Convergent synthesis of α-ketoamide inhibitors of Pin1

Article abstract of DOI:10.1021/ol9027013

(Chemical Equation Presented) A convergent synthesis of α-ketoamlde Inhibitors of Plni Is described. An a-hydroxyorthothloester derivative of Ser was reacted directly with an amine synthon. The reaction was catalyzed by HgO and HgCl₂ to form a-

Full text of DOI:10.1021/ol9027013

ORGANIC
LETTERS
2010
Vol. 12, No. 4
696-699
Convergent Synthesis of r-Ketoamide
Inhibitors of Pin1
Guoyan G. Xu and Felicia A. Etzkorn*
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061
fetzkorn@Vt.edu
Received November 23, 2009
ABSTRACT
A convergent synthesis of r-ketoamide inhibitors of Pin1 is described. An r-hydroxyorthothioester derivative of Ser was reacted directly with
an amine synthon. The reaction was catalyzed by HgO and HgCl2 to form r-hydroxyamide. Thus, hydrolysis and coupling were combined in
one step with 80% yield. Two diastereomers of a phospho-Ser-Pro r-ketoamide analogue were synthesized. The IC50 values of 100 and
200 µM were surprisingly weak for Pin1 peptidyl prolyl isomerase.
R-Ketoamides have been widely used in developing inhibi-
tors of peptidases,1 HDACs,2,3 and peptidyl prolyl isomerases
(PPIases).4 One of the best studied immunosuppressant drugs,
FK506, has an R-ketoamide, and it is a transition state analogue
inhibitor of FK506 binding proteins (FKBPs).5 R-Ketoamides
have been used to inhibit several classes of proteases, such as
serine proteases,6-10 cysteine proteases,11-14 and HIV and
FIV proteases.15
R-Ketoamides have an electron-deficient carbonyl due to
the effect of the neighboring electron-withdrawing amide.
R-Ketoamide-containing inhibitors have been used to elu-
cidate the mechanisms of enzymes because they serve as
transition state analogues to inhibit enzyme catalytic activity.
This occurs for serine or cysteine proteases through the
formation of a tetrahedral intermediate analogue (gem-diol
or hemiketal) with the enzyme upon binding.16 FK506, a
macrolide with an R-ketoamide functional group, has a
dihedral angle of 95° to 100° between the two carbonyl
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10.1021/ol9027013  2010 American Chemical Society
Published on Web 01/26/2010

groups.5 NMR studies of a 13C-labeled structure and a crystal
structure of FK506 showed that the two carbonyls were
orthogonal both in bound and unbound structures, indicating
that FK506 acts as a “twisted amide” transition state analogue
to inhibit FKBPs.5
Pin1, a PPIase enzyme belonging to the parvulin family,
specifically recognizes pSer/pThr-Pro containing substrates
and regulates many cellular events such as cell cycle
progression, transformation, and cell proliferation.17,18 Pin1
is overexpressed in many cancer cell lines and plays an
important role in oncogenesis.19,20 Pin1 is a potential
diagnostic and therapeutic anticancer target.21-23 Thus, we
are interested in developing potent and specific inhibitors of
Pin1.24,25
as Pin1 inhibitors. Our impetus for synthesizing these
challenging phosphorylated R-ketoamides was to investigate
the ability of these compounds to inhibit Pin1, the only
PPIase that regulates the cell cycle.
R-Ketoamides 1a and 1b were designed based on the
specificity of Pin1 for the pSer/pThr-Pro motif.18 An acetyl
was chosen for the N-terminus, since cocrystal structures of
Pin1-inhibitor complexes showed no electron density for
N-terminal residues, and to improve water solubility over
Fmoc derivatives.25,35,36 An aromatic naphthyl was intro-
duced at the C-terminus based on substrate and inhibitor
sequence specificities.18,36,37
Since R-ketoamides are an important class of enzyme
inhibitors, numerous synthetic methods have been reported,
including: cyano ylide coupling,26 transition-metal cataly-
sis,27 amidation of R-keto acids with amines,11,28 oxidation
of R-hydroxyamides with Dess-Martin periodinane6 or
TEMPO,29,30 and nucleophilic addition of a Grignard reagent
to a Weinreb amide.31 A solid-phase method for synthesis
of R-ketoamides has been developed.32 In this study, we have
developed a convergent route to synthesize R-ketoamides
by coupling a complex amine directly to an R-hydroxy-
orthothioester to form an R-hydroxyamide, which was then
oxidized to an R-ketoamide with the Dess-Martin periodi-
nane.33
Boc-Pro-OH and 2-(2-naphthyl)-ethylamine (NEA) were
coupled with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-
imide·HCl (EDC) to give Boc-Pro-NEA 2 (Scheme 1). Free
Scheme 1. Synthesis of H-Pro-NEA 3
We have reviewed Pin1 inhibitors, including synthetic
inhibitors, and natural products.4,23 R-Ketoamides are an
important class of inhibitor for PPIases (e.g., FK506). The
cocrystal structure of FK506 with FKBP was the main
evidence cited to support the “twisted amide” transition state
for PPIases,5,34 but R-ketoamides have not yet been reported
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amine 3 was prepared by TFA deprotection of 2, followed
by neutralization with aqueous NaHCO3.
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Aldehyde 4 was obtained by LiAlH4 reduction of the
Weinreb amide of protected Ser (Scheme 2).38 Lithio
trismethylthiomethane was added to aldehyde 4 to give
orthothioester 5.39 The two diastereomers of orthothioester
5 were not separated at this stage since the newly formed
stereocenter would be eliminated in the subsequent oxidation
of alcohol 7 to ketone 8. Orthothioester 5 was converted
directly to R-hydroxyamide 6 by aminolysis.
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Org. Lett., Vol. 12, No. 4, 2010
697

Alcohol 9 was phosphorylated with dibenzyl N,N-dieth-
ylphosphoramidite, followed by oxidation with t-BuOOH to
produce dibenzylphosphate 10.24,44 The diastereomers of 10
were isolated by reverse-phase semipreparative HPLC. The
ketone carbonyl for both diastereomers 10a and 10b appeared
in the IR, but not in the 13C NMR. The 84 ppm peak in the
Scheme 2. Synthesis of Inhibitors 1a and 1b
1
13C and the 4.74 ppm peak in the H NMR were assigned
by HMBC as the hydrated ketone (Supporting Information).
The ketone carbonyl stretch appeared in the IR at 1723 cm-1,
but much weaker than expected, indicating partial hydration.
Hydrogenolysis of each diastereomer of 10 under neutral
conditions furnished two diastereomers of the final com-
pound, 1a and 1b. Compounds 1a and 1b showed no ketone
carbonyl in both the 13C and IR spectra. Since we found
that both diastereomers were weak Pin1 inhibitors, vide infra,
we chose not to determine the absolute stereochemistry of
the inhibitors.
In our method, orthothioester 5 was directly reacted with
the fully formed right half of the molecule, the amine H-Pro-
NEA 3, as the nucleophile to produce amide 6 (Scheme 2).
Orthothioester 5 was used as a carboxylic acid synthon
instead of a nitrile to avoid the toxicity of NaCN or KCN.
In addition, conditions for the hydrolysis of a nitrile are
incompatible with amides.29
This synthesis of the target compound was convergent
compared with previous linear syntheses of R-ketoamides
via R-hydroxy acids. Previously, orthothioesters were either
hydrolyzed to a carboxylic acid and coupled with an amine
or methanolyzed to a methyl ester.39,45,46 Methyl esters
require the additional step of hydrolysis before coupling,
while R-hydroxy acids are difficult to isolate and purify.
Instead, the H-Pro-NEA amine was coupled in one step to
the R-hydroxyorthothioester. We attempted ozonolysis of a
cyano ylide to form the R-ketoamide functionality using
Wasserman’s method.9 However, due to the instability of
the R-ketoamide in subsequent Pro ester hydrolysis and
coupling to NEA, we chose to use aminolysis of thioester 5
with amine 3 to form the complete skeleton prior to oxidation
to the ketoamide.
The aminolysis was catalyzed by HgO and HgCl2, similar
to hydrolysis of orthothioesters.39,45 Thus, the R-hydroxy-
amide 6 was formed without additional coupling reagents
or steps. We propose that the key step in the reaction involves
Hg(II)-catalyzed hydrolysis of the orthothioester to a methyl
thioester intermediate 11 (Scheme 3).45,47 The THF used as
the solvent was wet, which provided the water for the
hydrolysis. Aminolysis with the amine nucleophile present
in situ then gave amide 6 directly. Thus, hydrolysis and
coupling were combined in a single step with 80% yield.
Although the overall yield in this 12-step synthesis is not
high (6% for the 9 linear steps), the aminolysis to form the
The Boc group of 6 was removed with TFA, and the
resulting amine was selectively acetylated with 1 equiv of
acetic anhydride in the presence of the free hydroxyl to give
7 (Scheme 2). Alcohol 7 was oxidized with the Dess-Martin
reagent to form R-ketoamide 8.6,33 Although the Dess-Martin
oxidation is known to produce the least epimerization alpha
to electrophilic ketones,6,40,41 epimerization occurred either
during oxidation or chromatography, but the diastereomers
(1:1) were not separated at this stage. This epimerization
occurs easily due to the strongly electron-withdrawing
ketoamide group and has been observed in several syntheses
of R-ketoamides.15,29 Epimerization of R-ketoesters has been
observed to occur in solution upon standing for one week.41
Both phospho-L-Thr and phospho-D-Thr containing inhibitors
of Pin1 have been reported, and the D-Thr inhibitors were
more potent.36,37 We wanted to test both diastereomers of 1
for inhibition, so we did not attempt to improve the
stereochemical purity of R-ketoamide 8.
The benzyl on the side chain of serine was removed with
BCl3 to give alcohol 9.42,43 Reverse-phase, semipreparative
HPLC purification gave a mixture of diastereomers of 9 in
a 1:1 ratio as determined by 1H NMR (Supporting Informa-
tion). The 86 ppm 13C NMR peak was assigned as the
hydrated ketone carbon based on the literature and the
assignment below for 10a.1 The ketone carbonyl stretch did
not appear in the IR at ca. 1720 cm-1, which also indicates
hydration of the ketone.
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Org. Lett., Vol. 12, No. 4, 2010

mediate mechanism.50 The hemiketal of an R-ketoamide has
been shown to act as a tetrahedral transition-state analogue
for a serine protease.16 We anticipated that the ketoamide
could act as a potent electrophile if the Cys-113 thiol of Pin1
was positioned for nucleophilic addition. The twisted amide
mechanism is not supported by this poor inhibition either.
Although a hydrated ketone would preclude either Cys-113
nucleophilic addition or the orthogonal carbonyl conforma-
tion necessary to mimic the twisted amide transition state,
as in FK506, the hydration is reversible. The ketone state
should be accessible for binding to the enzyme if it were a
favorable analogue of the transition state.
Scheme 3. Proposed Mechanism for Hydroxyamide 6 Formation
key intermediate for R-ketoamides should be very useful in
a variety of syntheses.
Although the two diastereomers of ketoamides were weak
inhibitors of Pin1, we have provided a convergent method
for synthesizing R-ketoamide peptidomimetics, which is a
very useful class of enzyme inhibitors, especially for
proteases. Many R-ketoamides have been developed as drugs,
and our convergent method contributes to the available
methods for their synthesis.
The R-chymotrypsin protease-coupled assay, with succi-
nyl-Ala-Glu-cis-Pro-Phe-p-nitroanilide (pNA) as the sub-
strate, was used to measure inhibition of Pin1 by the two
diastereomers of 1.24 The absorbance of pNA released from
the PPIase trans product by chymotrypsin was recorded
continuously at 390 nm by UV/vis. The IC50 values of the
two diastereomers were determined to be 100 ( 20 µM and
200 ( 20 µM.
Surprisingly, neither diastereomer was a potent inhibitor
of Pin1. These inhibitors were less potent than a similarly
substituted, ground-state analogue, Fmoc-pSer-Ψ[(Z)CHdC]-
Pro-(2)-N-(3)-ethylaminoindole (IC50 ) 28 µM).25 Since the
R-ketoamide of FK506 acts as a transition state analogue to
inhibit FKBPs,5 we expected more potent inhibition of Pin1
by at least one of the R-ketoamide stereoisomers. There are
two proposed mechanisms for Pin1 catalysis: the twisted-
amide mechanism5,48,49 and the Cys-113 tetrahedral inter-
Acknowledgment. We thank the NIH for Grant No. R01
CA110940 for financial support.
Supporting Information Available: Experimental pro-
cedures, HPLC, and spectroscopic data for compounds 1-10
and inhibition data for compound 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL9027013
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