Dipeptidyl α-fluorovinyl Michael acceptors: Synthesis and activity against cysteine proteases

Article abstract of DOI:10.1016/j.bmcl.2007.09.075

The synthesis of novel dipeptidyl α-fluorovinyl sulfones using a Horner-Wadsworth-Emmons approach on N-Boc-l-phenylalaninal is described. Inhibitory assays against a Leishmania mexicana cysteine protease (CPB2.8ΔCTE) revealed low biological activity. Relative rates of Michael additions of 2′-(phenethyl)thiol with vinyl sulfone and α-fluorovinyl sulfone were determined, and ab initio calculations on several Michael acceptor model structures were performed; both were in agreement with the biological testing results.

Full text of DOI:10.1016/j.bmcl.2007.09.075

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6563–6566
Dipeptidyl a-fluorovinyl Michael acceptors: Synthesis
and activity against cysteine proteases
Koen Steert,a Ibrahim El-Sayed,a Pieter Van der Veken,a Alisa Krishtal,b
Christian Van Alsenoy,b Gareth D. Westrop,c Jeremy C. Mottram,d Graham H. Coombs,c
Koen Augustynsa and Achiel Haemersa,*
aLaboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, BE-2610 Antwerp, Belgium
bDepartment of Chemistry, University of Antwerp, Universiteitsplein 1, BE-2610 Antwerp, Belgium
cDivision of Infection & Immunity, Institute of Biomedical and Life Sciences,
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK
dWellcome Centre for Molecular Parasitology and Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, UK
Received 23 August 2007; revised 21 September 2007; accepted 23 September 2007
Available online 23 October 2007
Abstract—The synthesis of novel dipeptidyl a-fluorovinyl sulfones using a Horner–Wadsworth–Emmons approach on N-Boc-L-
phenylalaninal is described. Inhibitory assays against a Leishmania mexicana cysteine protease (CPB2.8DCTE) revealed low biolog-
ical activity. Relative rates of Michael additions of 20-(phenethyl)thiol with vinyl sulfone and a-fluorovinyl sulfone were determined,
and ab initio calculations on several Michael acceptor model structures were performed; both were in agreement with the biological
testing results.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cysteine proteases are peptide bond-cleaving enzymes.
They make use of a nucleophilic thiol group that attacks
the peptide carbonyl group, giving rise to the free amine
and a peptidyl thioester—which after hydrolysis affords
the carboxylic acid part. They have been divided into
several clans, which are subdivided into several families
(http://merops.sanger.ac.uk/). Cysteine proteases are
very widespread in nature and hold cornerstone posi-
tions in the metabolism of both eukaryotic and prokary-
otic organisms. Several inhibitors are involved in drug
development programmes targeting human proteases
for diseases such as osteoporosis, arterial thrombosis,
rheumatoid arthritis, tumour invasion and metastasis
and Alzheimer’s disease.1,2 Cysteine proteases are also
considered as vital for protozoa such as Plasmodium,
Trypanosome and Leishmania and inhibitors of falcipain
and cruzain, two major parasitic cysteine proteases, are
under investigation as possible treatment of malaria and
Chagas’ disease.3
Most of these inhibitors are derived from the corre-
sponding peptide substrate of the target enzyme. They
are modified at the P1 position where the amide group
is substituted for a so-called warhead: an electrophilic
group with high affinity for the active site thiol group.
Frequently used warheads include ketones and nitriles
affording reversible tight binding inhibitors or epoxides
and Michael acceptors capable of reacting covalently
with the thiolate affording irreversible inhibitors.1
One of the most interesting Michael acceptor groups are
peptide vinyl sulfones and their analogues such as vinyl
sulfonamides and vinyl sulfonate esters.4 Vinyl sulfones
selectively inhibit several cysteine proteases in a low nM
range. Compounds such as 1 inhibit falcipains and cruz-
ain and are important tools in antiprotozoal drug design
and development (Fig. 1).5–7
Very few Michael acceptor inhibitors with substitutions
on the a-vinyl carbon have been reported. An a-acet-
oxymethyl group (Fig. 2) affords compounds showing
activity in an in vitro malaria model (IC50 = 10 nM). A
double addition–elimination mechanism of action is
claimed but detailed kinetic analysis of enzyme inhibi-
tion to verify this claim is not reported.8
Keywords: Cysteine proteases; a-Fluorovinyl Michael acceptors;
Inhibitors.
*
Corresponding author. Tel.: +32 38202717; fax: +32 38202739;
e-mail: achiel.haemers@ua.ac.be
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.075

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K. Steert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6563–6566
the commercially available diethyl [(phenylthio)-
methyl]phosphonate to the corresponding sulfone 5
afforded the HWE reagent. Reaction with N-tert-butox-
ycarbonyl-phenylalaninal13 4 yielded vinyl sulfone inter-
mediate 7.14,15 It has to be noted that only the E-isomer
1
of 7 was isolated (yield 52%) as revealed by H NMR
analysis (3JH = 15.2 Hz). After coupling the deprotected
vinyl sulfone 9 to carboxylic acid 11,16 reference target
compound 2 was obtained in an overall yield of 48%.
The fluorophenyl vinyl sulfone 3 was prepared (Scheme
1) by a WHE reaction of N-tert-butoxycarbonyl-phe-
nylalaninal 4 with the anion of diethyl 1-fluoro-1-(phen-
ylsulfonyl)methanephosphonate 6. The latter was
generated from fluoromethyl phenyl sulfone17 and
diethyl chlorophosphate using LiHMDS. This resulted
in both (E)- and (Z)-isomers 8.1 and 8.2, which were
separated on a small scale. Olefin geometries were as-
signed using coupling constant (3JHF = 32 Hz and
20.2 Hz) and the isomeric ratio was found to be approx-
imately 1:1. Racemisation was minimized by using reac-
tion times of 2 h and isolating the diethyl 1-fluoro-1-
(phenylsulfonyl)methanephosphonate 6 instead of the
literature-described in situ generation, thus avoiding
the use of two equivalents of strong base in the reaction
mixture.18 Coupling of carboxylic acid 11 with the
deprotected fluorovinyl sulfone 10 provided the (E)-
and (Z)-isomers 3.1 and 3.2 of our target compound.
Figure 1. Fluorinated and non-fluorinated vinyl sulfones cysteine
protease inhibitors.
Figure 2. Different substitutions on the a-carbon of Michael acceptors.
Vinyl sultams (Fig. 2) as conformationally restricted
analogues of vinyl sulfonamides were not active as
inhibitors of papain and demonstrated weak activity
against recombinant falcipain-2.9 Our interest in flu-
oro-olefins10 prompted us to evaluate the influence of
an a-fluorine in a Michael acceptor type cysteine prote-
ase inhibitor. In the thermodynamically favoured conju-
gate addition, the proposed mechanism involves the
rate-limiting reaction of a Michael acceptor olefin with
a rapidly formed complex between thiol and tertiary
base.11 The reactivity of the addition might indeed be
enhanced if the electrophilicity of the b-carbon of the
Michael acceptor is increased. The introduction of an
inductively electron withdrawing group on the a-carbon
such as fluorine will increase the electron deficiency at
Ca, but no prognosis can be made on how strong will
be the effect on Cb. On the other hand, the lone pairs
of fluorine might participate and show overlap with
the existing conjugate p-system, in this way contributing
as a mesomeric donor.
We used three approaches in order to obtain informa-
tion on the influence of a-fluorine substitution in a Mi-
chael addition substrate cysteine protease inhibitor: (i)
an in vitro chemical approach with a simplified amino
acid-derived fluoro-olefin 8; (ii) a theoretical approach
calculating the atomic charge distribution; and (iii) an
enzymatic evaluation of the peptide-derived fluoro-ole-
fin inhibitor 3 as a CPB inhibitor.
Inspired by the work of Reddick et al.,15 relative rates of
Michael additions of 20-(phenethyl)thiol with vinyl sul-
fone 7 and a-fluorovinyl sulfones 8 were determined
(Scheme 2).
We used the vinyl phenylsulfone acceptor system and
prepared reference compound 2 and its a-fluoro deriva-
tive 3. We found compound 2 to be a potent inhibitor of
the Leishmania cysteine protease CPB.12
The reactivity of these Michael acceptors towards nucle-
ophiles such as thiolates, serving as a model for the
Inhibitor 2 was synthesized (Scheme 1) using a Horner–
Wadsworth–Emmons (HWE) reaction. Oxidation of
Scheme 1. Synthesis of vinyl phenylsulfone 2 and phenyl a-fluorovinyl phenylsulfone 3. Reagents and conditions: (a) LiHMDS, THF, ꢀ78 ꢁC
(if R2 = F) or NaH, THF, 0 ꢁC (if R2 = H); (b) TFA, CH2Cl2 (1:1); (c) TBTU, NEt3, DMF.

K. Steert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6563–6566
6565
at the B3LYP/6-311++G** level of theory. Subse-
quently, the atomic charges of the atoms in the mole-
cules were calculated using the Hirshfeld method,20 as
implemented in the program STOCK.21 The atomic
charges of the backbones of the different molecules are
presented in Table 1.
A general trend in the atomic charge distribution of
the backbone is present. In the non-fluorinated com-
pounds, an alternating pattern of the atomic charges
is established, indicating the presence of a mesomeric
system in those a, b-unsaturated molecules. The car-
bonyl carbon in the aldehyde, acid and ester mole-
cules, as well as the sulfur atom in the sulfone
molecule, are positively charged, causing a further
polarization of the double bond between the a- and
the b-carbon. This mesomeric effect creates a slightly
positive charge on the b-carbon, making it suitable
for a nucleophilic attack. Although the values for
the charges on the b-carbon, presented in Table 1,
may seem rather small (0.01 a.u.), one must consider
the fact that the calculations are performed in the
gas phase, as solvation effects are difficult to take
explicitly into account. The effect of the mesomeric
system is expected to be larger in solution and a basic
environment, where the charge separation is further
stabilized by the solute molecules and the mesomeric
effect is strengthened by the presence of ionic mole-
cules (e.g. acid and base).
Scheme 2. Conjugate addition of thiolates to vinyl phenylsulfones.
active site cysteine residue, allows the evaluation of the
electronic modifications due to the presence of a fluorine
atom. Compared to the unsubstituted olefin geometry,
the steric differences due to the presence of the fluorine
atom will be limited because of its very small van der
˚
Waals radius (1.35 A).
Relative reaction rates of base-promoted Michael addi-
tions of 20-(phenethyl)thiol to acceptors 7 and 8 were
measured using 1H NMR spectroscopy. The integration
of the vinylic proton signal indicates the presence of
starting product, as a function of time several 1H
NMR spectra were taken in order to see the amount
of vinyl sulfone consumed.
Fluorovinyl sulfone compounds 8.1 and 8.2 underwent
no reaction in the given time. The vinylic protons
of 8 (d(E) = 6.1 ppm and d(Z) = 5.8 ppm) remained un-
changed during 1 h at room temperature. No difference
in reactivity was observed in the (E)- and (Z)-isomers.
The reference vinyl sulfone 7 however reacted to com-
pletion within 45 min; the identity of the addition prod-
uct was confirmed by MS. The addition reactions were
repeated afterwards and followed by TLC, starting
product fluorovinyl sulfone 8 could still be detected after
48 h.
Introducing a fluorine atom at the a-carbon results in a
surprising effect: the positive charge at the a-carbon im-
plies that the fluorine atom influences the charge density
by means of a field effect, rather than a mesomeric
donating effect. However, this field effect does not
extend further to the b-carbon. The bond between the
a-carbon and the b-carbon is further polarized, creating
a partial negative charge on the latter. As a result, the b-
carbon is no longer a suitable place for a nucleophilic at-
tack. This additional negative charge on the b-carbon
does not have an effect on the charge of the c-carbon.
Instead, the hydrogen atom on the b-carbon becomes
more positive.
In conclusion, a lower reactivity of the Michael acceptor
moiety towards thiolate nucleophiles is observed when
an a-fluorine is introduced. The problem urges a theo-
retical chemistry approach using ab initio calculations
to obtain charge densities on different atoms.
The negative charge on the b-carbon could also be ex-
plained by a mesomeric effect.22 In one of the possible
mesomeric forms, a double bond is formed between the
The effect of the a-fluorination of Michael substrates
was studied by ab initio methods. As model systems,
four different Michael acceptor groups were chosen:
vinyl aldehyde, vinyl acid, vinyl ester and vinyl sulfone
and their a-fluorinated derivatives (Fig. 3). The struc-
tures were optimized using the Gaussian03 program19
Table 1. The atomic charges (in a.u.) of the backbones of the studied
compounds
Figure 3. Michael acceptor model systems.

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K. Steert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6563–6566
Table 2. Inhibition of L. mexicana cysteine protease CPB2.8DCTE
with non-fluorinated (2) and fluorinated (3) Michael acceptor
inhibitors
References and notes
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*Mean standard deviation from three reactions.
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charge on the b-carbon. However, when bond lengths
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hydrogen atom by a fluorine atom, the changes being
smaller than 0.01 a.u., whereas a mesomeric effect
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a fluorine atom at the a-carbon.23 The total charge of
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Finally, the influence of an a-fluorine on the activity of
Michael acceptor cysteine protease inhibitors was deter-
mined using Leishmania CPB cysteine protease. Com-
pounds were tested as inhibitors of L. mexicana
cysteine protease CPB2.8DCTE, prepared and assayed
as described.24
Table 2 shows % inhibition of enzymatic activity at 20
and 2 lg/ml. Assays were carried out in 0.4 ml of
0.1 M sodium acetate, 2 mM EDTA, 1 mM DTT, pH
5.0. Enzyme (5 lg/ml) and inhibitor were added and
the mixture was incubated for 10 min at 30 ꢁC. The reac-
tion was started by addition of Z-FR-pNA (300 lM)
and the absorbance was monitored at 410 nm for
2 min at 30 ꢁC. The control reaction (no inhibitor) was
linear for > 2 min and the rate (change in absorbance
at 410 nm) was 1.29 · 10ꢀ3/s. Both isomers 3 were very
weak inhibitors. These results confirm the in vitro obser-
vations and the theoretical approach, where the intro-
duction of an a-fluorine in a Michael acceptor enzyme
inhibitor does not afford biologically interesting
compounds.
20. Hirshfeld, F. L. Theor. Chim. Acta 1977, 44, 129.
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Acknowledgment
24. Sanderson, S. J.; Pollock, K. G.; Hilley, J. D.; Meldal, M.;
Hilaire, P. S.; Juliano, M. A.; Juliano, L.; Mottram, J. C.;
Coombs, G. H. Biochem. J. 2000, 347(2), 383.
We are indebted to the Flemish Fund for Scientific Re-
search (FWO-Vlaanderen) for financial support.