Synthesis and antimycobacterial activity of calpinactam derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.09.069

Synthesis of calpinactam 1, a fungal antimycobacterial metabolite, utilizing solid-phase peptide synthesis is described. To explore the structure-activity relationships of 1, its derivatives with different amino acids were also synthesized on the basis of the same synthetic strategy. These derivatives were examined for antimycobacterial activity against Mycobacterium smegmatis. Among them, only peptide 6d having d-Ala in place of d-Glu showed moderate activity.

Full text of DOI:10.1016/j.bmcl.2012.09.069

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7739–7741
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and antimycobacterial activity of calpinactam derivatives
⇑
Kenichiro Nagai , Nobuhiro Koyama, Noriko Sato, Chisato Yanagisawa, Hiroshi Tomoda
Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2012
Revised 10 September 2012
Accepted 21 September 2012
Available online 12 October 2012
Synthesis of calpinactam 1, a fungal antimycobacterial metabolite, utilizing solid-phase peptide synthesis
is described. To explore the structure–activity relationships of 1, its derivatives with different amino acids
were also synthesized on the basis of the same synthetic strategy. These derivatives were examined for
antimycobacterial activity against Mycobacterium smegmatis. Among them, only peptide 6d having
in place of -Glu showed moderate activity.
D-Ala
D
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Calpinactam
Antimycobacterial activity
Solid-phase peptide synthesis
Tuberculosis
Tuberculosis (TB), an infectious disease caused by Mycobacte-
rium tuberculosis, remains the major cause of death in the world.
It was estimated that there were 8.8 million clinical cases, 1.1 mil-
lion deaths from TB and 0.35 million deaths from HIV-co-infected
TB in 2010.1 Furthermore, the emergence of multidrug-resistant
(MDR) TB is a major concern because it depends on the high risk
of death. Currently used drugs, namely, isoniazid, ethambutol, rif-
ampicin, and pyrazinamide for the treatment of TB were developed
over 40 years ago. Therefore, development of anti-TB drugs with
new modes of action and structural features is urgently needed.2,3
Calpinactam 1, isolated from the culture broth of Mortierella
alpina FKI-4905, exhibited potent and selective inhibitory activity
against Mycobacterium smegmatis and no activity of a panel of
microorganisms including gram-positive and gram-negative bacte-
ria, fungi, and yeast in paper disk assay.4,5 Furthermore, in a liquid
microdilution method, 1 inhibited the growth of M. smegmatis with
purification of intermediates was troublesome. To synthesize a
variety of derivatives for SAR study, a more efficient synthetic
route is needed. In this paper, we report synthesis of calpinactam
utilizing solid-phase peptide synthesis, which enables to prepare
its derivatives for elucidation of SAR.
Synthetic approach to calpinactam 1 is illustrated in Scheme 1.
We envisioned that 1 would be accessed through coupling of pep-
tide fragment 2 and commercially available (S)-3-aminoazepan-2-
one 3. Peptide fragment 2 would be achieved by solid-phase pep-
tide synthesis using a trityl linker. The Boc group was chosen for
N-terminal protection of 2 because it can be tolerant under mild
cleavage conditions and removed by TFA together with the N-trityl
and O-tert-butyl groups at the final stage.
Fmoc-D-allo-Ile-OH was attached to 2-chlorotrityl chloride re-
sin, and then removal of the Fmoc group was performed by treat-
ment with 2% DBU/DMF to give on-resin amino acid 4 (Scheme 2).
The loading yield was quantitative by determining quantity of dib-
enzofulvene generated from the Fmoc group.8 The amino acids for
the sequence of tetrapeptide 5 were elongated by sequential cou-
an MIC value of 0.78
be a linear hexapeptide composing of
-leucine -Leu), -histidine -His),
-allo-isoleucine ( -allo-Ile) and (S)-3-aminoazepan-2-one by ami-
l
g/mL. Its structure was determined to
-phenylalanine ( -Phe),
-glutamic acid -Glu),
D
D
L
(L
L
(
L
D
(D
D
D
pling of the Fmoc-amino acids, Fmoc-
His(Trt)-OH, and Fmoc-Leu-OH with PyBop and deprotection of
the Fmoc group. Next, Boc- -Phe-OH was smoothly coupled with
D-Glu(OtBu)-OH, Fmoc-
no acids analysis, NMR studies, and its total synthesis. Calpinactam
is structurally different from the clinically used antitubecular
drugs and far less complex than antimycobacterial peptides such
as lariatins6 and trichoderins.7 However, the mode of action of 1 re-
mains unclear. Thus, studies on structure–activity relationships
(SAR) of 1 would be useful for design of a chemical probe and
development of a new antimycobacterial agent. We have already
completed total synthesis of 1 in liquid-phase synthesis. However,
synthetic calpinactam was obtained in 6% overall yield because
D
5 to afford the corresponding pentapeptide, and following cleavage
from the resin was accomplished by treatment with 25% HFIP/DCM
to release the pentapeptide 2 without loss of the Boc, Trt, and t-Bu
groups.9 Finally, coupling of the peptide and (S)-3-aminoazepan-2-
one 3 using EDCIꢀHCl and HOBt followed by global deprotections
with TFA/TIPS/H2O (95/2.5/2.5) provided the crude product which
was purified by preparative reverse-phase HPLC to give calpinac-
tam in 37% overall yield. The 1H and 13C NMR spectra, optical rota-
tion, IR, and high-resolution MS were in complete accord with
those of the natural product.10
⇑
Corresponding author. Tel./fax: +81 3 5791 6261.
E-mail address: nagaik@pharm.kitasato-u.ac.jp (K. Nagai).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.069

7740
K. Nagai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7739–7741
N
O
N
O
NH
NTrt
O
O
O
O
O
O
H
N
H
N
H
N
H
H
H
H
NH
N
N
OH
NH
H2N
H2N
N
H
N
H
BocHN
N
N
H
+
H
O
O
O
O
2
3
CO2H
CO2tBu
Calpinactam (1)
Scheme 1. Synthetic approach of calpinactam.
N
NTrt
O
O
H
a
b-d
e,f
H
H
Cl
O
H2N
N
O
H2N
N
N
H
H
2-chlorotrityl
chloride resin
O
O
O
4
5
CO2tBu
N
N
NH
NTrt
O
O
O
O
O
H
H
H
H
H
H
H
g,h
NH
N
N
N
N
N
OH
H2N
N
H
N
H
BocHN
N
N
H
H
O
O
O
O
O
O
CO2H
CO2tBu
Calpinactam (1)
2
Scheme 2. Solid-phase synthesis of calpinactam. Reagents and conditions: (a) (i) Fmoc-
D
-allo-Ile-OH, DIPEA, DCM, rt, 1 h (ii) 2% DBU/DMF, rt, 5 min ꢁ 3; (b) (i) Fmoc-
D-Glu(O-
tBu)-OH, PyBop, DIPEA, DCM/DMF (4/1), rt, 1 h (ii) 2% DBU/DMF, rt, 5 min ꢁ 3; (c) (i) Fmoc- -His(Trt)-OH, PyBop, DIPEA, DCM/DMF (4/1), rt, 1 h (ii) 2% DBU/DMF, rt, 5 min ꢁ 3;
(d) (i) Fmoc-
L
-Leu-OH, PyBop, DIPEA, DCM/DMF (4/1), rt, 1 h (ii) 2% DBU/DMF, rt, 5 min ꢁL3; (e) Boc-
D-Phe-OH, PyBop, DIPEA, DCM/DMF (4/1), rt, 1 h; (f) HFIP/DMF (4/1), rt,
30 min; (g) (S)-3-aminoazepan-2-one 3, EDCI, HOBt, DIPEA, DMF, rt, 3 h; (h) TFA/TIPS/H2O (95/2.5/2.5), rt, 1 h. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DIPEA = N,N-
diisopropylethylamine, EDCIꢀHCl = 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, HOBt : 1-hydroxybenzotriazole, PyBop = benzotriazole-1-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate, HFIP = 1,1,1,3,3,3-hexafluoro-2-propanol, TIPS = triisopropylsilane.
Table 1
Antimicobacterial activity of calpinactam derivatives
N
NH
O
O
O
H
N
H
N
H
N
H
NH
H2N
N
H
N
H
O
O
O
CO2H
I
II
III
IV
V
VI
Peptide
I
II
III
IV
V
VI
Yield (%)
MIC (
0.78
>50.0
lg/mL)
1
3
3
3
3
3
3
37
71
38
37
52
52
82
55
48
48
35
48
26
95
47
66
57
34
39
D
D
D
D
D
D
D
-Phe
-Ala
-Phe
-Phe
-Phe
-Phe
-Phe
L
L
L
L
L
L
L
L
-Leu
L
L
L
L
L
L
L
L
L
-His
D
-Glu
-Glu
-Glu
-Glu
-Ala
-Glu
-Glu
-Glu
-Glu
-Glu
D
D
D
D
D
D
D
D
D
D
D
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
-Ala
6a
6b
6c
6d
6e
6f
-Leu
-Ala
-Leu
-Leu
-Leu
-Leu
-Leu
-His
-His
-Ala
-His
-His
-His
-His
-His
D
D
D
D
D
D
D
D
D
>50.0
>50.0
6.25
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
L-Ala
6g
6h
6i
3
L
-Phe
3
3
3
3
D
D
D
D
D
D
D
D
D
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
D-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
D-His
6j
L-His
L-His
L-His
L-His
L-His
L-His
L-His
L-His
L-His
L-Glu
6k
6l
D-Glu
D-Glu
D-Glu
D-Glu
D-Glu
D-Glu
D-Glu
D-Glu
L-Ile
ent-3
D
D
D
D
D
D
D
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
-allo-Ile
6m
6n
6o
6p
6q
6r
OH
L-Lys
(S)-3-Aminopiperid-2-one
Cycloheptylamine
H
3
3
N-Ac-D-Phe
-D-allo-Ile

K. Nagai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7739–7741
7741
New calpinactam derivatives 6a–r with different amino acids
were prepared by the same synthetic manner and subjected to
the assay. The assay results are summarized in Table 1. The MIC
values were determined by the previously reported method.4 Syn-
thetic calpinactam displayed antimycobacterial activity against M.
for Young Scientists B (23790019 to K.N.) from the Ministry of Edu-
cation, Culture, Sports, Science, and Technology of Japan.
Supplementary data
smegmatis with an MIC value of 0.78 lg/mL similar to the natural
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.069.
product. Our preliminary synthetic efforts were focused on studies
to investigate important amino acid residues. To determine
whether the side-chain of a specific residue plays an important role
in antimycobacterial activity, we designed a set of derivatives in
which each amino acid residue is replaced with alanine at one po-
sition. The alanine scan derivatives except for peptide 6d showed
no antimycobacterial activity at high concentrations, indicating
that these residues are essential for antimycobacterial activity.
References and notes
1. World Health Organization. Global Tuberculosis Control 2011; WHO: Geneva,
Switzerland, 2011.
2. Dover, L. G.; Coxon, G. D. J. Med. Chem. 2011, 54, 6157.
3. Gutierrez-Lugo, M.-T.; Bewley, C. A. J. Med. Chem. 2008, 51, 2606.
4. Koyama, N.; Kojima, S.; Nonaka, K.; Masuma, R.; Matsumoto, M.; Omura, S.;
Peptide 6d retained moderate activity (6.25 lg/mL).
¯
To determine the effect of stereochemistry on antimycobacteri-
al activity, we synthesized a series of derivatives where each con-
figuration of the amino acid residues is inverted at one position. All
of the stereoisomers 6g–l exhibited no inhibitory activity against
M. smegmatis, demonstrating the importance of the naturally
occurring configurations.
Next, we investigated the effect of varying N- and C-terminal
moieties on antimycobacterial activity. C-terminal deficient pep-
tide 6m and N-terminal deficient peptide 6q drastically reduced
the activity. Furthermore, compounds 6n–p which replaced the
Tomoda, H. J. Antibiot. 2010, 63, 183.
5. Koyama, N.; Kojima, S.; Fukuda, T.; Nagamitsu, T.; Yasuhara, T.; Omura, S.;
¯
Tomoda, H. Org. Lett. 2010, 12, 432.
6. Iwatsuki, M.; Tomoda, H.; Uchida, R.; Gouda, H.; Hirono, S.; Omura, S. J. Am.
¯
Chem. Soc. 2006, 128, 7486.
7. Pruksakorn, P.; Arai, M.; Kotoku, N.; Vilchèze, C.; Baughn, A. D.; Moodley, P.;
Jacobs, W. R., Jr.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2010, 20, 3658.
8. Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2002, 9, 203.
9. Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem. Soc., Chem.
Commun. 1994, 2559.
10. Synthetic calpincatam (1):
½
a 2D6
ꢂ
ꢃ 26 (c = 0.05, AcOH). ESI-MS
: calcd for
C
38H58N9O8: [M+H]+ 768.4408, found: 768.4402. IR (KBr) : 3418, 3281, 1678,
1630, 1525, 1437 cmꢃ1
.
1H NMR (600 MHz, DMSO-d6): d 8.63 (br s, 1H), 8.59 (d,
3-aminoazepan-2-one moiety with L-lysine, S-3-aminopiperid-2-
J = 8.0 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.94 (d, J = 8.0 Hz,
1H), 7.84 (dd, J = 5.0, 8.0 Hz, 1H), 7.80 (d, J = 7.0 Hz, 1H), 7.32ꢃ7.29 (ovlp m,
2H), 7.26ꢃ7.23 (ovlp m, 3H), 7.20 (br s, 1H), 4.57 (dt, J = 6.5, 8.0 Hz, 1H), 4.38
(m, 1H), 4.37 (m, 1H), 4.31 (dd, J = 5.5, 9.0 Hz, 1H), 4.25 (dt, J = 7.0, 7.5 Hz, 1H),
4.07 (t, J = 7.5 Hz, 1H), 3.15 (ddd, J = 5.0, 12.0, 14.0 Hz, 1H), 3.04 (dd, J = 6.0,
15.0 Hz, 1H), 3.03 (m, 1H), 3.02 (dd, J = 6.5, 13.5 Hz, 1H), 2.96 (dd, J = 7.5,
13.5 Hz, 1H), 2.85 (dd, J = 8.5, 15.0 Hz, 1H), 2.10ꢃ2.07 (ovlp m, 2H), 1.90ꢃ1.78
(ovlp m, 3H), 1.73ꢃ1.69 (ovlp m, 2H), 1.66 (m, 1H), 1.61 (m, 1H), 1.35 (m, 1H),
1.29ꢃ1.25 (ovlp m, 3H), 1.18 (m, 1H), 1.16 (m, 1H), 1.06 (m, 1H), 0.80 (t,
J = 7.5 Hz, 3H), 0.77 (d, J = 6.5 Hz, 3H), 0.75 (d, J = 6.5 Hz, 3H), 0.71 (d, J = 6.5 Hz,
3H). 13C NMR (150 MHz, DMSO-d6): d 174.1, 173.9, 171.6, 171.1, 169.9, 169.8,
167.9, 134.9, 134.0, 130.1, 129.5 (ꢁ2), 128.5 (ꢁ2), 127.2, 117.1, 56.2, 53.5, 52.0,
51.6, 51.4, 51.2, 40.8, 40.7, 37.3, 36.6, 31.2, 30.1, 28.8, 27.7, 27.6, 27.5, 25.7,
23.8, 23.1, 21.4, 14.5, 11.6.
one, and cycloheptylamine didn’t inhibit the growth of M. smegma-
tis, and N-acetyl calpinactam 6r was also inactive. These data
indicate that the entire peptide chain length plays an important
role in antimycobacterial activity as well as the N-terminal amino
group and C-terminal azepanone moiety.
In conclusion, we have demonstrated a synthetic route for calp-
inactam derivatives with different amino acids, which allowed
first-generation SAR analysis. The SAR revealed that the side
chains, stereochemistry, and entire peptide chain length are essen-
tial for antimicobacterial activity. In this study, peptide 6d where
D
-Glu is replaced with D-Ala retained moderate antimycobacterial
Natural calpinactam:
38H58N9O8: [M+H]+ 768.4408, found: 768.4411. IR (KBr) : 3432, 3288, 1673,
1633, 1540, 1446 cmꢃ1 1H NMR (600 MHz, DMSO-d6): d 8.79 (s, 1H), 8.57 (d,
½
a 2D5
ꢂ
ꢃ 23 (c = 0.05, AcOH). FAB-MS
: calcd for
activity. Thus, calpinactam seems to act on a limited space of the
active site, although its molecular target remains unclear. Our syn-
thetic strategy could readily provide a variety of derivatives. Thus,
we plan to synthesize a combinatorial library of calpinactam vary-
ing combination of unique amino acids on the basis of our result to
discover potent and structurally unique derivatives against M.
smegmatis and M. tuberculosis. This study is underway.
C
.
J = 8.0 Hz, 1H), 8.42 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 8.0 Hz,
1H), 7.83 (dd, J = 5.0, 7.0 Hz, 1H), 7.79 (d, J = 6.5 Hz, 1H), 7.34 (s, 1H), 7.30 (m,
2H), 7.26 (m, 1H), 7.24 (m, 2H), 4.55 (dt, J = 7.5, 8.0 Hz, 1H), 4.36 (m, 2H), 4.31
(dd, J = 6.0, 9.0 Hz, 1H), 4.26 (dt, J = 7.0, 8.0 Hz, 1H), 4.06 (t, J = 7.0 Hz, 1H), 3.14
(m, 1H), 3.02 (m, 1H), 3.01 (m, 1H), 3.00 (m, 1H), 2.96 (dd, J = 7.0, 14.0 Hz, 1H),
2.82 (dd, J = 8.0, 16.0 Hz, 1H), 2.07 (m, 2H), 1.85 (m, 1H), 1.84 (m, 1H), 1.80 (m,
1H), 1.71 (m, 1H), 1.70 (m, 2H), 1.60 (m, 1H), 1.34 (m, 1H), 1.27 (m, 1H), 1.26
(m, 2H), 1.17 (m, 1H), 1.16 (m, 1H), 1.05 (m, 1H), 0.80 (t, J = 7.0 Hz, 3H), 0.77 (d,
J = 6.0 Hz, 3H), 0.75 (d, J = 6.0 Hz, 3H), 0.72 (d, J = 6.0 Hz, 3H). 13C NMR
(150 MHz, DMSO-d6): d 174.1, 173.9, 171.5, 171.1, 170.0, 169.8, 167.9, 134.9,
134.2, 130.5, 129.5 (ꢁ2), 128.6 (ꢁ2), 127.2, 117.2, 56.2, 53.5, 52.2, 51.6, 51.4,
51.2, 40.8, 40.7, 37.3, 36.6, 31.2, 30.1, 28.8, 27.7, 27.6 (ꢁ2), 25.7, 23.8, 23.1,
21.4, 14.5, 11.6.
Acknowledgements
K. N. acknowledges a Kitasato University Research Grant for
Young Researchers. This study was supported by a Grant-in-Aid