Antiparasitic activities of novel, orally available fumagillin analogs

Article abstract of DOI:10.1016/j.bmcl.2009.07.029

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes,

Full text of DOI:10.1016/j.bmcl.2009.07.029

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5128–5131
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiparasitic activities of novel, orally available fumagillin analogs
a,
Christopher Arico-Muendel a, , , Paolo A. Centrella , Brooke D. Contonio a, Barry A. Morgan a,
,
*
Gary O’Donovan a, , Christopher L. Paradise a,à, Steven R. Skinner a, , Barbara Sluboski a,§
Jennifer L. Svendsen a, Kerry F. White a,–, Anjan Debnath b, Jiri Gut b, Nathan Wilson b,
James H. McKerrow b, Joseph L. DeRisi b, Philip J. Rosenthal b, Peter K. Chiang c
,
a Praecis Pharmaceuticals, 830 Winter Street, Waltham, MA 02451, United States
b University of California, San Francisco, CA 94158, United States
c Pharmadyn Inc., Sunnyvale, CA 94085-3528, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria par-
asites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic
profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed effi-
cacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin
based inhibitors against parasitic targets by inhibition of MetAP2.
Received 18 May 2009
Revised 20 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MetAP2
Fumagillin
TNP-470
Antiparasitic
Malaria
Trypanosomes
Amoeba
PPI-2458
The sesquiterpene fumagillin 1, produced by Aspergillus fumiga-
tus, has been extensively studied for its antimicrobial activity.1
While toxic, it is employed in veterinary applications and to a lim-
ited degree in humans, particularly in immuno-compromised indi-
viduals. A renewed interest in fumagillin, driven by recognition of
its anticancer properties, led to identification of its molecular tar-
get, methionine aminopeptidase-2 (MetAP2).2 MetAP2 is a cyto-
solic enzyme which removes methionine from the amino
terminus of newly synthesized proteins for subsequent post-trans-
lational modifications (e.g. myristoylation), which are required for
stability, activity, and intracellular localization.3,4 A related iso-
form, MetAP1, can process many of the same substrates, but its rel-
ative activity differs from that of MetAP2 based on the nature of
the amino acid residues flanking the cleavage site, primarily the
residue immediately adjacent to the N-terminal methionine. Fum-
agillin and structural analogues, such as TNP-470 (AGM-1470), 2a,
selectively inhibit MetAP2 enzymatic activity through irreversible,
covalent bond formation with histidine-231 in the active site of the
enzyme.5,6
* Corresponding author.
E-mail address: christopher.c.arico-muendel@gsk.com (C. Arico-Muendel).
Present address: GlaxoSmithKline, 830 Winter Street, Waltham, MA 02451,
United States.
à
Present address: University of Texas-Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75390, United States.
§
Present address: Provid Pharmaceuticals, 671 U.S. Rte. 1, North Brunswick, NJ
Previously, we showed that fumagillin and TNP-470 inhibited the
in vitro growth of Plasmodium falciparum and Leishmania donavani,
and speculated that their mode of action might stem from the
08902, United States.
Present address: Infinity Pharmaceuticals, 780 Memorial Drive, Cambridge, MA
02139, United States.
–
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.029

C. Arico-Muendel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5128–5131
5129
O
including the chloroquine resistant strains W2 and C2B. On average,
TNP-470 was approximately 10 times better as an inhibitor (IC50
0.3–1.66 ng/mL). These results suggested that the activity of fum-
agillin could be optimized further. We additionally described the
anti-trypanosomal activity of ovalicin, whose structure closely
resembles that of fumagillin, and ovalicin analogues.8 Evidence for
a direct interaction of PfMetAP2 with fumagillin was also recently
reported.9
H
O
H
O
OCH3
c
O
OCH3
X
O
O
R1
O
O
1, R1
=
OH
The concept of MetAP enzymes as anti-infective targets is fur-
ther validated by the data that a family of structurally related
inhibitors containing a 2-(2-pyridinyl)-pyrimidine core was effec-
tive therapeutically against malaria in vitro and in mouse mod-
els.10 These compounds target specifically PfMetAP1b. In this
Letter, we disclose new fumagillin analogs that show broad spec-
trum antiparasitic efficacy against malaria parasites, trypano-
somes, and amoebae. Moreover, two analogs showed in vivo
efficacy against the rodent malaria parasite P. berghei in mice.
The results validate fumagillin and its analogues as inhibitors of
these parasites, and provide initial SAR for further optimization.
We previously described a strategy to improve the pharmacoki-
netic and safety profile of fumagillin and TNP-470 by modifying
the structure of the substitutent at C6 on the cyclohexane ring,
which is a polyolefinic acid in fumagillin and a reactive chloroace-
tylcarbamate in TNP-470 (Scheme 1).11 Crystallographic studies
demonstrated that the primary interactions between fumagillin
a
b
O
O
3, R1 = H
O
NO2
O
O
or
N
4, R1
=
O
O
Scheme 1. Design and synthesis of fumagillin analogs. Reagents and conditions: (a)
NaOH, MeOH, H2O, 0 °C; (b) DSC or 4-NO2 phenylchloroformate, TEA, CH3CN, 25 °C;
(c) X, DIEA, CH3CN or EtOH, 25 °C.
inhibition of MetAP2.7 Moreover, we reported the cloning of the
MetAP2 gene of P. falciparum, the most lethal and common source
of malaria infection, and found a 40% sequence homology with
the murine and human enzymes. Importantly, fumagillin and
TNP-470 showed potent activity against four strains of P. falciparum,
Table 1
Effect of MetAP2 inhibitors on in vitro growth of Entamoeba histolytica, Plasmodium falciparum, and Trypanosoma brucei: initial compound set
Compound
Structurea
Growth IC50
P. falciparum (ng/mL)
b
E. histolytica (ng/mL)
T. brucei (mg/mL)
% Free MetAP2c
ND
O
1
5.0
99.6
12.3
7.74
39.4
20.5
36.7
26
HO
H
N
2
5
6
7
8
26.0
2.8
28
ND
6
Cl
O
O
H
N
10.0 0.1
1260 290
10.0 0.1
87.0 0.7
N
H
N
24
ND
3
N
O
HO
HO
42
H
N
O
N
BLQ
8
O
H
OH
O
OCH3
9
BLQ
BLQ
BLQ
90
O
H
N
O
H2N
O
a
Substituents correspond to X in Scheme 1.
Standard errors are <0.01 unless otherwise indicated. Abbreviations: BLQ, below limit of quantitation; ND, not determined.
Residual MetAP2 activity determined from Met-AMC based fluorescence following treatment of 80 nM MetAP2 with 800 nM compound for 8 h.
b
c

5130
C. Arico-Muendel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5128–5131
Table 2
Effect of MetAP2 inhibitors on in vitro growth of Entamoeba histolytica, Plasmodium falciparum, and Trypanosoma brucei: follow-up compound set
Structurea
Growth IC50
b
Compound
E. histolytica (ng/mL)
P. falciparum (ng/mL)
T. brucei (
l
g/mL)
% Free MetAP2c
ND
O
H
N
10
2540 340
BLQ
28.8
BLQ
N
N
O
H
N
N
11
64.3 0.3
55
ND
N
H
O
H
N
12
13
2280 310
1390 420
26.3
34.7
25
19
ND
5
N
N
H
O
H
N
N
N
H
O
H
N
HO
14
497 30
136
14
4
N
H
O
a
Substituents correspond to X in Scheme 1.
Standard errors are <0.01 unless otherwise indicated. Abbreviations: BLQ, below limit of quantitation; ND, not determined.
Residual MetAP2 activity determined from Met-AMC based fluorescence following treatment of 80 nM MetAP2 with 800 nM compound for 8 h.
b
c
and MetAP2 involve the reactive spiroepoxide moiety as well as
the prenyl containing substituent at C4, whereas the ester chain
at C6 extends through a narrow hydrophobic channel to the pro-
tein exterior.5 Thus, alterations at C6 should preserve the spiroep-
oxide scaffold forming the key inhibitory contacts while
modulating the physical properties of the molecule and allowing
additional interactions with the active site cleft and solvent. We
found that incorporation of alkyl carbamate functionalities at this
position yielded potent inhibitors of MetAP2 and HUVEC prolifera-
tion, resulting in the discovery of PPI-2458 (2b), an orally active
fumagillin analog effective in rat models of arthritis.12 The set of
compounds chosen for the current study contain basic and acidic
groups at a range of distances and orientations from the fumagillin
core. Synthesis was accomplished from commercially available
fumagillin as described previously.11
MetAP2 activity is seen. It is possible that the insertion of the
spacer residue lowers the permeability of the compounds in amoe-
ba. Finally, compounds 5–14 were also tested against Leishmania
donovani, but with the exception of 7, showed no activity at con-
centrations <100 lg/mL.
Compounds 12 and 13 (PPI-2788 and PPI-2791) were addition-
ally tested in mice infected with malaria P. berghei (Fig. 1). Controls
treated with PBS did not survive beyond 7 days post infection,
whereas both 12 and 13 were highly protective to 12 days. These
results show that significant in vivo efficacy can be achieved with
these prototype compounds.
This work builds on earlier studies demonstrating the efficacy of
fumagillin and ovalicin analogues against the parasites responsible
For our initial set (Table 1, 5–9) we examined whether the
activity of TNP-470 could be maintained in nonlabile, compact car-
bamate derivates containing a range of functional groups. With
few exceptions, compounds from this set potently inhibit all three
parasite classes at a level similar to that of fumagillin and TNP-470.
Hydrazide 5 and carboxylate 7 show the best activity across the
panel, indicating that either basic or acidic functionalities are toler-
ated. Both compounds are also highly efficient inhibitors of
hMetAP2, as determined by the level of residual free MetAP2 activ-
ity remaining following 8 h incubation with compound. In contrast,
hydration of the prenyl double bond in 9 causes nearly complete
loss of enzyme inhibition, and little to no antiparasitic activity is
observed.
A follow-up set of compounds (Table 2) extended the carbamate
moiety with conformationally constrained amino acyl residues as
spacers. The effect of this extension is marginal on T. brucei and
P. falciparum (D2 strain), but resulted in a 10–100 fold loss of activ-
ity against Entamoeba histolytica. However, little correlation with
Figure 1. Effect of MetAP2 inhibitors 12 and 13 on survival of mice infected with
malaria P. berghei. Following infection on Day 1, mice were dosed subcutaneously
with 25 mg/kg compound every 12 h, or with PBS as a control. Each data point was
the mean of triplicates.

C. Arico-Muendel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5128–5131
5131
for chloroquine resistant malaria and sleeping sickness. Based on
Supplementary data
the homology of human and Plasmodium MetAP2, we hypothesized
that fumagillin analogues would potently inhibit parasitic
MetAP2’s and demonstrate efficacy in vivo resulting from blockage
of an essential housekeeping enzyme. Separately, we showed that
the toxicity and poor pharmacokinetics of TNP-470, the most po-
tent inhibitor described for MetAP2, could be remedied by replace-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.029.
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work was supported in part by The Sandler Foundation and NIH
grant U01-AI53862 to J.L.D.
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