Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug

Article abstract of DOI:10.1038/s41429-019-0225-5

Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1–3, which converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.

Full text of DOI:10.1038/s41429-019-0225-5

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0225-5
SPECIAL FEATURE: ARTICLE
Synthesis and biological activity of analogs of CPZEN-45, a novel
antituberculosis drug
Yoshimasa Ishizaki1 Yoshiaki Takahashi1 Tomoyuki Kimura1 Michitaka Inoue1 Chigusa Hayashi1
Masayuki Igarashi1
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Received: 27 June 2019 / Revised: 2 August 2019 / Accepted: 6 August 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of
caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1–3, which
converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-
45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural
limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.
Introduction
To address this problem, we screened for novel anti-
mycobacterial substances having an effective spectrum of
activity with a new mode of action, from microbial pro-
ducts. As a part of this program, we discovered a structu-
rally analogous mixture of caprazamycins (CPZs), a group
of novel lipo-nucleoside antibiotics, in a culture broth of
Actinomycete strain Streptomyces sp. MK 730-62F2 [2, 3].
Its major component, caprazamycin B (CPZ-B), showed
excellent antimycobacterial activity in vitro against drug-
susceptible and multidrug-resistant Mycobacterium tuber-
culosis strains, although the compounds do have some
shortcomings, including its onerous separation from a
complex mixture by using HPLC and its extremely poor
water-solubility profile [4].
In the process of structural analysis of CPZs, two com-
pounds with the core structure, caprazene and caprazol,
were obtained very efficiently from the mixture, as shown in
Scheme 1 [3]. By using advanced medicinal chemistry with
core compounds, we aimed to create new derivatives that
overcome the disadvantages of CPZs. As a result, CPZEN-
45, which is expected to be a promising new anti-TB drug,
was discovered [4].
In reports from the World Health Organization, tuberculosis
(TB) is listed as one of the ten leading causes of death
worldwide in 2016 [1]. There are an estimated 10.4 million
new incidents of TB annually, and the number of deaths is
around 1.8 million. TB is one of the deadliest diseases
caused by a single infectious agent, and is the single largest
cause of death for individuals infected with human immu-
nodeficiency virus. The number of new multidrug-resistant
(MDR)-TB patients in 2016 was estimated to be 490,000, of
whom about 6.2% are infected with extensively drug-
resistant (XDR)-TB, against which existing anti-TB drugs
are ineffective [1]. There is a clear and urgent need to
develop new drugs for the treatment of this disease, in order
to reduce the worldwide spread of TB infections.
This article is dedicated to Dr. Kiyoshi Isono’s 88 years anniversary,
and his long-standing contribution to the study of antibiotics.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0225-5) contains supplementary
material, which is available to authorized users.
CPZEN-45, a derivative of caprazene, showed excellent
antibacterial activity against M. tuberculosis and MDR-TB
strains, and showed excellent therapeutic efficacy in a
murine TB model infected with an XDR-TB strain resistant
to ten drugs [5]. Currently, CPZEN-45 is under develop-
ment as an inhaled MDR/XDR-TB drug [6, 7].
* Yoshiaki Takahashi
takashow@bikaken.or.jp
* Masayuki Igarashi
igarashim@bikaken.or.jp
1
In previous research, we showed that CPZEN-45 exhibits
selective anti-TB activity by specifically inhibiting the
Institute of Microbial Chemistry (BIKAKEN), 3-14-23,
Kamiosaki, Shinagawa-ku, Tokyo, Japan

Y. Ishizaki et al.
1"'
COOH
3"'
O
N
O
N
6"'
5
NH
2
5'
N
1'
O
5"
O
H2N
O
O
O
O
1"
OH
O
O
O
HO
OH
HO
O
O
O
R
COOH
caprazene
O
O
O
N
O
N
NH
N
O
O
H2N
O
R
O
HO
COOH
HO
OH
A
B
C
D
E
F
HO
OH
O
N
O
N
NH
O
caprazamycins (CPZs)
N
O
O
H2N
O
HO
OH
HO
OH
caprazol
G
Scheme 1 Preparation of caprazene and caprazol from a mixture of caprazamycins A−G
Fig. 1 Biosynthetic pathway of arabinogalactan in M. tuberculosis (a),
teichoic acid in B. subtilis 168 (b), and bacterial peptidoglycan (c).
Reactions ‘a’ and ‘b’ surrounded by dashed lines indicate the reactions
observed in this report. P phosphate, GlcNAc N-acetyl-D-glucosamine,
Rha rhamnose, Galf galactofuranoside, Araf arabinofuranoside,
ManNAc N-acetyl-D-mannosamine, Gro glycerol, MurNAcX5 N-
acetyl-D-muramate pentapeptide, PBPs penicillin binding proteins.
Since radiolabeled UDP-GlcNAc was used as the substrate for the
enzyme assays in this report, MarY activity was evaluated with the
subsequent reaction by MurG
phospho-N-acetyl glucosaminyl transferase, WecA [8]
involved in arabinogalactan biosynthesis (Fig. 1) [9]. This
mechanism is different from that of the parent compounds,
CPZs, which acts on bacterial translocase I (also known as
MraY in various bacteria, or MurX in M. tuberculosis), a
paralog of WecA, which is involved in peptidoglycan bio-
synthesis (Fig. 1) [10]. WecA is a novel target as a drug for
tuberculosis. It is very interesting that CPZEN-45, the 1‴-
anilide derivative of caprazene, acquired WecA inhibitory
activity, although the starting material, caprazene, has no
enzyme inhibitory activity.
Because of its attractive biological activity and interest in
the synthetic chemistry, total synthesis of CPZEN-45 using
pure chemical methods has been reported [11, 12], but there

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug
has not yet been any reports on the structure−activity
relationships (SAR) of CPZEN-45. Herein, we describe the
SAR of five analogs, 1–5, of CPZEN-45 (Fig. 2), which
were prepared by focusing on the seven-membered ring and
an anilide moiety of CPZEN-45.
resulting in saturated compound 9 stereoselectively, with
high yield. The S configuration at C-2‴ position of 9 was
determined using X-ray crystallography (see Fig. S1 in
Supplemental). The expression of this stereoselectivity is
presumed to be due to the steric influence of uracil and N-
protected 5-amino-5-deoxy-D-ribose moiety located in the
beta face. Similar deprotection of the amino group as
described above proceeded smoothly to produce 4.
Results
Compound 5 was prepared using caprazol, which is one
of the core components obtained from a mixture of capra-
zamycins. Compound 5 corresponds to the 3‴-hydroxyl
derivative of 4. With respect to the seven-membered ring
moiety, 5 has a structure closer to the parent natural product
caprazamycins than CPZEN-45. As shown in Scheme 3, 5
was readily synthesized via 5″-N-t-butoxycarbonylation of
caprazol, introduction of 4-butylaniline through an amide-
linkage, and deprotection of the amino group.
Synthesis
As outlined in Scheme 2, compounds 2 and 3 were prepared
according to the synthetic method [4] of CPZEN-45. The
condensation reaction of N-Boc caprazene with 4-
propylaniline or 2-butylaniline was effectively attained
using 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmor-
pholinium chloride (DMT-MM) to produce amides 6 and 8.
Subsequent removal of the protecting group (Boc) from the
primary amino group by trifluoroacetic acid efficiently
produced analogs 2 and 3 of CPZEN-45.
Effects of the analogs of CPZEN-45 on enzyme
activity
On the other hand, treatment of 5″-N-Boc CPZEN-45 (7)
[4] with sodium borohydride induced the reduction of the
double bond on the seven-membered diazepinone ring,
To investigate the SAR of CPZEN-45 analogs, we used an
enzyme assay system we previously established, which uses
Fig. 2 Structures of CPZEN-45 and its analogs

Y. Ishizaki et al.
R
O
NH
COOH
O
NH
O
O
N
O
O
N
N
N
(a)
ref.4
NH
O
O
O
N
caprazene
N
O
O
O
O
O
O
HN
HN
O
O
HO
OH
HO
OH
HO
OH
HO
OH
N-Boc caprazene
R =
(CH2)2Me
(CH2)3Me
6
7
8
R =
R =
(CH2)3Me
O
N
H
2"'
O
N
N
(b)
NH
O
(b)
(c)
O
O
O
N
4
7
O
O
HN
O
HO
OH
2, 3
HO
OH
9
Scheme 2 Synthesis of 2–4 from caprazene. Reagents and conditions: a RNH2, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM), 95% aqueous 2-propanol, room temperature; b CF3COOH, MeOH, room temperature; c NaBH4, MeOH, room temperature
HO
N
COOH
O
N
(b)
NH
(a)
O
O
O
N
caprazol
O
O
O
HN
O
HO
OH
HO
OH
10
O
HO
N
N
H
O
N
(c)
NH
O
5
O
O
N
O
O
O
HN
O
HO
OH
HO
OH
11
Scheme 3 Synthesis of 5 from caprazol. Reagents and conditions:
a Boc2O, Et3N, aqueous 1,4-dioxane, room temperature; b 4-butyla-
chloride (DMT-MM), 95% aqueous 2-propanol, room temperature;
c CF3COOH, MeOH, room temperature
niline,
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug
cell lysates of Bacillus subtilis [9]. In addition to MraY,
which is widely distributed among bacteria, B. subtilis also
has a WecA ortholog, TagO, as the enzyme that catalyzes
the first step of the biosynthesis of teichoic acid (Fig. 1) [13].
The effects of the analogs of CPZEN-45 on the activity
of TagO were evaluated using sonicated cell lysate of
BKKZ2202 as the enzyme. The IC50 value of CPZEN-45
against TagO was 55 nM, the strongest activity among the
compounds tested. Compounds 1 and 2 were also effective,
but their activity was inferior to that of CPZEN-45. Com-
pound 3 showed moderate inhibition, but 4 and 5 were not
effective (Fig. 3 and Table 1). No compounds tested
inhibited MraY or MurG effectively, whereas caprazamycin
B inhibits MraY and/or MurG at an IC50 of less than
500 nM. One thing to be noted is that MraY activity was
evaluated by the subsequent reactions with MurG [14, 15]
(see Materials and Methods and Fig. 1c), because radi-
olabeled UDP-GlcNAc was used as the substrate of this
enzyme assay.
Effects of the analogs of CPZEN-45 on the viability
of B. subtilis 168-derived strains
To evaluate the contribution of tagO and mraY to the
resistance of the analogs of CPZEN-45, the MIC of these
compounds against B. subtilis strains were determined
(Table 1). CPZEN-45 showed the highest antibacterial
activity among all of the compounds tested against the
control strain BKZ2201, with an MIC of 8 µg/mL. The
activities of the analogs of CPZEN-45 were 2−32 times
lower than CPZEN-45. The tagO-overexpressing strain
BKKZ2202 was at least four times as resistant, compared
with BKKZ2201, as any of the analogs tested, except
compound 5. All of the analogs showed similar antibacterial
activity against the mraY-overexpressing strain BKKZ2203
and BKKZ2392 strain which overexpresses mraY and
murG, as BKKZ2201. The MICs of caprazamycin B against
BKKZ2203 and BKKZ2392 were quite different from those
against BKKZ2201.
a
b
CPZEN-45
125
100
75
50
25
0
1
2
3
4
5
10
100
1000
10000
Drug concentration (nM)
Drug concentration (nM)
Fig. 3 Effect of the analogs of CPZEN-45 on the TagO activity of cell lysates of B. subtilis BKKZ2202 strain. a Autoradiographic image of TLC,
developed and subjected to radioautography as described in Materials and methods, showing the effects of the analogs of CPZEN-45 on the
synthesis of the product of TagO. b TagO activities calculated from the autoradiographic intensity of the TagO products shown in (a). Circle,
CPZEN-45; triangle, 1; square, 2; diamond, 3; cross, 4; plus, 5
Table 1 Antibacterial activities
CPZEN-45
1
2
3
4
5
CPZ-B
against B. subtilis strains and
enzyme inhibition activities of
CPZEN-45 and its analogs
MIC (µg/mL)
BKKZ2201 (control)
8
32
16
32
256
>512
128
128
128
256
128
128
4
BKKZ2202 (tagO)
64
8
256
32
128
16
256
32
4
BKKZ2203 (mraY)
>128
>128
BKKZ2392 (mraY, murG)
8
64
32
64
IC50 (nM)
TagO
55
190
180
630
2,100
1,700
not tested
MraY+MurG
7900
>12,500 >12,500 >12,500 >12,500 >12,500 <500
CPZ-B represents caprazamycin B

Y. Ishizaki et al.
Table 2 Antibacterial activities of CPZEN-45 and its analogs against Mycobacterium strains
Test organisms
MIC (µg/mL)
CPZEN-45
1
2
3
4
5
Slowly growing mycobacteria
M. avium subsp. avium ATCC 25291T
M. avium subsp. paratuberculosis ATCC 43015
M. intracellulare JCM 6384T
M. bovis BCG Pasteur
0.5
0.25
0.125
2
16
8
8
64
1
>128
8
>128
16
0.5
0.5
8
1
1
4
16
4
8
32
128
>128
M. bovis BCG Tokyo 172–2
Rapidly growing mycobacteria
M. smegmatis ATCC 607T
1
4
4
8
128
32
128
64
64
>128
>128
128
64
M. fortuitum B-0701
M. vaccae ATCC 15483T
>128
32
>128
128
>128
64
>128
64
>128
64
Effects of the analogs of CPZEN-45 on the viability
of mycobacterial strains
We also checked the importance of the double bond on the
seven-membered ring. Compounds 4 and 5, with saturated
ring systems whose stereochemistry is the same as that of
caprazamycins, were prepared for the investigation of MraY
and TagO inhibitory activity. The inhibitory activities of these
compounds against TagO were notably lower than that of
CPZEN-45, suggesting that the double bond on the seven-
membered ring system is structurally important for binding to
TagO. This observation also suggests that the synthesis of
inhibitors of TagO or WecA utilizing caprazol, one of the core
compounds of CPZs, is less promising than anticipated.
With respect to MraY/MurG subsequent reactions, none
of the analogs tested inhibited these reactions effectively,
whereas caprazamycin B, the parent compound of CPZEN-
45, showed inhibitory effects against the subsequent reac-
tions. Data from the enzyme assay described above corre-
spond reasonably well with antibacterial activity against B.
subtilis strains.
These results indicate that the presence of double bond at
C-2‴ and C-3‴ on the seven-membered ring is crucial for
inhibitory activity against TagO, and the position and chain
length of the alkyl group attached to the anilide moiety is
also important. This observation suggests that there is a
strict structural limitation on the degree of inhibitory
activity of CPZEN-45 for TagO.
This finding appeared to be mostly applicable to WecA,
the ortholog of TagO, of slowly growing mycobacteria,
according to data on the antibacterial activity of the analogs
of CPZEN-45. In rapidly growing mycobacteria the analogs
of CPZEN-45 were hardly effective. This lack may be
because these compounds had lower affinity to WecA, or to
lower permeability of the cell membrane, or because they
were well recognized by an efflux pump in rapidly growing
mycobacteria. Even though CPZEN-45 was not so effec-
tive, it showed better inhibitory activity against M. smeg-
matis and M. vaccae than the other analogs, supporting the
superiority of this compound.
The antimycobacterial activity of CPZEN-45 and its analogs
is shown is Table 2. In slowly growing mycobacteria, 1, 2 and
3 showed similar or weaker activity at MIC values of 1−16
times higher, compared with CPZEN-45. 4 and 5 were not
effective at an MIC of ≥4 µg/mL. In rapidly growing bacteria,
all of the compounds tested were hardly effective, although
CPZEN-45 had better activity than the other analogs.
Discussion
Although caprazene was inactive, antibacterial activity was
restored for its 1‴-anilide derivatives, as exemplified by
CPZEN-45, which contains a 4-butylaniline. It has pre-
viously been shown that, in the process of discovery of
CPZEN-45, increasing the alkyl chain at the para-position
of aniline slightly improves the antimicrobial activity, but at
the same time enhances the hemolytic side effects [4].
Therefore, analogs 1 and 2 of CPZEN-45 with a shortened
alkyl chain were synthesized, and the effect of the small
structural change to CPZEN-45 on the antimycobacterial
activity and inhibition of TagO activity was evaluated in
sonicated cell lysates of B. subtilis. As the result, it is
revealed that as the carbon chain become shorter, from butyl
to propyl to ethyl, inhibitory activity against TagO and
antimycobacterial activity decreased.
Because 4-butylanilide was shown to have more effec-
tive inhibitory activity, we next tested the effect of the
position of the butyl group of the anilide moiety using data
from CPZEN-45 and 3. The inhibitory activity of 3 against
TagO and its antibacterial activity against Mycobacterium
strains were significantly reduced compared to that of
CPZEN-45, indicating that the para substitution of the butyl
group is superior to the ortho substitution.

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug
Although these observations should also be discussed
from the viewpoint of structural biology, the crystal struc-
ture of TagO/WecA orthologs has not been reported in any
species to date. Even for protein expression and purifica-
tion, there is only one report using the thermophilic bac-
terium Thermotoga maritima [16]. We are now trying to
overexpress WecA of M. tuberculosis using E. coli and M.
smegmatis, in order to perform subsequent structural ana-
lysis. If this trial successful, we will be able to confirm the
findings in this report, and elucidate the mode of mechan-
ism of CPZEN-45 against bacterial WecA, using the
structural information.
was washed with saturated aq sodium chloride, dried
(Na2SO4), and concentrated. The residue was purified by
silica gel column chromatography (7:1 CHCl3−MeOH) to
give 6 (516 mg, 84%) as a colorless solid. ESI-MS: m/z
calcd for C36H50N6NaO13 (M+Na)+ 797.3, found 797.3; 1H
NMR (600 MHz, DMSO-d6) δ 0.87 [3H, t, J = 7 Hz, CH3
(CH2)2C6H4NH], 1.31 [9 H, s, (CH3)3C−O], 1.56 (2H, m,
CH3CH2CH2−), 2.35 (3H, s, CH3N-5‴), 2.94 (3H, s,
CH3N-8‴), 5.02 (1H, s, H-1″), 5.59 (1H, d, J = 8 Hz, H-5),
5.63 (1H, slightly br d, J = ~2 Hz, H-1′), 6.39 (1H, t, J =
6 Hz, H-3‴), 7.13 and 7.52 [each 2H, d, J = 8.5 Hz, CH3
(CH2)2C6H4NH], 7.30 (1H, br s, HN-5″), 7.79 (1H, d, J =
8 Hz, H-6), 10.14 (1H, s, HN-1‴), 11.31 (1H, s, HN-3).
Materials and methods
5″-N-t-Butoxycarbonylcaprazene 2-butylanilide (8)
General information
To a solution of N-Boc caprazene (an addition salt of trie-
thylamine, 600 mg, 0.791 mmol) in 2-propanol−water
(19:1, 12 mL) were added 2-butylaniline (594 mg,
3.98 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM) (330 mg,
1.19 mmol) and the mixture was stirred for 2 h at room
temperature. Concentration gave a residue, which was dis-
solved in 5% aq potassium hydrogen sulfate (35 mL). The
aqueous solution was washed with EtOAc and neutralized
with sodium hydrogen carbonate (1.3 g). The precipitate
obtained was extracted with EtOAc. The organic solution
was washed with saturated aq sodium chloride, dried
(Na2SO4), and concentrated. The residue was purified by
silica gel column chromatography (7:1 CHCl3−MeOH) to
give 8 (359 mg, 58%) as a colorless solid. ESI-MS: m/z
calcd for C37H52N6NaO13 (M+Na)+ 811.3, found 811.3; 1H
NMR (600 MHz, DMSO-d6) δ 0.80 [3H, t, J = 7 Hz, CH3
(CH2)3C6H4NH], 1.19 (2H, m, CH3CH2CH2CH2−), 1.36
[9H, s, (CH3)3C–O], 1.41 (2H, m, CH3CH2CH2CH2−), 2.36
(3H, s, CH3N-5‴), 3.00 (3H, s, CH3N-8‴), 5.03 (1H, s, H-
1″), 5.60 (1H, d, J = 8 Hz, H-5), 5.61 (1H, slightly br d, J =
~2 Hz, H-1′), 6.47 (1H, t, J = 6 Hz, H-3‴), 7.37 (1H, br s,
HN-5″), 7.82 (1H, d, J = 8 Hz, H-6), 9.72 (1H, s, HN-1‴),
11.33 (1H, s, HN-3).
Melting point was determined on a Kofler block and is
uncorrected. NMR spectra were recorded on an AVANCE
III 600 spectrometer (1H at 600 and 13C at 150.9 MHz)
(Bruker, Billerica, MA, USA) at 300 K, unless stated
otherwise. The chemical shifts (δ) of H and 13C spectra
1
were measured downfield from internal Me4Si, and were
confirmed, when necessary, by shift-correlated 2D spectra.
The mass spectra were recorded using a Thermo Scientific
LTQ Orbitrap XL mass spectrometer (HRESI-MS) or
Thermo Scientific LTQ XL (ESI) spectrometer (Thermo
Fisher Scientific, San Jose, CA, USA). Thin layer chro-
matography (TLC) was performed on a Kieselgel 60 F254
(Merck, Darmstadt, Germany), and column chromato-
graphy was carried out on a Wakogel C-200 (Wako Pure
Chemical Industries, Ltd., Osaka, Japan). Elemental analy-
sis was performed using a Perkin Elmer PE 2400 II analyzer
(PerkinElmer Inc., Waltham, MA, USA). Optical rotations
were measured on a P-1030 polarimeter (JASCO Corpora-
tion, Tokyo, Japan).
Preparation of caprazene-1‴-anilides
5″-N-t-Butoxycarbonylcaprazene 4-propylanilide (6)
Caprazene 4-propylanilide (2)
To a solution of N-Boc caprazene [4] (an addition salt of
triethylamine, 602 mg, 0.793 mmol) in 2-propanol−water
(19:1, 12 mL) were added 4-propylaniline (323 mg,
2.39 mmol) and 4-(4, 6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM) (329 mg,
1.19 mmol) and the mixture was stirred for 3 h at room
temperature. Concentration gave a residue, which was dis-
solved in 5% aq potassium hydrogen sulfate (35 mL). The
aqueous solution was washed with EtOAc and neutralized
with sodium hydrogen carbonate (1.3 g). The precipitate
obtained was extracted with EtOAc. The organic solution
A solution of 6 (196 mg, 0.253 mmol) in trifluoroacetic acid
−methanol (4:1, 6 mL) was kept for 2 h at room tempera-
ture. The reaction solution was concentrated, and to the
resulting syrup was added diethyl ether. The precipitate
obtained was thoroughly washed with diethyl ether to give
2 bis-trifluoroacetate (218 mg, 94%) as a colorless solid,
[α]D20 + 83° (c 1, MeOH); HRESI-MS: m/z calcd for
C31H43N6O11 (M + H)+ 675.2984, found 675.2987; 1H
NMR (600 MHz, DMSO-d6) δ 0.87 (3H, t, J = 7 Hz,
CH3CH2CH2−), 1.56 (2H, m, CH3CH2CH2−), 2.39 (3H, br

Y. Ishizaki et al.
s, CH3N-5‴), 2.96 (3H, s, CH3N-8‴), 5.12 (1H, slightly br
s, H-1″), 5.59 (1H, d, J = 2 Hz, H-1′), 5.61 (1H, slightly br
d, J = 8 Hz, H-5), 6.40 (1H, br s, H-3‴), 7.14 and 7.52
[each 2H, d, J = 8.5 Hz, CH3(CH2)2C6H4NH], 7.67 (1H,
slightly br d, J = 8 Hz, H-6), 8.11 (3H, br s, NH3+), 10.17
(1H, s, HN-1‴), 11.33 (1H, s, HN-3). Anal Calc for
C31H42N6O11 2CF3COOH H2O: C 45.66, H 5.04, N 9.13.
Found: C 45.39, H 5.23, N 9.02.
−3.84 (2H, H-3′ and H-3″), 3.88 (1H, br, H-4″), 4.07 (1H, d,
J = 10 Hz, H-5′), 4.16 (1H, d, J = 8 Hz, H-4′), 4.33 (1H, t,
J = 4 Hz, H-2‴), 4.91 (1H, d, J = 5.5 Hz, HO-2″), 4.92 (1H,
d, J = 6 Hz, HO-3″), 4.96 (1H, s, H-1″), 5.03 (1H, s, H-1′),
5.07 (1H, d, J = 6 Hz, HO-3′), 5.38 (1H, d, J = 8 Hz, H-5),
5.43 (1H, d, J = 4 Hz, HO-2′), 6.87 and 7.36 [each 2H, d,
J = 8.5 Hz, CH3(CH2)3C6H4NH], 7.02 (1H, slightly br d, J =
8.5 Hz, HN-5″), 7.61 (1H, d, J = 8 Hz, H-6), 9.39 (1H, s,
HN-1‴), 11.16 (1H, s, HN-3); 13C NMR (150.9 MHz,
DMSO-d6) δ 13.7 (CH3CH2CH2CH2−), 21.3 (C-3‴), 21.6
(CH3CH2CH2CH2−), 28.1 [(CH3)3C−O], 32.8 (CH3CH2
CH2CH2−), 33.6 (CH3N-5‴), 34.1 (CH3CH2CH2CH2−),
36.6 (CH3N-8‴), 41.8 (C-5″), 53.5 (C-4‴), 61.6 (C-2‴), 63.7
(C-6‴), 68.7 (C-3′), 71.1 (C-3″), 74.2 (C-5′), 74.6 (C-2′),
75.4 (C-2″), 77.5 [(CH3)3C-O], 82.1 (C-4′), 83.1 (C-4″), 89.0
(C-1′), 100.1 (C-5), 109.9 (C-1″), 120.5, 127.3, 135.8, and
137.4 (aromatic), 139.1 (C-6), 149.6 (C-2), 155.8 (COHN-
5″), 163.1 (C-4), 167.4 (C-1‴), 171.0 (C-7‴). Anal Calc for
C37H54N6O13 1.5H2O: C 54.34, H 7.02, N 10.28. Found: C
54.22, H 7.23, N 10.19.
Caprazene 2-butylanilide (3)
A solution of 8 (226 mg, 0.286 mmol) in trifluoroacetic acid
−methanol (4:1, 6.8 mL) was kept for 2 h at room tem-
perature. The reaction solution was concentrated, and to the
resulting syrup was added diethyl ether. The precipitate
obtained was thoroughly washed with diethyl ether to give
3 bis-trifluoroacetate (253 mg, 94%) as a colorless solid,
[α]D20 + 76° (c 1, MeOH); HRESI-MS: m/z calcd for
C32H45N6O11 (M + H)+ 689.3141, found 689.3146; 1H
NMR (600 MHz, DMSO-d6) δ 0.81 (3H, t, J = 7 Hz,
CH3CH2CH2CH2−), 1.20 (2H, m, CH3CH2CH2CH2−),
1.42 (2H, m, CH3CH2CH2CH2−), 2.39 (3H, br s, CH3N
-5‴), 3.03 (3H, s, CH3N-8‴), 5.13 (1H, slightly br s, H-1″),
5.59 (1H, d, J = 2 Hz, H-1′), 5.66 (1H, slightly br d, J =
7 Hz, H-5), 6.47 (1H, br s, H-3‴), 7.70 (1H, slightly br d, J
= 7 Hz, H-6), 8.14 (3H, br s, NH3+), 9.72 (1H, slightly br s,
HN-1‴), 11.35 (1H, s, HN-3). Anal Calc for C32H44N6O11
2CF3COOH H2O: C 46.26, H 5.18, N 8.99. Found: C
45.95, H 5.56, N 8.99.
3‴-Deoxycaprazol 4-butylanilide (4)
A solution of 9 (223 mg, 0.273 mmol) in trifluoroacetic acid
−methanol (4:1, 6.7 mL) was kept for 2 h at room tem-
perature. The reaction solution was concentrated, and to the
resulting syrup was added diethyl ether. The precipitate
obtained was thoroughly washed with diethyl ether to give
4 bis-trifluoroacetate (250 mg, 98%) as a colorless solid,
[α]D20 + 22° (c 1, MeOH); HRESI-MS: m/z calcd for
C32H47N6O11 (M + H)+ 691.3297, found 691.3313; 1H
NMR (600 MHz, DMSO-d6) δ 0.88 (3H, t, J = 7 Hz,
CH3CH2CH2CH2−), 1.27 (2H, m, CH3CH2CH2CH2−),
1.47 (2H, m, CH3CH2CH2CH2−), 2.14 (3H, slightly br s,
CH3N-5‴), 2.44 (2H, m, CH3CH2CH2CH2−), 3.08 (3H, s,
CH3N-8‴), 4.40 (1H, s, H-2‴), 4.93 (1H, s, H-1′), 4.99 (1H,
s, H-1″), 5.46 (1H, slightly br d, J = 7.5 Hz, H-5), 7.45 (1H,
slightly br d, J = 7.5 Hz, H-6), 8.03 (3H, br s, NH3+), 9.47
(1H, s, HN-1‴), 11.18 (1H, s, HN-3). Anal Calc for
C32H46N6O11 2CF3COOH H2O: C 46.16, H 5.38, N 8.97.
Found: C 46.29, H 5.49, N 8.90.
5″-N-t-Butoxycarbonyl-3‴-deoxycaprazol 4-butylanilide (9)
To an ice-cooled solution of 7 (986 mg, 1.25 mmol) in
methanol (20 mL) was added sodium borohydride (711 mg,
18.7 mmol). After the mixture was stirred for 3 h at room
temperature, the reaction was quenched by the addition of
dry-ice. Concentration gave a residue, which was extracted
with 1-butanol. The organic solution was washed with water
and concentrated. The resulting solid was purified by column
chromatography (upper layer of 5:1:1 EtOAc−1-BuOH
−20% aq AcOH) to give 9 (831 mg, 81%) as a colorless
solid. An analytical sample was prepared by crystallization
from methanol−water, m.p. 163~164 °C; [α]D20 − 9° (c 1,
MeOH); ESI-MS: m/z calcd for C37H54N6NaO13 (M+Na)+
Preparation of caprazol-1‴-anilide
5″-N-t-Butoxycarbonylcaprazol (10)
1
813.4, found 813.4; H NMR (600 MHz, DMSO-d6) δ 0.88
(3H, t, J = 7 Hz, CH3CH2CH2CH2−), 1.27 (2H, m,
CH3CH2CH2CH2−), 1.34 [9H, s, (CH3)3C-O], 1.47 (2H, m,
CH3CH2CH2CH2−), 1.95−2.07 (2H, m, H-3‴), 2.12 (3H, s,
CH3N-5‴), 2.44 (2H, m, CH3CH2CH2CH2−), 2.88 (1H,
slightly br d, J = 14.5 Hz, H-4‴a), 3.00 (1H, slightly br d,
J = 14 Hz, H-5″a), 3.06 (3H, s, CH3N-8‴), ~3.35 (1H, H-5″
b), 3.40 (1H, slightly br t, J = 12 Hz, H-4‴b), 3.71 (1H, d,
J = 10 Hz, H-6‴), 3.73−3.77 (2H, H-2′ and H-2″), 3.78
To a suspension of caprazol [4] (tetrahydrate, 346 mg,
0.534 mmol) in water−1, 4-dioxane (2:1, 10 mL) were
added triethylamine (145 mg, 1.43 mmol) and di-t-butyl
dicarbonate (167 mg, 0.765 mmol) and the mixture was
stirred for 1 h at room temperature. To the resulting solution
was added 28% aq ammonia (0.1 mL) and the solution was
concentrated to give a colorless solid of 10 (421 mg, 97%)

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug
as an addition salt of triethylamine. This solid was used in
the next reaction without further purification.
temperature. The reaction solution was concentrated, and to
the resulting syrup was added diethyl ether. The precipitate
obtained was thoroughly washed with diethyl ether to give
5 bis-trifluoroacetate (209 mg) as a colorless solid. The solid
was dissolved in 1-BuOH and the solution was washed
successively with 5% aq sodium hydrogen carbonate and
water. Concentration gave a solid, which was chromato-
graphed (upper layer of 2:1:1 EtOAc−1-PrOH−H2O) to
give 5 trifluoroacetate (114 mg, 59%) as a colorless solid;
[α]D20 + 17° (c 0.5, MeOH); HRESI-MS: m/z calcd for
C32H47N6O12 (M+H)+ 707.3246, found 707.3249; ESI-MS:
m/z 819.3 (M+CF3COOH−H)−; 1H NMR (600 MHz,
DMSO-d6) δ 0.88 (3H, t, J = 7 Hz, CH3CH2CH2CH2−),
1.26 (2H, m, CH3CH2CH2CH2−), 1.46 (2H, m, CH3
CH2CH2CH2−), 2.38 (3H, s, CH3N-5‴), 2.43 (2H, m,
CH3CH2CH2CH2−), 3.00 (1H, slightly br dd, J = ~2 and
14 Hz, H-4‴a), 3.05 (1H, slightly br dd, J = ~2 and ~13 Hz,
H-5″a), 3.14 (1H, slightly br d, J = ~13 Hz, H-5″b), 3.15
(3H, s, CH3N-8‴), 3.22 (1H, slightly br d, J = 14 Hz, H-4‴
b), 3.58 (1H, d, J = 10 Hz, H-6‴), 3.66 (1H, ddd, J = 4.5,
6.5 and 9 Hz, H-3′), 3.73 (1H, t, J = 4.5 Hz, H-2′), 3.76
(1H, t, J = 4 Hz, H-2″), 3.98−4.04 (2H, m, H-3″ and H-4″),
4.09 (1H, d, J = 10 Hz, H-5′), 4.19 (1H, d, J = 9 Hz, H-4′),
4.37 (1H, m, H-3‴), 4.42 (1H, d, J = 5 Hz, H-2‴), 4.83 (1H,
s, H-1′), 4.96 (1H, s, H-1″), 5.15 (1H, d, J = 6.5 Hz, HO-
3′), 5.22 (1H, d, J = 5.5 Hz, HO-3″), 5.24 (2H, d, J = 4 Hz,
HO-2″ and HO-3‴), 5.44 (1H, d, J = 8 Hz, H-5), 5.52 (1H,
d, J = 4 Hz, HO-2′), 6.85 and 7.38 [each 2H, d, J = 8.5 Hz,
CH3(CH2)3C6H4NH], 7.42 (1H, d, J = 8 Hz, H-6), 8.02 (3H,
Compound 10 as an addition salt of triethylamine; ESI-
MS m/z calcd for C27H41N5NaO15 (M+Na)+ 698.3, found
1
698.3; H NMR (600 MHz, DMSO-d6) δ 1.01 [9H, t, J =
7 Hz, (CH3CH2)3N], 1.34 [9H, s, (CH3)3C–O], 2.31 (3H, s,
CH3N-5‴), 2.89 (1H, d, J = 15 Hz, H-4‴a), 2.98 (3H, s,
CH3N-8‴), 3.25 (1H, d, J = 15 Hz, H-4‴b), 3.66 (1H, d,
J = 10 Hz, H-6‴), 4.94 (1H, s, H-1″), 5.55 (1H, d, J = 8 Hz,
H-5), 5.59 (1H, d, J = 2.5 Hz, H-1′), 7.02 (1H, d, J =
8.5 Hz, HN-5″), 7.92 (1H, d, J = 8 Hz, H-6), 11.32 (1H, br
s, HN-3). Anal Calc for C27H41N5O15 Et3N 2H2O: C 48.76,
H 7.44, N 10.34. Found: C 48.55, H 7.58, N 10.05.
5″-N-t-Butoxycarbonylcaprazol 4-butylanilide (11)
To a solution of 10 (an addition salt of triethylamine, 2.28 g,
2.80 mmol) in 2-propanol−water (10:1, 44 mL) were added
4-butylaniline (480 mg, 3.22 mmol) and 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-
MM) (1.06 g, 3.83 mmol) and the mixture was stirred at
room temperature. The same amounts of 4-butylaniline and
DMT-MM were added after 5 and 15 h, and the mixture
was further stirred for 2 days. Concentration gave a syrup,
which was extracted with 5% aq potassium hydrogen sul-
fate (150 mL) and EtOAc (50 mL). The aqueous solution
was washed with EtOAc and neutralized with sodium
hydrogen carbonate (8.18 g). The precipitate obtained was
extracted with EtOAc and the organic solution was washed
with saturated aq sodium chloride, dried (Na2SO4), and
concentrated. The residue was purified by silica gel column
chromatography (6:1 CHCl3−MeOH) to give 11 (1.03 g,
44%) as a colorless solid; [α]D20 − 11° (c 0.5, MeOH); ESI-
MS: m/z calcd for C37H54N6NaO14 (M+Na)+ 829.4, found
br s, NH3+), 9.48 (1H, s, HN-1‴), 11.16 (1H, s, HN-3); 13
C
NMR (150.9 MHz, DMSO-d6) δ 13.7 (CH3CH2CH2CH2−),
21.7 (CH3CH2CH2CH2−), 32.7 (CH3CH2CH2CH2−), 34.0
(CH3CH2CH2CH2−), 36.3 (CH3N-5‴), 38.8 (CH3N-8‴),
39.6 (C-5″), 58.2 (C-4‴), 63.0 (C-6‴), 66.5 (C-2‴), 67.3 (C-
3‴), 68.4 (C-3′), 69.6 (C-3″), 74.2 (C-2″), 74.4 (C-2′), 74.8
(C-5′), 78.0 (C-4″), 81.4 (C-4′), 89.6 (C-1′), 99.9 (C-5),
109.6 (C-1″), 117.3 (CF3, q, JC, F = 302 Hz), 120.4, 127.2,
135.6, and 137.6 (aromatic), 138.9 (C-6), 149.5 (C-2),
157.6 (CF3COOH, q, JC, F = 30 Hz), 163.2 (C-4), 167.2 (C-
1‴), 169.8 (C-7‴). Anal Calc for C32H46N6O12 CF3COOH
3H2O: C 46.68, H 6.11, N 9.61. Found: C 46.56, H 6.07,
N 9.39.
1
829.3; H NMR (600 MHz, DMSO-d6) δ 0.88 [3H, t, J =
7 Hz, CH3(CH2)3C6H4NH], 1.27 (2H, m, CH3CH2CH2
CH2−), 1.37 [9H, s, (CH3)3C–O], 1.46 (2H, m,
CH3CH2CH2CH2−), 2.37 (3H, s, CH3N-5‴), 2.97 (1H, d,
J = 15 Hz, H-4‴a), 3.13 (3H, s, CH3N-8‴), 3.43 (1H, d,
J = 15 Hz, H-4‴b), 3.65 (1H, d, J = 10 Hz, H-6‴), 4.08
(1H, d, J = 10 Hz, H-5′), 4.18 (1H, d, J = 8 Hz, H-4′), 4.37
(2H, s, H-2‴ and H-3‴), 4.87 (1H, s, H-1′), 4.94 (1H, s,
H-1″), 5.35 (1H, d, J = 8 Hz, H-5), 6.85 and 7.35 [each 2H,
d, J = 8.5 Hz, CH3(CH2)3C6H4NH], 6.92 (1H, slightly br d,
J = 8.5 Hz, HN-5″), 7.58 (1H, d, J = 8 Hz, H-6), 9.38
(1H, s, HN-1‴), 11.14 (1H, s, HN-3). Anal Calc for
C37H54N6O14 2H2O: C 52.72, H 6.94, N 9.97. Found: C
52.95, H 7.24, N 9.99.
Construction of B. subtilis 168 derivatives
overexpressing tagO, mraY and murG
The Bacillus subtilis-Escherichia coli shuttle vector
pHYermA was constructed using pHY300PLK (Takara bio
Inc. Shiga, Japan) [17], as a source of replication origins,
and erythromycin-resistant ermA gene from a Staphylo-
coccus aureus strain. Fragments of tagO and mraY genes of
B. subtilis were cloned into pHYermA, and the resulting
plasmids were designated as pHYermA-tagO and
Caprazol 4-butylanilide (5)
A solution of 11 (187 mg, 0.222 mmol) in trifluoroacetic
acid−methanol (4:1, 5 mL) was kept for 2 h at room

Y. Ishizaki et al.
Table 3 Bacillus subtilis strains and plasmids used in this study
sonicated five times for 60 s each with 60 s breaks on ice,
and centrifuged at 3000 rpm to remove unbroken cells. The
supernatant, which was used as the enzyme source, was
stored at –80 °C until use. For the evaluation of enzyme
activity, the reaction mixture, contained 250 ng of total
protein/µL, 125 µM undecaprenyl phosphate (Larodan AB,
Solna, Sweden), 4 µCi/mL uridine diphosphate N-Acetyl-
[1-14C] D-Glucosamine (American Radiolabeled Chemicals
Inc., St. Louis, MO), and 0.1% CHAPS in a total volume of
0.05 mL of buffer A, followed by incubation for 2 h at
30 °C. The reaction was stopped by adding 0.25 mL of
chloroform/methanol (2:1), mixed vigorously, centrifuged
(3000 rpm, 15 min), and the resulting lower phase con-
taining the radiolabeled product was washed with 50 µL of
water and subsequent vigorous mixing. The resulting mix-
ture was centrifuged, and the lower phase was chromato-
graphed on a silica gel Kieselgel 60 F254 (Merck) by
developing with CHCl3 /MeOH/H2O, ≈14 M NH4OH
(56:42:6.8:2.7) and subsequent autoradiography (Typhoon
9400, GE Healthcare). The intensity of the autoradiographic
image was quantified using ImageJ software (National
Institutes of Health).
Strains/plasmids
Description
B. subtilis
168
trpC2
ATCC
BKKZ2201
BKKZ2202
BKKZ2203
BKKZ2392
Plasmids
168/pHYermA
This work
This work
This work
This work
168/pHYermA-tagO
168/pHYermA-mraY
168/pHYermA-mraYmurG
pHY300PLK
B. subtilis-E. coli shuttle
vector, p15A and pAMα1
replicons, AmpR, TetR
Takara
Bio Inc.
pHYermA
p15A and pAMα1 replicons, This work
EMR
pHYermA-tagO
pHYermA-mraY
pHYermA carrying a tagO
fragment
This work
pHYermA carrying an mraY This work
fragment
pHYermA-
mraYmurG
pHYermA carrying an mraY- This work
murG fragment
AmpR ampicillin resistant, TetR tetracycline resistant, EMR erythromy-
cin resistant
The enzyme activity of MraY was evaluated by the
subsequential reactions with another transglycosylase
MurG, which catalyzes the addition of N-acetyl-D-glucosa-
mine to the MraY product (Fig. 1). In this reaction, the same
procedure as used in the TagO reaction was applied, but cell
lysates of the BKKZ2392 strain, instead of the
BKKZ2203 strain, were used, and 100 µM UDP-MurNAc-
pentapeptide (UK Bacterial Cell Wall Biosynthesis Net-
work, University of Warwick, Coventry, UK) was added. In
this experiment, caprazamycin B was used as a control.
pHYermA-mraY, respectively. For the construction of
plasmid pHYermA-mraYmurG, a DNA fragment contain-
ing mraY-murD-spoVE-murG genes (part of an operon
consisting of ten genes) was amplified by PCR, inserted into
pHYermA, and the murD-spoVE region was cut out using
the restriction enzyme HindIII. E. coli HST08 strain (Takara
Bio Inc., Shiga, Japan) was used for plasmid construction.
Transformation of B. subtilis 168 strains using the con-
structed plasmids was performed as previously described
[9]. Erythromycin at concentrations of 500 and 5 µg/mL
was used for the selection of transformants of E. coli and B.
subtilis, respectively. The resulting strains (Table 3) were
used for MIC evaluation of CPZEN-45 analogs by micro-
broth dilution in LB medium, Miller (Thermo Fisher Sci-
entific Kabushiki Kaisha, Kanagawa, Japan). The strains
were incubated for 16 h at 37 °C, and the MIC values were
evaluated.
Antimycobacterial activity
The MICs against bacteria of CPZEN-45 and its analogs
(Table 2) were measured using a serial agar dilution method
using Middlebrook 7H10 agar (Thermo Fisher Scientific
Kabushiki Kaisha) with glycerol and OADC enrichment
(Thermo Fisher Scientific Kabushiki Kaisha). MIC values
were measured after 2−14 days at 37 °C.
Evaluation of enzyme activity of MraY and TagO of
B. subtilis
Acknowledgements We are grateful to Dr. Kiyoko Iijima of the
Institute of Microbial Chemistry (BIKAKEN) for assistance with the
NMR spectrometry experiments.
The enzyme activity of TagO was measured as previously
described, with some modifications [9]. Log phase cell
culture (OD600 ≈ 0.4) of B. subtilis BKKZ2202 was col-
lected, washed twice with ice-cold buffer A (50 mM
MOPS-KOH (pH 7.9), 10% Glycerol, 5 mM MgCl2, 5 mM
3-mercapto-1,2-propanediol) and resuspended in 1/40
volume of buffer A. The cell suspensions were probe-
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug
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