Elative rates of Michael reactions of 2′-(phenethyl)thiol with vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides relevant to vinyl sulfonyl cysteine protease inhibitors

Article abstract of DOI:10.1021/ol034555l

(Matrix presented) The relative rates of Michael additions of 2′-(phenethyl)thiol to representative vinyl sulfonyl Michael acceptors were measured. The dependence of the reactivity of the Michael acceptor on the nature of the sulfonyl R substituent was determined in order to evaluate the effect of these substituents on the inactivation kinetics of comparably substituted vinyl sulfonyl cysteine protease inhibitors. The rates of these Michael additions vary over 3 orders of magnitude, with phenyl vinyl sulfonate esters (R = OPh) being ca. 3000-fold more reactive than N-benzyl vinyl sulfonamides (R = NHBn).

Full text of DOI:10.1021/ol034555l

ORGANIC
LETTERS
2003
Vol. 5, No. 11
1967-1970
Relative Rates of Michael Reactions of
2′-(Phenethyl)thiol with Vinyl Sulfones,
Vinyl Sulfonate Esters, and Vinyl
Sulfonamides Relevant to Vinyl Sulfonyl
Cysteine Protease Inhibitors
Jason J. Reddick, Jianming Cheng, and William R. Roush*
Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1055
roush@umich.edu
Received March 31, 2003
ABSTRACT
The relative rates of Michael additions of 2′-(phenethyl)thiol to representative vinyl sulfonyl Michael acceptors were measured. The dependence
of the reactivity of the Michael acceptor on the nature of the sulfonyl R substituent was determined in order to evaluate the effect of these
substituents on the inactivation kinetics of comparably substituted vinyl sulfonyl cysteine protease inhibitors. The rates of these Michael
additions vary over 3 orders of magnitude, with phenyl vinyl sulfonate esters (R ) OPh) being ca. 3000-fold more reactive than N-benzyl vinyl
sulfonamides (R ) NHBn).
Cysteine proteases are essential to the life cycles of the
parasites that cause malaria, Chagas’ disease, and leishma-
niasis, making them attractive targets for the treatment of
these diseases.1-3 Numerous groups have made important
contributions toward the development of cysteine protease
inhibitors, and several classes of potent, irreversible inhibitors
are now available (including those based on Michael accep-
tors that target the active site cysteine residue).4,5 Following
up on leads provided by the work of Hanzlik and Palmer,6,7
our laboratory has developed potent irreversible vinyl sul-
fonyl inhibitors of the cysteine proteases associated with
several parasites.8-10 The active site cysteine reacts with the
vinyl sulfonyl unit of the inhibitors by conjugate addition,11
leading to irreversible binding of the inhibitor in the active
(5) Alvarez-Hernandez, A.; Roush, W. R. Curr. Opin. Chem. Biol. 2002,
6, 459.
(6) Liu, S.; Hanzlik, R. P. J. Med. Chem. 1992, 35, 1067.
(7) Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bro¨mme, D. J. Med. Chem.
1995, 38, 3193.
(8) Roush, W. R.; Gwaltney, S. L.; Cheng, J.; Scheidt, K. A.; McKerrow,
J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994.
(9) Roush, W. R.; Cheng, J.; Knapp-Reed, B.; Alvarez-Hernandez, A.;
McKerrow, J. H.; Hansell, E.; Engel, J. C. Bioorg. Med. Chem. Lett. 2001,
11, 2759.
(1) McKerrow, J.; Sun, E.; Rosenthal, P.; Bouvier, J. Annu. ReV.
Microbiol. 1993, 47, 821.
(2) McKerrow, J. H. In PerspectiVes in Drug DiscoVery and Design;
Anderson, P. S., Kenyon, G. L., Marshall, G. R., Eds.; ESCOM Science
Publishers: Leiden, 1994; Vol. 2, p 437.
(3) Robertson, C. D.; Coombs, G. H.; North, M. J.; Mottram, J. C. In
PerspectiVes in Drug DiscoVery and Design; Anderson, P. S., Kenyon, G.
L., Marshall, G. R., Eds.; ESCOM Science Publishers: Leiden, 1996; Vol.
6, p 99.
(10) Shenai, B. R.; Lee, B. J.; Alvarez-Hernandez, A.; Chong, P. Y.;
Emal, C. D.; Neitz, R. J.; Roush, W. R.; Rosenthal, P. J. Antimicrob. Agents
Chemother. 2003, 47, 154.
(4) Powers, J. X.; Asgian, J. L.; Ikici, O¨ . D.; James, K. E. Chem. ReV.
2002, 102, 4639.
(11) Simpkins, N. S. Tetrahedron 1990, 46, 6951.
10.1021/ol034555l CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/01/2003

Scheme 1. Synthesis of Michael Addition Substrates
Figure 1. Michael acceptors employed in this study.
site.12 This mechanism was unambiguously demonstrated for
cruzain, the major cysteine protease associated with Trypa-
nosoma cruzi (Chagas’ disease), by three-dimensional struc-
tures of several enzyme-inhibitor complexes solved by
X-ray crystallography, which show the active site Cys-25
covalently bound in 1,4-fashion with respect to the vinyl
sulfonyl group of the inhibitor.12
Several vinyl sulfonyl inhibitors exhibit strikingly potent
inhibition kinetics against cruzain, while also demonstrating
potent in ViVo activities against T. cruzi.8,9 In fact, one vinyl
sulfone inhibitor targeting cruzain is capable of curing mice
infected by T. cruzi.13 A continuing goal of our collaboration
with the McKerrow and Rosenthal groups at UCSF is to
develop viable therapeutics for Chagas’ disease and malaria
by designing improved inhibitors that have better bioavail-
ability, lower host toxicity, and increased selectivity for the
parasites.
To understand the structural effects that give rise to very
potent inhibition of cruzain and related cysteine protases, it
is necessary to separate the binding constant (Ki) of the
inhibitor scaffold and rate constant of the irreversible
chemical step of inhibition (kinact) of various vinyl sulfonyl
derivatives. However, very fast inhibition rates for many of
our vinyl sulfonyl cysteine protease inhibitors have precluded
determination of kinact/Ki values; the majority of inhibition
of 2-hydroxyethanethiol to N-methyl-N-phenylvinylsulfona-
mide have also been reported.15 However, we are not aware
of any kinetic evaluation of the full array of vinyl sulfonyl
units reported in the cysteine protease inhibition literature.
An analysis of the Michael reactivity of the vinyl sulfonyl
derivatives should allow us to anticipate the magnitude of
kinact in the design of future generations of cysteine protease
inhibitors. We also expect that the data reported here may
be useful in situations where the Michael acceptor reactivities
of different vinyl sulfonyl functional groups can be exploited
in organic synthesis.11,17
The vinyl sulfonyl derivatives used in this study were
synthesized as summarized in Scheme 1, using chemistry
patterned after our syntheses of vinyl sulfonyl cysteine
protease inhibitors.8-10 Enone, enoate, and vinyl sulfonyl
groups were installed by stabilized Wittig and Horner-
Wadsworth-Emmons reactions to furnish compounds 2, 3,
4, and 7. The phenyl sulfonate ester 1 and the sulfonamides
5, 6, 8, and 9 were installed via base-promoted coupling of
phenol or the appropriate amine with the sulfonyl chloride
11.
constants from our previous work have been reported as kassoc
14
values,8,9 from which it is impossible to extract Ki and kinact
.
To assess the intrinsic reactivities of the Michael acceptors,
which presumably contribute to kinact for the enzyme inhibi-
tion reaction, we have carried out pseudo-first-order kinetic
analyses of Michael additions of 2′-(phenethyl)thiol to
inhibitor analogues 1-9 (Figure 1).
Mechanistic studies of base-catalyzed conjugate additions
of alcohols15 and the uncatalyzed addition of amines16 to
vinyl sulfones and vinyl sulfonamides have previously been
reported. The kinetics of the base-catalyzed Michael addition
(12) Brinen, L. S.; Hansell, E.; Cheng, J.; Roush, W. R.; McKerrow, J.
H.; Fletterick, R. J. Structure 2000, 8, 831.
(13) Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp. Med.
1998, 188, 725.
(14) Bieth, J. G. Methods Enzymol. 1995, 248, 59.
(15) Davies, W. G.; Hardisty, E. W.; Nevell, T. P.; Peters, R. H. J. Chem.
Soc. B 1970, 998.
Relative rates of base-promoted Michael additions of 2′-
phenethylthiol to acceptors 1-9 were measured by 1H NMR
(16) Davies, W. G.; Hardisty, E. W.; Nevell, T. P.; Peters, R. H. J. Chem.
Soc. B 1970, 1004.
(17) Morris, J.; Wishka, D. G. J. Org. Chem. 1991, 56, 3549.
Org. Lett., Vol. 5, No. 11, 2003
1968

Table 1. (A) Relative Pseudo-First-Order Rate Constants of Michael Addition of 2′-(Phenethyl) Thiol to Various Michael Acceptors.
(B) Relative Apparent Second-Order Rate Constants for Inhibition of Cruzain by Inhibitors 12
a Relative pseudo-first-order rate constant. b Apparent second-order rate constants of inhibition of cruzain for known inhibitors.8,9 c Data for the appropriate
inhibitor 12 with R ) SO2NHBn (Bn ) CH2Ph) are not available; indicated data are for the N-phenyl vinyl sulfonamide (R ) SO2NHPh).9
spectroscopy (Table 1A). 2′-(Phenethyl)thiol and triethyl-
amine served as the most convenient model for the practical
time scale. The reaction solvent was methanol-d4, and
CH2Cl2 was used as an internal integration standard. Reaction
rates were measured by integration of a vinylic proton signal
(δ ) 6.7 ppm) of the starting material against the CH2Cl2
internal standard. A large excess (10 equiv.) of 2′-(phenethyl)-
thiol was employed to ensure pseuo-first-order kinetics for
consumption of the Michael acceptor.
The data in Table 1A demonstrate that the pseudo-first-
order rates of Michael addition to this class of compounds
vary by up to 3 orders of magnitude as a function of the
nature of the sulfonyl unit. Vinyl sulfonamide 9 was the least
reactive substrate, while the phenyl vinyl sulfonate ester 1
underwent conjugate addition at a rate ca. 3000-fold higher.
We also tested the N-methylsulfonamides 5 and 8 since we
were concerned that triethylamine would deprotonate the
sulfonamide units of 6 and 9, artificially depressing the
apparent conjugate addition rates. The data for compound
pairs 5/6 and 8/9 show that this effect depresses the rates by
a factor of 3. We have also included data for enone 2 and
enoate 7 to establish the reactivities of vinyl sulfonyl
compounds compared to conventional enoyl Michael accep-
tors.
electrophilicity of the enoyl â-carbon, which tracks the
energy of the transition state for formation of the initial
enolate intermediate upon addition of the nucleophile to the
Michael acceptor. The relative energies of the enolate species
is highly dependent on the inductive and/or resonance effects
of the substituents bonded to the carbonyl carbon.19,20
However, the situation differs for conjugate addition
reactions of vinyl sulfonyl acceptors. As these compounds
undergo conjugate addition, the reactive intermediate is an
R-carbanion, which is stabilized by the sulfur atom of the
sulfonyl unit. The origin of this R-stabilization has been
attributed to the polarizability of the sulfur atom and by σ
effects of antibonding orbitals on the sulfur atom.21-24 In
contrast to enoyl Michael acceptors, electronic effects arising
from the linking oxygen or nitrogen atoms of the sulfonate
ester and sulfonamide groups are exclusively inductive, since
poor sp3/d orbital overlap between oxygen or nitrogen with
sulfur precludes an electron-donating resonance effect.
Therefore, strong σ-electron-withdrawing inductive effects
would be expected to stabilize R-carbanion formation.
Indeed, sulfonate ester 1 is a highly reactive Michael
acceptor, comparable to that of conventional enone electro-
In polar protic solvents, the triethylamine-catalyzed ad-
dition of thiols to enoyl compounds involves formation of
an enolate ion intermediate, followed by protonation by the
triethylammonium conjugate acid.18 The relative rates of
Michael addition to R,â-unsaturated esters, amides, and
ketones are readily understood by consideration of the
(19) Bernardi, F.; Bottoni, A.; Rossi, I.; Robb, M. A. J. Mol. Struct.
1993, 300, 157.
(20) Rosenberg, R. E. J. Org. Chem. 1998, 63, 5562.
(21) Bernardi, F.; Csizmadia, I. G.; Mangini, A.; Schlegel, H. B.;
Whangbo, M.-H.; Wolfe, S. J. Am. Chem. Soc. 1975, 97, 2209.
(22) Bernardi, F.; Bottoni, A.; Venturini, A.; Mangini, A. J. Am. Chem.
Soc. 1986, 108, 8171.
(23) Borden, W. T.; Davidson, E. R.; Anderson, N. H.; Denniston, A.
D.; Epiotis, N. D. J. Am. Chem. Soc. 1978, 100, 1604.
(24) Lehn, J.-M.; Wipff, G. J. Am. Chem. Soc. 1976, 98, 7498.
(18) Hiemstra, H.; Wynberg, H. J. Am. Chem. Soc. 1981, 103, 417.
Org. Lett., Vol. 5, No. 11, 2003
1969

philes (see 2). Our results also show that the vinyl sulfona-
mides 5, 6, 8, and 9 are substantially less reactive Michael
acceptors than the sulfonate ester 1 and have absolute
reactivity comparable to that of R,â-unsaturated ester 7. Since
the NH of sulfonamide 9 is reasonably acidic (pKa of phenyl
sulfonamide ) 10.1 and pKa of triethylammonium ion )
11.0),25 9 is likely in equilibrium with the anionic N-
deprotonated form, thereby suppressing the rate of Michael
addition by destabilizing the corresponding R-carbanion
product of the initial Michael addition step. This effect should
be absent in the N-methyl vinyl sulfonamide 8, which still
shows substantially lower reactivity as a Michael acceptor
than the vinyl sulfonate esters. This may be attributed to the
lower electronegativity of nitrogen relative to oxygen.26 An
identical effect is seen in the N-alkoxy sulfonamides 5 and
6, which have higher Michael reactivities than 8 and 9,
possibly due to an additive electron-withdrawing effect of
the N-alkoxy group. However, it is unclear at present why
the vinyl sulfonamides 8 and 9 are so much less reactive
than the phenyl vinyl sulfone 3. On the basis of the inductive
effect arguments (O > N > C), we would have expected
that 3 would be the least reactive substrate in this series.26
Normalized second-order rate constants of inactivation of
cruzain for a known set of inhibitors containing the vinyl
sulfonyl substituents examined in this study are given in
Table 1B. The data summarized in Table 1B clearly indicate
that changes in the inhibitor P3 substituent, distal to the site
of the enzymatic Michael addition, often result in a dramatic
change in the inhibition kinetics. This points toward the
fundamentally important role of enzyme binding (Ki) in the
inhibition event. In addition, changes in the P1′ substituent
of the inhibitor (“R” in structure 12) may also affect enzyme
binding and ultimately also the absolute potency as an
enzyme inhibitor (best approximated as kinact/Ki values). We
hope that kinact values for our highly potent inhibitors can be
obtained by using stopped-flow or rapid-quench kinetic
techniques.
Whether the relative reactivity profile of the various vinyl
sulfonyl derivatives examined in this study ultimately cor-
relates with kinact values for enzyme inhibition remains to be
determined. Nevertheless, we expect that the reactivity effects
demonstrated here will be useful as a general preliminary
gauge of Michael reactivity in the design of new cysteine
protease inhibitors containing the vinyl sulfonyl functional
groups.
At the outset of these investigations, we had hoped that it
might be possible to correlate the relative Michael reactivity
of 1-9 with the kinact values for analogously substituted
inhibitors 12 against the cysteine protease cruzain. However,
we have not been able to obtain kinact data for most of our
inhibitors;8-10 instead, we have second-order kassoc values.14
Acknowledgment. We thank the National Institutes of
Health (Program Project Grant No. AI 35707) for generous
financial support of this work.
Supporting Information Available: Details of the kinet-
ics measurements and procedures for synthesis of 1-9 are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
(25) Gordon, A. J.; Ford, R. A. The Chemist’s Companion: A Handbook
of Practical Data, Techniques, and References; John Wiley & Sons: New
York, 1972.
(26) Ceppi, E.; Eckhardt, W.; Grob, C. A. Tetrahedron Lett. 1973, 14,
3627.
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