0 Hz), 7.48 (1H, d, J = 6.0 Hz), 7.47 (1H, d, J = 6.0 Hz),
7.20 (1H, dd, J = 8.7, 2.5 Hz), 7.13 (0.5H, d, J = 2.5 Hz), 7.11
(0.5H, d, J = 2.5 Hz), 6.797 (0.5H, d, J = 8.7 Hz), 6.792 (0.5H, d,
J = 8.7 Hz), 5.85 (1H, brd, J = 8.4 Hz), 5.32 (0.5H, d, J = 17.4
Hz), 5.29 (0.5H, d, J = 17.4 Hz), 4.39-4.30 (1H, m), 4.17 (1H, d,
J = 17.4 Hz), 3.82 (1.5H, s), 3.81 (1.5H, s), 3.80-3.65 (1H, m),
3.35-3.30 (2H, m), 3.22-3.12 (1H, m), 2.66-2.56 (1H, m), 2.09-
1.98 (1H, m), 1.89-1.79 (1H, m), 1.08-1.00 (3H, m); MS (ESI):
488 (M+H)+
1
5.1.24. to obtain the title compound. Yield: 58.8%; H NMR
(DMSO-d6) : 9.83 (0.5H, s), 9.81 (0.5H, s), 9.45 (0.5H, d, J =
7.4 Hz), 9.41 (0.5H, d, J = 7.4 Hz), 7.65 (1H, d, J = 2.9 Hz),
7.316 (0.5H, s), 7.311 (0.5H, s), 7.24 (0.5H, d, J = 8.8 Hz), 7.23
(0.5H, d, J = 8.8 Hz), 6.98 (1H, d, J = 8.8 Hz), 6.91-6.87 (2H, m),
6.64 (1H, d, J = 7.8 Hz), 6.23 (1H, d, J = 7.8 Hz), 5.02 (2H, s),
4.78 (0.5H, d, J = 17.1 Hz), 4.77 (0.5H, d, J = 17.1 Hz), 4.50 (1H,
d, J = 17.1 Hz), 4.45-4.37 (1H, m), 3.92-3.81 (1H, m), 3.77 (3H,
s), 3.19-3.08 (1H, m), 3.07-2.91 (2H, m), 2.68-2.60 (1H, m),
1.82-1.65 (2H, m), 0.89-0.82 (3H, m); MS (ESI): 502 (M+H)+
5.1.31. 2-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}benzoic acid
(18I)
5.1.35. N-{(1R)-1-[(4-
Aminoanilino)carbonyl]propyl}-6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
carboxamide (18M)
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 1-tert-butoxycarbonyl-
1,4-phenylenediamine was used for the similar procedure as in
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 2-aminobenzoic acid
tert-butyl ester was used for the similar procedure as in 5.1.24. to
obtain the title compound. Yield:21.3%; 1H NMR (DMSO-d6) :
13.78-13.22 (1H, br), 11.4 3(0.5H, s), 11.37 (0.5H, s), 9.53 (0.5H,
d, J = 6.5 Hz), 9.46 (0.5H, d, J = 6.6 Hz), 8.46-8.40 (1H, m), 7.95

5.1.24. to obtain the title compound. Yield:56.9%; 1H NMR
(DMSO-d6) : 9.72 (0.5H, s), 9.70 (0.5H, s), 9.43 (0.5H, d, J =
7.4 Hz), 9.39 (0.5H, d, J = 7.5 Hz), 7.65 (1H, d, J = 3.2 Hz), 7.31
(0.5H, s), 7.30 (0.5H, s), 7.24 (0.5H, d, J = 8.9 Hz), 7.23 (0.5H, d,
J = 8.9 Hz), 7.17 (2H, d, J = 8.6 Hz), 6.98 (1H, d, J = 8.9 Hz),
6.48 (1H, d, J = 8.6 Hz), 6.46 (1H, d, J = 8.6 Hz), 4.847 (1H, s),
4.842 (1H, s), 4.79 (0.5H, d, J = 17.1 Hz), 4.77 (0.5H, d, J = 17.1
Hz), 4.50 (1H, d, J = 17.1 Hz), 4.39-4.32 (1H, m), 3.91-3.81 (1H,
m), 3.77 (3H, s), 3.18-3.07 (1H, m), 3.03-2.92 (2H, m), 2.68-2.60
(1H, m), 1.80-1.62 (2H, m), 0.89-0.81 (3H, m); MS (ESI): 502
(M+H)+
5.1.39. 4-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}-1H-pyrrole-
2-carboxylic acid (18Q)
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, 4-aminopyrrole-2-
carboxylic acid tert-butyl ester was used for the similar procedure
as in 5.1.24. to obtain the title compound. Yield:53.3%; 1H NMR
(DMSO-d6) : 12.30-12.10 (1H, br), 11.46 (1H, s), 10.10 (0.5H,
s), 10.07 (0.5H, s), 9.44 (0.5H, d, J = 7.4 Hz), 9.39 (0.5H, d, J =
7.4 Hz), 7.659 (0.5H, s), 7.651 (0.5H, s), 7.315 (0.5H, s), 7.310
(0.5H, s), 7.24 (0.5H, d, J = 8.9 Hz), 7.23 (0.5H, d, J = 8.9 Hz),
7.15 (1H, s), 6.98 (1H, d, J = 8.9 Hz), 6.61 (1H, s), 4.78 (0.5H, d,
J = 17.1 Hz), 4.76 (0.5H, d, J = 17.1 Hz), 4.50 (1H, d, J = 17.1
Hz) 4.37-4.29 (1H, m), 3.91-3.80 (1H, m), 3.77 (3H, s), 3.12
(0.5H, t, J = 13.2 Hz), 3.11 (0.5H, t, J = 12.8 Hz), 3.03-2.92 (2H,
m), 2.66-2.51 (1H, m), 1.80-1.62 (2H, m), 0.86-0.80 (3H, m); MS
(ESI): 520 (M+H)+
5.1.36. 2-Amino-5-{[(2R)-2-({[6-(5-chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}benzoic acid
hydrochloride (18N)
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, tert-butyl 2,5-
diaminobenzoate was used for the similar procedure as in 5.1.24.
to obtain the title compound. Yield: 38.8%; 1H NMR (DMSO-d6)
: 9.92 (0.5H, s), 9.89 (0.5H, s), 9.45 (0.5H, d, J = 7.4 Hz), 9.40
(0.5H, d, J = 7.4 Hz), 7.95-7.92 (1H, m), 7.66 (0.5H, s), 7.61
(0.5H, s), 7.427 (0.5H, d, J = 8.7 Hz), 7.421 (0.5H, d, J = 8.7 Hz),
7.31 (1H, s), 7.24 (0.5H, d, J = 8.8 Hz), 7.23 (0.5H, d, J = 8.8
Hz), 6.98 (1H, d, J = 8.8 Hz), 6.71 (1H, d, J = 8.7 Hz), 4.78
(0.5H, d, J = 17.1 Hz), 4.77 (0.5H, d, J = 17.1 Hz), 4.50 (1H, d, J
= 17.1 Hz), 4.39-4.32 (1H, m), 3.91-3.79 (1H, m), 3.77 (3H, s),
3.17-3.07 (1H, m), 3.03-2.92 (2H, m), 2.68-2.60 (1H, m), 1.83-
1.65 (2H, m), 0.89-0.81 (3H, m); MS (ESI): 546 (M+H)+
5.1.40. 6-(5-Chloro-2-methoxybenzyl)-3,7-dioxo-
N-{(1R)-1-[(1H-tetrazol-5-
ylamino)carbonyl]propyl}-1,4-diazepan-1-
carboxamide (18R)
Instead of the starting material compound of 5.1.23., that is,
methylamine, 5-amino-1H-tetrazole was used for the similar
procedure as in 5.1.23. to obtain the title compound.
Yield:58.1%; 1H NMR (DMSO-d6) : 15.90 (1H, brs), 12.10
(1H, brs), 9.52-9.47 (1H, m), 7.67 (1H, br), 7.31 (1H, s), 7.249
(0.5H, d, J = 8.8 Hz), 7.243 (0.5H, d, J = 8.8 Hz), 6.9 8(1H, d, J
= 8.8 Hz), 4.75 (1H, d, J = 17.2 Hz), 4.56-4.46 (1H, m), 4.52 (1H,
d, J = 17.2 Hz), 3.94-3.84 (1H, m), 3.77 (3H, s), 3.18-2.90 (3H,
m), 2.68-2.58 (1H, m), 1.89-1.63 (2H, m), 0.90-0.75 (3H, m); MS
(ESI): 479 (M+H)+
5.1.37. 5-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}-2-
hydroxybenzoic acid (18O)
5.1.41. (6R)-6-(5-chloro-2-methoxybenzyl)-3,7-
dioxo-N-{(1R)-1-[(1H-tetrazol-5-
ylamino)carbonyl]propyl}-1,4-diazepan-1-
carboxamide ((R)-18R)
Instead of the starting material compound of 5.1.24., that is, the
glycine tert-butyl ester hydrochloride, tert-butyl 4-amino-2-
hydroxybenzoate was used for the similar procedure as in 5.1.24.
to obtain the title compound. Yield: 56.7%; 1H NMR (DMSO-d6)
: 13.78-13.22 (1H, br), 11.43 (0.5H, s), 11.37 (0.5H, s), 9.53
(0.5H, d, J = 6.5 Hz), 9.46 (0.5H, d, J = 6.6 Hz), 8.46-8.40 (1H,
m), 7.95 (1H, d, J = 7.5 Hz), 7.70-7.60 (1H, m), 7.57 (1H, t, J =
7.5 Hz), 7.35-7.31 (1H, m), 7.28-7.22 (1H, m), 7.15 (1H, t, J =
7.5 Hz), 6.99 (1H, d, J = 8.8 Hz), 4.77 (0.5H, d, J = 17.3 Hz),
4.73 (0.5H, d, J = 17.3 Hz), 4.53 (1H, d, J = 17.3 Hz), 4.31-4.25
(1H, m), 3.95-3.84 (1H, m), 3.77 (3H, s), 3.15-3.06 (1H, m),
3.05-2.91 (2H, m), 2.69-2.61 (1H, m), 1.94-1.72 (2H, m), 0.95-
0.89 (3H, m); MS (ESI): 547 (M+H)+
To the crude compound (R)-17 in methylene chloride (5 ml)
solution, 5-amino-1H-tetrazole monohydrate (180 mg),
triethylamine (2 ml), and n-propyl phosphoric acid anhydride
(50% ethyl acetate solution) (1.5 ml) were added and the mixture
was stirred at room temperature for 15 hours. Then, 2M hydrogen
chloride aqueous solution was added to the mixture at 0C, and
the resultant mixture was extracted with ethyl acetate. The
organic layer was successively washed with saturated saline,
dried over with anhydrous sodium sulfate, then concentrated. The
residue
was
purified
by
preparative
TLC
(chloroform/methanol/acetic acid = 10/1/0.1). Hexane and ethyl
acetate were added to obtain the compound, and the appeared
precipitate was collected by filtration to obtain the title
compound (15 mg). 1H NMR (CDCl3) : 9.46 (1H, d, J = 7.7 Hz),
7.71 (1H, br), 7.34 (1H, s), 7.27 (1H, d, J = 8.5 Hz), 7.01 (1H, d,
J = 8.5 Hz), 4.80 (1H, d, J = 17.5 Hz), 4.57 - 4.45 (2H, m), 3.93 -
3.85 (1H, m), 3.80 (3H, s), 3.20 - 3.09 (1H, m, J = 14.2 Hz), 3.06
- 2.91 (2H, m), 2.69 - 2.60 (1H, m), 1.87 - 1.66 (2H, m, J = 7.3
Hz), 0.89 (3H, t, J = 7.1 Hz); MS (ESI): 479 (M+H)+
5.1.38. 3-amino-5-{[(2R)-2-({[6-(5-Chloro-2-
methoxybenzyl)-3,7-dioxo-1,4-diazepan-1-
yl]carbonyl}amino)butanoyl]amino}benzoic acid
(18P)
Instead of the starting material compound of 5.1.24., that is,
the glycine tert-butyl ester hydrochloride, tert-butyl 5-amino-3-
tert-butoxycarbonylaminobenzoate was used for the similar
procedure as in 5.1.24. to obtain the title compound.
Yield:54.8%; 1H NMR (DMSO-d6) : 12.73-11.90 (1H, br), 10.1
(0.5H, s), 10.0 (0.5H, s), 9.46 (0.5H, d, J = 7.3 Hz), 9.42 (0.5H, d,
J = 7.4 Hz), 7.65 (1H, d, J = 3.2 Hz), 7.32-7.22 (3H, m), 7.10
(0.5H, s), 7.09 (0.5H, s), 6.99 (0.5H, s), 6.97 (0.5H, s), 6.87
(0.5H, s), 6.86 (0.5H, s), 5.32 (2H, brs), 4.78 (1H, d, J = 17.3 Hz),
4.50 (1H, d, J = 17.1 Hz), 4.42-4.38 (1H, m), 3.90-3.82 (1H, m),
3.77 (3H, s), 3.17-3.07 (1H, m), 3.04-2.93 (2H, m), 2.67-2.62
(1H, m), 1.85-1.60 (2H, m), 0.88-0.81 (3H, m); MS (ESI): 546
(M+H)+
5.1.42. (S)-tert-Butyl 6-(1-
hydroxypropyl)nicotinate(23a)
To 6-chloronicotinic acid 19a (10 g) in methylene chloride
(100 ml) solution, N,N'-diisopropyl-O-tert-butylisourea (50 g)
was added and the mixture was stirred under heating and reflux
for 16 hours. The insoluble compound was filtered out, and the
filtrate was concentrated. The residue was purified by silica gel
column chromatography (chloroform/ethyl acetate = 6/1) to
obtain the compound 20a (11.66 g). To the compound 20a (6.0 g)

in
dimethylacetoamide
(30
ml)
solution,
= 6.8 Hz), 3.84 (2H, s), 1.90-1.74 (2H, m), 1.57 (9H, s), 0.79 (3H,
tris(dibenzylideneacetone)dipalladium (0.51 g), zinc cyanide
(0.22 g), diphenylphosphinoferrocene (0.62 g), and zinc powder
(1.98 g) were added and the mixture was stirred under argon
atmosphere at 120C for 3 hours. The reaction solution was
filtered by sellite, which was then washed with ethyl acetate.
Then, the filtrate was washed with distilled water and saturated
saline, then the organic layer was dried over with anhydrous
sodium sulfate and concentrated. The obtained residue was
purified by silica gel column chromatography (hexane/ethyl
acetate = 5/1) to obtain the compound 21a (3.59 g). To copper (I)
iodide (0.93 g) in tetrahydrofuran (50 ml) suspension,
ethylmagnesium bromide (0.86M tetrahydrofuran solution) (12.5
ml) was added dropwise under cooling at 0C, the mixture was
stirred at 0C for 30 minutes, then the compound 21a (1.0 g)
obtained above in tetrahydrofuran (10 ml) solution was added at
the -20C. After stirring for 30 minutes at that temperature, 28%
ammonia solution in water and ethyl acetate were added to the
mixture and the solution separated. The aqueous layer was
extracted with ethyl acetate, while the combined organic layer
was washed with saturated saline, then was dried over with
anhydrous sodium sulfate and concentrated. The obtained residue
was purified by silica gel column chromatography (hexane/ethyl
acetate = 8/1) to obtained the compound 22a (0.47 g). To the
compound 22a (200 mg) in tetrahydrofuran (1 ml) solution, (-)-
B-chlorodiisopinocampheylborane (65% hexane solution) (0.92
ml) was added dropwise under cooling at -20C, then the mixture
was stirred at that temperature for 18 hours. Then, ether and
distilled water were added to the reaction mixture, and the
solution was separated. The organic layer was extracted with
distilled water and 1M hydrogen chloride. Then, the combined
aqueous layer was neutralized by sodium hydrogen carbonate,
and extracted with ethyl acetate. Organic layer was washed with
saturated saline, then was dried over with anhydrous sodium
sulfate and concentrated to obtained the title compound (150 mg,
84%ee) . The optical purity was determined using CHIRALCEL
t, J = 7.5 Hz); MS (ESI): 237 (M+H)+
5.1.44. tert-Butyl 2-[(1R)-1-aminopropyl]-
isonicotinate D-tartrate (25b)
Instead of the starting material compound of 5.1.42., that is, 6-
chloronicotinic acid, 2-chloroisonicotinic acid was used for the
similar procedure as in 5.1.42. and 5.1.43. to obtain the title
1
compound. H NMR (DMSO-d6) : 8.81 (1H, d, J = 4.9 Hz),
7.90 (1H, d. J = 1.5 Hz), 7.77 (1H, dd, J = 4.9, 1.5 Hz), 4.39 (1H,
t, J = 6.8 Hz), 3.83 (2H, s), 1.90-1.76 (2H, m), 1.57 (9H, s), 0.80
(3H, t, J = 7.5 Hz); MS (ESI): 237 (M+H)+
5.1.45. tert-Butyl 5-[(1R)-1-aminopropyl]-
nicotinate D -tartrate (25c)
Instead of the starting material compound of 5.1.42., that is, 6-
bromonicotinic acid, 5-bromonicotinic acid was used for the
similar procedure as in 5.1.42. and 5.1.43. to obtain the title
1
compound. H NMR (DMSO-d6) : 8.99 (1H, d, J = 2.0 Hz),
8.81 (1H, d, J = 2.1 Hz), 8.33 (1H, t, J = 2.0 Hz), 7.75-6.95 (3H,
br), 4.31 (0.5H, d, J = 2.6 Hz), 4.18 (1H, t, J = 6.4 Hz), 4.04-3.99
(0.5H, br), 3.86 (1H, s), 1.95-1.71 (2H, m), 1.57 (9H, s), 0.78 (3H,
t, J = 7.4 Hz); MS (ESI): 237 (M+H)+
5.1.46. tert-Butyl 5-[(1S)-1-hydroxypropyl]-2-
furancarboxylic acid (28)
To 5-formyl-2-furancarboxylic acid 26 (10 g) in methylene
chloride (200 ml) solution, N,N'-diisopropyl-O-tert-butylisourea
(42 g) was added and the mixture was stirred under heating and
reflux for 12 hours. The insoluble compound was filtered out,
then the filtrate was concentrated. The residue was purified by
silica gel column chromatography (hexane/ethyl acetate = 6/1) to
obtain the compound 27 (10.97 g). To the (1S,2R)-2-di-n-
butylamino-1-phenyl-1-propanol (81 mg) in hexane (5 ml)/
toluene (5 ml) solution, 27 (1 g) was added and the mixture was
stirred at room temperature for 30 minutes. Next, diethylzinc in
1N hexane solution (11.3 ml) was added to the reaction solution
under ice cooling and the mixture was stirred at that temperature
for 16 hours. Saturated ammonium chloride aqueous solution was
added to the reaction solution, the mixture was stirred at 20
minutes, and 1N hydrochloric acid was added. The obtained
mixed solution was extracted with ethyl acetate. The organic
layer was dried over with anhydrous sodium sulfate, then
concentrated to obtain the title compound (1.53 g, 85%ee). The
optical purity was determined using CHIRALCEL AD-H (Daicel
Chemical Industries) (movement phase: hexane/ethanol = 98/2,
AD-H
(Daicel
Chemical
Industries)
(movement
phase:hexane/ethanol = 98/2, 1.0ml/min) . 1H NMR (CDCl3) :
9.10 - 9.08 (1H, m), 8.23 (1H, dd, J = 2.2, 8.1 Hz), 7.32 (1H, d, J
= 8.1 Hz), 4.78 - 4.73 (1H, m), 4.00 (1H, d, J = 5.7 Hz), 1.95 -
1.85 (1H, m), 1.80 - 1.67 (1H, m), 1.64 - 1.59 (9H, m), 0.94 (3H,
t, J = 7.3 Hz)
5.1.43. tert-Butyl 6-[(1R)-1-aminopropyl]-
nicotinate D -tartrate (25a)
1
To the compound 23a (1.33 g) in tetrahydrofuran (27 ml)
solution, triphenylphosphine (4.41 g), diethyl azodicarboxylate
(2.65 ml) and phtalimide (1.24 g) were added under cooling at
0C, then the mixture was stirred at room temperature for 1 hour.
The mixture was concentrated, the residue was purified by silica
gel column chromatography (chloroform/ethyl acetate = 6/1) to
obtain the compound 24a (1.34 g). To the compound 24a (1.37 g)
in methanol (14 ml), hydrazine monohydrate (0.9 ml) was added
under warming at 60 C, then the mixture was stirred at that
temperature for 2 hours. Then, ethyl acetate and distilled water
were added to the mixture. Then, the organic layer and the
aqueous layer were separated, subsequently the aqueous layer
was extracted with ethyl acetate. The combined organic layer was
washed with saturated saline, then was dried over with anhydrous
sodium sulfate and concentrated. To the obtained compound
(0.94 g) in ethyl acetate (9.4 ml) solution, D-tartaric acid in
ethanol 1M solution (3.74 ml) was added, then the appeared
precipitate was collected by filtration to obtained title compound
1.0ml/min). H NMR (CDCl3) : 6.98 (1H, d, J = 3.4 Hz), 6.30
(1H, d, J = 3.4 Hz), 4.65 (1H, br), 1.99-1.80 (2H, m), 1.55 (9H, s),
0.96 (3H, t, J = 7.4 Hz)
5.1.47. tert-Butyl 5-[(1R)-1-aminopropyl]-2-
furancarboxylate D -tartrate (31)
Instead of the starting material compound of 5.1.43., that is,
the compound 23a, the compound 28 was used for the similar
1
procedure as in 5.1.43., to obtain the title compound. H NMR
(DMSO-d6) : 8.25-7.30 (3H, br), 7.16 (1H, d, J = 3.5 Hz), 6.59
(1H, d, J = 3.5 Hz), 4.15 (1H, t, J = 6.8 Hz), 3.95 (2H, s), 1.89-
1.70 (2H, m), 1.51 (9H, s), 0.85 (3H, dt, J = 7.3, 2.3 Hz); MS
(ESI): 226 (M+H)+
5.1.48. tert-Butyl 5-[(1S)-1-hydroxypropyl]-3-
furancarboxylate (34a)
To 3-furancarboxylic acid (1.12 g), nitromethane (10 ml),
indium (III) trifluoromethane sulfonate (56 mg), lithium
perchlorate (1.06 g), and propionic anhydride (1.28 ml) were
added and the mixture was stirred at 50C for 3 hours. Water was
added to the reaction solution and the solution separated. The
aqueous layer was extracted with ethyl acetate. The combined
1
(1.28 g). H NMR (DMSO-d6) : 9.04 (1H, d, J = 2.1 Hz), 8.29
(1H, dd. J = 8.1, 2.1 Hz), 7.62 (1H, d, J = 8.1 Hz), 4.34 (1H, t, J

organic layer was washed with saturated saline, dried over with
anhydrous sodium sulfate, and concentrated to obtain 5-
propionyl-3-furancarboxylic acid (1.11 g) To 5-propionyl-3-
furancarboxylic acid (1.10 g) in methylene chloride (22 ml)
solution, N,N'-diisopropyl-O-tert-butylisourea (8 ml) was added
and the mixture was stirred under heating and reflux for 3 hours.
The insoluble compound was filtered out, then the filtrate was
concentrated. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 5/1). Then, hexane was
added to the obtained compound, and the appeared precipitate
was collected by filtration to obtained the compound 33a (0.71 g).
The mother liquid was concentrated to obtain the compound 33 a
(0.37 g). The compound 34a was synthesized in twice. To the
compound 33a (0.1 g , 0.90 g) in tetrahydrofuran (1 ml, 9 ml)
temperature for 1 hour. Then, bromotrichloromethane (2.8 ml)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (4.2 ml) were added to
the mixture. After stirring at 0C for 6 hours, saturated sodium
hydrogencarbonate aqueous solution was added to the mixture,
and the resultant mixture was extracted with ethyl acetate. The
organic layer was successively washed with saturated saline,
dried over with anhydrous sodium sulfate, and concentrated.
Then, the residue was successively purified by silica gel column
chromatography (hexane/ethyl acetate = 8/2 to 7/3) to give the
compound 39 (1.0 g). To the compound 39 (0.67 g) in methanol
(10 ml) solution, 4M sodium hydroxide aqueous solution (1.18
ml) was added, and the mixture was stirred at room temperature
for 3 hours. The methanol was removed by evaporation, and the
residue was diluted water, and the resultant solution was washed
with ethyl acetate. The aqueous layer was acidified by 6M
hydrogen chloride aqueous solution, and the resultant solution
was extracted with ethyl acetate. The organic layer was
successively washed with saturated saline, dried over with
anhydrous sodium sulfate, and concentrated to give the
compound 40 (0.52 g). To the compound 40 (0.52 g) in
methylene chloride (10 ml) solution, 1-(3-dimethylaminopropyl)-
solution
cooled
at
-20C,
65%(-)-B-
chlorodiisopinocampheylborane in hexane solution (0.26 ml, 4.8
ml) was added dropwise, then the mixture was stirred at that
temperature for overnight. Then, water was added to the each
reaction mixture, and the obtained solutions were combined. The
resultant mixture was extracted with ethyl acetate. The organic
layer was washed with saturated saline, then was dried over with
anhydrous sodium sulfate and concentrated. The residue was
purified by silica gel column chromatography (hexane/ethyl
acetate = 3/1) to obtain the compound 34a. (1.33 g, 91%ee) The
optical purity was determined using CHIRALCEL AD-H (Daicel
Chemical Industries) (movement phase:hexane/ethanol = 98/2,
1.0ml/min) . 1H NMR (CDCl3) : 7.86 (1H, s), 6.53 (1H, s), 4.63
- 4.55 (1H, m), 4.11 - 4.02 (1H, m), 1.75 - 1.71 (1H, m), 1.71 -
1.67 (1H, m), 1.54 (9H, s), 1.00 - 0.93 (3H, m)
3-ethylcarbodiimide
hydrochloride
(0.44
g),
4-
dimethylaminopyridine (23 mg) and 2,2,2-trichloroethanol (0.28
ml) were added and the mixture was stirred at room temperature
for 2.5 hours. The reaction solution was evaporated. The residue
was diluted with ethyl acetate, then the solution was successively
washed with distilled water, saturated potassium hydrogensulfate
aqueous solution, saturated sodium hydrogencarbonate aqueous
solution and saturated saline, dried over with anhydrous sodium
sulfate, then concentrated. Then, the residue was successively
purified by silica gel column chromatography (hexane/ethyl
acetate = 6/1 to 3/1) to give the compound 41 (0.46 g). A 4M
hydrogen chloride/ethyl acetate solution (5 ml) was added to the
compound 41 (0.75 g), and the mixture stirred at room
temperature for 2 hours. Then, the reaction solution was
concentrated to obtain the title compound (0.51 g). 1H NMR
(CDCl3) : 9.50 - 9.37 (2H, m), 8.39 (1H, s), 4.97 - 4.89 (2H, m),
4.84 - 4.75 (1H, m), 2.37 - 2.28 (2H, m), 1.11 (3H, t, J = 7.5
Hz,); MS (ESI): 301 (M+H)+
5.1.49. tert-Butyl 5-[(1R)-1-aminopropyl]-3-
furancarboxylate D -tartrate (36a)
Instead of the starting material compound of 5.1.43., that is,
the compound 23a, the compound 34a was used for the similar
1
procedure as in 5.1.43., to obtain the title compound. H NMR
(DMSO-d6) : 8.28 (1H, s), 6.65 (1H, s), 4.12 (1H, t, J = 6.3 Hz),
3.91 (2H, s), 1.89-1.72 (2H, m), 1.50 (9H, s), 0.82 (3H, t, J = 7.4
Hz); MS (ESI): 209 (M-NH2)+
5.1.50. tert-Butyl 5-[(1R)-1-aminopropyl] -3-
thiophenecarboxylate D -tartrate (36b)
5.1.52. (R)-2-(1-Aminopropyl)thiazole-4-
carboxylic acid 2,2,2-trichloroethyl ester
hydrochloride(48)
Instead of the starting material of 5.1.48., that is, 3-
furancarboxylic acid, 3-thiophenecarboxylic acid was used for
the similar procedure as with 5.1.48. and 5.1.49. to obtain the
To (R)-1-amino-1-thioxobutane-2-ylcarbamic acid tert-butyl
ester (2.82 g) in dimethoxyethane (60 ml) solution cooled at 0C,
potassium hydrogen carbonate (10.3 g) and ethyl bromopyruvate
(4.9 ml) were added ,and the mixture was stirred at that
temperature for 30 min. Then, the mixture was warmed to room
temperature. After stirring at room temperature for 18hours, the
reaction mixture was evaporated. The residue was diluted with
CHCl3, then the solution was successively washed with distilled
water and saturated saline, dried over with anhydrous sodium
sulfate, then concentrated to obtain the compound 44. To the
compound 44 in dimethoxyethane (60 ml) solution cooled at 0C,
trifluoroacetic anhydride (3.6 ml) and pyridine (4.7 ml) were
added, and the mixture was stirred at that temperature for 1 hour.
Then, the reaction mixture was evaporated, the residue was
diluted with ethyl acetate. Then, the solution was successively
washed with saturated potassium hydrogensulfate aqueous
solution, saturated sodium hydrogencarbonate aqueous solution
and saturated saline, dried over with anhydrous sodium sulfate,
then concentrated. Then, the residue was successively purified by
silica gel column chromatography (hexane/ethyl acetate = 9/1 to
17/3) to give the compound 45 (4.25 g). To the compound 45
(2.1 g) in ethanol (40 ml) solution , 4M sodium hydroxide
aqueous solution (3.3 ml) was added , and the mixture was stirred
1
title compound. H NMR (DMSO-d6) : 8.17 (1H, s), 7.41 (1H,
s), 4.35 (1H, t, J = 7.8 Hz), 3.93 (2H, s), 1.92-1.70 (2H, m), 1.51
(9H, s), 0.84 (3H, t, J = 7.3 Hz); MS (ESI): 225 (M+H)+
5.1.51. (R)-2-(1-Aminopropyl)oxazole-4-
carboxylic acid 2,2,2-trichloroethyl ester
hydrochloride (42)
To (R)-2-(tert-butoxycarbonylamino)butyric acid (5 g) in
methylene chloride (100 ml) solution, serine methyl ester
hydrochloride (5.7 g), 1-hydroxybenzotriazole (5 g), N,N-
diisopropylethylamine (6.4 ml), and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (7.1 g) were added and the
mixture was stirred at room temperature for 3 hours. The reaction
solution was diluted with ethyl acetate, then was successively
washed with distilled water, saturated potassium hydrogensulfate
aqueous solution, saturated sodium hydrogencarbonate aqueous
solution and saturated saline, dried over with anhydrous sodium
sulfate, then concentrated. Then, the residue was successively
purified by silica gel column chromatography (hexane/ethyl
acetate = 4/6 to 3/7) to give the compound 38 (5.27 g). To the
compound 38 (2.46 g) in methylene chloride (50 ml) solution
cooled at -20C, N,N- diethylaminosulfur trifluoride (1.17 ml)
was dropwisely added, and the mixture was stirred at that

at room temperature for 2 hours. Then, water and ethylacetate
were added to the mixture, and the organic layer and the aqueous
layer was separated. The aqueous layer was acidified by 6M
hydrogen chloride aqueous solution, and the resultant solution
was extracted with ethyl acetate. The organic layer was
successively washed with saturated saline, dried over with
anhydrous sodium sulfate, and concentrated to give the
compound 46 (1.47g). To the compound 46 (1.47 g) in methylene
chloride (30 ml) solution, 1-(3-dimethylaminopropyl)-3-
deglycosylation. Due to the removal of EndoHf, the sample was
re-loaded onto the same column. After the elution of the sample,
the amino-terminal extra region was cleaved by enterokinase
(Roche Applied Science) for the activation as the enzyme.
Sample was concentrated to 15mg/ml in 20 mM MES buffer (pH
6.0) and saturated compounds. Crystallization was performed by
the hanging-drop vapor diffusion method. Crystals were obtained
at 20°C after a few days in 22% PEG8000, 5 mM ZnCl2, and 100
mM MES buffer (pH 6.8).
ethylcarbodiimide
hydrochloride
(1.18
g),
4-
5.5. X-ray data processing and structure solution
dimethylaminopyridine (63 mg) and 2,2,2-trichloroethanol (0.74
ml) were added and the mixture was stirred at room temperature
for 3 hours. The reaction solution was evaporated. The residue
was diluted with ethyl acetate, then the solution was successively
washed with distilled water, saturated potassium hydrogensulfate
aqueous solution, saturated sodium hydrogencarbonate aqueous
solution and saturated saline, dried over with anhydrous sodium
sulfate, then concentrated. Then, the residue was successively
purified by silica gel column chromatography (hexane/ethyl
acetate = 5/1) to give the compound 47 (1.42 g). A 4M hydrogen
chloride/ethyl acetate solution (5 ml) was added to the compound
47 (1.42 g), and the mixture stirred at room temperature for 3
hours. Then, the reaction solution was concentrated to obtain the
The X-ray data sets were collected at room temperature on an
R-Axis IV++ imaging plate detector (Rigaku) using a MicroMax
007 rotating anode generator (Rigaku). Data processing was
carried out with HKL2000.23 Co-crystal structures of human
chymase were solved by the molecular replacement method. The
previously reported chymase structure (PDB code: 1T3124) was
used as the search model. Phase refinement and model
improvement were carried out with refmac25 and coot.26
Stereochemistry checks indicate that the refined protein model is
in good agreement with expectations within each resolution range.
Acknowledgements
1
title compound (0.7 g). H NMR (CDCl3) : 9.43 - 9.30 (3H, m),
Authors thanks Dr. Y. Tomimori, Dr. M. Terakawa for
performing enzyme assay. Dr. T. Nishihara, Dr. Y. Fukuda and
Dr. T. Nakatsuka are acknowledged for support and
encouragement throughout this study.
8.37 (1H, s), 5.00 - 4.93 (3H, m), 2.42 - 2.27 (2H, m), 1.10 (3H, t,
J = 7.3 Hz); MS (ESI): 316 (M+H)+
5.2. Human chymase assay
References and Notes
The human chymase assay was performed as follows:
recombinant human chymase4 was preincubated with the test
compounds for 10 min in 50 mM Tris/HCl buffer (pH7.5)
containing 1 M NaCl and 0.01% Triton X-100. The enzyme
reaction was initiated with substrate Suc-Ala-Ala-Pro-Phe-MCA
(Peptide Institute), supplied at 100 lM, and was stopped with
30% acetic acid after 10 minincubation. The intensity of the
fluorescence of the AMC produced by chymase was measured
(extinction380 nm, emission 460 nm), and the IC50 value
wascalculated from the inhibition of AMC formation at
eachconcentration of the tested compound.
1. Schwartz, L. B.; Austen, K. F. Enzymes of the mast cell
granule. J. Invest. Dermatol. 1980, 74, 349.
2. Urata, H.; Kinoshita, A.; Misono, K. S.; Bumpus, F. M.;
Husain, A. Identification of a highly specific chymase as the
major angiotensin II-forming enzyme in the human heart. J.
Biol. Chem. 1990, 265, 22348.
3. Tomimori, Y.; Muto, T.; Fukami, H.; Saito, K.; Horikawa,
C.; Tsuruoka, N.; Saito, M.; Sugiura, N.; Yamashiro, K.;
Sumida, M.; Kakutani, S.; Fukuda, Y. Chymase participates
in chronic dermatitis by inducing eosinophil infiltration. Lab.
Invest. 2002, 82, 789.
5.3. Inhibitory effects on the enzymatic activity of serine
proteases
4. Tomimori, Y.; Muto, T.; Fukami, H.; Saito, K.; Horikawa,
C.; Tsuruoka, N. Yamashiro, K.; Saito, M.; Sugiura, N.;
Sumida, M.; Kakutani, S.; Fukuda, Y. Mast cell chymase
regulates dermal mast cell number in mice. Biochem.
Biophys. Res. Commun. 2002, 290, 1478.
The inhibitory effects on the enzymatic activity of
serineproteases were evaluated by a similar procedure as in 5.2.
The enzymes and substrates used for the assays were asfollows:
Suc-Ala-Ala-Pro-Phe-MCA (Peptide Institute) for bovine
pancreatic a-chymotrypsin (Sigma) and humanneutrophil
cathepsin G (Calbiochem); Boc-Gln-Ala-Arg-MCA (Peptide
Institute) for bovine pancreatic trypsin(Nacalai Tesque);
MeOSuc-Ala-Ala-Pro-Val-pNA (Bachem) for human neutrophil
elastase (Calbiochem).The assay buffer was as follows: 0.1 M
Tris/HCl (pH 7.5)containing 1M KCl and 0.01% Triton X-100
for achymotrypsin;50 mM Tris/HCl buffer (pH 7.5) containing1
M NaCl and 0.01% Triton X-100 for cathepsin G andelastase; 50
mM Tris/HCl buffer (pH 7.5) containing 20 mM CaCl2 for
trypsin.
5. Mao, X. Q.; Shirakawa, T.; Yoshikawa, T.; Yoshikawa, K.;
Kawai, M.; Sasaki, S.; Enomoto, T.; Hashimoto, T.;
Furuyama, J.; Hopkin, J. M.; Morimoto, K. Association
between genetic variants of mast-cell chymase and eczema.
Lancet 1996, 348, 581.
6. Tanaka, K.; Sugiura H.; Uehara, M.; Sato, H.; Hashimoto-
Tamaoki, T.; Furuyama, J. Association between mast cell
chymase genotype and atopic eczema: comparison between
patients with atopic eczema alone and those with atopic
eczema and atopic respiratory disease. Clin. Exp. Allergy
1999, 29, 800.
5.4. Preparation of the crystal
Human chymase, which substituted Cys22 with Ser and
Phe127 with Lys for high-yield production21 and the avoidance of
self-cleavage22, respectively, was expressed by Pichia pastoris.
The supernatant with the secretory product was loaded onto a
heparin sepharose 6FF column (GE healthcare), and were eluted
by NaCl. Collected sample was concentrated to 15 mg/ml, and
treated with EndoHf (New England Biolabs) for the
7. Weidinger, S.; Rummler, L.; Klopp, N.; Wagenpfeil, S.;
Baurecht, H. J.; Fischer, G.; Holle, R.; Gauger, A.; Schafer,
T.; Jakob, T.; Ollert, M.; Behrendt, H.; Wichmann, H. E.;
Ring, J.; Illig, T. Association study of mast cell chymase
polymorphisms with atopy. Allergy 2005, 60, 1256.

8. Tanaka, T.; Muto, T.; Maruoka, H.; Imajo, S.; Fukami,
H.; Tomimori, Y.; Fukuda, Y.; Nakatsuka, T. Identification
of 6-substituted 4-arylsulfonyl-1,4-diazepane-2,5-diones as a
novel scaffold for human chymase inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 3431.
17. The two enantioselective reactions16, 18 for the synthesis of
23a-c, 28, 34a-b which are highly reliable and widely used
gave R configuration. We confirmed that the absolute
configurations at ethyl group of 1 synthesized by the same
procedure was R form according to the X-ray crystal
structure of compound 1 within the human chymase.
9. Maruoka, H.; Tanaka, T.; Muto, T.; Imajo, S.; Fukami, H.;
Tomimori, Y.; Fukuda, Y.; Nakatsuka, T. Development of
6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-
diones as orally active human chymase inhibitors. Bioorg.
Med. Chem. Lett. 2007, 17, 3435.
18. Soai, K.; Yokoyama, S.; Hayasaka, T. Chiral N,N-
dialkylnorephedrines as catalysts of the highly
enantioselective addition of dialkylzincs to aliphatic and
aromatic aldehydes. The asymmetric synthesis of secondary
aliphatic and aromatic alcohols of high optical purity. J. Org.
Chem. 1991, 56, 4264
10. Watanabe, N.; Tomimori, Y.; Terakawa, M.; Ishiwata, K.;
Wada, A.; Muto, T.; Tanaka, T.; Maruoka, H.; Nagahira, K.;
Nakatsuka, T.; Fukuda, Y. Oral administration of chymase
inhibitor improves dermatitis in NC/Nga mice. J. Invest.
Dermatol. 2007, 127, 971.
19. Chapman, C. J.; Frost, C. G.; Hartleya, J. P.; Whittleb, A. J.
Efficient aromatic and heteroatom acylations using catalytic
indium complexes with lithium perchlorate. Tetrahedron
Lett. 2001, 42 773.
11. Matsuda, H.; Watanabe, N.; Geba, G. P.; Sperl, J.; Tsudzuki,
M.; Hiroi, J.; Matsumoto, M.; Ushio, H.; Saito, S.; Askenase,
P. W.; Ra, C. Development of atopic dermatitis like skin
lesion with IgE hyperproduction in NC/Nga mice. Int.
Immunol 1997, 9, 461.
20. Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D.
R. Synthesis of Functionalized Oxazolines and Oxazoles
with DAST and Deoxo-Fluor. Org. Lett. 2000, 2, 1165
21. Wang, Z.; Walter, M.; Selwood, T.; Rubin, H.; Schechter,
M. Recombinant Expression of Human Mast Cell Proteases
Chymase and Tryptase., Biol. Chem. 1998, 379, 167.
12. Tsudzuki, M.; Watanabe, N.; Wada, A.; Nakana, Y.; Hiroi,
J.; Matsuda, H. Genetic analysis for dermatitis and IgE
hyperproduction in NC/Nga mouse. Immunogenetics 1997,
47, 88.
22. McGrath, M. E.; Osawa, A. E.; Barnes, M. G.; Clark, J. M.;
Mortara, K. D.; Schmidt, B. F. Production of Crystallizable
human Chymase from a Bacillus subtilis System. FEBS Lett.
1997, 413, 486.
13. Terakawa, M.; Fujieda, Y.; Tomimori, Y.; Muto, T.; Tanaka,
T.; Maruoka, H.; Nagahira, K.; Ogata, A.; Nakatsuka, T.;
Fukuda, Y. Oral chymase inhibitor SUN13834 ameliorates
skin inflammation as well as pruritus in mouse model for
atopic dermatitis. Eur. J. Pharmacol. 2008, 601, 186
23. Otwinowski, Z.; Minor, W.
" Processing of X-ray
Diffraction Data Collected in Oscillation Mode ", Methods
in Enzymology 1997, 276: Macromolecular Crystallography,
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Academic Press (New York).
14. Rikken, G.; Gertner, J. J. Invest. Dermatol. 2010, 130
(supplement 2S), 40th Annual Meeting of the ESDR,
Abstract 410
24. de Garavilla, L.; Greco, M. N.; Sukumar, N.; Chen, Z.W.;
Pineda, A. O.; Mathews, F. S.; Di Cera, E.; Giardino, E.
C.; Wells, G. I.; Haertlein, B. J.; Kauffman, J. A.;
Corcoran, T. W.; Derian, C. K.; Eckardt, A. J.; Damiano, B.
P.; Andrade-Gordon, P.; Maryanoff, B. E. A novel, potent
dual inhibitor of the leukocyte proteases cathepsin G and
chymase: molecular mechanisms and anti-inflammatory
activity in vivo. J. Biol. Chem. 2005, 280, 18001.
15. The absolute configuration of (R)-12 was determined by the
X-ray crystal structure analysis of 49 and 50 which were
described in our previous paper.9 The absolute configuration
at 6-position of 1,4-diazepane core of 50 was determined as
R form on the base of the absolute configuration of 49 which
was determined as R form on the base of L-tartaric acid. An
epimerization was not detected in that procedure, so the
absolute configuration at 6-position of 1,4-diazepane core of
(R)-12 was determined as R form.
25. Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N.
S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.;
Vagin, A. A. REFMAC5 for the Refinement of
Macromolecular Crystal Structures Acta Crystallogr. 2011,
D67, 355.
26. Emsley, P.; Lohkamp, B.; Scottc, W. G.; Cowtand, K.
Features and development of Coot. Acta Crystallogr. D66,
486.
16. Brown, C. H.; Chandrasekharan, J.; Ramachandran, V. P.
Chiral synthesis via organoboranes. 14. Selective reductions.
41. Diisopinocampheylchloroborane, an exceptionally
efficient chiral reducing agent. J. Am. Chem. Soc. 1988, 110,
1539

Graphical Abstract
Discovery of novel series of 6-benzyl
substituted 4-aminocarbonyl-1,4-diazepane-
2,5-diones as human chymase inhibitors
using structure-based drug design
Leave this area blank for abstract info.
Taisaku Tanaka, Hajime Sugawara, Hiroshi Maruoka, Seiichi Imajo and Tsuyoshi Muto
Asubio Pharma Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan