Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2010.06.088

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis

Full text of DOI:10.1016/j.bmcl.2010.06.088

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4317–4319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and
structure–activity relationships
Seung Kyu Lee a,d, Kwang Hyun Choi a, Sang Jae Lee a, Se Won Suh b, B. Moon Kim c, Bong Jin Lee a,d,
*
a Promeditech Ltd, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
b Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea
c Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
d Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly syn-
thesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic
strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide
inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity rela-
tionships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity
against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 17 May 2010
Revised 14 June 2010
Accepted 15 June 2010
Available online 19 June 2010
Keywords:
PDF
SAR
Antibacterial
Retro-amide
The increased prevalence of antibiotic resistance resulting from
evolution of bacterial genes is becoming one of major health con-
cerns. After an extensive search for new antibiotics, many effective
antibacterial drugs have been developed since the 1940s. However,
many of these agents share the same target and the actual number
of antibacterial targets is very limited. Industrial and academic
antibacterial research groups have been trying to find inhibitors
that interfere with new targets and thus can shed a light in solving
the antibiotic resistance problems.
All proteins produced in prokaryotic translation process begin
with N-formyl methionine, which is commonly removed in a mul-
ti-step process beginning with deformylation. Peptide deformylase
(PDF), which is an essential enzyme in protein synthesis in bacte-
ria, has been recognized as a potent and useful target for antibac-
terial therapy.1 It is metalloprotease, which has a function of
removing the formyl group at the N-terminal methionine residue
of nascent proteins.2 The naturally occurring antibiotic actinonin,
which was first isolated in 1962 from an actinomycete,3 is a potent
inhibitor of PDF.4 There have been many reports on the PDF inhib-
itors containing peptide5 or non-peptide6 scaffolds, and peptide
scaffold inhibitors5 based on actinonin have shown potent effects
on both PDF enzyme inhibition and cell-based antibacterial activ-
ity. Among various peptidomimetic scaffolds, retro-amide is one
of possible bioisosteres of amide function.7 To the best of our
knowledge, there have been no reports on PDF inhibitors having
N-alkyl retro-amide at P01 site. We undertook a comprehensive
structure–activity relationship (SAR) study on PDF inhibitors
equipped with the retro-amide scaffold, particularly focusing on
the modification of P01, P20 , and P03 residues. Our SAR study led to
the discovery of 7a that possesses a promising antibacterial activ-
ity (Fig. 1). Here, we describe the synthesis of retro-amide inhibi-
tors and corresponding SAR around P01, P20 , and P30 positions.
The synthesis of PDF inhibitors used in this study is outlined in
Schemes 1 and 2.8 Compound 2 was prepared from alkylation of
glycine ethyl ester hydrochloride using corresponding alkyl tosyl-
ate in the presence of NaHCO3. In this step, employment of a stron-
R1
O
R1 R2
N
O
H
N
R3
MBG
MBG
N
H
N
H
R3
O
R2
O
O
O
OH
O
O
H
N
HO
N
HO
N
H
N
H
N
N
H
N
H
O
O
O
Actinonin
7a
* Corresponding author. Address: Bong-Jin Lee, College of Pharmacy, Seoul
National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Republic of
Korea. Tel.: +82 2 880 7869; fax: +82 2 872 3632.
Figure 1. Pharmacophore and structure of PDF inhibitor actinonin and retro-amide
7a.
E-mail address: lbj@nmr.snu.ac.kr (B.J. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.088

4318
S. K. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4317–4319
R1 R2
N
Table 1
O
R1
PDF inhibition and in vitro antibacterial activities for P01 side chain
a
b
O
O
Boc
NH3+Cl-
NH
O
N
H
O
O
O
1
2
3
O
R1
N
O
HO
O
R1 R2
N
O
R1 R2
N
O
N
H
N
H
N
H
c
d, e
HO
O
O
O
NH3+Cl-
N
H
NH R3
O
O
5
Compound
R1
IC50 (nM)
MIC (lg/ml)
4
P. aer PDF
S. pne
H. inf
M. cat
Scheme 1. Reagents and conditions: (a) R1OTs, NaHCO3, acetonitrile, 60 °C, 18 h;
7a
7b
7c
7d
7e
Cyclopentylmethyl
Cyclopenylethyl
i-Pentyl
Cyclopropylmethyl
Cyclobutylmethyl
29
31
21
15
13
0.2
0.4
0.8
12.5
0.4
0.2
0.8
0.8
6.3
0.4
0.1
0.1
0.1
1.6
0.1
(b) N-Boc-L
-amino acid (introduction of R2), HOBt, EDC, diethylamine, dichloro-
methane, rt, 1 h; (c) 4 M HCl in 1,4-dioxane, rt, 18 h; (d) R3COCl, triethylamine,
dichloromethane; (e) hydroxylamine, MeOH, rt, 30 min.
O
O
R1 R2
N
O
R1 R2
N
O
a
b
O
O
NH3+Cl-
O
NH
N
N
Table 2
PDF inhibition and in vitro antibacterial activities for P02 side chain
O
O
4
8
c
R1 R2
N
O
O
R1 R2
N
O
d
HO
NH NR4
N
H
NH NR4
O
O
O
O
HO
N
[AA]
6
7
N
H
N
H
N
H
O
O
Scheme 2. Reagents and conditions: (a) carbonyl diimidazole, N,N-diisopropyleth-
ylamine, dichloromethane, rt, 4 h; (b) R4NH, toluene, reflux, 18 h; (c) R4NCO,
triethylamine, dichloromethane, rt, 18 h; (d) hydroxylamine, MeOH, rt, 30 min.
Compound
AA
IC50 (nM)
P. aer PDF
MIC (lg/ml)
S. pne
H. inf
M. cat
7a
7f
7g
7h
7i
t-Leucine
Valine
Alanine
Phenylalanine
i-Leucine
Leucine
29
13
42
61
32
0.2
0.8
6.3
25.0
3.2
3.2
0.2
0.8
12.5
25.0
1.6
0.1
0.1
12.5
12.5
0.2
ger base such as K2CO3 led to lower yield due to hydrolysis of the
product. The corresponding alkyl tosylate was obtained conven-
tionally from alkyl alcohol through reaction with tosyl chloride
in the presence of DMAP and triethylamine. Compound 2 was cou-
7j
7k
36
116
3.2
200.0
0.8
50.0
Proline
200.0
pled to N-Boc-L-amino acid through standard peptide coupling
methodology using EDC and HOBt. Removal of the N-Boc group
with 4 M HCl solution in 1,4-dioxane gave ammonium chloride salt
4. For the synthesis of bis(retro-amide) 5, compound 4 was treated
with corresponding acid chloride in the presence of triethylamine
and subsequent substitution with hydroxylamine produced the fi-
nal amide 5. For the synthesis of retro-amide—urea derivatives,
compound 4 was directly coupled with corresponding isocyanate
to provide compound 6. When the corresponding isocyanate was
not commercially available, compound 4 was treated with car-
bonyl diimidazole9 and the resulting imidazolide 8 was coupled
with the corresponding amine in toluene to give compound 6.
The final urea 7 was obtained through treatment of compound 6
with a solution of hydroxylamine in ethyl alcohol.10
Table 3
PDF inhibition and in vitro antibacterial activities for P03 retro-amides
O
O
HO
N
N
H
N
H
R3
O
Compound
R3
IC50 (nM)
MIC (lg/ml)
P. aer PDF
S. pne
H. inf
M. cat
Compounds were tested using a Pseudomonas aeruginosa Ni-PDF
enzyme assay and primary in vitro antibacterial activity was tested
against Streptococcus pneumoniae, Haemophilus influenzae, and Mor-
axella catarrhalis. These strains are known to cause respiratory
tract-associated infection. Data from these assays on PDF inhibitors
are summarized in Tables 1–3.
5a
5b
5c
5d
i-Propyl
Phenyl
Thiophene-2-yl
Ph(2-MeO)
52
41
19
11
100.0
12.5
12.5
50.0
50
0.8
0.1
0.2
0.1
3.2
1.6
0.4
The size of the ring substituted at P01 side chain appears to have
little effect on the enzyme inhibition activity, but showed differ-
ence in antibacterial activity against tested strains (Table 1). Intro-
duction of the cyclopropyl at P01 site (7d) caused decrease in
antibacterial activity compared to other analogs (7a and 7e). As
the ring size was increased from a three-membered ring to a
five-membered ring, antibacterial activity was improved, and
cyclopentylmethyl analog (7a) showed optimal activity. Incorpora-
tion of cyclopentylethyl (7b) resulted in slightly reduced activity
relative to that of cyclopentylmethyl derivative (7a), which shows
that the cyclopentylmethyl group is optimal for the P01 binding
pocket. Branched alkyl chain (7c) is also tolerated in hydrophobic
P01 binding pocket, but compound 7c displayed reduced antibacte-
rial activity compared to 7a.
Variation in P02 amino acids displayed significant effect on en-
zyme inhibition and antibacterial activity. From the assay data
summarized in Table 2, preferred side chain for P02 site appears to
be a branched ethyl group because valine and t-leucine derivatives
(7a and 7f, respectively) revealed potent inhibition against enzyme
and bacterial strains. Amino acid derivatives (7g–7j) with a shorter
or longer side chain than valine displayed reduced activity. Inter-
estingly proline derivative 7k showed significantly reduced anti-
bacterial activity. The loss in activity can be explained by the

S. K. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4317–4319
4319
Table 4
in the reduction of antibacterial activity. For urea derivatives,
methoxy group substitution at the 2-position of the phenyl ring
(7a) significantly increased the antibacterial activity against the
tested strains.
PDF inhibition and in vitro antibacterial activities for P03 ureas
We conducted a modeling study based upon the X-ray crystal
structure of actinonin/P. aeruginosa Zn–PDF complex11 to gain an
insight on the structural differences between 7a and actinonin in
their binding modes. The compound 7a fits into the active site of
P. aeruginosa Zn–PDF and the resulting 3D structure of 7a is super-
imposed with X-ray crystal structure of actinonin/P. aeruginosa Zn–
PDF complex (Fig. 2). Superimposition of these structures yields a
close overlap between the P01 and P02 side chains of 7a and that of
actinonin.
An extensive SAR study of retro-amide PDF inhibitors led us to
discover 7a, which displays a good in vitro antibacterial activity
against pathogens associated with respiratory tract infection. Mod-
eling study showed that the P01 and P02 side chains of 7a are very ni-
cely overlapped with those of actinonin. Our results show that the
retro-amide scaffold can be used as an excellent bioisostere of
amide group for peptide inhibitors in the PDF inhibitor design.
O
O
HO
N
N
H
N
H
NR4
O
Compound
NR4
IC50 (nM)
MIC (lg/ml)
P. aer PDF
S. pne
H. inf
M. cat
7a
7l
NH-Ph(2-MeO)
NH-Ph
Piperidine-1-yl
N(Me)-Ph
29
17
47
50
28
55
42
43
54
65
49
0.2
3.2
12.5
50.0
25.0
6.3
0.2
0.4
12.5
25.0
12.5
6.3
6.3
0.4
0.8
6.3
0.1
0.1
0.8
0.4
0.2
0.1
0.1
0.1
0.1
0.2
0.1
7m
7n
7o
7p
7q
7r
7s
7t
7u
Morpholin-4-yl
NH-cHex
NH-ethyl(2-MeO)
NH-thiazol-2-yl
NH-pyridin-2-yl
NH-pyridin-4-yl
NH-pyridin-3-yl
6.3
12.5
12.5
25.0
6.3
3.2
Acknowledgements
This work was supported by a Grant (01-PJ11-PG9-01BT09-
0001) of the Ministry of Health & Welfare, Republic of Korea. This
work was also supported by the National Research Foundation of
Korea (NRF) Grant funded by the Korea government (MEST) (No.
20100001707).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.088.
References and notes
1. Yuan, Z.; Trias, J.; White, R. J. Drug Discovery Today 2001, 6, 954.
2. Mazel, D.; Coïc1, E.; Blanchard, S.; Saurin, W.; Marlière, P. J. Mol. Biol. 1997, 266,
939.
3. Gordon, J.; Kelly, B. K.; Miller, G. A. Nature 1962, 195, 701.
4. Chen, D. Z.; Patel, D. V.; Hackbarth, C. J.; Wang, W.; Dreyer, G.; Young, D. C.;
Margolis, P. S.; Wu, C.; Ni, Z. J.; Trias, J.; White, R. J.; Yuan, Z. Biochemistry 2000,
39, 1256.
5. (a) Chen, D.; Yuan, Z. Expert Opin. Invest. Drugs 2005, 14, 1107; (b) Johnson, K.
W.; Lofland, D.; Moser, H. E. Curr. Drug Targets Infect. Disord. 2005, 5, 39; (c)
Ramanathan-Girish, S.; McColm, J.; Clements, J. M.; Taupin, P.; Barrowcliffe, S.;
Hevizi, J.; Safrin, S.; Moore, C.; Patou, G.; Moser, H. Antimicrob. Agents
Chemother. 2004, 48, 4835.
Figure 2. Compound 7a (green) is superimposed with an X-ray crystal structure of
actinonin (yellow) that is bound to Pseudomonas aeruginosa Zn–PDF (RCSB protein
data bank ID: 1LRY). The Zn2+ atom is colored in red. The accessible surface of the
binding site is shown in purple.
6. (a) Apfel, C.; Banner, W. D.; Bur, D.; Dietz, M.; Hubschewerlen, C.; Locher, H.;
Marlin, F.; Masciadri, R.; Pirson, W.; Stalder, H. J. Med. Chem. 2001, 44, 1847; (b)
Teo, J. W. P.; Thayalan, P.; Beer, D.; Yap, A. S. L.; Nanjundappa, M.; Ngew, X.;
Duraiswamy, J.; Liung, S.; Dartois, V.; Schreiber, M.; Hasan, S.; Cynamon, M.;
Ryder, N. S.; Yang, X.; Weidmann, B.; Bracken, K.; Dick, T.; Mukherjee, K.
Antimicrob. Agents Chemother. 2006, 50, 3665.
7. Lipinsky, C. A.; Hutson, N. J. Annu. Rep. Med. Chem. 1989, 32, 2493.
8. Experimental procedures for synthesis and biological evaluation of compounds
are available in the Supplementary data.
9. Batey, A. R.; Santhakumar, V.; Yoshina-Ishii, C.; Taylor, D. S. Tetrahedron Lett.
1998, 39, 6267.
10. (a) Pirrung, M. C.; Chau, J. H. L. J. Org. Chem. 1995, 60, 8084; (b) Thouin, E.;
Lubell, W. D. Tetrahedron Lett. 2000, 41, 457.
absence of hydrogen bonding of NH hydrogen with a nearby water
molecule or a carbonyl oxygen in the enzyme backbone. One of
major differences between actinonin and our retro-amide inhibi-
tors is the number of backbone atoms between P01 and P20 side
chains. In spite of this difference in backbone chain length, it ap-
pears that side chains of retro-amide inhibitors may be properly lo-
cated in the binding pocket.
The introduction of urea derivative (7l) at P03 site (Table 4) led to
compounds having more potent antibacterial activity than the cor-
responding amide compound 5b (Table 3). Phenyl urea (7l) was
found to be a preferred substituent for the P03 position. Introduction
of a heterocyclic ring (7r–7u) instead of the phenyl group resulted
11. Guilloteau, J. P.; Mathieu, M.; Giglione, C.; Blanc, V.; Dupuy, A.; Chevrier, M.;
Gil, P.; Famechon, A.; Meinnel, T.; Mikol, V. J. Mol. Biol. 2002, 320, 951. RCSB
Protein Data Bank accession number is 1LRY.