Synthesis and antibacterial activity of alaremycin derivatives for the porphobilinogen synthase

Article abstract of DOI:10.1016/j.bmcl.2011.03.106

The preparation and the antibacterial activity of alaremycin derivatives such as their CF₃-derivatives and (R)- and (S)-4-oxo-5- acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described. The IC₅₀ values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were determined using PBGS assay.

Full text of DOI:10.1016/j.bmcl.2011.03.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2812–2815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial activity of alaremycin derivatives for the
porphobilinogen synthase
⇑
⇑
Noritaka Iwai , Kyosuke Nakayama, Jumpei Oku, Tomoya Kitazume
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation and the antibacterial activity of alaremycin derivatives such as their CF3-derivatives and
(R)- and (S)-4-oxo-5-acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described.
The IC50 values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were
determined using PBGS assay.
Received 7 March 2011
Revised 24 March 2011
Accepted 28 March 2011
Available online 7 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Porphobilinogen synthase
Assay
CF3–alaremycin derivatives
IC50
The development of multi-drug-resistant pathogens has be-
come a serious problem in the chemotherapy of bacterial infec-
tions diseases. One of the strategies to overcome this problem is
to find a new drug with a molecular target. For this purpose, Wachi
and co-workers have reported a new antibiotic, which is structur-
ally related to 5-aminolevulinic acid, a precurser of heme biosyn-
thesis, and named alaremycin, was isolated from the culture
broth of Streptomyces sp. A012304.1 The antibacterial activity of
alaremycin was enhanced in the presence of 5-aminolevulinic
acid (ALA). It is reported that alaremycin may affect the heme
biosynthetic pathway.1 Two ALA molecules would be condensed
by porphobilinogen synthase (hemB protein; PBGS) to produce
porphobilinogen reported by Frere et al.2
In the above reaction system, ALA first forms a Schiff base with
the enzyme. Alaremycin might inhibit this reaction by competing
with ALA. If so, the addition of an excess amount of ALA to the
medium should relieve the lethal effect of alaremycin. Alaremycin
contains amide group and methylidene group which are not con-
tained in known PBGS inhibitors, so alaremycin might be expected
as the novel backbone of PBGS inhibitor.3–11 In 2010, the co-crys-
tallization of P. aeruginosa phorphobiinogen synthase with alare-
mycin have been reported to understand the molecular basis of
alaremycin’s antibiotic activity at the atomic level. The crystal
structure have revealed that the antibiotic efficiently blocked the
active site of phorphobiinogen synthase. Further, in the above Let-
ter, it is suggested that 4-oxo-5-acetylaminohexanoic acid derived
from a reduced derivative of 4-oxo-5-acetylaminohexenoic acid
(alaremycin) is important material to understand the alaremycin’s
antibiotic activity for P. aeruginosa phorphobiinogen synthase.12
In general, it is well known the introduction of fluorine atoms
into the target hydrocarbon materials for the purpose of the
improvement of biological activities.13–16 As it is interesting in
the introduction of fluorine atoms into the alaremycin and their
derivatives to improve the activity against porphobilinogen syn-
thase (PBGS), we have designed the introduction of fluorine atoms
into the alaremycin and its derivatives.
In this Letter, we would like to describe the synthesis of
CF3–alaremycin derivatives, (R)- and (S)-4-oxo-5-acetylamino-
hexanoic acid and their fluorinated derivatives. Further, we
describe the IC50 values of the antibacterial activity of the obtained
derivatives for the inhibitor of porphobilinogen synthase (PBGS).
For the purpose of the development of the potent PBGS inhibitor
for Pseudomonas sp. including the human pathogen P. aeruginosa,
we have designed several kinds of alaremycin derivatives and their
CF3-derivatives as synthetic candidates as shown in Figure 1. Espe-
cially, we have challenged to search the improvement of antibacte-
rial activity based on the effect of fluorine atoms, methyidene
group and the carbon length.
At first, according to a total synthetic strategy devised by
retrosynthetic analysis, alaremycin derivatives were successfully
synthesized. In Scheme 1, we have examined the synthesis of
4-oxo-5-acetylaminopentanoic acid 1 which is the absence of
methylidene group in alaremycin.
To transform from the methylidene group to methyl group, we
have designed the synthetic route as shown in Scheme 2. To syn-
thesize (R)- and (S)-4-oxo-5-acetylaminohexanoic acid 3, the
⇑
Corresponding authors. Tel.: +81 45 924 5754; fax: +81 45 924 5780 (T.K.).
E-mail addresses: kitazume.t.aa@m.titech.ac.jp, tkitazum@bio.titech.ac.jp
(T. Kitazume).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.106

N. Iwai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2812–2815
2813
O
O
O
O
HN
HN CF3
HN
HN
HO2C
HO2C
HO2C
HO2C
O
O
O
O
1
4
(S)-3
(R)-3
O
O
O
O
CF3
HN
CF3
HN
HN CF3
HN CF3
HO2C
HO2C
HO2C
HO2C
O
O
O
O
8
(S)-5
7
(R)-5
O
O
O
O
CF3
HN
CF3
HN CF3
HN
HN
HO2C
HO2C
HO2C
HO2C
O
O
O
O
Alaremycin
12
10
11
Figure 1. Structures of synthetic candidate.
O
O
O
HN
HN
HN
CF3
NH2 HCl
CF3
b
a
tBuO2C
a)
2
HO2C
HO2C
HO2C
O
O
O
O
1
(S)-5
Scheme 1. Reagents and conditions: (a) Ac2O, Na2CO3, H2O, 0 °C, 1 h then rt, 36 h,
yield 28%.
Scheme 4. Reagents and conditions: (a) (CF3CO)2O, Et3N, CH2Cl2, rt; (b) H2O, rt.
methylidene group of alaremycin was replaced by the chiral
methyl group, and then (R)- and/or (S)-3 was prepared from (R)-
and/or (S)-alanine as a starting material. In the synthetic route to
(R)- and/or (S)-3, the step b using Stetter reaction was the key step.
Further, as Kobayashi and co-workers have reported the
synthetic route to alaremycin via azide intemediate,17 we have
designed the introduction of fluorine atoms into alaremycin to
search the improvement of the activity. The CF3–alaremycin
(4-oxo-5-(trifluoroacetylamino)-5-hexenoic acid 4) as the target
material was prepared by the following Scheme 3.
To prepare the fluorinated derivatives of (R)- and (S)-4-4-oxo-5-
acetylaminohexanoic acid 3, we have examined the synthesis of
(R)- and (S)-4-oxo-5-(trifluoroacetylamino)hexanoic acid 5 derived
from (R)- and/or (S)-alanine as shown in Scheme 4.
In the next step, to search the alaremycin derivatives with
highly activity, we have designed the expansion of carbon length
NHBoc
NHBoc
NH2
NHBoc
ClH
a
tBuO2C
b
OHC
MeO2C
MeO2C
O
O
O
NH2
CF3CO2H
HN
HN
tBuO2C
c
tBuO2C
d
e
HO2C
O
O
O
(S)-3
2
Scheme 2. Reagents and conditions: (a) DIBAL-H, toluene, À78 °C, 2 h, yield 53%; (b) 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide, tert-butyl acrylate, DBU,
MS4A, THF, 50 °C, 10 h, yield 33%; (c) TFA, CH2Cl2, 0 °C, yield 43%; (d) (CH3CO)2O, Et3N, CH2Cl2, rt, yield 86%; (e) H2O, TFA, rt, yield 92%.
O
O
N3
HN
CF3
HN
CF3
MeO2C
a
b
HO2C
MeO2C
O
O
O
4
Scheme 3. Reagents and conditions: (a) NaReO4, CF3SO3H, (CF3CO)2O, CCl4, 50 °C, 12 h, yield 60%; (b) 1N LiOH, H2O, THF, 0 °C, 1 h, yield 50%.

2814
N. Iwai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2812–2815
NH2 CF3CO2H
OtBu
NHBoc
NHBoc
OtBu
a
b
tBuO2C
tBuO2C
OtBu
OHC
O
O
6
O
O
CF3
CF3
OtBu
HN
HN
d
c
tBuO2C
HO2C
CF3
OH
O
O
O
HN
d
HO2C
O
7
Scheme 5. Reagents and conditions: (a) 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide, methyl acrylate, DBU, MS4A, THF, 50 °C, 20 h, yield 41%; (b) 20% TFA in
CH2Cl2, 0 °C, 2 h, yield 67%; (c) CF3CH2COCl, Et3N, CH2Cl2, rt, overnight, yield 65%; (d) TFA, H2O, rt, 3 h, yield 91%; (e) (Boc)2O, DMAP, MeCN, rt, overnight then TMG, rt, 2 h,
yield 43%.
O
O
CF3
HN
HN
CF3
a
a
tBuO2C
tBuO2C
OtBu
9
6
O
O
O
O
CF3
HN
b
HN
CF3
HO2C
b
c
HO2C
O
O
8
11
Scheme 8. Reagents and conditions: (a) CF3CH2CO2H, (COCl)2, CH2Cl2, DMF, Et3N,
yield 74%; (b) TFA, H2O, rt, 3 h, yield 88%.
Scheme 6. Reagents and conditions: (a) CF3CH2CH2COCl, Et3N, CH2Cl2, rt, over-
night, yield 63%; (b) TFA, H2O, 0 °C, 3 h, yield 88%; (c) (Boc)2O, DMAP, MeCN, rt
overnight then TMG, rt, 24 h, yield 52%.
O
CF3
HN
a
NHBoc
tBuO2C
9
NHBoc
tBuO2C
NHBoc
tBuO2C
a
b
O
MeO2C
OHC
O
O
CF3
HN
b
O
HO2C
NH2 CF3CO2H
c
d
CF3
HN
O
e
HO2C
12
O
O
Scheme 9. Reagents and conditions: (a) CF3CH2CH2CO2H, (COCl)2, CH2Cl2, DMF,
Et3N, yield 44%; (b) TFA, H2O, rt, 3 h, yield 71%.
10
9
Scheme 7. Reagents and conditions: (a) DIBAL-H, toluene, À78 °C, 2 h, yield 82%;
(b) 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide, tert-butyl acrylate,
DBU, MS4A, THF, reflux, 12 h, yield 35%; (c) TFA in CH2Cl2; (d) CF3CH2CO2H, Et3N,
CH2Cl2, DMF, (COCl)2, yield 75%; (e) TFA, H2O, rt, 3 h, yield 55%.
Obviously, from the results of the IC50 values as shown in Figure
3 and Table 1, we have found that the PBGS inhibitory activity of
(R)-3 and (S)-3 have been improved up to about two and/or four
times more than that of alaremycin. Further, it is interesting the
difference of antibacterial activity between (R)-3 and (S)-3. How-
ever, the activity of compound 5 as fluorinated modification of
compound 3 is not higher more than that of alaremycin as shown
in Figure 5 and Table 1. Further, compound 1 which is the absence
of methylidene group does not improve the activity, and its
CF3-derivative 10 is also no activity as shown in Figures 3 and 5
and Table 1. However, the IC50 values as shown in Figures 4 and
5 and Table 1 suggest the effect of the introduction of fluorine
atoms for the improvement of antibacterial activity. Especially,
we have found that CF3–alaremycin derivatives (compounds 4, 7,
8, 11 and 12) inhibit the PBGS in Pseudomanas fluorescens, and that
of the trifluoroacetylamido group in CF3–alaremycin. At first, we
have prepared 4-oxo-5-[(3,3,3-trifluoro-1-oxopropyl)amino]-5-
hexenoic acid 7 and 4-oxo-5-[(4,4,4-trifluoro-1-oxobutyl)amino]-
5-hexenoic acid 8 as shown in Schemes 5 and 6.
To prepare CF3-derivative of compound 1 and the related deriv-
atives, we have developed the synthetic routes for the introduction
of fluorine atoms into the target materials (10–12) using the fol-
lowing Schemes 7–9.
The IC50 values of alaremycin and alaremycin derivatives were
determined using PBGS assay.18 The protocol is summarized in
Figure 2.

N. Iwai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2812–2815
2815
Protocol for PBGS assay
Alaremycin
8 µg/ml PBGS ( pf HemB2)
derivative
0.1 mM ~ 10 mM
buffer A (50 mM Na-HEPES, 5 mM MgCl2, pH 8.0)
37 °C 15 min
5 mM ALA
1/10 vol. 50% trichloroacetic acid
4 °C 15 min
Figure 5. PBGS activity of compounds (R)-5, (S)-5, 10 and 11.
15000 rpm, 10 min at 4 °C
Sup
same vol. of Ehrlich's reagent
the PBGS inhibitory activity of compounds 4 and 8 have been im-
proved up to about 100 and/or 50 times more than that of
alaremycin.
(p-dimethylaminobenzaldehyde)
incubation 15 min at rt
In conclusion, we have established the synthetic route for CF3–
alaremycin derivatives. Further, we have found the PBGS inhibitory
activity of obtained CF3–alaremycin derivatives have been in-
creased up to about 50 and/or 100 times more than that of alare-
mycin. This is the first example for the preparation and the
antibacterial activity for the porphobilinogen synthase (PBGS) of
CF3–alaremycin derivatives.
Abs. 555 nm
Figure 2. The IC50 values using PBGS assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.106.
References and notes
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Biochemistry 2006, 45, 8243.
3. Jaffe, E. K. J. Bioenerg. Biomembr. 1995, 27, 169.
Figure 3. PBGS activity of compounds (R)-3, (S)-3 and 1.
4. Senior, N. M.; Brocklehurst, K.; Cooper, J. B.; Wood, S. P.; Erskine, P.; Shoolingin-
Jordan, P. M.; Thomas, P. G.; Warren, M. J. Biochem. J. 1996, 320, 401.
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Table 1
IC50 values of compounds
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Compound no.
Activity IC50 (mM)
Compound no.
Activity IC50 (mM)
1
2.5
0.7
1.1
0.022
4.1
7
8
10
11
0.9
0.051
8.0
0.5
1.3
(R)-3
(S)-3
4
(R)-5
(S)-5
12
3.0
Alaremycin
2.6
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Figure 4. PBGS activity of compounds 4, 7 and 8.