Parallel solution-phase synthesis of mechanism-based cysteine protease inhibitors

Article abstract of DOI:10.1021/ol0166496

Figure presented A seven-step parallel solution-phase synthesis has been developed for access to ketone-containing mechanism-based cysteine protease inhibitors. The use of liquid-liquid extractions, volatile or solid-supported reagents, and resin-bound sc

Full text of DOI:10.1021/ol0166496

ORGANIC
LETTERS
2001
Vol. 3, No. 23
3707-3709
Parallel Solution-Phase Synthesis of
Mechanism-Based Cysteine Protease
Inhibitors
Alice Lee and Jonathan A. Ellman*
Center for New Directions in Organic Synthesis, Department of Chemistry,
UniVersity of California, Berkeley, California 94720
jellman@uclink4.berkeley.edu
Received August 25, 2001
ABSTRACT
A seven-step parallel solution-phase synthesis has been developed for access to ketone-containing mechanism-based cysteine protease
inhibitors. The use of liquid−liquid extractions, volatile or solid-supported reagents, and resin-bound scavengers eliminates the need for
intermediate column chromatographic purification during this synthesis sequence.
Cysteine proteases are important pharmaceutical targets
because of their role in the pathogenesis of many diseases.1
Characterized by a conserved cysteine residue in the active
site, this class of proteases includes the calpains,2 which have
been implicated in neurodegenerative disorders, cathepsin
K,3 which has been linked to osteoporosis, and the caspase
family of proteases,4 which are involved in programmed cell
death.
active site cysteine residue upon the amide carbonyl (Figure
1a). A common feature of virtually all cysteine protease
Cysteine proteases catalyze the hydrolysis of amide bonds
in peptides and proteins through nucleophilic attack of the
(1) (a) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000,
43, 305-341. (b) Otto, H.-H.; Schirmeister, T. Chem. ReV. 1997, 97, 133-
171.
Figure 1. (a) Peptide proteolysis via cysteine protease activity.
(b) Ketone-based cysteine protease inhibitors (X ) S, O, C, NH,
NR, O2C).
(2) Suzuki, K.; Sorimachi, H. FEBS Lett. 1998, 433, 1-4.
(3) Bossard, M. J.; Tomaszek, T. A.; Thompson, S. K.; Amegadzie, B.
Y.; Hanning, C. R.; Jones, C.; Kurdyla, J. T.; McNulty, D. E.; Drake, F.
H.; Gowen, M.; Levy, M. A. J. Biol. Chem. 1996, 271, 12517-12524.
(4) Thornberry, N. A. Chem. Biol. 1998, 5, R97-R103.
(5) See also the Symposium-in Print on cysteine protease inhibitors
featured in Bioorg. Med. Chem. 1999, 7, 571-644.
inhibitors is an electrophilic functionality that can react with
the nucleophilic cysteine residue.1,5 The display of function-
ality about the electrophilic ketone carbonyl has proven to
be a particularly powerful strategy for accessing both
reversible and irreversible inhibitors.6
(6) (a) Marquis, R. W.; Ru, Y.; LoCastro, S. M.; Zeng, J.; Yamashita,
D. S.; Oh, H.-J.; Erhard, K. F.; Davis, L. D.; Tomaszek, T. A.; Tew, D.;
Salyers, K.; Proksch, J.; Ward, K.; Smith, B.; Levy, M.; Cummings, M.
D.; Haltiwanger, R. C.; Trescher, G.; Wang, B.; Hemling, M. E.; Quinn,
C. J.; Cheng, H.-Y.; Lin, F.; Smith, W. W.; Janson, C. A.; Zhao, B.;
McQueney, M. S.; D′Alessio, K.; Lee, C.-P.; Marzulli, A.; Dodds, R. A.;
Blake, S.; Hwang, S.-M.; James, I. E.; Gress, C. J.; Bradley, B. R.; Lark,
M. W.; Gowen, M.; Veber, D. F. J. Med. Chem. 2001, 44, 1380-1395. (b)
Mjalli, A. M.; Chapman, K. T.; Zhao, J. J.; Thornberry, N. A.; Peterson, E.
P.; MacCoss, M. Bioorg. Med. Chem. Lett. 1995, 5, 1405-1408. (c) Krantz,
A.; Copp, L. J.; Coles, P. J.; Smith, R. A.; Heard, S. B. Biochemistry 1991,
30, 4678-4687.
We have previously reported a strategy for the solid-phase
synthesis of ketone-containing mechanism-based cysteine
protease inhibitors (Figure 1b) that allows for display of
diverse functionality on both sides of the ketone carbonyl.7
In this method, the P1 side chain8 is introduced as part of a
10.1021/ol0166496 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/19/2001

Scheme 1a
a (a) i. (S)-tert-butanesulfinamide, Ti(OEt)4, CH2Cl2; ii. 2:1 sand/Na2SO4‚10H2O; (b) i. vinyl Grignard/Et2O, CH2Cl2; ii. aqueous workup;
(c) i. 4 N HCl/dioxane, MeOH; ii. 1 M KOH; iii. sulfonic acid resin, CH2Cl2; iv. saturated NH3/MeOH; (d) acyl transfer resin (preloaded
with R2CO2H), CH2Cl2; (e) DMDO, acetone; (f) i. HSR3, P-TBD, THF, reflux, 6-12 h; ii. isocyanate resin, CH2Cl2, reflux, 12 h; (g) i.
Dess-Martin periodinane, CH2Cl2; ii. thiosulfate resin, tertiary amine resin.
chloromethyl ketone scaffold, which is prepared from the
corresponding amino acid. For this reason, the P1 side chains
that can readily be introduced are limited by the commercial
availability of the appropriate amino acids.
obtained in diastereomer ratios that range from 88:12 to 94:6
(Table 1). As previously observed for Grignard additions to
sulfinyl imines, ether is important for high diastereoselec-
tivities.9
Herein we report an alternative route to access ketone-
based inhibitors that enables the introduction of diverse
hydrocarbon functionality at the P1 position using readily
available aldehyde inputs (Scheme 1). First, the asymmetric
synthesis of allylic amines 4 is accomplished using the tert-
butanesulfinamide chiral auxiliary.9 The allylic amines 4 are
then further functionalized to provide the desired ketones 8.
The key feature of the synthesis sequence is the complete
reliance on volatile or solid-supported reagents and support-
bound scavengers,10 thereby eliminating the need for inter-
mediate column chromatographic purification during the
synthesis sequence.
The synthesis of the allylic amine 4 proceeds through the
initial condensation of tert-butanesulfinamide and aldehyde
1 in the presence of Ti(OEt)4 which serves as both a Lewis
acid and water scavenger. Titanium adducts are removed by
incubation of the reaction mixture with a finely crushed
mixture of 2:1 sand/Na2SO4‚10H2O in methylene chloride.
Simple filtration and solvent removal leads to the sulfinimine
product 2, which is used immediately in the next step.
Excess amounts of vinyl Grignard in diethyl ether are used
for 1,2-addition to sulfinimine 2 to provide sulfinamide 3.
Quenching with a solution of aqueous ammonium chloride
and brine followed by extraction of the reaction mixture
removes the excess Grignard reagent. Sulfinamide 3 is
Sulfinamide 3 is cleaved under acidic methanol conditions,
and the resultant ammonium salt is treated with aqueous
potassium hydroxide. The free amine 4 is scavenged onto
sulfonic acid resin. Rinsing of the resin removes Grignard
reaction side products that are not captured by the resin.
Elution with ammonia and methanol provides amine 4 in
approximately 50% yield for the three steps as calculated
1
by H NMR analysis using p-xylene calibration (Table 1).
Amide 5 is obtained in 80-100% yield by reaction of
allylic amine 4 with a carboxylic acid preloaded onto
hydroxynitrobenzophenone resin.11 Subsequent epoxidation
is carried out with dimethyldioxirane. Excesses of this
volatile reagent and the acetone byproduct are removed under
reduced pressure to afford high yields of epoxide 6 (Table
1
1) in excellent purity as determined by H NMR analysis.
Epoxide 6 can be opened with a variety of nucleophiles.12
For our inhibitor development efforts, we are most interested
in the mercaptomethyl ketone inhibitor class13 and therefore
have focused on thiol nucleophiles. Thiol addition to the
epoxide is accomplished using a support-bound guanidine
base (P-TBD)14 in refluxing THF. The excess thiol is
removed by subsequent scavenging with a resin-bound
isocyanate.15 Filtration of the resins and solvent removal
affords the alcohol 7. Unfortunately, introduction of impuri-
1
ties occurs during this step as observed by H NMR, and
these impurities are carried into the final step. A high-loading
tertiary amine base (PS-DIEA, Argonaut) and Amberlyst
(7) Lee, A.; Huang, L.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121,
9907-9914.
(8) Nomenclature for the substrate amino acid preference is Pn, ..., P2,
P1, P1′, P2′,..., Pm′. Amide bond hydrolysis occurs between P1 and P1′ (see
Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1968, 27, 157-
162).
(9) (a) Liu, G.; Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc. 1997,
119, 9913-9914. (b) Cogan, D. A.; Liu, G.; Kim, K.; Backes, B. J.; Ellman,
J. A. J. Am. Chem. Soc. 1998, 120, 8011-8019.
(10) For a recent review on solid-supported reagents and scavengers,
see: Ley, S. V.; Baxendale, I. R.; Ream, R. N.; Jackson, P. S.; Leach, A.
G.; Longbottom, D. A.; Nesi, M.; Scott, J. S.; Storer, R. I.; Taylor, S. J. J.
Chem. Soc., Perkin Trans. 1 2000, 3815-4195.
(11) Cohen, B. J.; Karoly-Hafeli, H.; Patchornik, A. J. Org. Chem. 1984,
49, 922-924.
(12) Rao, A. S.; Paknikar, S. K.; Kirtane, J. G. Tetrahedron 1983, 39,
2323-2367.
(13) Huang, L.; Lee, A.; Ellman, J. A. Submitted.
(14) (a) Iijima, K.; Fukuda, W.; Tomoi, M. J. Macromol. Sci., Pure Appl.
Chem. 1992, A29, 249. (b) Xu, W.; Mohan, R.; Morrissey, M. M.
Tetrahedron Lett. 1997, 38, 7337-7340.
(15) Booth, R. J.; Hodges, J. C. J. Am. Chem. Soc. 1997, 119, 4882-
4886.
3708
Org. Lett., Vol. 3, No. 23, 2001

Table 1. Yields of Intermediates (Scheme 1)
entry
R1
3, dr
4 (%)
R2
5 (%)
6 (%)
R3
8 (%)
1
2
3
4
5
6
7
CH(CH2)5
88:12
51
CH2CH2C6H5
CH2CH2CH3
C6H5
CH2CH2C6H5
C6H5
100
80
85
100
100
100
100
93
100
100
100
100
100
100
CH2CH2CO2CH2CH3
CH2CH2C6H5
CH2CH2C6H4-p-t-Bu
CH2CH2CO2CH2CH3
CH2CH2C6H5
30
60
33
56
33
42
31
CH2CH(CH2)5
CH2CH2C6H5
88:12
94:6
50
50
CH2CH2C6H5
CH2CH2CH3
CH2CH2CO2CH2CH3
CH2CH2C6H4-p-t-Bu
a Yield was based on p-xylene calibration in NMR analysis. b Yield over two steps (ring opening and oxidation) was calculated on the basis of isolated
pure product.
A-21 ion-exchange resin (Aldrich) gave lower yields, while
the polymer-bound BEMP (Aldrich) and macroporous tet-
raalkylammonium carbonate (Argonaut) gave comparable
yields and purity to that observed with P-TBD. In addition,
the presence of diastereomers complicates the NMR analysis
of the alcohol product, preventing accurate yield determina-
tions for this step by p-xylene calibration.
Oxidation of alcohol 7 to the desired mercaptomethyl
ketone product 8 is accomplished using Dess-Martin pe-
riodinane in reagent grade methylene chloride.16 The oxidant
and byproducts are then scavenged using resin-bound thio-
sulfate and tertiary amine resin.17 Column chromatography
of the final product 8 furnishes pure ketone in moderate yield
(30-60%) over the final two steps (Table 1). Chiral HPLC
analyses of the products indicate that no epimerization occurs
during the synthesis sequence within the limits of detection.18
A parallel synthesis strategy has been developed in the
solution phase for access to mechanism-based cysteine
protease inhibitors incorporating P1 diversity from readily
available aldehydes. The sequence combines the use of
liquid-liquid extractions, volatile or solid-supported reagents,
and support-bound scavengers to eliminate the need for
intermediate column chromatographic purification. Seven
mercaptomethyl ketones 8 have been synthesized using this
seven-step sequence to incorporate both branched and
unbranched nonproteinogenic side chains at the P1 position,
demonstrating the viability of the method.
Acknowledgment. This work was supported by the NIH
(R01GM54051). The Center for New Directions in Organic
Synthesis is supported by Bristol-Myers Squibb as Sponsor-
ing Member. A.L. gratefully acknowledges BMS for a
graduate fellowship.
(16) “Wet” methylene chloride is known to accelerate the Dess-Martin
oxidation reaction (see: Meyer, S. D.; Schreiber, S. L. J. Org. Chem. 1994,
59, 7549-7552).
Supporting Information Available: Experimental pro-
cedures for compounds 4-8 and characterization for com-
pounds 8, entries 1-7. This material is available free of
charge via the Internet at http://pubs.acs.org.
(17) (a) Parlow, J. J.; Case, B. L.; South, M. S. Tetrahedron 1999, 6785-
6796. (b) South, M. S.; Dice, T. A.; Parlow, J. J. Biotechnol. Bioeng. 2000,
71, 51-57.
(18) See entries 1, 2, and 4 in the Supporting Information for chiral HPLC
analysis data.
OL0166496
Org. Lett., Vol. 3, No. 23, 2001
3709