Identification of potent type I MetAP inhibitors by simple bioisosteric replacement. Part 1: Synthesis and preliminary SAR studies of thiazole-4-carboxylic acid thiazol-2-ylamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.05.055

A series of thiazole-4-carboxylic acid thiazol-2-ylamide (TCAT, 4) derivatives were designed and synthesized according to simple bioisosteric replacement from previously reported pyridine-2-carboxylic acid thiazol-2-ylamide (PCAT) MetAP inhibitors. The preliminary SAR studies demonstrated that these TCAT series of compounds showed different activity and selectivity compared with those of the corresponding PCAT compounds. These findings provide useful information for the design and discovery of more potent inhibitors of type I MetAPs.

Full text of DOI:10.1016/j.bmcl.2005.05.055

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3732–3736
Identification of potent type I MetAP inhibitors by
simple bioisosteric replacement. Part 1: Synthesis
and preliminary SAR studies of thiazole-4-carboxylic acid
thiazol-2-ylamide derivatives
Yong-Mei Cui,a Qing-Qing Huang,a Jie Xu,a Ling-Ling Chen,a Jing-Ya Li,a
Qi-Zhuang Ye,a,b Jia Lia,* and Fa-Jun Nana,*
aChinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, 189 Guoshoujing Road,
Zhangjiang Hi-Tech Park, Shanghai, 201203, PR China
bHigh-Throughput Screening Laboratory, University of Kansas, Lawrence, KS 66047, USA
Received 17 March 2005; revised 14 May 2005; accepted 18 May 2005
Available online 29 June 2005
Abstract—A series of thiazole-4-carboxylic acid thiazol-2-ylamide (TCAT, 4) derivatives were designed and synthesized according to
simple bioisosteric replacement from previously reported pyridine-2-carboxylic acid thiazol-2-ylamide (PCAT) MetAP inhibitors.
The preliminary SAR studies demonstrated that these TCAT series of compounds showed different activity and selectivity compared
with those of the corresponding PCAT compounds. These findings provide useful information for the design and discovery of more
potent inhibitors of type I MetAPs.
Ó 2005 Elsevier Ltd. All rights reserved.
The methionine aminopeptidases (MetAPs) represent a
unique class of proteinases that are responsible for
removing the N-terminal methionine residue from nas-
cent polypeptide chains.1 There are two major classes
of MetAPs (type I and type II), each of which can be
subdivided into two subclasses, those with N-terminal
extensions and those without it. Eubacteria (for exam-
ple, Escherichia coli, Bacillus subtilis, and Salmonella
typhimurium) have only type I MetAPs, while archaea
(for example, Methanobacterium thermoautotrophicum,
Sulfolobus solfataricus, and Pyrococcus furiosis) have
only type II MetAPs. Eukaryotic cells contain both type
I and type II MetAPs.2 Although the enterohemorrhagic
E. coli strain O157:H7 has more than 20% of its 5416
genes different from the E. coli laboratory strain K-
12,3 it has exactly the same MetAP sequence. Deletion
of the MetAP gene from E. coli4 or S. typhimurium5
proved to be lethal, demonstrating the essential role of
MetAP enzymes in bacteria. Deletion of either type I
or type II MetAP from the yeast Saccharomyces
cerevisiae produced a slow-growth phenotype, and
removal of both MetAPs made the yeast nonviable.6
Our previous work has also indicated that the selective
ScMetAP1 inhibitor significantly inhibited ScMetAP1
deletion strain growth, but had no effect on ScMetAP2
deletion strain growth.7 Therefore, MetAPs are poten-
tial targets for developing antibacterial and antifungal
drugs, and the inhibitors against MetAPs offer new hope
for treatment of bacterial and fungal infections.8
Various inhibitors have been identified for MetAPs.
Natural product, fumagillin, and its derivatives9 are
potent inhibitors of type II MetAP in humans, and cova-
lently modified the enzyme. Until recently, only weak
inhibitors of type I MetAPs, such as fumagillin,10 phos-
phorus-based Met analogs,11 and bestatin-derived
peptide inhibitors, have been identified.12 Various 1,2,
4-triazole-based derivatives have recently been reported
to represent a new class of more potent inhibitors for
Staphylococcus aureus MetAP-I (SaMetAP-I).13
*
Corresponding authors. Tel.: +86 21 50801313, ext. 132; fax: +86 21
50800721 (J.L.); tel.: +86 21 50800954; fax: +86 21 50800721
(F.-J.N.); e-mail addresses: jli@mail.shcnc.ac.cn; fjnan@mail.shcnc.
ac.cn
In our previous work,14 starting with compound 1, with
an IC50 of 5.0 lM for EcMetAP1, which was identified
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.055

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3732–3736
3733
Table 1. Inhibitory activity of TCAT derivatives on EcMetAP1 and
ScMetAP1a
as a hit in a random screening of a diverse molecule li-
brary of 4500 compounds for EcMetAP1 inhibitors,
we have subsequently carried out successful structural
modifications and structure–activity relationship
(SAR) analyses and obtained potent inhibitors (for
example, for 2a, IC50 = 130 nM for EcMetAP1; for 2b,
IC50 = 140 nM for ScMetAP1; for 3, IC50 = 53 nM for
EcMetAP1). They represent small-molecule MetAP
inhibitors with novel structures different from those of
alkylating fumagillin derivatives and peptidic bestatin-
based MetAP inhibitors.
Compound
R
IC50 (lM)
EcMetAP1
ScMetAP1
1b
4
—
—
H
5.0 0.8
0.11 0.02
1.97 0.37
7.0 0.1
2.26 0.38
13.21 2.19
5a
5b
5c
5d
0.06 0.01
0.081 0.011
0.054 0.014
4.62 0.36
0.22 0.01
2.63 0.61
5e
5f
0.044 0.014
0.042 0.007
0.040 0.009
0.024 0.002
0.37 0.05
6.38 1.05
0.25 0.03
0.13 0.02
To find new MetAP inhibitors with improved potency
and selectivity, good specificity in vitro, and good
efficacy in vivo, we set out to explore the SAR of the
above inhibitors.
5g
5h
Our previous work revealed that the pyridine-2-car-
bonyl unit is very important in pyridine-2-carboxylic
acid thiazol-2-ylamide (PCAT) inhibitors. It was
regarded as the key chelator for the cobalt ion of the
enzyme. The 1,3-thiazole ring is a classical isostere of
pyridine, and therefore, a logical first step was to de-
sign new analogs in which the pyridine ring in 1 was
replaced by a thiazole moiety, which gave the thia-
zole-4-carboxylic acid thiazol-2-ylamide (TCAT) 4 with
greatly enhanced in vitro potency toward EcMetAP1
(Table 1). The potency toward EcMetAP1 increased
about 50-fold compared with the lead compound 1,
with an IC50 value of 110 nM. The introduction of
the 3-N-acyl group greatly enhanced the activity
against EcMetAP1 and ScMetAP1 compared with that
of PCAT itself in the PCAT series of compounds, by
analogy with which two series of new compounds A
and B were designed, bearing a 5-N-acyl group or an alkyl
group, respectively (Fig. 1).
5i
0.19 0.01
3.32 0.34
5j
0.076 0.003
0.11 0.02
0.99 0.02
0.48 0.05
5k
5l
0.27 0.04
0.19 0.01
1.83 0.24
1.81 0.40
5m
5n
5o
5p
5q
0.076 0.011
0.092 0.016
0.029 0.003
0.088 0.013
2.65 0.36
1.53 0.21
1.12 0.24
4.91 0.65
Initially, a series of 5-N-acyl derivatives 5a–l were
synthesized (Table 1). These molecules shared the
substructural diversity of the 5-position of the thia-
zole ring of TCAT as that of fatty acylamino (with-
out substituent or with short saturated or
unsaturated side chain, compounds 5a–h) and aro-
matic acylamino (compounds 5i–l). Compounds 5c–l
were readily available from common intermediate 5a
and various carboxylic acids or chlorides, as shown
in Scheme 1. Ethyl 5-aminothiazole-4-carboxylate 8
was prepared according to the literature.15 Treatment
of 8 with Boc2O, followed by base hydrolysis, affor-
ded 10. Condensation of compound 10 with 2-amino-
thiazole in the presence of DCC in DMF, followed
by amine deprotection and acylation with various
carboxylic acids or chlorides under neutral or basic
conditions, afforded 5-N-acyl-substituted compounds
5c–l.
5r
5s
0.066 0.002
0.30 0.02
0.67 0.09
10.50 0.74
5t
0.16 0.02
0.14 0.02-
0.11 0.01
0.86 0.07
1.45 0.16
4.71 0.63
5u
5v
a Assays were performed, as previously described.14a
b See Ref. 14a.

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3732–3736
between MetAP1 and inhibitors. The preparation of
these compounds is summarized in Scheme 2. Direct
amidation of 5a produced 5n–p and11. Compound 5m
was obtained by the base hydrolysis of 5n. Compound
5a reacted with malonic acid diethyl ester to give com-
pound 5r, the base hydrolysis of which afforded com-
pound 5q. Compounds 5s–v were prepared via
aminolysis of 11 by the corresponding amines.
As shown in Table 1, all of the 10 compounds showed
good inhibition of EcMetAP1, with IC50 values less than
0.3 lM. The hydroxy derivative 5m was apparently less
active than the other derivatives (5n–p) against
EcMetAP1, indicating that the free hydroxy group is
unfavorable on the side chain of these compounds.
However, all these four derivatives (5m–p) showed mod-
erate activity against ScMetAP1. In addition, although
similar in inhibiting EcMetAP1, the carboxyl-substitut-
ed derivative 5q was apparently less active than 5r
against ScMetAP1, indicating that a free carboxyl on
the side chain of TCAT is unfavorable in inhibiting
ScMetAP1 and may reflect subtle differences in the ac-
tive sites of EcMetAP1 and ScMetAP1. Surprisingly,
the amino-derived compounds (5s–v) showed less activ-
ity against the enzymes than the corresponding PCAT
derivatives, which were the best inhibitors of PCAT
against MetAP1. This may suggest that simple replace-
ment of an ethylenic group of a pyridine ring by its isos-
teric S atom significantly affects the interaction of the
inhibitor with the enzymes.
Figure 1.
Scheme 1. Reagents and conditions: (a) (i) Boc2O, DMAP, CH3CN,
(ii) ButOH, reflux, 95%; (b) LiOH, MeOH–H2O, 100%; (c) 2-
aminothiazole, DCC, HOBt, DMAP, DMF, 81%; (d) TFA, CH2Cl2,
100%; (e) appropriate acyl chloride, Py, AcOEt; (f) 2-butenic acid,
pivaloyl chloride, CH2Cl2, Py.
From the above results, we can see that simple replace-
ment of the pyridine ring of original PCAT inhibitors
Simple replacement of a pyridine ring of the original
inhibitor with a 4-thiazolyl unit usually gave compounds
that were more active against the enzymes than the cor-
responding PCAT inhibitors, the results of which are
shown in Table 1. Most of the 12 compounds tested
showed excellent inhibition of EcMetAP1 (IC50 values
in the range of tens to hundreds of nanomolar). Saturat-
ed or unsaturated alkyl acylamino derivatives (5c–e)
showed excellent activity against EcMetAP1, with IC50
values less than 100 nM. Derivatives with three-, four-,
and five-membered rings (5f–h) also retained good activ-
ity to EcMetAP1, indicating that the small-sized hydro-
phobic group in this position was involved in the
interaction of the inhibitor with EcMetAP1. On the
other hand, the four compounds with aromatic groups
directly connected to the carbonyl carbon (5i–l) showed
less significant activity against the enzymes, except for
the F-containing derivatives. Inhibition of ScMetAP1
was not as outstanding as that of EcMetAP1, but the re-
sults were satisfactory, except for 5d and 5f, which
showed that similar cyclopropyl and allyl groups inhib-
ited the interaction with ScMetAP1.
It has been pointed out that the hydrogen bond formed
between the enzyme and 3-position substituents of
PCAT contributed to the binding affinity. Similar to
PCAT series of compounds, we synthesized compounds
5m–v for the purpose of forming extra hydrogen bonds
Scheme 2. Reagents and conditions: (a) appropriate acyl chloride, Py,
AcOEt; (b) LiOH, dioxane–H2O; (c) diethyl malonate, heat; (d) LiOH,
MeOH–H2O; (e) chloroacetyl chloride, Py, AcOEt; (f) appropriate
amine, dioxane.

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3732–3736
Table 2. Inhibition of EcMetAP1 and ScMetAP1a
3735
Compound
R
IC50 (lM)
EcMetAP1
ScMetAP1
1b
4
—
—
5.0 0.8
0.11 0.02
0.18 0.03
7.0 0.1
2.26 0.38
9.82 0.43
6a
Scheme 3. Reagents and conditions: (a) NaOMe, Et2O; (b)
NH2CSNH2, MeOH, reflux; (c) NaNO2, H3PO2; (d) LiOH, MeOH–
H2O; (e) 2-aminothiazole, DCC, HOBt, DMF.
6b
6c
6d
0.18 0.03
0.22 0.02
0.10 0.02
7.64 0.99
7.20 0.54
0.99 0.08
gave a new type of potent MetAP1 inhibitor, with IC50
values between tens to hundreds of nanomolar against
EcMetAP1. Systematic SAR studies of introducing 5-
N-acyl substituents demonstrated that these TCAT ser-
ies of compounds showed different activity and selectiv-
ity compared with those of the corresponding PCAT
compounds. This may suggest that simple replacement
of an ethylenic group of a pyridine ring by its isosteric
S atom significantly affects the interaction of the inhibi-
tor with the enzymes. Therefore, to obtain more potent
MetAP1 inhibitors, we synthesized several derivatives of
the original TCAT compound 4 with simple alkyl mod-
ifications at the 5-position.
6e
6f
0.16 0.02
0.17 0.00
0.98 0.10
2.11 0.22
6g
6h
76.50 11.70
0.21 0.02
> 100
3.41 0.44
6i
6j
0.17 0.03
3.80 0.74
0.90 0.11
0.082 0.009
6k
7a
0.065 0.006
0.074 0.008
0.66 0.10
0.88 0.08
As shown in Scheme 3, the syntheses of 5-alkyl-substi-
tuted compounds 6a–k could be effected by the conden-
sation of 5-alkyl-substituted-thiazole-4-carboxylic acid,
which was obtained by several transformations from
the corresponding aldehyde and ethyl dichloroacetate,16
with 2-aminothiazole in the presence of DCC or EDC in
DMF.
7b
0.089 0.006
0.50 0.05
a Assays were performed, as previously described.14a
b See Ref. 14a.
Derivatives of the original TCAT compound 4 with sim-
ple alkyl modifications at the 5-position are given in Ta-
ble 2. Small electron-donating substituents, such as
methyl, ethyl, isopropyl, isobutyl, and n-pentyl, had lit-
tle effect on the activity against EcMetAP1 (compounds
6a–e), with IC50 values between 110 and 220 nM. In
addition, unsaturation had no significant effect on the
activity of these alkyl derivatives against EcMetAP1
(6f). However, these compounds demonstrated a differ-
ent activity trend toward ScMetAP1; the short
straight-chain alkyl derivatives 6a and 6b showed de-
creased activity against ScMetAP1, and the derivative
with an unsaturated side chain (6f) showed a similar
activity to 4. Although the branched side-chain deriva-
tive 6c showed weak activity against ScMetAP1, inser-
tion of a methylene spacer between the thiazole and
the isopropyl groups significantly improved the activity
(6d vs. 6c). Although not so apparent, this trend also
persisted between these two compounds against EcMe-
tAP1. In addition, moderate chain length alkyl deriva-
tive 6e showed good activity against ScMetAP1, with
an IC50 value less than 1.0 lM. This may be attributed
to appropriate space and shape of the pockets with
which ScMetAP1 accommodates its inhibitor. Consider-
ing the substrate specificity of MetAPs, too long or a too
large substituent chain may decrease the binding affinity,
as is the case with compound 6g, almost with no inhib-
itory activity against EcMetAP1 or ScMetAP1. At the
same time, we synthesized several aryl-group-containing
derivatives (6h–k). From the results, we can see that sub-
stitution by a phenyl ring (6h) or benzyl group (6i)
showed lower activity than 4 against both EcMetAP1
and ScMetAP1. However, introduction of an electron-
withdrawing 2-chloro-4-fluoro group to the phenyl
group of the benzyl substituent (6j) or an additional
insertion of a methylene spacer (6k) dramatically in-
creased the activity. From the above results, we deduce
that substituents at the 5-position with appropriate size
and spatial arrangement may contribute to activity
against the enzymes. The SAR analyses may reflect sub-
tle differences in the active site of EcMetAP1 and ScMe-
tAP1, and the difference in the mode of interaction
between the inhibitors and the enzymes.
During our process of synthesizing the above 5-alkyl-
substituted compounds, we first intended to introduce
an unsaturated alkyl group by using an a,b-unsaturated
aldehyde, such as crotonaldehyde; however, we finally
obtained the b-methoxy product 7a, as the Michael

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of ethyl dichloroacetate and the a,b-unsaturated alde-
hyde with NaOMe in Et2O.16 Using the same method,
we obtained 7b from the corresponding a,b-unsaturated
aldehyde, 4-methyl-2-pentenyl. The inhibitory activities
of these two compounds to EcMetAP1 and ScMetAP1
are shown in Table 2. Both showed good inhibition of
EcMetAP1 with IC50 values less than 100 nM. Although
not so outstanding as those for EcMetAP1, the inhibi-
tion activities against ScMetAP1 are satisfying, com-
pared with the simple 5-alkyl-substituted compounds
(6a–c, 6g–i). This strongly indicates that the introduc-
tion of b-methoxy dramatically improved activity
against the enzymes.
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In conclusion, we obtained a new series of potent Me-
tAP1 inhibitors through simple bioisosteric replacement
from the PCAT series of compounds. Preliminary sys-
tematic SAR studies of introducing 5-N-acyl substitu-
ents demonstrated that these TCAT series of
compounds showed different activity and selectivity
compared with those of the corresponding PCAT com-
pounds. The introduction of b-methoxy unexpectedly
during the syntheses of 5-alkyl-substituted compounds
improved the inhibitory activity against the enzymes
dramatically. These findings provide a new starting
point for the design and discovery of more potent Me-
tAP1 inhibitors.
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Acknowledgments
This work was supported by the National Natural Sci-
ence Foundation of China Grants 30271528 (F.-J.N.),
the Qi Ming Xing Foundation of Shanghai Ministry of
Science and Technology Grant 02QB14013 (F.-J.N.).
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