Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.02.066

The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.

Full text of DOI:10.1016/j.bmcl.2008.02.066

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2373–2376
Synthesis of barbiturate-based methionine
aminopeptidase-1 inhibitors
Manas K. Haldar,a Michael D. Scott,a Nitesh Sule,b
D. K. Srivastavab,* and Sanku Mallika,*
aDepartment of Pharmaceutical Sciences, North Dakota State University, 1401 Albrecht Boulevard, Fargo, ND 58105, USA
bDepartment of Chemistry, Biochemistry and Molecular Biology, North Dakota State University, Fargo, ND 58105, USA
Received 18 December 2007; revised 26 February 2008; accepted 27 February 2008
Available online 4 March 2008
Abstract—The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1
(MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.
Ó 2008 Elsevier Ltd. All rights reserved.
Cellular protein synthesis starts with an N-terminal
methionine in the eukaryotic cells or with a formylmethi-
onine in the prokaryotic cells. Removal of the methionine
residue is essential for proper folding, post-translational
modifications and translocation of the synthesized pro-
teins.1 The metalloenzymes methionine aminopeptidases
(MetAPs) catalyze the hydrolytic removal of the N-termi-
nal methionine residue from the newly synthesized
proteins.1 While the prokaryotic cells contain only the
type-1 enzyme, eukaryotic cells contain both the type-1
and -2 enzymes in the cytosol.2 Deletion of the MetAP
is shown to be lethal in various bacteria.2
In contrast to MetAP-2, there are only a few reports in
the literature on inhibition of human MetAP-1. Peptide
hydroxamic acids are reported to be competitive inhibi-
tors for human MetAP-1 with moderate potency.7
Substituted pyridines were identified as another class
of human MetAP-1 inhibitors using high-throughput
screening of 175,000 small organic molecules.3b Ovali-
cin, the highly-potent, non-competitive, natural product
inhibitor for MetAP-2, was found to bind human Me-
tAP-1 weakly.8 We have recently initiated a research
program to design and synthesize selective inhibitors
for bacterial MetAP-1 as antibacterial agents and selec-
tive inhibitors for human MetAP-1 as anti-cancer
agents.
Since the discovery that the irreversible MetAP-2 inhib-
itor fumagillin prevents angiogenesis, both reversible
and irreversible inhibitors for this enzyme have been
extensively studied as potential anti-angiogenesis and
anti-cancer agents.3 Selective inhibition of MetAP-1 in
bacteria (or in parasites) over the human enzymes has
been shown to be a promising approach in designing
new antibacterial4 and antimalarial5 agents. The role
of MetAP-1 in human cell cycle progression has been
elucidated recently.6 Selective inhibition of the human
MetAP-1 (over MetAP-2) led to the arrest of cell
division and induction of apoptosis in leukemia cells.6
Under physiological conditions, the metal ions in the ac-
tive site of MetAPs are not firmly established. The active
site binds to two divalent transition metal ions and the
residues responsible for binding to the metal ions are
conserved.4 One of these two bound metal ions under-
goes a rapid exchange with free metal ions in solution.4
Since Co (II) ions activate all MetAPs, usually the inhib-
itors are screened using Co (II) form of the enzyme. Be-
sides Co (II), Mn (II), Fe (II), Ni (II) and Zn (II) are
proposed as possible metal ions under physiological
conditions.4 To complicate inhibitor design, the potency
of MetAP inhibitors depend on the identity of the metal
ions in the active site of the enzyme.9 In addition, recent
NMR spectroscopic studies have demonstrated that in
the absence of any substrate or inhibitor, the enzyme
in solution has several structures in dynamic equilib-
rium.4 For our studies, we added CoCl2 (100 lM) in
Keywords: Type-1 MetAP inhibitors; Barbiturates.
*
Corresponding authors. Tel.: +1 701 231 7888; fax: +1 701 231 8331
(S.M.); e-mail addresses: dk.srivastava@ndsu.edu; sanku.mallik@
ndsu.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.066

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M. K. Haldar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2373–2376
the assay buffer; hence the reported compounds are Co
(II) coordinating inhibitors.
was determined using a BCA protein assay kit from
Pierce, with BSA as the standard.
All of the reported competitive inhibitors for MetAPs
contain a Lewis base to coordinate to the transition
metal ions in the active sites of the enzymes. In pursuit
of designing new types of MetAP inhibitors, we noted
that barbiturate-based potent inhibitors are known for
the matrix metalloproteinases10 and for other metallo-
enzymes.11 Herein, we report that potent inhibitors for
MetAP-1 can be synthesized in one or two high-yielding
steps starting from barbituric acid. To the best of our
knowledge, this is the first report of MetAP inhibition
by the barbituric acid derivatives.
The inhibitory potencies of the synthesized compounds
were determined by using the reported chromogenic
substrate for MetAPs, Met-Pro-p-nitroanilide.16 Proline
aminopeptidase was used as the coupling enzyme and
we ascertained that the synthesized compounds are not
inhibiting this enzyme.17 The yields obtained during
the syntheses of the barbiturate derivatives and the
measured inhibition constants with E. Coli and human
MetAP-1 are shown in Table 1.
We observed that the barbituric acid derivatives were
more potent inhibitors of MetAP-1 compared to the
derivatives of 2-thiobarbituric acid or 1,3-dimethylbar-
bituric acid. Under the enzyme assay conditions
(25 mM HEPES buffer, pH 7.5), the barbituric and thio-
barbituric acid derivatives are expected to exist as the
corresponding conjugate bases.18 It is likely that the
conjugate bases from the compounds 1–20 are the active
forms of these inhibitors. From our preliminary model-
ing studies, it is anticipated that the oxygen atom at C-2
of the barbituric acid moiety is binding to the active site
metal ion of MetAP-1.
We observed that barbituric acid, 2-thiobarbituric acid
and 1,3-dimethylbarbituric acid were weak inhibitors
for recombinant Escherichia Coli and human MetAP-1
(Ki > 100 lM). Preliminary docking studies suggested
that barbituric acid is binding to one of the Co (II) ions
in the active site through the oxygen atom on C-2. Our
goal was to introduce substituents on the barbiturate
moiety to produce more potent inhibitors. Knoevenegal
condensation of aromatic aldehydes appeared to be an
attractive method to achieve this goal as the products
will have alkenes conjugated to the aromatic benzene
ring. The general structures and the syntheses of the bar-
biturate inhibitors are shown in Scheme 1.
The barbituric acid derivatives 1–9 showed moderate to
excellent potency in inhibiting the enzyme. Some of these
inhibitors were found to be selective for the human Me-
tAP-1 over the E. Coli enzyme. For example, compound
4 was 67 times more potent in inhibiting the human en-
zyme (Ki = 5 lM) compared to the E. Coli MetAP-1
(Ki = 335 lM). In order to determine the effect of an
additional potential coordinating atom to the active site
Co (II) atoms of MetAP, we synthesized the barbiturate
derivative of 2-hydroxy-4-methoxybenzaldehyde. How-
ever, the resultant compound was found to be a weak
inhibitor for both E. Coli and human MetAP-1
(Ki > 100lM for both enzymes) (data now shown).
We performed the Knoevenegal condensation by heating
to reflux the barbituric acid (or thiobarbituric acid) and
the aromatic aldehydes in water.12 For 1,3-dimethylbar-
bituric acid, the condensation reaction was more conve-
niently performed by grinding the two solid reactants at
room temperature in the presence of amidosulfonic
acid.13 The residue was dissolved in dimethylsulfoxide
and poured into water to precipitate the crude products.
The solids obtained were recrystallized from dimethyl-
formamide to give the pure products.
Escherichia Coli and human MetAP-1 were expressed
and purified as described previously, from expression sys-
tems kindly provided by Dr. Anthony Addlagatta14 and
Dr. Brian Matthews.15 No attempt was made to remove
the His-tags from either protein. Purity of the proteins
was confirmed by SDS–PAGE. Protein concentration
Inhibitors with substituents on the benzene ring were
more effective compared to the molecule containing
the unsubstituted benzene ring. In general, for E. Coli
MetAP-1, compounds with electron releasing groups
at the para-position of the benzene ring showed higher
inhibitory potency compared to compounds with elec-
tron withdrawing groups on the aromatic ring. We did
not observe any such trend for the inhibition of the
human MetAP-1. Based on our calculations employing
the semi-empirical PM3 force field (Spartan 06, Wave-
function Inc.), the charge densities at the oxygen atoms
of the barbituric acid moiety are not perturbed by the
nature of the substituents on the benzene ring. Cur-
rently, we are performing quantitative structure activity
relationship studies with the synthesized inhibitors and
MetAP-1 to determine the origin of this observed
selectivity.
X
X
H2O
HN
NH
HN
NH
+ ArCHO
+ ArCHO
Reflux
O
O
O
O
X
= O, S
Ar
O
O
N
H3C
O
CH3
O
H3C
O
CH3
O
NH2SO3H
Grind
N
N
N
All of the compounds excepting 10 were competitive
inhibitors for both E. Coli and human MetAP-1. Com-
pound 10 was the most effective inhibitor synthesized
(Ki = 50 and 10 nM for the E. Coli and human
MetAP-1, respectively) and it demonstrated a mixed
Ar
Scheme 1. General structures and the syntheses of the barbiturate-
based MetAP-1 inhibitors.

M. K. Haldar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2373–2376
2375
Table 1. Structures, synthetic yields and the inhibition constants of the barbiturate derivatives
X
R
O
R
O
N
N
Y
Product
X
R
Y
Yield (%)
Ki (lM), MetAP-1
Human
162 13
E. Coli
1
2
O
O
O
O
O
O
O
O
O
O
S
H
C6H5
85
86
61
79
86
89
96
94
88
83
67
75
56
23
94
78
96
50
89
63
42
56
87
89
69
87
89
54
70
45
517 63
105 18
H
C6H4-pNO2
C6H4-mNO2
C6H4-pCO2H
C6H4-pOH
C6H4-pF
8
1
3
H
29
2
335 47
2
0.3
1
4
H
5
5
H
4
89 12
1
10
2
2
6
H
0.2
2
7
H
C6H4-pNMe2
C6H4-pOMe
13
25
12
1
2
3
10
2
8
H
0.4
2
9
H
CH@CH2C6H5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
CH@CH2C6H4-pNMe2
C6H5
C6H4-pNO2
0.05 0.03
>100
>100
0.01 0.002
>100
>100
H
S
H
S
H
C6H4-mNO2
C6H4-pCO2H
C6H4-pOH
>100
>100
>100
>100
>100
S
H
S
H
>100
>100
S
H
C6H4-pF
6 0.6
>100
> 100
S
H
C6H4-pNMe2
C6H4-pOMe
CH@CH2C6H5
CH@CH2C6H4-pNMe2
C6H5
>100
87 31
>100
S
H
S
H
98
1
4
0.1
S
H
17
1
O
O
O
O
O
O
O
O
O
O
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
> 100
>100
C6H4-pNO2
C6H4-mNO2
C6H4-pCO2H
C6H4-pOH
C6H4-pF
C6H4-pNMe2
C6H4-pOMe
CH@CH2C6H5
CH@CH2C6H4-pNMe2
mode of inhibition for both of the enzymes. In fact, 10 is
one of the most potent inhibitors reported for human
MetAP-1 so far. In addition to any electronic effect,
the hydrophobic alkene moieties also possibly contrib-
ute to the excellent inhibitory potency exhibited by com-
pound 10. Structurally, it appears that the addition of
the alkenyl spacer to compound 7 (i.e., compound 10)
leads to substantial improvement in the inhibitory
potency. A similar trend was observed for the inhibitors
1 and 9. Currently, we are evaluating the effect of this
structural modification on the inhibitory potency for
the compounds 2–6 and 8.
basis for this lack of inhibition exhibited by this series
of compounds. The compounds 21–30 cannot deproto-
nate to generate the conjugate base under the enzyme
assay conditions. In addition, the two methyl groups
on the barbiturate moiety may sterically interfere with
the binding process in the enzyme-active site. Either
of these two factors (or a combination) may lead to
the lack of inhibition of MetAP-1 by this class of
compounds.
In conclusion, we have synthesized several derivatives of
barbituric acid and demonstrated their effectiveness in
inhibiting MetAP-1. All of the reported inhibitors were
synthesized in one step from commercially available
starting materials. One of the synthesized compounds
(e.g., 10) showed excellent inhibitory potency for
E. Coli and human MetAP-1 (Ki < 100 nM). Another
moderate inhibitor (e.g., 4) showed very good selectivity
(>60) for human MetAP-1 compared to the E. Coli
enzyme. Detailed molecular modeling and cellular assay
studies are currently in progress and these results will be
reported later.
In contrast, most of the thiobarbiturate derivatives
synthesized did not inhibit MetAP-1. The thiobarbitu-
rate derivatives 18 and 20 showed weak inhibition of
E. Coli MetAP-1 (Ki = 87 and 17 lM, respectively).
Compounds 16 and 20 demonstrated moderate and
selective inhibition of human MetAP-1 (Ki = 6 and
1 lM, respectively). The synthesized derivatives of
1,3-dimethylbarbituric acid (21–30) failed to inhibit
the enzyme. We do not yet understand the molecular

2376
M. K. Haldar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2373–2376
9. Huang, M.; Xie, S. X.; Ma, Z. Q.; Hanzlik, R. P.; Ye, Q.
Acknowledgment
Z. Biochem. Biophys. Res. Commun. 2006, 339, 506.
10. (a) Hutchings, S.; Liu, W.; Radinov, R. Heterocycles 2006,
67, 763; (b) Kim, S. H.; Pudzianowski, A. T.; Leavitt, K.
J.; Barbosa, J.; McDonnell, P. A.; Metzler, W. J.; Rankin,
B. M.; Liu, R.; Vaccaro, W.; Pitts, W. Bioorg. Med. Chem.
Lett. 2005, 5, 1101; (c) Daniewski, A. R.; Liu, W.; Okabe,
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This research was supported by the NIH Grant 1R01
CA113746 and NSF DMR-0705767 to S.M. and D.K.S.
Supplementary data
11. (a) Suzuki, H.; Kneller, M. B.; Rock, D. A.; Jones, J. P.;
Trager, W. F.; Rettie, A. E. Arch. Biochem. Biophys. 2004,
429, 1; (b) Soong, C. L.; Ogawa, J.; Sakuradani, E.;
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Experimental details and characterization data for the
synthesis of the compounds reported in Table 1 are
available as Supplementary data. Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2008.02.066.
12. Deb, M. L.; Bhuyan, P. L. Tetrahedron Lett. 2005, 46,
6453, A representative procedure is provided here. To a
solution of barbituric acid or thiobarbituric acid (3 mmol)
in hot water (20 mL), the aromatic aldehyde (3 mmol) was
added. After stirring at room temperature for 30 min the
solution was heated to reflux for 1 h. The precipitated
solid was filtered, washed with methanol and dried under
vacuum. The crude product was recrystallized from N,N-
dimethylformamide and was characterized by 1H, 13C
NMR and mass spectroscopy.
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