Serine protease inhibition by a silanediol peptidomimetic

Article abstract of DOI:10.1021/ol301933n

Silanediol peptidomimetics are demonstrated to inhibit a serine protease. Asymmetric synthesis of the inhibitor was accomplished using Brown hydroboration and CBS reduction of an acylsilane intermediate. The silanediol product was found to inhibit the ser

Full text of DOI:10.1021/ol301933n

ORGANIC
LETTERS
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Vol. XX, No. XX
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Serine Protease Inhibition by a Silanediol
Peptidomimetic
Swapnil Singh and Scott McN. Sieburth*
Department of Chemistry, Temple University, 1901 North 13th Street, Philadelphia,
Pennsylvania 19122, United States
scott.sieburth@temple.edu
Received July 12, 2012
ABSTRACT
Silanediol peptidomimetics are demonstrated to inhibit a serine protease. Asymmetric synthesis of the inhibitor was accomplished using Brown
hydroboration and CBS reduction of an acylsilane intermediate. The silanediol product was found to inhibit the serine protease chymotrypsin with
a Ki of 107 nM. Inhibition of the enzyme may involve exchange of a silane hydroxyl with the active site serine nucleophile, contrasting with previous
silanediol protease inhibitors.
Design of protease inhibitors is a focus of many phar-
maceutical research programs because of the myriad of
biological processes that these enzymes mediate.1 More
than 30 protease inhibitors have been approved for
clinical use, and many of these have reached the
marketplace.2 Protease inhibitors are used to control
hypertension,3 AIDS, thrombosis,4 and cancer5 but
have the potential for many additional applications.
Each inhibitor is tailored to the protease class, based on
the active site mechanism, and employ the recognition
elements that flank the active site to give the inhibitors
specificity and potency.
Within each protease class, certain functionally relevant
replacements for the tetrahedral intermediate have found
broad utility.6 Typical inhibitors of metalloproteases uti-
lize zinc ion coordinating groups.7 For aspartic proteases,
a carbinol is used to hydrogen bond with the active site
carboxylic acids.8 Cysteine protease inhibitors often em-
ploy electrophilic functional groups to capture the cysteine
nucleophile.9 Serineproteasesare inhibitedbyelectrophilic
groups that interact with the active site serine alcohol
nucleophile. Useful electrophiles include R-fluoro ketones
and 1,2-dicarbonyls.10 A similar effect is found with the
recently introduced anticancer agent bortezomib, which
inhibits the closely related threonine protease found in the
proteasome. This inhibition is effected by coordination of
the active site hydroxyl by a Lewis acidic boronic acid.11
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DePriest, S. A.; Mayer, D.; Naylor, C. B.; Marshall, G. R. J. Am. Chem.
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r
10.1021/ol301933n
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Each of these functional groups have steric, electronic, and
metabolic consequences that present challenges and op-
portunities in pharmaceutical design.
The central Alaꢀ[Si]ꢀPhe dipeptide of 2 had been pre-
viously prepared as part of a study of inhibitors of an
angiotensin-converting enzyme, but this synthesis employed
Serine proteases are contemporary targets for treatment
of Alzheimer’s disease, cancer, obesity, diabetes, throm-
bosis, and more.12 Novel functionality for serine protease
inhibitor design may therefore have broad utility.
Silanols have potential as serine protease inhibitors
because a silanol hydroxyl willrapidlyexchangewithwater
and with alcohols (Scheme 1).13 A covalent interaction
between the silicon and the enzyme would contrast with
previously described silanediol inhibitors that have been
inhibitors of aspartic and metallo proteases where water is
the nucleophilic group.14,15
a lengthy sequence.21 In this original preparation, the P1
0
stereogenic center of 2 was derived from the commercially
available, enantiomerically pure Roche ester.22 Subsequent
separation of diastereomers led to the correct stereochem-
istry at P1. We have recently reported an asymmetric method
0
for constructing the P1 methyl stereogenic center, and
we employed this chemistry to prepare 2 (Scheme 2).23
Magnesium-mediated cycloaddition of isoprene with di-
chlorodiphenylsilane can be performed on a >100 g scale.24
Hydroboration of 5 with monoisopinocampheylborane
at ꢀ20 °C leads to silacyclopentane 6 in 93% ee. Warming
this β-hydroxysilane with a 3:1 mixture of 48% HF in
ethanol leads to Peterson fragmentation and formation of
fluorosilane 7 in 90% distilled yield.19
Scheme 1. Silanols Readily Exchange with Alcohols
23
0
Scheme 2. Setting the P1 Methyl Stereochemistry
Chymotrypsin is an archetype serine protease, charac-
terized by a requirement for an aromatic side chain at P1
(Figure 1).16 The use of the phenylalanine isostere 1 by
Imperiali and Abeles underscored the importance of this
aromatic substituent in chymotrypsin inhibitor design.17
To evaluate a silanediol as an inhibitor of this enzyme, we
elected to utilize 2, an Alaꢀ[Si]ꢀPhe dipeptide analog. The
0
choice of alanine substitution for P1 in 2 was based on its
effective use as a steric shield for the silanediol to minimize
oligomerization.18 Chymotrypsin is relatively insensitive
toward substitution at P1 in substrates,19 and methyl
Construction of the R-aminosilane component utilized a
BrookꢀCorey acylsilane approach (Scheme 3).25 Addition
0
substitution was effective in phosphoramide inhibitors
developed by Bartlett et al.20 Termination of 2 with a
primary amide ensures that the polarity and hydrogen
bonding capabilities of the substrate are retained. Evalua-
tion of 2 as an inhibitor used the commercially available
chymotrypsin substrate 3 (vide infra).
(12) Bachovchin, D. A.; Cravatt, B. F. Nat. Rev. Drug Discovery
2012, 11, 52–68.
(13) Tacke, R.; Linoh, H.; Ernst, L.; Moser, U.; Mutschler, E.; Sarge,
S.; Cammenga, H. K.; Lambrecht, G. Chem. Ber. 1987, 120, 1229–1237.
(14) A silanediol inhibitor of the serine protease elastase was re-
ported: Showell, G. A.; Montana, J. G.; Chadwick, J. A.; Higgs, C.;
Hunt, H. J.; MacKenzie, R. E.; Price, S.; Wilkinson, T. J. Silicon diols,
effective inhibitors of human leukocyte elastase. In Organosilicon Chem
VI, [Eur Silicon Days], 2nd ed.; Auner, N., Weis, J., Eds.; Wiley-VCH
Verlag GmbH & Co. KGaA: 2005; pp 569ꢀ574. This silanediol has also
ꢀ
been prepared by Skrydstrup et al.: Hernandez, D.; Lindsay, K. B.;
Nielsen, L.; Mittag, T.; Bjerglund, K.; Friis, S.; Mose, R.; Skrydstrup, T.
J. Org. Chem. 2010, 75, 3283–3293.
(15) Sieburth, S. McN.; Chen, C.-A. Eur. J. Org. Chem. 2006, 311–
322.
(16) Schecter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967,
27, 157–162.
(17) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760–3767.
(18) Mutahi, M. wa; Nittoli, T.; Guo, L.; Sieburth, S. McN. J. Am.
Chem. Soc. 2002, 124, 7363–7375.
(19) Schellenberger, V.; Braune, K.; Hofmann, H.; Jaubke, H. Eur. J.
Biochem. 1991, 199, 623–636.
(20) Bartlett, P. A.; Lamden, L. A. Bioorg. Chem. 1986, 14, 356–377.
(21) Kim, J.; Hewitt, G.; Carroll, P.; Sieburth, S. McN. J. Org. Chem.
2005, 70, 5781–5789.
(22) Cohen, N.; Eichel, W. F.; Lopresti, R. J.; Neukom, C.; Saucy, G.
J. Org. Chem. 1976, 41, 3505–3511.
(23) Sen, S.; Purushotham, M.; Qi, Y.; Sieburth, S. McN. Org. Lett.
2007, 9, 4963–4965.
(24) Manuel, G.; Mazerolles, P.; Lesbre, M.; Pradel, J.-P. J. Orga-
nomet. Chem. 1973, 61, 147–165.
Figure 1. Inhibition of the serine protease chymotrypsin requires
an aromatic substitutent at P1.
(25) Brook, A. G.; Duff, J. M.; Jones, P. F.; Davis, N. R.
J. Am. Chem. Soc. 1967, 89, 431–434. Corey, E. J.; Seebach, D.; Freedman,
R. J. Am. Chem. Soc. 1967, 89, 434–436.
B
Org. Lett., Vol. XX, No. XX, XXXX

of 2-lithio-1,3-dithiane to fluorosilane 7, followed by
deprotonation and alkylation with benzyl bromide,
gave 8. Hydrolysis of the dithiane then gave the silyl
ketone 9. Reduction of this ketone using the (S)-B-Me-
CBS reagent gave a clean reduction with a diastereos-
electivity of >90% dr (NMR).26 The resulting alcohol
10 underwent Mitsunobu inversion with phthalimide to
produce 11.
Scheme 4. Completion of the Inhibitor Synthesis
Scheme 3. Asymmetric Construction of the R-Amino Silane
Completion of the inhibitor (Scheme 4) began with
ozonolysis of the terminal alkene, followed by Pinnick
oxidation of the resulting aldehyde. Acid 12 was
then converted to the primary amide through the
mixed anhydride using ethyl chloroformate. Hydro-
lysis of the phthalimide with hydrazine gave the
corresponding amine, which was then coupled with
Boc-protected proline using HATU to give tripeptide
analog 14.
Hydrolysis of diphenylsilane 14 to silanediol 15 used our
protocol developed in earlier work.27 Treatment of 14 with
triflic acid in methylene chloride at 0 °C cleaved the
Siꢀphenyl bonds (as well as the Boc group). This was
followed by dilution with ammonium hydroxide. The
resulting product was then treated with 48% aqueous
HF, to form the difluorosilane. This monomeric product
is readily hydrolyzed to the corresponding silanediol under
mildly basic aqueous conditions.
Figure 2. Dixon plot showing inhibition of chymotrypsin by 15,
with concentration of substrate 3 at 50 μM (b) and 45 μM (2).
Evaluation of 15 as an inhibitor of chymotrypsin em-
ployed the method of Bartlett et al. in which hydrolysis of
substrate 3 was followed by the increase in UV absorbance
at 410 nm.28,29 Chymotrypsin is inhibited by 15 in a
concentration-dependent manner. The results are shown
in a Dixon plot (Figure 2). These experiments found the Ki
for this inhibition to be 107 nM.
Inhibition of chymotrypsin by 15 is consistent with
exchange of a silanediol hydroxyl group with the nucleo-
philic active site serine hydroxyl of the enzyme. It is likely
that this hydroxyl exchange occurs through a pentavalent
intermediate.30
(30) Lickiss, P. D.; Rappoport, Z.; Apeloig, Y. Polysilanols. The
Chemistry of Organic Silicon Compounds (3); John Wiley & Sons: New
York, 2001; pp 695ꢀ744. Trivalent species have also been postulated:
Oostendorp, D. J.; Bertrand, G. L.; Stoffer, J. O. J. Adhes. Sci. Technol.
1992, 6, 171–191. It is unlikely however that a pentavalent silanol is the
inhibitor: Karsch, H. H.; Bienlein, F.; Sladek, A.; Heckel, M.; Burger, K.
J. Am. Chem. Soc. 1995, 117, 5160–5161. Bassindale, A. R.; Parker,
D. J.; Taylor, P. G.; Auner, N.; Herrschaft, B. Chem. Commun. 2000, 7,
565–566.
(26) Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998, 37, 1986–
2012.
(27) Kim, J.; Sieburth, S. McN. J. Org. Chem. 2004, 69, 3008–3014.
(28) Delmar, E. G.; Largman, C.; Brodrick, J. W.; Geokas, M. C.
Anal. Biochem. 1979, 99, 316–320.
(29) Hansen, K. B.; Grosch, B.; Greiveldinger-Poenaru, S.; Bartlett,
P. A. J. Org. Chem. 2003, 68, 8465–8470.
Org. Lett., Vol. XX, No. XX, XXXX
C

Synthesis of this inhibitor took advantage of an asym-
metric hydroboration and an asymmetric borane reduction
to control the two stereogenic centers flanking the silicon. The
inhibition of chymotrypsin illustrates the use of silanediols as
inhibitors of a third family of proteases, adding to metallo
and aspartic examples.15 Additional studies are in progress.
of Health (R01 GM076471) for generous support for this
program.
Supporting Information Available. Experimental de-
tails and characterization data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. The authors thank Yingmei Qi
for preliminary studies and the National Institutes
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX