Design and synthesis of macrocyclic peptidyl hydroxamates as peptide deformylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.011

Macrocyclic peptidyl hydroxamates were designed, synthesized, and evaluated as peptide deformylase (PDF) inhibitors. The most potent compound exhibited tight, slow-binding inhibition of Escherichia coli PDF (K_I^* = 4.4 nM) and had pot

Full text of DOI:10.1016/j.bmcl.2007.12.011

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3060–3063
Design and synthesis of macrocyclic peptidyl hydroxamates
as peptide deformylase inhibitors
Gang Shen, Jinge Zhu, Anthony M. Simpson and Dehua Pei*
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA
Received 14 October 2007; revised 30 November 2007; accepted 6 December 2007
Available online 21 February 2008
Abstract—Macrocyclic peptidyl hydroxamates were designed, synthesized, and evaluated as peptide deformylase (PDF) inhibitors.
The most potent compound exhibited tight, slow-binding inhibition of Escherichia coli PDF ðKꢀI ¼ 4:4 nMÞ and had potent antibac-
terial activity against Gram-positive bacterium Bacillus subtilis (MIC = 2–4 lg/mL).
Ó 2007 Elsevier Ltd. All rights reserved.
Peptide deformylase (PDF), an essential enzyme for bac-
terial protein biosynthesis and maturation, catalyzes the
removal of N-formyl group from newly synthesized
polypeptides.1 PDF is being actively pursued as a target
for designing novel antibiotics to treat antibiotic resis-
tant pathogens.2,3 Over the past decade, numerous
PDF inhibitors have been reported; two of the inhibitors
have already advanced to phase I clinical trials for
treatment of upper respiratory tract infections.4
We have previously reported that macrocyclic N-
formylhydroxylamine 1 (Fig. 1) and its congeners are
highly potent, selective inhibitors against PDF (e.g.,
KꢀI ¼ 0:33 nM for 1 against Escherichia coli PDF) and
have broad-spectrum antibacterial activity against clini-
cally significant pathogens.5 However, synthesis of com-
pound 1 was somewhat lengthy (11 steps), primarily due
to an inefficient preparation of the N-formylhydroxyl-
amine moiety (eight steps). In this work, we attempt to
replace the N-formylhydroxylamine moiety with an-
other metal chelating group, the synthetically more
accessible hydroxamate. Thus, macrocyclic hydroxa-
mates 2a–c (Fig. 1) were designed, synthesized, and eval-
uated as potential PDF inhibitors. Compound 2a is
isosteric to inhibitor 1 except that the N-formylhydr-
oxylamine moiety in the latter is replaced by a hydroxa-
mate. Compound 2b contains a proline as the P20
residue, because previous studies have shown that the
proline confers excellent potency and pharmacokinetic
properties.6 Compound 2c should be synthetically very
accessible because its chiral hydroxamate group can be
readily prepared from D-aspartic acid.
Retrosynthetic analysis shows that the macrocycles can
be conveniently prepared via olefin metathesis from
diene 3, which in turn can be prepared from acid 4
and amine 5 (Scheme 1). This synthetic strategy allows
for the convergent synthesis of macrocycles of different
ring sizes and specificity determinants by using the com-
mon acid 4 and varying the structure of the alkenyl
amine 5.
Synthesis of acid 4 started from the commercially
available 5-hexenoic acid, which was stereoselectively
alkylated at the C-2 position with a t-butoxycarbonylm-
ethyl group using 4S-benzyloxazolidin-2-one as the chi-
O
O
O
H
N
H
N
H
N
H
N
OH
N
H
N
H
HO
O
O
O
1
2a
O
O
H
H
H
H
N
N
N
N
N
HO
N
HO
H
O
O
O
HN
O
O
Keywords: Peptide deformylase; Antibacterial; Macrocycle; Inhibition;
Hydroxamate.
2b
Figure 1. Structures of PDF inhibitors.
2c
*
Corresponding author. Fax: +1 614 292 1532; e-mail: pei.3@osu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.011

G. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3060–3063
3061
ral auxiliary group7 (Scheme 2). Removal of the auxil-
iary group by hydrolysis produced the desired acid 4,
which was used as the common building block for the
preparation of various cyclic hydroxamates.
a, b
NHBoc
CN
8
O
H
H
N
O
N
The 6-heptenylamine needed for the preparation of
amine 5 was obtained by reducing 5-hexenyl nitrile with
LiAlH4 (Scheme 3). The resulting amine was protected
with a Boc group to facilitate its isolation. Removal of
the Boc group by TFA, condensation with N-Boc-tert-
leucine, and deprotection with TFA afforded amine 5.
Amine 5 was coupled to the common acid 4 to give diene
9. The terminal alkenes were crosslinked using Grubbs’s
ruthenium catalyst8 followed by catalytic hydrogenation
(Pd/C and H2) to produce the 15-membered macrocycle
10. The t-butyl ester was cleaved with TFA treatment
and the resulting acid was coupled with O-t-butylhydr-
oxylamine to give the t-butyl protected hydroxamate
group. Finally, the t-butyl group on the hydroxamate
was removed by treatment with TFA and aqueous
HCl to give hydroxamate 2a.9 Compound 2b was syn-
thesized in a similar manner except that N-Boc-proline
was used instead of N-Boc-tert-leucine.
c-e
H2N
N
H
f
O
O
O
5
9
O
H
N
O
i, j, k
g, h
N
H
2a
O
O
10
Scheme 3. (a) LiAlH4, Et2O; (b) (Boc)2O, NaOH aq, 85% (two steps);
(c) TFA, CH2Cl2; (d) N-Boc-L-tert-Leu-OH, EDC, HOAt, Et3N,
CH2Cl2, 78%; (e) TFA, CH2Cl2; (f) 4, EDC, Et3N, CH2Cl2, 42%; (g)
2nd-generation Grubbs’s catalyst, CH2Cl2, reflux, 55%; (h) H2, 10% Pd/
C, MeOH, EtOAc; (i) TFA; (j) t-BuONH2 ÆHCl, EDC, Et3N, HOAt,
CH2Cl2, 40%; (k) TFA, 37% HCl, 48%.
Compound 2c was synthesized from N-Fmoc-Asp(t-
Bu)-OH, which was condensed with amine 5 (Scheme
4). After removal of the N-terminal Fmoc group, the
resulting amine 11 was acylated with 4-pentenoic acid
and EDC. Cyclization of the diene 12 was achieved with
the second-generation Grubbs’s catalyst, which afforded
better yields than the first-generation catalyst. The con-
figuration of the ring C@C bond in intermediate 13 was
not determined. To install the hydroxamate group, al-
kene 13 was treated with TFA to cleave the t-butyl ester
O
O
O
O
O
OH
H
a, b
N
H2N
N
H
Fmoc-NH
O
O
11
O
O
O
H
N
O
H
O
O
N
H
N
c
d
O
HN
NH
O
OH
O
HN
O
O
H
N
O
O
O
O
N
H
4
O
O
2a
+
H
N
13
H2N
12
O
5
3
O
H
H
N
N
Scheme 1. Retrosynthetic analysis of 2a.
BnO
N
H
e, f
g
O
HN
O
O
2c
O
O
O
O
a, b
14
HN
Bn
O
+
N
O
OH
Scheme 4. (a) tert-Leu-NH(CH2)5CH@CH2, EDC, DIPEA, CH2Cl2,
64%; (b) 20% piperidine; (c) CH2@CHCH2CH2CO2H, EDC, Et3N,
CH2Cl2, 71%; (d) second-generation Grubbs’s catalyst, 76%; (e) TFA;
(f) BnONH2, EDC, Et3N, CH2Cl2, 54%; (g) H2, 10% Pd/C, MeOH,
EtOAc, 24%.
Bn
6
O
O
O
c
OH
d
N
O
O
O
Bn
and the resulting acid was coupled with O-benzylhydr-
oxylamine to give cycloalkene 14. Catalytic hydrogena-
tion of 14 reduced the ring C@C bond and
simultaneously removed the benzyl group from the
hydroxamate moiety. Unfortunately, the hydroxamate
was unstable under the hydrogenolysis condition and
O
O
7
4
Scheme 2. (a) Pivaloyl chloride, TEA, THF; (b) LiCl, 86% (two steps);
(c) LDA, THF, t-butylbromoacetate, 50%; (d) H2O2, LiOH, H2O,
THF, 96%.

3062
G. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3060–3063
Table 1. Inhibition constants of Co-EcPDF by compounds 1 and 2a–c
Compound KI, nM
KꢀI , nM k5, minꢂ1 k6, minꢂ1
of 4.4 nM against EcPDF. The corresponding N-form-
ylhydroxylamine (comꢀpound 1) is also a slow-binding
inhibitor and has a KI value of 0.33 nM.5 Compound
2b, which has a proline at the P02 position, is also a
slow-binding but significantly weaker inhibitor
ðKꢀI ¼ 1400 nMÞ. Compound 2c, which was syntheti-
cally most accessible, was a very poor inhibitor
(KI > 20,000 nM). This is not unexpected, because previ-
ous studies have shown that PDF has stringent require-
ment for a hydrophobic residue such as methionine and
norleucine at the P01 site.13 Thus, both N-formylhydr-
oxylamine and hydroxamate are effective metal-chelat-
ing groups when attached to proper macrocycles.
1
109 5a
210 30
0.33 0.15a 1.2a
4.4 0.7 12
0.0038 0.0010a
0.26 0.02
0.30 0.01
ND
2a
2b
2c
16,000 4000 1400 200
>20,000
NDb
3.1
ND
a Data from Ref. 5.
b ND, no slow binding detected.
1.5
1.0
0
1
2
4
8
Next, compounds 2a and 2b were tested for antibacterial
activities in vitro. Bacillus subtilis and E. coli overnight
cultures were diluted sixfold into fresh LB medium con-
taining 0–24 lg/mL inhibitor and cell growth was mon-
itored at 600 nm. Compound 2a had potent antibacterial
activity against B. subtilis, with a minimal inhibitory
concentration (MIC) of 2–4 lg/mL. Interestingly, in
contrast to most of the reported PDF inhibitors, which
are usually bacteriostatic, compound 2a exhibited bacte-
ricidal activity. When added to cell cultures in the expo-
nential phase, the cell density continued to increase for
several hours and then gradually decreased to near zero
as a result of cell lysis (Fig. 2a). It had only weak activity
against the Gram-negative E. coli cells (MIC > 24 lg/
mL) (Fig. 2b). Compound 2b showed weak activity
against B. subtilis (MIC > 24 lg/mL) and was inactive
against E. coli cells (not shown).
16
24
μg/mL
0.50
0.0
0
5
10
15
20
25
Time (h)
2.5
2.0
0 μg/mL
1.5
1.0 μg/mL
2.0 μg/mL
4.0 μg/mL
8.0 μg/mL
16.0 μg/mL
24.0 μg/mL
1.0
In summary, a novel class of macrocyclic peptidyl
hydroxamates has been prepared from commercially
available 5-hexenoic acid (nine steps). One of the inhib-
itors showed potent inhibition of EcPDF and bacterici-
dal activity against Gram-positive bacteria. Further
optimization of the ring size and P02 side chain may lead
to highly potent, selective PDF inhibitors.
0.50
0
5
10
15
20
25
Time (h)
Figure 2. Antimicrobial activity of compound 2a against B. subtilis (a)
and E. coli (b).
even under carefully controlled conditions, substantial
reduction of the hydroxamate to the corresponding
amide was observed. As a result, the hydrogenation step
had rather poor yields (24%).
Acknowledgment
This work was supported by grants from the National
Institutes of Health (AI40575 and AI62901).
Compounds 2a–c were tested for inhibition of
cobalt(II)-substituted E. coli PDF (Co-EcPDF)10 using
a continuous assay and peptide N-formyl-Met-Leu-p-
nitroanilide (f-ML-pNA) as substrate.11 Compounds
2a and 2b exhibited slow-binding inhibition12 that can
be described by equation
References and notes
1. Meinnel, T.; Mechulam, Y.; Blanquet, S. Biochimie 1993,
75, 1061.
2. Yuan, Z.; White, R. J. Biochem. Pharmacol. 2006, 71,
1042.
3. Leeds, J. A.; Dean, C. R. Curr. Opin. Pharmacol. 2006, 6,
445.
KI
k5
E þ I ꢀ E ꢁ I ꢀ E ꢁ Iꢀ
k6
4. (a) Jain, R.; Chen, D.; White, R. J.; Patel, D. V.; Yuan, Z.
Curr. Med. Chem. 2005, 12, 1607; (b) Chen, D.; Yuan, Z.
Expert Opin. Invest. Drugs 2005, 14, 1107.
5. (a) Hu, X.; Nguyen, K. T.; Verlinde, C. L. M. J.; Hol, W.
G. J.; Pei, D. J. Med. Chem. 2003, 46, 3771; (b) Hu, X.;
Nguyen, K. T.; Jiang, V. C.; Lofland, D.; Moser, H. E.;
Pei, D. J. Med. Chem. 2004, 47, 4941.
where KI is the equilibrium constant for the formation
of the initial EÆI complex, whereas k5 and k6 are the for-
ward and reverse rate constants for the slow intercon-
version of EÆI and EÆI*, respectively. The overall
potency of the inhibitor is described by the equilibrium
constant KꢀI ¼ KI ꢁ k6=ðk5 þ k6Þ. The equilibrium and
rate constants for 2a and 2b are listed in Table 1. Com-
pound 2a is a highly potent inhibitor, having a KꢀI value
6. (a) Chen, D.; Hackbarth, C.; Ni, Z. J.; Wu, C.; Wang, W.;
Jain, R.; He, Y.; Bracken, K.; Weidmann, B.; Patel, D. V.;

G. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3060–3063
3063
Trias, J.; White, R. J.; Yuan, Z. Antimicrob. Agents
Chemother. 2004, 48, 250; (b) Jain, R.; Sundram, A.;
Lopez, S.; Neckermann, G.; Wu, C.; Hackbarth, C.; Chen,
D.; Wang, W.; Ryder, N. S.; Weidmann, B.; Patel, D.;
Trias, J.; White, R.; Yuan, Z. Bioorg. Med. Chem. Lett.
2003, 13, 4223.
28.7 (d), 28.6 (d), 28.2, 27.8, 27.1, 26.8, 26.5. ESI-HRMS:
Calcd for C19H35N3O4Na+ ([M+Na]+), 392.2520; found,
392.2537. 2b: 1H NMR (400 MHz, CD3OD): d 8.37 (br s,
0.4H), 4.25 (br s, 1H), 3.40–3.24 (m, 3H), 2.85–2.81 (m,
2H), 2.45–2.37 (m, 2H), 2.06–1.83 (m, 4H), 1.54–1.33 (m,
14H). 13C NMR (100 MHz, CD3OD): d 177.4, 174.2,
169.4, 61.3, 47.5, 40.8, 38.2, 32.5, 32.0, 31.3, 30.4, 30.3 (d),
30.2, 27.8, 27.3. ESI-HRMS: Calcd for C18H31N3O4H+
([M+H]+), 354.2387; found, 354.2399.
7. Evans, D. A.; Rieger, D. L.; Bilodeau, M. T.; Urpi, F.
J. Am. Chem. Soc. 1991, 113, 1047.
8. Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett.
1999, 1, 953.
10. Rajagopalan, P. T. R.; Grimme, S.; Pei, D. Biochemistry
2000, 39, 779.
11. Wei, Y.; Pei, D. Anal. Biochem. 1997, 250, 29.
12. Morrison, J. F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201.
13. Hu, Y. J.; Wei, Y.; Zhou, Y.; Rajagopalan, P. T. R.; Pei,
D. Biochemistry 1999, 38, 643.
9. 2a: 1H NMR (400 MHz, CD3OD): d 8.04 (br s, 0.8H),
4.32–4.27 (m, 1H), 3.69–3.64 (m, 1H), 2.89–2.81 (m, 2H),
2.38–2.32 (m, 1H), 2.17–2.11 (m, 1H), 1.59–1.22 (m, 16H),
1.02 (s, 4.5H), 0.99 (s, 4.5H). 13C NMR (100 MHz,
CD3OD): d 176.6, 172.6, 170.7, 62.1, 43.7, 38.9, 35.5, 29.0,