Orally active fumagillin analogues: Transformations of a reactive warhead in the gastric environment

Article abstract of DOI:10.1021/ml3003633

Semisynthetic analogues of fumagillin, 1, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, 3 (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, 3 underwent ring-opening addition of water and HCl, leading to four products, 4-7, which were characterized in detail. The chlorohydrin, but not the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism. In agreement, chlorohydrin 6 was shown to revert rapidly to 3 in rat plasma. In an ex vivo assay, rats treated with purified acid degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together, the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a pharmacologically useful level of MetAP2 inhibition.

Full text of DOI:10.1021/ml3003633

Letter
pubs.acs.org/acsmedchemlett
Orally Active Fumagillin Analogues: Transformations of a Reactive
Warhead in the Gastric Environment
Christopher C. Arico-Muendel,*,† Heather Blanchette,‡ Dennis R. Benjamin,§ Teresa M. Caiazzo,∥
Paolo A. Centrella,⊥ Jennifer DeLorey,@ Elisabeth G. Doyle,# Steven R. Johnson,∇ Matthew T. Labenski,○
Barry A. Morgan,● Gary O’Donovan,† Amy A. Sarjeant,◆ Steven Skinner,† Charles D. Thompson,◇
Sarah T. Griffin,▲ William Westlin,○ and Kerry F. White△
Praecis Pharmaceuticals, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1420, United States, and Department of Chemistry,
Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
S
* Supporting Information
ABSTRACT: Semisynthetic analogues of fumagillin, 1, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the
clinic for the treatment of cancer. An optimized fumagillin analogue, 3 (PPI-2458), was found to be orally active, despite
containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, 3 underwent ring-
opening addition of water and HCl, leading to four products, 4−7, which were characterized in detail. The chlorohydrin, but not
the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism.
In agreement, chlorohydrin 6 was shown to revert rapidly to 3 in rat plasma. In an ex vivo assay, rats treated with purified acid
degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together,
the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a
pharmacologically useful level of MetAP2 inhibition.
KEYWORDS: Fumagillin, MetAP2, PPI-2458, angiogenesis inhibitor, irreversible inhibitor
rugs that contain reactive functional groups (e.g., Michael
acceptors, epoxides, and β-lactams) and that inhibit their
criteria. Another potential liability of this drug class results from
in vivo processes that may degrade the warhead before it
reaches its target. Specialized formulations, prodrugs, or
nonoral routes of administration may be required to ensure
bioavailability. Despite these challenges, drugs that act through
a covalent mechanism have been approved for a diverse
spectrum of diseases over the years, and a recent survey
indicates a healthy pipeline of such molecules in the clinic.1
The sesquiterpenoid fumagillin 1 (Scheme 1) contains a
spiroepoxide moiety that forms a covalent linkage with an
active site histidine residue in the enzyme methionine
aminopeptidase-2 (MetAP2).3 Serendipitously, the scaffold of
this natural product, in particular the C4 side chain, mimics the
D
molecular targets by forming stable covalent linkages represent
extreme cases of slow off-rate binding, where drug occupancy
may become governed by protein turnover or other cellular
processes that occur on an extended time scale.1 A particular
advantage of such drugs is that provided the on rate is
sufficiently rapid, a pharmacological effect may be achieved
even without high or continuous exposure. Because of their
reactivity, irreversible inhibitors are often thought to carry an
enhanced risk of off-target toxicity, for example, by hapteniza-
tion of proteins leading to an autoimmune response. Although
side effects have been associated with well-known drugs of this
type, notably allergic responses to penicillins, few cases of
mechanism-related toxicity have been established in detail.2
Inclusion of in vivo testing at an early stage in the discovery
process can help ensure that a drug candidate acting through an
irreversible mechanism meets appropriate specificity and safety
Received: October 26, 2012
Accepted: February 22, 2013
Published: February 22, 2013
© 2013 American Chemical Society
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resulted in improved safety and pharmacokinetic properties, in
particular oral bioavailability and a lack of CNS effects.11
Further studies demonstrated the utility of 3 in rodents as a
disease-modifying agent for the treatment of rheumatoid
arthritis.11−17 While highly desirable for treating chronic
disease, the oral activity of 3 was surprising given the expected
acid lability of the epoxide groups. Here, we describe the
behavior of 3 in acidic solutions as a model for the gastric
environment. The major products of acidic degradation were
identified and characterized with respect to their ability to
inhibit MetAP2, delineating a mechanism for the oral activity of
3.
Scheme 1. Structures of Compounds 1−3, with C4 and C6
Side Chains Marked
Treatment of 3 with 6 mM HCl (pH 2) resulted in three
products (4−6) that could be resolved by HPLC. The early
eluting peaks corresponding to 4 and 5 showed a mass increase
of 18 amu (+H2O), whereas the late eluting peak due to 6
displayed a mass increase of 36 amu (+HCl, confirmed from
the 37Cl isotopomer). In simulated gastric fluid [0.7% (v/v)
HCl and 0.2% NaCl (pH 1.2)], additional species were evident.
In particular, another HCl adduct, 7, could be discerned eluting
near 3 at 1.75 min. Over a 20 h time span, the quantity of 7
gradually increased relative to the quantity of 6 (Figure 1b).
On a preparative scale, compounds 4 and 5 were synthesized
by treatment of 3 with aqueous HCl, whereas a more
convenient route to 6 and 7 was found using anhydrous HCl
in dioxane. Under these conditions, the formation of 7 was
rapid, and trapping 6 in useful quantities required neutralization
of the reaction mixture after 5 min at room temperature. Both 6
and 7 reacted with KOtBu with loss of HCl, again yielding
products that could be resolved by HPLC. In the case of 6, this
material was isolated and shown to be identical with 3; the
isomer formed from 7 was identified as a new compound, 8.
Isolated compounds 4−8 were characterized by NMR
spectroscopy, using previous analyses of 3 as a reference.
Proton assignments were made using two-dimensional COSY,
N-methionyl residue of polypeptide chains that makes up the
endogenous substrate of MetAP2.4 Noncovalent interactions
arising from active site complementarity appear to play an
important role in the effectiveness of fumagillin as an inhibitor:
reversible fumagillin derivatives have been shown to inhibit
MetAP2,5 whereas the simple cyclohexane spiroepoxide core is
inactive.6 Long used as a veterinary antibiotic, fumagillin has
attracted additional interest over the past 20 years because of its
antiangiogenic effects.7 In particular, semisynthetic analogue 2
(TNP-470),8 in which the polyolefinic chain at C6 was
truncated, was efficacious in animal models for cancer9 and
entered human clinical trials.10 More recently, we disclosed
compound 3 {(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-
3-(3-methylbut-2-en-1-yl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-
yl [(R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, PPI-
2458},6 in which replacement of the chloroacetimide of 2
Figure 1. (a) Acid degradation of 3 in 6 mM HCl (pH 2). (b) Conversion of 6 (retention time of 2.14 min) to 7 (retention time of 1.75 min) at pH
1.
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Scheme 2. Summary of Structures and Pathways for Formation of Compounds 4−8
TOCSY, and ROESY correlations and are summarized in Table
S1 of the Supporting Information.
1
As was the case with their HPLC retention times, the H
NMR spectra of 4 and 5 were similar, but markedly different
from that of 3. Notably, the resonance of H17, whose
anomalously upfield chemical shift was interpreted in terms
of its proximity to the spiroepoxide function, migrated
downfield into the region of H14−H16. Significant chemical
shift changes were also apparent for H6, H8, and H9. These
changes were consistent with opening of the spiroepoxide ring.
Oxidation of 4 and 5 with TPAP resulted in a loss of 2 amu and
the disappearance of H6 as opposed to H8 or H9. Taken
together, the data suggested that 4 and 5 could be assigned to
isomeric perhydrobenzofuran structures (Scheme 2). In
accordance with the guidelines for ring closure, the oxidation
data excluded the alternative formation of a six-membered ring,
which would have generated a tertiary alcohol.18 The absolute
stereochemistry of both 4 and 5 was confirmed by
crystallography (details in the Supporting Information),
revealing the compounds as epimers at C3 (Figure 2). A
plausible mechanism leading to 4 and 5 involves SN1 opening
of the spiroepoxide and formation of diastereomeric diols,
followed by SN2-type ring formation (Scheme 2).
Figure 2. ORTEP plots of one molecule of 4 (left) and 5 (right).
Displacement parameters are drawn at the 50% occupancy level.
by ring opening of the side chain epoxide. Finally, while the 1H
NMR spectrum of 8 still exhibited the signature of the allylic
alcohol in 7, other signals characteristic of 3, such as the
upfield-shifted resonance of H17, were visible. This was
consistent with re-formation of the spiroepoxide functionality
in 8. Taken together, the data suggest that near pH 2,
degradation proceeds through limited pathways featuring rapid
opening of the more labile spiroepoxide by two distinct
mechanisms for water and HCl. At lower pH values, processes
involving the C4 side chain epoxide also contribute, leading to
additional products.
Compounds 4−8 were tested for their ability to inhibit
MetAP2 and HUVEC proliferation (Table 1).6 The enzyme
assay monitors loss of MetAP2 activity with Met-AMC
substrate over an 8 h window following treatment with a 10-
fold excess of compound (200 nM), whereas the HUVEC assay
records endothelial cell growth over 3 days in the presence of
0.02 nM to 2 μM compound. In the MetAP2 assay, compounds
4 and 5 failed to show measurable inhibition of MetAP2 over
an 8 h time period and likewise did not attenuate growth of
HUVEC cells at up to micromolar concentrations. Chlorohy-
drins 6 and 7, on the other hand, were active in both assays, as
The NMR spectrum of compound 6 was characterized by
large changes in the chemical shifts of H6, Me7, H8, and H9, all
of which moved downfield at least 0.4 ppm from their locations
in the spectrum of 3. Together with the facile reversion of 6 to
3, the data support assignment of 6 as a chlorohydrin (Scheme
1
2). The H NMR spectrum of its isomer, 7, was quite distinct
from that of 6, the most striking difference being the
disappearance of the signal from Me7 and the emergence of
a two-proton olefinic resonance. A weak coupling is seen
between this peak and H6 in the TOCSY experiment, and a
through-space correlation is observed to H4/5 in the ROESY
experiment, supporting its assignment as the methylene formed
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Finally, we investigated the ability of the purified degradants
to inhibit MetAP2 in vivo. Following oral dosing in rats, cells
from whole blood, liver, and thymus were harvested and treated
with a biotinylated form of 3.11 Free MetAP2 was thus labeled
with an affinity tag, captured on streptavidin beads, and
quantitated in an ELISA format. As shown in Figure 4, a single
Table 1. Inhibition of MetAP2 Activity and HUVEC
Proliferation by Compounds 3−8
a
compd
MetAP2 (% remaining activity)
HUVEC IC50 (nM)
3
4
5
6
7
8
3
3
0.18
>30000
>30000
0.15
107
105
6
6
6
2
58
27
3.3
3.1
a
Presented as residual MetAP2 activity after treatment with the
compound for 8 h.
was epoxide 8. MetAP2 and HUVEC inhibition were
correlated, with 6 nearly as efficacious as 3, whereas 7 and 8
were somewhat less active. The results underscore the
importance of C4 side chain structure, insofar as comparatively
minor changes resulting from the opening of the side chain
epoxide in 7 and 8 can be accommodated, whereas the major
rearrangement yielding the bicyclic structures of 4 and 5 is not
compatible with the active site of MetAP2.
The similarity between the activity of the two spiroepoxides
and their corresponding chlorohydrins prompted a closer study
of the inhibitory mechanism. Progress curves for the inhibition
of MetAP2 in HEPES buffer (pH 7.5) showed rapid
inactivation by 3, whereas that of 6 proceeded more gradually
(Figure 3a). When isomeric species 7 and 8 were also
considered, the rates of inhibition decreased in the following
order: 3 > 6 > 8 > 7 (not shown). The activity of 6 was also
highly pH sensitive: in MES buffer (pH 6.5), no inhibition was
observed, whereas 3 was equally effective under both conditions
(Figure 3b). This behavior is consistent with a mechanism in
which the chlorohydrins lack intrinsic activity but revert to
epoxides under slightly basic conditions as an obligatory step
toward inhibition. In agreement, we determined that 6 was
∼50% converted to 3 after 8 h in HEPES but was largely
unchanged in MES. Because no reversion from 8 to 3 has been
observed, we conclude that 8 is a direct, albeit less optimal,
inhibitor of MetAP2.
Separately, we tested the stability of 6 in rat plasma. Exposure
for 1 h at 4 °C resulted in quantitative reversion to 3 (Table S2
of the Supporting Information), whereas the amount in solvent
controls is negligible. Close to 10% reversion occurs within the
first minute. Thus, the kinetics of reversion of 6 to 3 in plasma
greatly exceeds that observed in simple pH 7.5 buffer, possibly
because of protein binding. One may surmise that in vivo,
active species (3 and probably 8) are efficiently regenerated
upon absorption from the gut.
Figure 4. Free MetAP2 levels in liver, thymus, and whole blood
following a single PO (3 mg/kg) dose of 3−8 as a percentage of
vehicle control. Data are averages of triplicate experiments with
percentage uncertainties shown.
dose of 3 at 3 mg/kg effectively inhibited MetAP2 in all three
tissues, whereas perhydrobenzofurans 4 and 5 were inactive.
Compounds 6−8, while less effective than 3, nonetheless
significantly inhibited MetAP2 in all compartments studied.
Thus, the level of MetAP2 inhibition in vivo tracks potency in
the biochemical assay. Notably, compounds 3 and 6−8 had
reduced activity in solid tissues, including thymus, with a
disparate drop in activity noted with compounds 6 and 7. While
tissue concentrations were not determined, these findings may
suggest that the distribution of these compounds into tissues
may be variable compared to that in plasma. Plasma analysis
following dosing with purified degradants confirmed the
presence of 4, 5, and 8 in vivo, with measurements at 2 and
24 h indicating that their exposure levels matched or exceeded
that of 3 when administered at an equal dose (Table S3 of the
Supporting Information). Because the LC−MS/MS protocol
specifically quantitated levels of the parent molecule, the
exposure to active species (which in the case of 3 could include
Figure 3. Time dependence of MetAP2 activity (% relative to DMSO or ethanol control) in the presence of (a) compounds 3−6 in HEPES (pH
7.5) and (b) 3 and 6 in HEPES (pH 7.5) or MES (pH 6.5).
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ACS Medicinal Chemistry Letters
Letter
8, as well as cytochrome P450-derived metabolites) is probably
underestimated.
∇Rutgers, The State University of New Jersey, Mario School of
Pharmacy, 160 Frelinghuysen Rd., Piscataway, NJ 08854.
○Celgene Avilomics Research, 45 Wiggins Ave., Bedford, MA
01730.
Previous work has demonstrated some tolerance for
modifications to the C4 side chain that are compatible with
MetAP2 inhibition. CYP3A metabolism of 3 yields diastereo-
meric triepoxides as well as an allylic alcohol that contribute
significantly to MetAP2 inhibition in vivo.19 While most drug
discovery efforts have focused on other regions of the molecule,
in particular the C6 chain, a highly potent inhibitor in which the
C4 chain is replaced by a benzyl oxime has been reported by
Pyun et al.20 Compounds 4 and 5 also have precedent,
beginning with Tarbell’s extensive studies on fumagillin
chemistry.21 More recently, an analogous derivative of
fumagillin (fumagiringillin) was isolated from the fungus
Aspergillus fumigatus, apparently as a single diastereomer.22
Studies of the earlier semisynthetic drug 2, which is rapidly
metabolized in vivo, identified a perhydrobenzofuran as the
major product of the metabolic pathway.23
The results presented here, together with findings from CYP
metabolism studies, contribute to a detailed picture of the
ADME properties of 3.24 In an acidic milieu modeling the
environment of the stomach, two of the four major degradants
retain MetAP2 inhibitory activity and have been characterized
as ring-opened chlorohydrins. Animals orally dosed with parent
compound 3, its isomer 8, or the corresponding chlorohydrins
6 and 7 exhibit significant inhibition of MetAP2 in blood and
tissue. The degree of inhibition correlates with the biochemical
efficacy of each species, with allylic alcohols 7 and 8 showing an
overall reduction in potency relative to 3 and 6. Activity in vivo
appears to require re-formation of the spiroepoxide, which is
likely to occur upon passage into the neutral environment of
the gut, or immediately upon absorption into the bloodstream.
Thus, among the inhibitory compounds, only parent molecule
3 and its isomer 8 are detectable in plasma. Loss of 3 to inactive
species such as 4 and 5 is compensated by its irreversible
mechanism of action. The correlation of in vitro and in vivo
SAR highlights the specific nature of MetAP2 inhibition by 3,
providing support for its utility as a therapeutic candidate.
●237 Prospect St., Franklin, MA 02038.
◆Department of Chemistry, Northwestern University, 2145
Sheridan Rd., Evanston, IL 60208.
◇Merck & Co. Inc., West Point, PA 19486.
▲Chemical Research and Development, Pfizer, Eastern Point
Road, Groton, CT 06340.
△Infinity Pharmaceuticals, 780 Memorial Dr., Cambridge, MA
02139.
Notes
The authors declare the following competing financial
interest(s): C.C.A.-M., G.O., and S.S. are employees of
GlaxoSmithKline, which now owns rights to PPI-2458.
ACKNOWLEDGMENTS
■
We thank Drs. Roy Copley and Randy Rutkowske of
GlaxoSmithKline for careful review of the crystallography
data and for obtaining the 13C NMR spectra, respectively.
ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
AMC, 7-amino-4-methylcoumarin; BLQ, below limit of
quantitation; CNS, central nervous system; CYP, cytochrome
P450; DIEA, diisopropylethylamine; DQF-COSY, double-
quantum-filtered correlated spectroscopy; ELISA, enzyme-
linked immunosorbent assay; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HPCD, hydroxypropylcyclodex-
trin; HUVEC, human umbilical vascular endothelial cell; IV,
intravenous; MES, 2-(N-morpholino)ethanesulfonic acid;
MetAP2, methionine aminopeptidase-2; PO, orally dosed;
ROESY, rotating frame Overhauser spectroscopy; TOCSY,
total correlation spectroscopy; TPAP, tetrapropylammonium
perruthenate; WFI, water for injection.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures for the synthesis of compounds 4−8, NMR
resonance assignments, details of the crystal structures of
compounds 4 and 5 (with CIF files), procedures for
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: christopher.c.arico-muendel@gsk.com.
Present Addresses
†GlaxoSmithKline, 830 Winter St., Waltham, MA 02451−1420.
‡Cubist Pharmaceuticals, 65 Hayden Ave., Lexington, MA
02421.
§Seattle Genetics, Inc., 21823 30th Dr. SE, Bothell, WA 98021.
∥Pfizer Inc., One Burtt Rd., Andover, MA 01810.
⊥X-Chem, Inc., 100 Beaver St., Waltham, MA 02453.
@175 Oakdale St., Attleboro, MA 02703.
#Vertex Pharmaceuticals, 130 Waverly St., Cambridge, MA
02139.
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