Small ring constrained peptidomimetics. Synthesis of epoxy peptidomimetics, inhibitors of cysteine proteases

Article abstract of DOI:10.1021/jo000961w

Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of PhSeCl gave a series of different α-phenylselenyl aldehydes. Mukajiama reaction with silylketene acetals gave an intermediate product that was finally transformed into the desired oxiranyl peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-dependent reversible inhibitors of cysteine protease.

Full text of DOI:10.1021/jo000961w

J . Org. Chem. 2001, 66, 697-706
697
Sm a ll Rin g Con str a in ed P ep tid om im etics. Syn th esis of Ep oxy
P ep tid om im etics, In h ibitor s of Cystein e P r otea ses
Mariangela Demarcus,† Maria Luisa Ganadu,† Giovanni Maria Mura,† Andrea Porcheddu,†
Laura Quaranta,‡ Gianna Reginato,‡ and Maurizio Taddei*,†
Dipartimento di Chimica, Universita` degli Studi di Sassari, Via Vienna 2, 07100 Sassari, Italy, and
Centro di Studio del CNR Chimica dei Composti Eterociclici e loro Applicazioni, Via G. Capponi 9,
50121 Firenze, Italy
mtad@ssmain.uniss.it
Received J une 26, 2000
Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were
prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed
into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of
PhSeCl gave a series of different R-phenylselenyl aldehydes. Mukajiama reaction with silylketene
acetals gave an intermediate product that was finally transformed into the desired oxiranyl
peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters
starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS
on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover
the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small
library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-
dependent reversible inhibitors of cysteine protease.
The chemistry of peptidomimetics has grown rapidly
in the past decade. Starting from the pioneering works
where the CO-N bond of an oligopeptide was substituted
with a CH2OH group,1 several different functions have
been introduced in a peptidic sequence to obtain mol-
ecules able to mimic a peptidic messenger or recognize
different protein structures.2
One of the most important fields of action for peptido-
mimetics has been enzyme inhibition as in, for example,
the preparation of protease inhibitors. The most common
strategy has been to introduce inside to a peptidic
sequence that can be recognized from the enzyme, a
functional group that can interact with the reactive
center present in the active site. As proteases are
nucleophiles, the peptidomimetic inhibitor has been
designed as an electrophile. Thus ketones,3 fluoroke-
tones,4 aldehydes,5 hydoxamates,6 vinyl sulfones,7 and
aziridines8 have been reported as effective inhibitors of
proteases. Epoxides also have been found to be effective
protease inhibitors, and several papers reported the
synthesis and the biological activity of epoxy peptidomi-
metics.9 We communicated the preparation of peptido-
mimetics containing the oxirane ring where the hetero-
cycle mimics exactly the peptidic bond and the other parts
of the molecule remains virtually unaltered.10 The ret-
rosynthesis of these products is reported in Scheme 1.
As they are potentially interesting as enzyme inhibitors
or as valuable intermediates for the preparation of other
kinds of peptidomimetics, we tried to apply this procedure
to the preparation of products suitable for solid-phase
peptide synthesis or for the preparation of libraries.
We report here the application of the procedure to the
preparation of N-Fmoc epoxy acids, their introduction in
(5) Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin, Y.;
Fuhrman, S. A.; Matthews, D. A.; Love, R. A.; Hendrickson, T. F.;
Patick, A. K.; Meador, J . W., III; Ferre, R. A.; Brown, E. L.; Ford, C.
E.; Bindord, S. L.; Worland, S. T. J . Med. Chem. 1998, 41, 2786.
Yasuma, T.; Oi, S.; Choh, N.; Nomura, T.; Furuyama, N.; Nishimura,
A.; Fujisawa, Y.; Sohda, T. J . Med. Chem. 1998, 41, 4301.
(6) Hanessian, S.; Bouzbouz, S.; Boudon, A.; Tucker, G. C.; Peyrou-
lan, D. Bioorg. Med. Chem. Lett. 1999, 9, 1691 and references therein.
(7) Palmer, J . T.; Rasnick, D.; Klaus, J .; Bro¨mme, D. J . Med. Chem.
1995, 38, 3193.
* Fax: +39-079-229559.
(8) Schirmeister, T. J . Med. Chem. 1999, 42, 560 and references
therein.
† Universita` degli Studi di Sassari.
‡ Centro di Studio del CNR Chimica dei Composti Eterociclici e loro
Applicazioni.
(1) Spatola, A. F. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York,
1983; Vol. 7, p 267.
(2) For recent leading reviews, see: Goodman, M.; Zhang J .
Chemtracts-Org. Chem. 1997, 10, 629. Hanessian, S.; Mcnaughton-
Smith, G.; Lombart, H. G.; Lubell, W. D. Tetrahedron 1997, 53, 12789.
Methods in Molecular Medicine: Vol. 30, Peptidomimetics Protocols;
Kazmierski, W. M., Walker, J . M., Eds.; Human Press: Totowa, NJ ,
1999.
(3) Semple, G.; Ashworth, D. M.; Batt, A. R.; Baxter, A. J .; Benzies,
D. W. M.; Elliot, L. H.; Evans, D. M.; Franklin, R. J .; Hudson, P.;
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Bioorg. Med. Chem. Lett. 1998, 8, 959.
(9) J ohnson, R. L. Pept. Struct. Funct. Proc. Am. Pept. Symp. 8th
1983, 587. Kaltenbronn, J . S.; Hudspeth, J . P.; Lunney, E. A.;
Michniewicz, B. M.; Nicolaides, E. D.; Repine, J . T.; Roark, W. H.; Stler,
M. A.; Tinney, F. J . J . Med. Chem. 1990, 33, 838. Marchand-Bryneert,
J .; Ferroud, D.; Serchod-Porcin, B.; Ghosez, L. Bull. Soc. Chim. Belg.
1990, 99, 1075. Albeck, A.; Persky, R.; Kliper, S. Bioorg. Med. Chem.
Lett. 1995, 5, 1767. Meara, J . P.; Rich, D. H. J . Med. Chem. 1996, 39,
3357. Lee, C. S.; Choy, N.; Park, C. C. H.; Son, Y. C.; Kim, S.; Ok, J .
H.; Yoon, H.; Kim, S. C. Bioorg. Med. Chem. Lett. 1996, 6, 589. Choy,
N.; Choi, H.; J ung, W. H.; Kim, C. R.; Yoon, H.; Kim, S. C.; Lee, T. G.;
Koh, J . S. Bioorg. Med. Chem. Lett. 1997, 7, 2635. Ro, S. Baek, S-G.;
Lee, B.; Park, C.; Choy, N.; Lee, C. S.; Son, Y. C.; Choi, H.; Koh, J . S.;
Yoon, H.; Kim, S. C.; Ok, J . H. Bioorg. Med. Chem. Lett. 1998, 8, 2423.
Roush, W. R.; Gonzales, F. V.; McKerrow, J . H.; Hansell, E. Bioorg.
Med. Chem. Lett. 1998, 8, 2809. Perlman, N.; Livneh, M.; Albeck, A.
Tetrahedron 2000, 56, 1505.
(4) Morris, T. S.; Frormann, S.; Shechosky, S.; Lowe, C.; Lall, M.
S.; Gauss-Mu¨ller, V.; Purcell, R. H.; Emerson, S. U.; Vederas, J . C.;
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(10) Mann, A.; Quaranta, L.; Reginato, G. Taddei, M. Tetrahedron
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10.1021/jo000961w CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/13/2001

698 J . Org. Chem., Vol. 66, No. 3, 2001
Demarcus et al.
Ta ble 1. P r ep a r a tion of Selen yl Ald eh yd es
Sch em e 1
alkene
(yield)a
selenyl aldehyde
(yield)a [de]b
starting amino acid
N-Fmoc-Ala-OH
N-Fmoc-Phe-OH
N-Fmoc-Val-OH
N-Fmoc-(OBn)Ser-OH
5 (71%)
6 (82%)
7 (70%)
8 (66%)
9 (70%) [85%]
10 (73%) [95%]
11 (79%) [95%]
12 (73%) [90%]
a
Yields of products isolated after column chromatography.
Determined by 1H NMR (δ 9.0-9.5).
b
Sch em e 3a
Sch em e 2a
a
(a) SOCl2, 50 °C, followed by t-BuSH, Et3N, CH2Cl2. (b) LDA,
THF, -78 °C, Me3SiCl.
this effort was unnecessary as the mixture of E and Z
isomers gave a single diastereoisomer in the next step.
To get a good level of stereoselectivity in the addition
of PhSeCl to alkenes 5-8, we coordinated first the
starting material with Ti(Oi-Pr)4 and then added the
selenyl chloride at -78 C.13 To preclude the side forma-
tion of the mixed acetal 13 we must add LiCl to the
reaction mixture (at least 10 equiv) as a source of
nucleophilic chloride to compete with isopropoxyde in the
attack to the selenonium intermediate.
a
(a) 2-Chloro-4,6-dimethoxy[1,3,5]triazine, NMM, DME, fol-
lowed by H2 and Pd/C (10%). (b) MeOCH2PPh3Br, KN(SiMe3)2,
THF, 0 °C. (c) PhSeCl, Ti(Oi-Pr)4, LiCl, Na2CO3, CH2Cl2, -78 °C.
a polypeptidic frame, and the solid-supported synthesis
of analogous epoxy derivatives for the preparation of a
small library.
Two different strategies were possible: build the
epoxide on a growing oligopeptide or try to assemble an
epoxidic monomer and introduce it as a simple amino acid
during the preparation of the peptide. In the first strategy
we may have the problem of the chemistries working on
the oligopeptidic frame, and in the second case we must
to be sure that the epoxide ring would be stable to the
conditions of deprotection and couplings. Looking more
carefully to the first strategy we thought that a possible
intermediate was an oligopeptidic aldehyde. Although the
preparation of a peptidic aldehyde has been previously
described11 we observed that during the activation of the
carboxylic group a possible racemization of the stereo-
center of the amino acid may occur through the genera-
tion of an azalactone formed by attack of the amide
oxygen to the activated carbonyl. For this reason we
decided to follow the other strategy.
Thus the N-Fmoc amino acids 1-4 were transformed
into the corresponding aldehydes by reduction of the 4,6-
dimethoxy[1,3,5]triazin-2-yl esters with H2 and Pd/C in
THF (Scheme 2).12 The solution obtained from the
hydrogenation, filtered from the Pd on carbon, was
directly added to the THF solution containing the ylide
formed from methoxymethyl triphenylphoshonium bro-
mide and NaN(SiMe3)2. This addition was carried out at
0 °C; the deep red solution of the ylide discolored rapidly
and the products 5-8 were obtained as a mixture of E
and Z isomers after a hydrolytic workup and filtration
of the formed triphenylphosphine oxide. In the case of
compounds 5 and 6 it was possible to separate the
isomers by column chromatography on silica gel although
When the reaction was carried out without the Ti(Oi-
Pr)4 we obtained a mixture of diastereoisomers as evi-
1
denced by the well separated aldehyde signals in the H
NMR at δ 9.0-9.5. Consequently the diastereoisomeric
purity of products 9-12 could be simply determined by
comparison of this part of the 1H NMR spectra. When
the reactions were carried out in the presence of the
Lewis acid and LiCl, the purity resulted always included
between 95% and 85%.
Diastereoisomerically pure aldehydes 9-12 were then
reacted with silyl thio-ketene acetals 22-25 prepared
from the acids 14-17 as described in Scheme 3.
The aldol-type reaction14 was carried out in the pres-
ence of 1.5 equiv of BF3OEt2, and the best results were
obtained by precomplexation of the aldehyde with the
Lewis acid at -78°, addition of 2.5 equiv of the silyl
thioketene acetal, and aqueous workup at -20 °C.
Products 26-37 were obtained after column chroma-
tography on silica gel in high diastereoisomeric excess
as revealed by 1H and 13C NMR and HPLC analysis
(Scheme 4).15 Unfortunately attempts to transform com-
pounds 26-37 into the corresponding Mosher’s esters,
to verify the enantiomeric purity, were unsuccessful as
(13) Toshimitsu, A.; Aoai, T.; Owada, H.; Uemura, S.; Okano, M. J .
Chem. Soc., Chem. Commun. 1980, 412. Demarcus, M.; Filigheddu, S.
N.; Mann, A.; Taddei, M. Tetrahedron Lett. 1999, 40, 4417.
(14) Annunziata, R.; Cinquini, M.; Cozzi, F.; Cozzi, P. G.; Consolandi,
E. J . Org. Chem. 1992, 57, 456.
(15) When compound 5 was reacted with PhSeCl, in the absence of
Ti(Oi-Pr)4, aldehyde 9 was obtained as a mixture of two diasteroisomes.
When this mixture was submitted to Mukayiama-type reaction,
exclusively two isomers were observed for compound 26 (HPLC
analysis). When the same reaction was carried out on diastereoiso-
merically pure 9, the HPLC trace of 26, obtained after column
chromatography, showed the presence of a predominant peak (at least
95%) that was attributed to a single diastereoisomer.
(11) O’Donnel, M. J .; Drew, M. D.; Pottorf, R. S.; Scott, W. L. J .
Comb. Chem. 2000, 2, 172 and references therein.
(12) Falorni, M.; Giacomelli, G.; Porcheddu, A.; Taddei, M. J . Org.
Chem. 1999, 64, 8962.

Small Ring Constrained Peptidomimetics
J . Org. Chem., Vol. 66, No. 3, 2001 699
Ta ble 2. P r ep a r a tion of N-F m oc-ep oxy P ep tid om im etics
products:
aldol product epoxide
selenyl aldehyde
silylketene acetal
22 R2 ) R3) CH3
yielda [%]
deb [%]
90
9 R1 ) CH3
26
38
27
39
28
40
29
41
30
42
31
43
32
44
33
45
34
46
35
47
36
48
37
49
67
74
71
78
66
80
71
81
75
73
77
75
70
86
68
88
75
86
65
75
67
80
71
73
9 R1 ) CH3
23 R2 ) R3 ) -(CH2)5-
24 R2 ) Me2CHCH2CH2, R3 ) H
25 R2 ) PhCH2OCH2CH2-, R3 ) H
22 R2 ) R3 ) CH3
92
91
9 R1 ) CH3
9 R1 ) CH3
90
10 R1 ) PhCH2
10 R1 ) PhCH2
10 R1 ) PhCH2
10 R1 ) PhCH2
11 R1 ) Me2CH
11 R1 ) Me2CH
12 R1 ) PhCH2OCH2
12 R1 ) PhCH2OCH2
90
23 R2 ) R3 ) -(CH2)5-
24 R2 ) Me2CHCH2CH2, R3 ) H
25 R2 ) PhCH2OCH2CH2-, R3 ) H
22 R2 ) R3 ) CH3
92
89
91
>95
>95
90
23 R2 ) R3 ) -(CH2)5-
22 R2 ) R3 ) CH3
23 R2 ) R3 ) -(CH2)5-
92
a
b
Yields of products isolated after column chromatography. de determined by HPLC analysis of the pure compounds.
Sch em e 4a
Sch em e 5a
a
(a) BF3OEt2, CH2Cl2, -78 °C. (b) mCPBA, MeOH, Na2CO3,
-15 °C.
a
(a) (R)-MTPA-Cl, pyridine, CCl4, 50 °C. (b) NaOH 0.2 M, THF,
the classical conditions to generate the MTPB esters gave
always substantial amounts of the undesired alkenes (see
Scheme 5). For this reason we postponed the analysis of
the presence of enantiomers to the next step.
followed by HCl. (c) CH2N2, Et2O, -15 °C. (d) TBAF, THF, 0 °C.
The configuration assignment was performed by trans-
formation of epoxides 40, 41, and 44 into the alcohols
50-52 by treatment with TBAF in THF as reported for
analogous compounds.16 In such transformation the con-
figuration at C4 and the relative stereochemistry of the
substituents at C2 and C3 are preserved.17 As the
mechanism proposed for the aldol type reaction is ex-
pected to predominantly give the anti product (at C2-
C3),14 the resulting alkenes would be the E isomer as
The â-hydroxyselenyl derivatives were transformed
into the desired epoxides 38-49 by treatment with
MCPBA and K2CO3 in MeOH at -15 °C. The N-Fmoc
group was remarkably stable under this conditions
provided that the temperature never exceed -10°. The
trans arrangement of the epoxide was determined by the
1
value of the coupling constant of the H signal at 2.5-
1
confirmed by H and 13C NMR data.18 Alcohols 50-52
3.2 ppm. In the case of compounds 38, 39, 42, 43, and
46-49 the value could be simply read on the spectrum
as the proton is part of an AM system, whereas for the
other compounds we must carry out decoupling experi-
ments after a full assignment of all the resonances by
COSY experiments.
were also easily transformed into the MTPA esters to
(16) J enmalm, A.; Berts, W.; Li, Y.-L.; Luthman, K.; Cso˜regh, I.;
Hacksell, U. J . Org. Chem. 1994, 59, 1139.
(17) Li, J .-L.; Luthman, K.; Hacksell, U. Tetrahedron Lett. 1992,
33, 4487.

700 J . Org. Chem., Vol. 66, No. 3, 2001
Demarcus et al.
Sch em e 6a
verify the enantiomeric integrity of the products pre-
pared, which was always very good.
Finally, the thioesters could be deprotected (aqueous
NaOH 0.2 M) to give the corresponding epoxy acids. The
acids are mainly solids that are stable if stored as pure
compounds but are likely to decompose if stored in CDCl3
solutions. For this reason we decided to store the tert-
butyl thioesters and prepare the corresponding acids just
before the use in the coupling experiments.
Compounds 38-49 can be considered as doubly pro-
tected amino acids and we decided to verify if they could
be used in solid-phase peptide synthesis (SPPS).
Thus a Wang-type resin was loaded with N-Fmoc
alanine using standard protocols and subsequently depro-
tected with piperidine in DMF. The coupling with the
acid obtained from 38 was carried out using a classical
procedure (PyBOP, HOBT, DIPEA, NMP) and was
complete (Kaiser’s test)19 after 1 h. The coupling step of
the acid was not particularly troublesome as it could be
carried out successfully also using our alternative het-
erogeneous coupling agent (DMTMM, DIPEA in NMP).20
First cleavage of product 56 with TFA/H2O 95/5 gave
a brownish waxy material that we could not identify as
the desired product. Better results were obtained using
TFA 50% in CH2Cl2 in the presence of Et3SiH. After
filtration and evaporation of the solvent under high
vacuum, the crude resulted formed by at least four
products. After separation by PTLC we could isolate
compound 57 in 36% yield. A previous deprotection of
the N-Fmoc group followed by mild acid cleavage was
again unsuccessful. The HPLC analysis of the crude
showed six peaks and the expected molecular ion was
not found in the mass spectra.
As we could imagine from the behavior of compounds
38-49 at the prolonged storage in CDCl3, epoxides are
not stable in acidic solutions. For SSPS we had to found
a linker that could be cleaved under non-acidic conditions.
Nevertheless we tried the SASRIN linker (3-methoxy-4-
hydroxy-methyl phenol)21 and we were pleased to find
that when the cleavage was carried out under the
standard conditions (1% TFA/CH2Cl2) followed by neu-
tralization of the filtrate with 2 equiv of Et3N, compound
57 was recovered in good yield and with an acceptable
level of purity (see Experimental Section). Working on
SASRIN it was possible to carry out the deprotection of
the Fmoc followed by a second coupling (DMTMM,
DIPEA) with an amino acid to give a product as 58.
Deprotection of the Fmoc and cleavage from the resin
gave product 59 in 61% yield (Scheme 6). With the
previously described procedure we demonstrated that our
epoxy amino acids could be successfully inserted inside
to a peptide sequence as a mimic of a dipeptide frame
(as, for example, the protease cleavage site). Moreover
compounds 38-49 could be used in the preparation of
a
(a) 25% Piperidine in DMF. (b) Acid obtained from 38 in NaOH
0.2 M, THF followed by HCl, DMTMM, DIPEA, NMP. (c) TFA 1%
in CH2Cl2 followed by Et3N. (d) N-Fmoc-Val-OH, DMTMM,
DIPEA, NMP.
peptide libraries. On the other hand, these compounds
have been prepared with a “classical” organic synthesis
protocol that limits the possibility to prepare libraries of
epoxy acids to test their activity.
To reach this goal we decided to design a possible
strategy to prepare the desired epoxy amino acids on a
solid support to exploit the potential of a truly solid-phase
synthesis for the preparation of libraries. Looking to
Scheme 2, the route proposed to build the structure on
the carboxylic part of the amino acid is the wrong one in
the growing of a peptide-type molecule on a resin.
Although the preparation of aldehydes anchored to the
resin through the nitrogen have been previously de-
scribed,11,22 we excluded this possibility from unsatisfac-
tory previous experiences of our laboratory.
We took advantage of the work of Kobajashi on the
preparation of silyl thioketene acetal loaded on a resin23
to accomplish a synthesis on solid phase. A Merrifield-
type resin was loaded with potassium thioacetate, and
the thioester obtained was reduced with LiBH4 to give
the thiol free resin (positive Ellman’s test)24 that was
loaded with the acyl chloride derived from acid 14.
Formation of the thioester was indicated by a negative
Ellman’s test and by IR spectra showing a strong car-
bonyl stretching around 1690 cm-1. The silyl thioketene
acetal was prepared treating the beads with LDA in THF
at -40° followed by Me3SiCl. After washing the beads
with dry NMP and pyridine, the aldol-type reaction with
aldehyde 9 was carried out at -40 °C in CH2Cl2 and in
the presence of TMSOTf (20 mol %). Any other Lewis acid
employed gave unsatisfactory results except for Sc(OTf)3,
successfully employed by Kobajashi, which gave the same
results (in our hands) as TMSOTf. In the presence of BF3-
OEt2, employed in the homogeneous phase, no reaction
at all was observed. The formation of product 63 (Scheme
7) was determined with a visual test for primary alcohols
(2,4,6-trichloro[1,3,5]-triazine and Alizarin R, TCT-AliR)
(18) For compounds 50 and 52 we did not observe any NOE effect
between CH(4) and the CH2 of the isobutyl group in position 2 as
expected in the case of the Z isomer. Moreover the 13C resonance at
C-3 was around δ 131. From reported data16 the E isomer is expect to
have a chemical shift lower than δ 133.
(19) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(20) Falchi, A.; Giacomelli, G.; Porcheddu, A.; Taddei, M. Synlett,
2000, 275.
(21) SASRIN is a registered trademark of BACHEM. For leading
references, see: Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron
1993, 49, 11065. Mergler, M.; Nyfeler, R.; Tanner, R.; Gosteli, J .; Grogg,
P. Tetrahedron Lett. 1988, 29, 4009. Mergler, M.; Gosteli, J .; Grogg,
P.; Nyfeler, R.; Tanner, R. Chimia 1999, 53, 29.
(22) Rotella, D. P. J . Am. Chem. Soc. 1996, 118, 12246.
(23) Kobayashi, S. Chem. Soc. Rev. 1999, 28, 1 and references
therein.
(24) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

Small Ring Constrained Peptidomimetics
J . Org. Chem., Vol. 66, No. 3, 2001 701
Sch em e 7a
Sch em e 8
a
(a) Ref 23 (b) Me2CHCOCl, CH2Cl2, DIPEA. (c) LDA, THF,
Me3SiCl, -78 °C. (d) 9, TMSOTf, CH2Cl2, -40 °C. (e) MCPBA,
CH2Cl2, Na2CO3, 0 °C. (f) 25% Piperidine in DMF followed by NH3
(dry) in MeOH at 50 °C.
developed in our laboratory.25 After 5 h at -40 °C not all
of the beads selected showed a homogeneous red color.
Thus the temperature was raised to 0 °C and after 2 h
at this temperature, the visual test suggested that the
reaction had proceeded further. After several washings
with CH2Cl2, pyridine, and THF, the epoxidation took
place using a solution of MCPBA in DMP/CsCO3. When
the beads were clear at the TCT-AliR test, we tried first
to cleave the product from the beads using KOH in
dioxane/MeOH but we obtained very low yields of the
desired acid. Different nucleophilic cleavage conditions
with hydroxides were attempted, but results were always
disappointing. When we tried to cleave the product with
a solution of NH3 in dry methanol at 50 °C, we obtained
an almost complete removal of the organic product from
the resin.26 The crude consisted of a mixture of the Fmoc-
protected and deprotected compounds. Thus we carried
out first the deprotection of the Fmoc group (piperidine
in DMF) and then the cleavage. After evaporation of the
solvent product 64 was isolated by separation from
diethyl ether in 67% yield and with a purity higher than
80% (HPLC analysis). Analytical samples of the epoxy
amides were recovered by PTLC. An accurate 1H and 13C
NMR analysis showed that we obtained product 64 as a
mixture of diasteroisomers in approximately a 2/1 ratio.27
A small library of epoxy amides were prepared by
splitting the resin in nine tubes, adding the acyl chlorides
reported in Scheme 8, then mixing together the linked
thioester, and finally adding the aldehyde 9. This small
libray of nine compounds was analyzed with a ES-MS
and showed an almost equal distribution of the expected
molecular ions. To verify if some of the compounds
prepared in this work could be considered effective
inhibitors of proteases, we tested them as inhibitors of
the cysteine protease papain. This enzyme is the most
studied cysteine protease and can be considered an easily
available model for the search of inhibitors of cysteine
proteases involved in different pathologies.28
BAPA29 both in the absence and in the presence of the
epoxides at different concentrations, usually in the range
from 1 to 500 mM. During the proteolitic assay, described
in the Experimental Section, a concentration of papain
lower than 10 nmol/min/mL of hydrolyzed L-BAPA was
used. The low concentration of papain in the assay allows
a reproducible measure of the initial rate of reaction at
a practically constant substrate concentration, if the
starting L-BAPA concentration (1 mM) is lower than the
Km value of papain for this substrate (2.5 mM, deter-
mined in our experimental conditions). Therefore, the
initial rate of reaction was calculated with a linear fit,
with an error attributable to the substrate disappearance
less than 5%.
From the products submitted to the assay, reproducible
results were obtained only with the stable thio-esters or
with the free amino acids. The values of the inhibition
constant were calculated by Dixon plots,. The inhibition
of papain was observed with compounds 38, 39, 58, 60,
and 64.
In Table 3 the inhibition constants calculated for the
different compounds are listed. To verify the hypothesis
of a slow irreversible reaction between papain and the
epoxides, the enzyme was incubated with the different
epoxides at a concentration of 200 mM. Aliquots were
assayed, after a 20-fold dilution in cuvette, immediately
and after 24 and 48 h of incubation at 4 °C. A significant
decrease of the papain activity is not evident after 24 and
48 h with respect to the starting of incubation. Therefore,
in our conditions, we do not observe irreversible inhibi-
tion of papain, neither during the spectrophotometric
assay nor after a prolonged incubation.
The activity of commercial papain was determined
spectrophotometrically following the hydrolysis of L-
(25) Attardi, M. E.; Falchi, A.; Taddei, M. Tetrahedron Lett. 2000,
41, 7395.
(26) We were not able to find the conditions for a hydrolityc clevage
from the resin to obtain a carboxylic acid containing the intact epoxide.
(27) The formation of a mixture of diasteroisomers may be expected
on the basis of the sterochemical model proposed in ref 14.
(28) Yabe, Y.; Guillaume, D.; Rich, D. H. J . Am. Chem. Soc. 1988,
110, 4043. Albeck, A.; Fluss, S.; Persky, R. J . Am. Chem. Soc. 1996,
118, 3591. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
Although the inhibition potency of our product is poor,
these data confirm that the activity is strictly dependent
(29) L-BAPA: benzoylarginyl p-nitroanilide.

702 J . Org. Chem., Vol. 66, No. 3, 2001
Demarcus et al.
Ta ble 3. In h ibition Con sta n ts for th e
Non -Tim e-Dep en d en t In h ibition of P a p a in
over anhydrous Na2SO4. After evaporation of the solvent
product 5 (2.3 g, 71% yield) was purified by column chroma-
1
tography on silica gel. H NMR (of the major isomer, CDCl3)
compound
Ki (µM)
δ 1.35 (d, 3H, J ) 7 Hz), 3.5 (s, 3H), 4.20-4.3 (m, 2H), 4.70
(dd, J ) 16, 7 Hz, 1H), 4.8 (m, 2H7, 6.31 (d, J ) 16 Hz, 1H),
6.8 (bd, 1H), 7.20-7.90 (m, 8H). 13C NMR (of the major isomer,
CDCl3). 21.6, 37.9, 46.2, 55.4, 73.0, 98.7, 126.8, 128.0, 129.7,
136.5, 140.7, 145.7, 159.9.
38
39
42
43
46
47
48
49
24 ( 1.
106 ( 4
>500
>500
>500
>500
>500
>500
(1-Ben zyl-3-m eth oxy-a llyl)-ca r ba m ic Acid 9H-F lu or en -
1
9-yl Meth yl Ester 6. H NMR (of the major isomer, CDCl3) δ
2.31 (m, 1H), 2.40 (m, 1H), 3.50 (s, 3H), 4.56-4.60 (m, 2H),
4.70 (m, 2H), 4.88 (dd, J ) 16, 7 Hz, 1H), 6.40 (d, J 16 Hz,
1H), 6.72 (bd, 1H7, 7.2-7.9 (m, 13 H). 13C NMR (of the major
isomer, CDCl3) δ 36.8, 40.6, 51.2, 53.4, 76.7, 95.5, 126-129,
137.3, 141.9, 140.8, 158.8.
58
60
64
48 ( 6
298 ( 11
2 ( 1
H-Ala-Aib-OH 65
>500
(1-Isop r op yl-3-m eth oxy-a llyl)-ca r ba m ic Acid 9H-F lu o-
r en -9-yl Meth yl Ester 7. 1H NMR (of the major isomer,
CDCl3) δ 1.35 (d, J ) 7 Hz, 3H), 1.41 (d, J ) 7 Hz, 3H), 2.01
(m, 1H), 3.50 (s, 3H), 4.56-4.60 (m, 2H), 4.65 (m, 2H), 4.80
(dd, J ) 16, 7 Hz, 1H,), 6.45 (d, J 16 Hz, 1H), 6.60 (bd, 1H,),
7.3-7.9 (m, 8H,). 13C NMR (of the major isomer, CDCl3) δ 17.8,
18.8, 30.3, 37.7, 53.2, 56.9, 75.5, 99.0, 126-129, 136.5, 140.9,
144.7, 159.6.
on the presence of the epoxide as demonstrated by the
absence of activity for compound 65.
We have described an effective stereoselective synthe-
sis of epoxy peptidomimetics that provides N-Fmoc-
protected fragments that can be successfully employed
in SPPS. We also applied the synthesis to the solid-phase
preparation of the compounds suitable for the prepara-
tion of libraries. Finally the epoxy peptidomimetics
obtained were effective inhibitors of papaine, suggesting
that an analogous approach can be used for the search
for new inhibitors for other proteases involved in impor-
tant pathologies.
(1-Ben zyloxym eth yl-3-m eth oxy-a llyl)-ca r ba m ic Acid
9H-F lu or en -9-yl Meth yl Ester 8. 1H NMR (of the major
isomer, CDCl3) δ 3.50 (m, 3H), 4.4-4.8 (m, 7 H), 5.20 (d-like,
2H), 6.0 (bd, 1H), 6.7 (d, J ) 17 Hz, 1H), 7.2-7.9 (m, 13 H).
13C NMR (of the major isomer, CDCl3) δ 37.8, 48.9, 53.9, 74.7,
76.0, 77.1,95.7, 126.1-129, 136.5, 137.0, 141.0, 144.7, 159.8.
(1-Meth yl-3-oxo-2-ph en ylselan yl-pr opyl)-car bam ic Acid
9H-F lu or en -9-yl Meth yl Ester 9. Gen er a l P r oced u r e. To
a solution of 5 (1 g, 3.0 mmol) in CH2Cl2 (30 mL) cooled to
-78 °C were added Ti (Oi-Pr)4 (0.85 g, 3 mmol) and LiCl (1.2
g, 30 mmol). After 3 h of stirring at -78 °C, PhSeCl (0.59 g,
3.1 mmol) was added followed by Na2CO3 (0.63 g, 6 mmol).
The mixture was vigorously stirred at -78 °C for 2 h and then
10 mL of an aqueous solution of NaHCO3 10% was added. The
mixture was stirred until room temperature was reached and
the stirred for and additional 1 h. CH2Cl2 was added and the
organic layer was separated and washed three times with HCl
1 M and with water. After drying over anhydrous Na2SO4, the
solvent was evaporated and product 9 (0.97 g, 70% yield) was
isolated by column chromatography on silica gel (eluent EtAc/
light petroleum 1/6). 1H NMR (CDCl3) δ 1.32 (d, J ) 7 Hz,
3H), 3.21 (dd, J ) 9, 2 Hz, 1H), 4.20 (m, 1H), 4.50 (m, 1H),
4.70 (m, 2H), 6.50 (bd, 1H), 7.2-7.9 (m, 13H), 9.73 (d, J ) 2
Hz, 1H). 13C NMR (CDCl3) δ 22.0, 36.7, 37.9, 60.5, 73.9, 126-
129, 135.8, 136.9, 141.9, 157.8, 197.4. We were not able to
obtain a microanalitical sample of the selenyl aldehydes.
(1-Ben zyl-3-oxo-2-ph en ylselan yl-pr opyl)-car bam ic Acid
Exp er im en ta l Section
All solvents employed in this work have been previously
dried over activated molecular sieves (4 Å). When THF or ether
were used in reactions where a carbanionic species was formed,
they were distilled under N2 over Na and subsequently over
LiAlH4 just before the use. All of the reactions in homogeneous
phase were carried out under N2 athmosphere. The reactions
on solid phase were carried out in a reactor with a sinter where
the mixing was done by bubbling N2. The reactor had a cooling
jacket with inlet and outlet sidearms to allow the cooling (or
the heating) of the reaction mixture by connection with a
thermostatic circulating bath. Papain from Carica papaya (P
3125) and Na-benzoyl-L-arginine p-nitroanilide (L-BAPA) (B
3279) were purchased from Sigma. The NMR spectra were
recorded using a 300 MHz spectrometer. The HPLC were
carried out using the following general conditions: column
C-18-Lichrospher 100, 5 mm, 250 mm × 4 mm, eluent MeCN/
H2O from 80/20 to 20/80 in 35 min. Detector UV lamp, λ 254
nm, flow 1 mL/min.
1
9H-F lu or en -9-yl Meth yl Ester 10. H NMR (CDCl3) δ 2.2-
(S,E)-(3-Meth oxy-1-m eth yl-a llyl)-ca r ba m ic Acid 9H-
F lu or en -9-yl Meth yl Ester (5). Gen er a l P r oced u r e. To a
solution of 2-chloro-4,6-dimethoxy-[1,3,5]triazine (1.75 g, 10
mmol) in DME (60 mL) cooled to 0 °C was added N-methyl-
morpholine (1.12 g, 10.8 mmol). A white precipitate was
immediately formed and to this mixture was slowly added a
solution of (S)-N-Fmoc alanine (3.1 g, 10 mmol) dissolved in
DME (15 mL). After 3 h of stirring at 0 °C, the solid formed
was filtered off on Celite and the solution containing the
activated ester was transferred into a flask containing Pd/C
10% (0.20 g, 0.1 mmol of active Pd) dispersed in DME (20 mL).
The flask was connected with a buret containing H2. After
stirring at room temperature for 3 h (or until TLC analysis
showed the disappearance of the activated ester spot, Rf 0.6
with eluent AcOEt/hexane 4/6) the catalyst was filtered on
Celite and the solution added to a flask containing methoxy-
methylene-triphenyl-λ5-phosphane in THF (50 mL) (prepared
from methoxymethy-triphenyl-phpshonium chloride, 3.78 g,
12.1 mmol and NaHMDS, 13 mL of a 1 M solution in THF, 13
mmol) cooled to 0 °C. The mixture was stirred at room
temperature for 4 h and then poured into 150 mL of ether.
The eventually formed precipitate was filtered off and the
solution was washed three times with HCl 1 M and three times
with NaHCO3 5%. The ethereal layer was separated and dried
2.4 (m, 2H), 3.31 (dd, J ) 9, 3 Hz, 1H), 4.29 (m, 1H), 4.51 (m,
1H), 4.70 (m, 2H), 6.50 (bd, 1H), 7.2-7.9 (m, 18H), 9.78 (d,
J ) 3 Hz, 1H). 13C NMR (CDCl3) δ, 35.7, 37.9, 42.8, 61.5, 73.9,
126-129, 135.8, 136.9, 140.5, 141.9, 157.9, 196.4.
(1-Isop r op yl-3-oxo-2-p h en ylsela n yl-p r op yl)-ca r b a m -
ic Acid 9H-F lu or en -9-yl Meth yl Ester 11. 1H NMR (CDCl3)
δ 1.32 (d, J ) 7 Hz, 3H), 1.38 (d, J ) 7 Hz, 3H), 2.02 (m, 1H),
3.28 (dd, J ) 9, 1 Hz, 1H), 4.16 (m, 1H), 4.51 (m, 1H), 4.77 (m,
2H), 6.00 (bd, 1H), 7.20-7.90 (m, 13H), 9.65 (d, J ) 1 Hz, 1H).
13C NMR (CDCl3) δ 17.3, 17.9, 32.8, 35.7, 37.9, 61.5, 72.9, 126-
129, 135.8, 136.9, 141.9, 157.9, 195.4.
(1-Ben zyloxym et h yl-3-oxo-2-p h en ylsela n yl-p r op yl)-
ca r ba m ic Acid 9H-F lu or en -9-yl Meth yl Ester 12. 1H NMR
(CDCl3) δ 3.31 (dd, J ) 9, 2 Hz, 1H), 4.06 (m, 1H), 4.50 (m,
1H), 4.60-4.70 (m, 4H), 5.03 (d-like, 2H), 6.35 (bd, 1H), 7.20-
7.90 (m, 18H), 9.67 (d, J ) 2 Hz, 1H). 13C NMR (CDCl3) δ 39.7,
40.5, 61.5, 70.7, 71.9, 73.9, 126-129, 135.8, 136.9, 141.9, 143.8,
158.0, 193.0.
(1-ter t-Bu tylsu lfan yl-2-m eth yl-pr open yloxy)-tr im eth yl-
sila n e 22. Gen er a l P r oced u r e. Isobutyryl chloride (2 g, 9.4
mmol) was dissolved in dry CH2Cl2 (25 mL) and cooled to 0
°C. tert-Butyl thiol (0.9 g, 10 mmol) was added followed by
Et3N (1.5 g, 15 mmol). The mixture was stirred at room
temperature overnight. After hydrolytic workup, the organic

Small Ring Constrained Peptidomimetics
J . Org. Chem., Vol. 66, No. 3, 2001 703
layer was dried and the solvent distilled away using a small
Vigreaux column (the thioester boils at 60 °C at 25 mmHg).
The residue was dried over molecular sieves (4 Å). Product 18
(1.6 g, 10 mmol) was dissolved in dry THF (25 mL) at -78 °C
and to this solution was slowly added LDA (11 mL of a 1 M
solution in THF) followed, after 20 min, by TMSCl (1.3 g, 12
mmol). The mixture was allowed to reach room temperature
under stirring, the solid formed was filtered away using a
small Celite pad, and the liquor was evaporated to dryness.
To the residue was added dry ether (15 mL) to induce the
additional formation of a white precipitate that was filtered
off. The filtrate was evaporated again and the addition of ether
was repeated until no more precipitate was formed. Evapora-
tion of the solvent gave product 22 sufficiently pure to be used
in the next step: 2.12 g, 91% yield. 1H NMR (CDCl3) δ 0.08 (s,
9H), 1.45 (s, 9H), 1.76 (s, 3H), 1.80 (s, 3H). 13C NMR (CDCl3)
δ 1.0, 18.7, 31.8, 33.7, 89.3, 155.9.
135.6, 138.8, 141.9, 158.2, 200.8. After an additional PTLC:
Anal. Calcd for C35H43NO4SSe (652.75) C, 64.40; H, 6.64; N,
2.15. Found: C, 64.54; H, 6.70; N, 2.10.
2-Ben zyloxym eth yl-5-(9H-flu or en -9-ylm eth oxyca r bon -
yla m in o)-3-h yd r oxy-4-p h en ylsela n yl-h exa n eth ioic Acid
1
S-ter t-Bu tyl Ester 29. H NMR (CDCl3) δ 1.22 (d, J ) 7 Hz,
3H), 1.35 (s, 9H), 2.76 (m, 1H), 2.80 (bs, 1H), 3.20 (dd, J ) 8,
2 Hz, 1H), 3.60-3.79 (m, 3H), 4.23 (m, 1H), 4.21 (m, 1H), 4.60
(s-like, 2H), 4.77 (m, 2H), 6.17 (bd, 1H), 7.20-7.90 (m, 18H).
13C NMR (CDCl3) δ 22.3, 27.8, 37.8, 39.8, 41.1, 43.7, 60.3, 65.5,
67.8, 74.6, 77.8, 126-129, 132.6, 138.8, 141.9, 142.0, 157.8,
200.0. After an additional PTLC: Anal. Calcd for C39H43NO5-
SSe (716.79) C, 65.35; H, 6.05; N, 1.95. Found: C, 66.00; H,
6.03; N, 1.93.
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-3-h ydr oxy-
2,2-dim eth yl-6-ph en yl-4-ph en ylselan yl-h exan eth ioic Acid
1
S-ter t-Bu tyl Ester 30. H NMR (CDCl3) δ 1.20 (s, 3H), 1.22
(s, 3H), 1.39 (s, 9H), 2.20 (m, 1H), 2.31 (m, 1H), 2.89 (bs, 1H),
3.06 (dd, J ) 6, 4 Hz, 1H), 3.54 (bd, 1H), 4.12 (m, 1H), 4.21
(m, 1H), 4.77 (m, 2H), 6.75 (bd, 1H), 7.20-8.00 (m, 18 H). 13C
NMR (CDCl3) δ 17.3, 18.5, 29.0, 36.8, 37.9, 38.9, 59.0, 61.8,
67.6, 69.9, 73.0, 126-129, 136.7, 139.9, 141.0, 158.0, 203.3.
After an additional PTLC: Anal. Calcd for C39H43NO4SSe
(700.79) C, 66.84; H, 6.18; N, 2.00. Found: C, 66.76; H, 6.13;
N, 2.04.
(ter t-Bu tylsu lfan yl-cycloh exyliden e-m eth oxy)-tr im eth -
yl-sila n e 23. 1H NMR (CDCl3) δ 0.08 (s, 9H), 1.40 (s, 9H), 1.6-
2.0 (m, 6H), 2.2-2.4 (m, 4H). 13C NMR (CDCl3) δ 0.9, 26.6,
28.9, 31.9, 34.7, 35.0, 100.7, 153.0.
(1-ter t-Bu tylsu lfan yl-4-m eth yl-pen t-1-en yloxy)-tr im eth -
1
yl-sila n e 24. H NMR (CDCl3) δ 0.08 (s, 9H), 1.08 (d, J ) 7
Hz, 3H), 1.12 (d, J ) 7 Hz, 3H), 1.40 (s, 9H), 1.80-2.07 (m,
3H), 5.59 (dd, J 7, 4 Hz, 1H). 13C NMR (CDCl3) δ 0.9, 21.2,
22.0, 28.1, 30.8, 31.8, 33.6, 89.2, 161.9.
1-[3-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-1-h ydr ox-
y-4-p h en yl-2-p h en ylsela n yl-b u t yl]-cycloh exa n eca r b o-
(3-Ben zyloxy-1-ter t-b u t ylsu lfa n yl-p r op en yloxy)-t r i-
m eth yl-sila n e 25. 1H NMR (CDCl3) δ 0.09 (s, 9H), 1.40 (s,
9H), 4.04 (d, J ) 7 Hz, 2H), 4.93 (s, 2H), 5.12 (t, J ) 7 Hz,
1H), 7.20 (s-like, 5H). 13C NMR (CDCl3) δ 0.7, 31.8, 34.0, 60.8,
76.8, 89.9, 126-127, 139.0, 161.4.
1
th ioic Acid S-ter t-Bu tyl Ester 31. H NMR (CDCl3) δ 1.40
(s, 9H), 1.62-2.40 (m, 12H), 2.80 (bs, 1H), 3.10 (dd, J ) 10, 4
Hz, 1H), 3.55 (bd, 1H), 4.10 (m, 1H), 4.20 (m, 1H), 4.77 (m,
2H), 6.15 (bd, 1H), 7.20-8.00 (m, 18 H). 13C NMR (CDCl3) δ,
24.8, 26.8, 29.0, 30.8, 35.8, 37.7, 38.5, 59.5, 62.8, 67.4, 69.0,
73.2, 126-129, 136.8, 139.2, 140.0, 157.6, 202.9. After an
additional PTLC: Anal. Calcd for C42H47NO4SSe (740.85) C,
68.09; H, 6.39; N, 1.89. Found: C, 68.12; H, 6.38; N, 1.90.
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-3-h ydr oxy-
2,2-d im eth yl-4-p h en ylsela n yl-h exa n eth ioic Acid S-ter t-
Bu tyl Ester 26. Gen er a l P r oced u r e. To a solution of
aldehyde 9 (1 g, 2.1 mmol) dissolved in dry CH2Cl2 (25 mL)
cooled to -78 °C was added BF3OEt2 (0.61 g, 4.3 mmol) slowly
with a syringe and the mixture stirred at -78 °C for 30 min.
Compound 22 (1.21 g, 5.25 mmol) was added and the mixture
was stirred at -78 °C for 2 h. Then the temperature was raised
to -20° and the mixture was stirred at this temperature for 2
h. A pH 7 phosphate buffer (10 mL) was added and the mixture
was warmed to room temperature. Additional CH2Cl2 (50 mL)
was added and the organic layer was washed several times
with saturated solutions of NH4Cl, NaHCO3, and brine. After
evaporation of the solvent, product 26 (0.9 g, 69% yield) was
isolated by column chromatography on silica gel (eluent EtAc/
light petroleum 1/2). 1H NMR (CDCl3) δ 1.12 (d, J ) 7 Hz,
3H), 1.28 (s, 3H), 1.30 (s, 3H), 1.35 (s, 9H), 2.80 (bs, 1H), 3.20
(dd, J ) 6, 2 Hz, 1H), 3.73 (bd, 1H), 4.03 (m, 1H), 4.21 (m,
1H), 4.74 (m, 2H), 6.07 (bd, 1H), 7.20-7.90 (m, 13H). 13C NMR
(CDCl3) δ 14.6, 16.7, 17.9, 25.8, 38.8, 39.8, 41.1, 43.7, 63.3,
69.8, 75.6, 126-129, 132.6, 138.8, 141.9, 157.2, 200.3. After
an additional PTLC: Anal. Calcd for C33H39NO4SSe (624.69)
C, 63.45; H, 6.29; N, 2.24. Found: C, 63.98; H, 6.31; N, 2.19.
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-3-h ydr oxy-
2-isobu tyl-6-p h en yl-4-p h en ylsela n yl-h exa n eth ioic Acid
1
S-ter t-Bu tyl Ester 32. H NMR (CDCl3) δ 1.20 (d, J ) 7 Hz,
3H), 1.24 (d, J ) 7 Hz), 1.39 (s, 9H), 1.90-2.30 (m, 6H), 2.69
(bs, 1H), 3.09 (dd, J ) 7, 2 Hz 1H), 3.64 (m, 1H), 4.09 (m, 1H),
4.21 (m, 1H), 4.77 (m, 2H), 6.75 (bd, 1H), 7.20-8.00 (m, 18
H). 13C NMR (CDCl3) δ 17.3, 18.5, 26.8, 29.0, 34.8, 36.8, 37.9,
38.9, 48.7, 59.0, 67.6, 69.9, 73.0, 126-129, 136.7, 139.9, 141.0,
158.0, 203.3. After an additional PTLC: Anal. Calcd for C41H47
-
NO4SSe (728.84) C, 67.56; H, 6.50; N, 1.92. Found: C, 67.49;
H, 6.45; N, 1.99.
2-Ben zyloxym eth yl-5-(9H-flu or en -9-ylm eth oxyca r bon -
yla m in o)-3-h yd r oxy-6-p h en yl-4-p h en ylsela n yl-h exa n e-
th ioic Acid S-ter t-Bu tyl Ester 33. 1H NMR (CDCl3) 1.38
(s, 9H), 1.90-2.30 (m, 2H), 2.50 (bs, 1H), 3.08-19 (m, 2H),
3.60 (m, 1H), 4.09 (m, 1H), 4.21-4.40 (m, 5H), 4.77 (m, 2H),
6.70 (bd, 1H), 7.20-8.00 (m, 23 H). 13C NMR (CDCl3) δ, 26.8,
29.0, 34.8, 36.8, 37.9, 38.9, 47.7, 50.8, 58.8, 59.0, 67.6, 69.9,
73.0, 126-129(very intense), 136.8, 139.9, 141.0, 146.8, 158.1,
200.6. After an additional PTLC: Anal. Calcd for C45H47NO5-
SSe (792.88) C, 68.17; H, 5.97; N, 1.77. Found: C, 68.00; H,
5.77; N, 1.78.
1-[3-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-1-h ydr ox-
y-2-p h en ylsela n yl-bu tyl]-cycloh exa -n eca r both ioic Acid
1
S-ter t-Bu tyl Ester 27. H NMR (CDCl3) δ 1.18 (d, J ) 7 Hz,
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-3-h ydr oxy-
2,2,6-tr im eth yl-4-p h en ylsela n yl-h ep ta n eth ioic Acid S-
3H), 1.35 (s, 9H), 1.61-2.01 (m, 10H), 2.81 (bs, 1H), 3.32 (dd,
J ) 7, 6 Hz, 1H), 3.70 (bd, 1H), 4.10 (m, 1H), 4.21 (m, 1H),
4.70 (m, 2H), 6.17 (bd, 1H), 7.20-7.90 (m, 13H). 13C NMR
(CDCl3) δ 14.0, 22.6, 26.7, 29.9, 25.8, 38.8, 38.8, 42.1, 43.8,
61.3, 67.8, 75.9, 126-129, 131.6, 139.8, 141.9, 158.0, 200.7.
After an additional PTLC: Anal. Calcd for C36H43NO4SSe
(664.76) C, 65.04; H, 6.52; N, 2.11. Found: C, 65.00; H, 6.50;
N, 2.10.
1
ter t-Bu tyl Ester 34. H NMR (CDCl3) δ 1.18 (d, J ) 7 Hz),
1.21 (d, J ) 7 Hz, 3H), 1.30 (bs, 6H), 1.35 (s, 9H), 1.90 (m,
1H), 2.64 (bs, 1H), 3.21 (dd, J ) 7, 2 Hz, 1H), 3.65 (m, 1H),
4.10 (m, 1H), 4.32 (m, 1H), 4.78 (m, 2H), 6.70 (bd, 1H), 7.20-
7.80 (m, 13 H). Anal. Calcd for C35H43NO4SSe (652.75): C,
64.40; H, 6.64; N, 2.15. Found: C, 64.30; H, 6.70; N, 2.10.
1-[3-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-1-h ydr ox-
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-3-h ydr oxy-
2-isobu tyl-4-p h en ylsela n yl-h exa n eth ioic Acid S-ter t-Bu -
tyl Ester 28. H NMR (CDCl3) δ 1.03 (d, J ) 7 Hz, 3H), 1.10
(d, J ) 7 Hz, 3H), 1.22 (d, J ) 7 Hz, 3H), 1.35 (s, 9H), 1.71-
2.22 (m, 4 H), 2.90 (bs, 1H), 3.30 (dd, J ) 8, 2 Hz, 1H), 3.65
(m, 1H), 4.12 (m, 1H), 4.21 (m, 1H), 4.75 (m, 2H), 6.07 (bd,
1H), 7.20-7.90 (m, 13H). 13C NMR (CDCl3) δ 19.6, 22.7, 22.9,
25.8, 29.0, 32.8, 37.9, 38.8, 43.8, 44.1, 56.7, 75.6, 126-129,
y-4-m et h yl-2-p h en ylsela n yl-p en t yl]-cycloh exa n eca r b o-
th ioic Acid S-ter t-Bu tyl Ester 35. H NMR (CDCl3) δ 1.16
(d, J ) 7 Hz, 3H), 1.21 (d, J ) 7 Hz, 3H), 1.35 (s, 9H), 1.4-
1.90 (m, 11H), 2.60 (bs, 1H), 3.20 (dd, J ) 7, 3 Hz, 1H), 3.40
(m, 1H), 4.11 (m, 1H), 4.30 (m, 1H), 4.78 (m, 2H), 6.70 (bd,
1
1
1H), 7.2-7.8 (m, 13H). Anal. Calcd for
C38H47NO4SSe
(692.81): C, 65.88; H, 6.84; N, 2.02; Found: C, 65.93; H, 6.80;
N, 2.09.

704 J . Org. Chem., Vol. 66, No. 3, 2001
Demarcus et al.
6-Ben zyloxy-5-(9H-flu or en -9-ylm eth oxycar bon ylam in o)-
200.0. Anal. Calcd for C33H37NO4S (543.72): C, 72.90; H, 6.86;
N, 2.58. Found: C, 72.67; H, 6.78; N, 2.45.
3-h yd r oxy-2,2-d im et h yl-4-p h en ylsela n yl-h exa n et h ioic
1
Acid S-ter t-Bu tyl Ester 36. H NMR (CDCl3) δ 1.25 (s, 3H),
1-{3-[1-(9H -F lu or e n -9-ylm e t h oxyca r b on yla m in o)-2-
p h en yl-eth yl]-oxir a n yl}-cycloh exa n eca r both ioic Acid S-
1.30 (s, 3H), 1.38 (s, 9H), 2.60 (bs, 1H), 3.19 (dd, J ) 7 and 4
Hz, 1H), 3.60-4.20 (m, 6H,) 4.40 (m, 1H,), 4.77 (m, 2H), 6.70
(bd, 1H), 7.20-8.01 (m, 18H). After an additional PTLC: Anal.
Calcd for C40H45NO5SSe (730.82) C, 65.74; H, 6.21; N, 1.92.
Found: C, 65.60; H, 6.20; N, 1.90.
1
ter t-Bu tyl Ester 43. H NMR (CDCl3) δ 1.33 (s, 9H), 1.41-
1.95 (m, 10H), 2.43 (m, 1H), 2.63 (d, J ) 3 Hz, 1H), 2.78 (m,
1H), 3.10 (dd, J ) 8, 3 Hz, 1H), 4.26 (m, 1H), 4.50 (m, 1H),
4.77 (m, 2H), 6.21 (bs, 1H), 7.20-7.80 (m, 13H). 13C NMR
(CDCl3) δ 18.2, 29.2, 27.4, 28.3, 38.9, 38.0, 45.3, 52.7, 57.9,
58.9, 65.8, 75.0, 125.6, 126.5, 127.8, 128.9, 136.5, 139.9, 141.3,
159.9, 201.9. Anal. Calcd for C36H41NO4S (583.78): C, 74.07;
H, 7.08; N, 2.40. Found: C, 74.20; H, 7.18; N, 2.46.
1-[4-Ben zyloxy-3-(9H-flu or en -9-ylm eth oxycar bon ylam i-
n o)-1-h yd r oxy-2-p h en ylsela n yl-b u t yl]-cycloh exa n eca r -
both ioic Acid S-ter t-Bu tyl Ester 37. 1H NMR (CDCl3) δ 1.40
(s, 9H), 1.80-2.20 (m, 10H), 2.60 (bs, 1H), 3.25 (dd, J ) 7 and
4 Hz, 1H), 3.60-4.20 (m, 7H) 4.77 (m, 2H), 6.70 (bd, 1H), 7.20-
8.00 (m, 18H). After an additional PTLC: Anal. Calcd for
2-{3-[1-(9H -F lu or e n -9-ylm e t h oxyca r b on yla m in o)-2-
p h en yl-eth yl]-oxir a n yl}-4-m eth yl-p en ta n eth ioic Acid S-
ter t-Bu tyl Ester 44. 1H NMR (CDCl3) δ 1.11 (d, J ) 7 Hz,
3H), 1.22 (d, J ) 7 Hz, 3H), 1.40 (s, 9H), 1.54-1.80 (m, 3H),
2.50-2.80 (m, 3H), 2.70 (dd, J ) 8, 2 Hz 1H), 3.11 (dd J ) 9,
2 Hz, 1H), 4.20 (m, 1H), 4.43 (m, 1H), 4.71 (m, 2H), 6.34 (bs,
1H), 7.20-7.80 (m, 13H). 13C NMR (CDCl3) δ, 23.5, 24.7, 26.6,
29.9, 36.7, 38.0, 39.4, 44.0, 49.9, 58.6, 59.9, 65.2, 74.2, 125.7,
126.2, 127.8, 128.3, 136.2, 140.1, 141.1, 157.8, 201.7. Anal.
Calcd for C35H41NO4S (571.77): C, 73.52; H, 7.23; N, 2.45.
Found: C. 73.65; H, 7.30; N, 2.40
C
43H49NO5SSe (770.88) C, 67.00; H, 6.41; N, 1.82. Found: C,
66.97; H, 6.37; N, 1.80.
2-{3-[1-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-eth yl]-
oxir a n yl}-2-m eth yl-th iop r op ion ic Acid S-ter t-Bu tyl Es-
ter 38. Gen er a l P r oced u r e. To a solution of compound 26
(0.62 g, 1 mmol) dissolved in MeOH (10 mL, cooled to -15 °C,
was added K2CO3 (0.2 g). After 10 min of stirring at -15 °C,
MCPBA (0.34 g of a 60% active sample, 1.2 mmol) was added
and the mixture was stirred for 30 min at -15 °C. Water was
added followed by diethyl ether. The ethereal layer was
separated and washed with a 10% solution of Na2S2O4 and a
10% solution of NH4Cl. After separation and drying (Na2SO4)
the solvent was evaporated and product 38 was isolated by
column chromatography on silica gel (eluent light petroleum/
EtOAc 3/1): 0.34 g, 74% yield.1H NMR (CDCl3) δ 1.18 (s, 3H),
1.21 (s, 3H), 1.30 (d, J ) 7 Hz, 3H), 1.41 (s, 9H), 2.33 (d, J )
2 Hz, 1H), 3.03 (dd, J ) 9, 2 Hz, 1H), 4.10 (m, 1H), 4.40 (m,
1H), 4.75 (m, 2H), 6.7 (bs, 1H), 7.2-7.8 (m, 8H). 13C NMR
(CDCl3) δ 16.3, 19.2, 20.1, 26.3, 37.9, 39.9, 44.3, 53.7, 61.0,
63.2, 75.0, 126.5, 127.9, 128.9, 136.3, 141.7, 157.9, 201.0. Anal.
Calcd for C27H33NO4S (467.62): C, 69.35; H, 7.11; N, 3.00.
Found: C. 69.76; H, 7.31; N, 3.09
1-{3-[1-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-eth yl]-
oxir a n yl}-cycloh exa n eca r both ioic Acid S-ter t-Bu tyl Es-
ter 39. 1H NMR (CDCl3) δ 1.25 (d, J ) 7 Hz, 3H), 1.33 (s, 9H),
1.4-1.9 (m, 10H), 2.63 (d, J ) 3 Hz, 1H), 3.07 (dd, J ) 8, 3
Hz, 1H), 4.16 (m, 1H), 4.51 (m, 1H), 4.71 (m, 2H), 6.71 (bs,
1H), 7.02-7.84 (m, 8H). 13C NMR (CDCl3) δ 16.2, 29.2, 21.5,
27.4, 28.3, 37.9, 38.1, 44.3, 51.7, 58.6, 66.8, 75.2, 126.5, 127.8,
128.9, 136.5, 141.3, 159.9, 201.6. Anal. Calcd for C30H37NO4S
(507.69) C. 70.97; H, 7.35; N, 2.76. Found: C. 70.67; H, 7.30;
N, 2.72.
3-Ben zyloxy-2-{3-[1-(9H-flu or en -9-ylm eth oxyca r bon yl-
a m in o)-2-p h en yl-eth yl]-oxir a n yl}-th iop r op ion ic Acid S-
1
ter t-Bu tyl Ester 45. H NMR (CDCl3) δ 1.49 (s, 9H), 2.60-
2.80 (m, 4H), 3.10 (dd, J ) 8, 2 Hz, 1H), 3.76 (d-like, 2H), 4.09
(m, 1H), 4.50-4.70 (m, 5H), 6.13 (bs, 1H), 7.20-7.90 (m, 18H).
13C NMR (CDCl3) δ, 26.0, 38.2, 39.0, 42.4, 51.6, 52.7, 58.7, 64.8,
67.9, 71.2, 77.1, 125.7, 126.5, 127.6, 127.9, 128.4, 128.9, 136.5,
137.2, 139.9, 141.9, 159.6, 201.1. Anal. Calcd for C39H41NO5S
(635.81): C. 73.67; H, 6.50; N, 2.20. Found: C. 73.6O; H, 6.40;
N, 2.12.
2-{3-[1-(9H -F lu or e n -9-ylm e t h oxyca r b on yla m in o)-2-
m eth yl-p r op yl]-oxir a n yl}-2-m eth yl-th iop r op ion ic Acid
1
S-ter t-Bu tyl Ester 46. H NMR (CDCl3) δ 1.13 (s, 3H), 1.22
(s, 3H), 1.25-1.28 (m, 6H), 1.40 (s, 9H), 1.93 (m, 1H), 2.39 (d,
J ) 2 Hz, 1H), 3.13 (dd, J ) 8, 2 Hz, 1H), 4.21 (m, 1H), 4.43
(m, 1H), 4.77 (m, 2H), 6.47 (bs, 1H), 7.20-7.80 (m, 8H). 13C
NMR (CDCl3) δ 16.3, 18.7, 19.8, 20.0, 25.3, 33.9, 36.7, 38.9,
44.8, 52.7, 60.0, 66.2, 75.7, 126.5, 127.9, 128.9, 136.3, 141.7,
157.9, 200.8. Anal. Calcd for C29H37NO4S (495.67): C. 70.27;
H, 7.52; N, 2.83. Found: C. 70.17; H, 7.58; N, 2.80.
1-{3-[1-(9H -F lu or e n -9-ylm e t h oxyca r b on yla m in o)-2-
m eth yl-p r op yl]-oxir a n yl}-cycloh exa n e-ca r both ioic Acid
1
S-ter t-Bu tyl Ester 47. H NMR (CDCl3) δ 1.20 (d, J ) 7 Hz,
9H-F lu or en -9-ylm eth oxyca r bon yla m in o)-eth yl]-oxir a -
n yl}-4-m eth yl-p en ta n eth ioic Acid S-ter t-Bu tyl Ester 40.
1H NMR (CDCl3) δ 1.10 (d, J ) 7 Hz, 3H), 1.20 (d, J ) 7 Hz,
3H), 1.36 (d, J ) 7 Hz, 3H), 1.40 (s, 9H), 1.54-1.80 (m, 3H),
2.60 (m, 1H), 2.78 (dd, J ) 8, 2 Hz, 1H), 3.18 (dd J ) 9, 2 Hz,
1H), 4.10 (m, 1H), 4.40 (m, 1H), 4.70 (m, 2H), 6.18 (bs, 1H),
7.20-7.80 (m, 8H). 13C NMR (CDCl3) δ 16.7, 22.5, 23.7, 25.6,
29.0, 36.3, 37.0, 39.0, 45.0, 49.4, 54.6, 64.2, 73.2, 126.2, 127.8,
128.3, 136.2, 140.1, 157.8, 200.7. Anal. Calcd for C29H37NO4S
(495.67): C, 70.27; H, 7.52; N, 2.83. Found: C, 70.05; H, 7.42;
N, 2.68
3H), 1.24 (d, J ) 7 Hz, 3H), 1.33 (s, 9H), 1.40-1.90 (m, 11H),
2.65 (d, J ) 3 Hz, 1H), 3.09 (dd, J ) 8, 3 Hz, 1H), 4.21 (m,
1H), 4.65 (m, 1H), 4.74 (m, 2H), 6.79 (bs, 1H), 7.20-7.80 (m,
8H). 13C NMR (CDCl3) δ 17.6, 18.2, 19.9, 29.2, 20.5, 26.4, 28.4,
33.4, 37.0, 39.1, 45.3, 50.7, 58.9, 67.0, 77.2, 126.5, 127.8, 128.9,
136.5, 141.3, 158.9, 200.1. Anal. Calcd for
C32H41NO4S
(535.74): C. 71.74; H, 7.71; N, 2.61. Found: C. 71.54; H, 7.67;
N, 2.56.
2-{3-[2-Ben zyloxy-1-(9H-flu or en -9-ylm eth oxyca r bon yl-
a m in o)-eth yl]-oxir a n yl}-2-m eth yl-th iop r op ion ic Acid S-
1
ter t-Bu tyl Ester 48. H NMR (CDCl3) δ 1.19 (s, 3H), 1.21 (s,
3-Ben zyloxy-2-{3-[1-(9H-flu or en -9-ylm eth oxyca r bon yl-
a m in o)-eth yl]-oxir a n yl}-th iop r op ion ic Acid S-ter t-Bu tyl
3H), 1.40 (s, 9H), 2.31 (d, J ) 2 Hz, 1H), 3.10 (dd, J ) 9, 2 Hz,
1H), 3.95-4.10 (m, 5H), 4.42 (m, 1H), 4.75 (m, 2H), 6.32 (bs,
1H), 7.20-7.80 (m, 13H). 13C NMR (CDCl3) δ 17.3, 19.0, 26.7,
38.9, 38.9, 44.3, 52.7, 60.0, 62.2, 70.3, 74.5, 76.0, 125.5, 126.5,
127.9, 128.9, 136.3, 140.9, 141.7, 159.9, 199.6. Anal. Calcd for
1
Ester 41. H NMR (CDCl3) δ 1.30 (d, J ) 7 Hz, 3H), 1.41 (s,
9H), 2.71-2.85 (m, 2H), 3.15 (dd, J ) 8, 2 Hz, 1H), 3.70 (d-
like, 2H), 4.08 (m, 1H), 4.50-4.80 (m, 5H), 6.38 (bs, 1H), 7.20-
7.90 (m, 13H). 13C NMR (CDCl3) δ 16.3, 27.0, 37.2, 39.9, 43.4,
50.6, 53.7, 64.0, 66.9, 73.2, 75.1, 126.5, 127.6, 127.9, 128.4,
C
34H39NO5S (573.74): C. 71.18; H, 6.85; N, 2.44. Found: C.
71.22; H, 6.84; N, 2.44.
128.9, 136.5, 137.2, 141.9, 159.6, 200.1. Anal. Calcd for C33H37
-
NO5S (559.72): C, 70.81; H, 6.66; N, 2.50. Found: C, 70.58;
H, 6.60; N, 2.59.
1-{3-[2-Ben zyloxy-1-(9H-flu or en -9-ylm eth oxyca r bon yl-
a m in o)-eth yl]-oxir a n yl}-cycloh exa n eca r bo-th ioic Acid
S-ter t-Bu tyl Ester 49. 1H NMR (CDCl3) δ 1.43 (s, 9H), 1.40-
1.95 (m, 10H), 2.60 (d, J ) 3 Hz, 1H), 3.00 (dd, J ) 8, 3 Hz,
1H), 4.10-4.61 (m, 6H), 4.76 (m, 2H), 6.65 (bs, 1H), 7.20-7.80
(m, 13H). 13C NMR (CDCl3) δ 16.2, 20.5, 26.4, 28.9, 36.9, 39.8,
42.3, 50.0, 55.6, 61.8, 69.8, 76.8, 77.7, 125.7, 126.5, 127.8, 128.9,
136.5, 141.0, 141.3, 159.0, 200.6. Anal. Calcd for C37H43NO5S
(613.81): C. 72.40; H, 7.06; N, 2.28. Found: C. 72.50; H, 7.11;
N, 2.21.
2-{3-[1-(9H -F lu or e n -9-ylm e t h oxyca r b on yla m in o)-2-
p h en yl-eth yl]-oxir a n yl}-2-m eth yl-th iop r op ion ic Acid S-
1
ter t-Bu tyl Ester 42. H NMR (CDCl3) δ 1.19 (s, 3H), 1.22 (s,
3H), 1.45 (s, 9H), 2.33 (d, J ) 2 Hz, 1H), 2.45 (m, 1H), 2.59
(m, 1H), 3.13 (dd, J ) 9, 2 Hz, 1H), 4.11 (m, 1H), 4.46 (m, 1H),
4.75 (m, 2H), 6.12 (bs, 1H), 7.20-7.80 (m, 13H). 13C NMR
(CDCl3) δ 16.3, 26.3, 38.9, 39.9, 47.3, 53.0, 54.5, 61.0, 64.2,
75.9, 125.3, 126.4, 126.5, 127.9, 128.9, 136.3, 141.7, 157.9,

Small Ring Constrained Peptidomimetics
J . Org. Chem., Vol. 66, No. 3, 2001 705
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-4-h ydr oxy-
2-isobu tyl-h ex-2-en oic Acid Meth yl Ester 50. To a solution
of compound 40 (100 mg, 0.2 mmol) dissolved in dry THF (1
mL) was added 2 mL of a 0.2 M solution of NaOH and the
mixture stirred at room temperature until the TLC analysis
showed the disappearance of the starting material. Ethyl
acetate (10 mL) was added, the mixture was cooled to 0 °C,
and HCl 0.5 M was added to red litmus paper. The ethereal
layer was separed and dried over anhydrous Na2SO4 and the
solvent was evaporated. THF (5 mL) was added, the solution
was cooled to -15 °C, and a solution containing diazomethane
in diethyl ether (prepared using the Diazald kit) was added
until persistence of the yellow-green solution. The excess of
diazomethane was destroyed adding one drop of acetic acid
and a saturated solution of NaHCO3 was added. The mixture
was warmed to room temperature and the organic layer
separated and washed with water and brine. After drying (Na2-
SO4), to this solution was added TBAF (52 mg, 0.2 mmol) and
the mixture was heated gently at 35 °C for 4 h. The solvent
was evaporated and product 50 was isolated by PTLC (eluent
light petroleum/EtAc 4/1): 35 mg, 80% yield. 1H NMR (CDCl3)
δ 1.10 (d, J ) 7 Hz, 3H), 1.21 (d, J ) 7 Hz, 3H), 1.31 (d, J )
8 Hz, 3H), 1.80 (m, 1H), 1.92 (AB part of an ABX system, 2H),
3.01(bs, 1H), 3.51 (s, 3H), 3.98 (m, 1H), 4.01 (m, 1H), 4.41 (m,
1H), 4.70 (m, 2H), 6.09 (bs, 1H), 6.89 (d, J ) 6 Hz, 1H), 7.20-
7.80 (m, 8H). 13C NMR (CDCl3) δ 16.2, 22.7, 23.4, 25.7, 36.2,
39.2, 50.2, 56.8, 71.0, 74.2, 126.5, 127.9, 128.9, 131.4, 136.5,
139.8, 143.7, 157.9, 169.9. Anal. Calcd for C26H31NO5 (437.53):
C. 71.37; H, 7.14; N, 3.20. Found 71.27; H, 7.10; N, 3.23
2-Ben zyloxym eth yl-5-(9H-flu or en -9-ylm eth oxyca r bon -
yla m in o)-4-h yd r oxy-h ex-2-en oic Acid Meth yl Ester 51. 1H
NMR (CDCl3) δ 1.27 (d, J ) 7 Hz, 3H), 3.00 (bs, 1H), 3.65 (s,
3H), 3.89 (m, 1H), 4.06 (AB system, 2H), 4.30-4.70 (m, 6H),
6.60 (bs, 1H), 7.02 (d, J ) 9 Hz, 1H), 7.20-7.90 (m, 13H). 13C
NMR (CDCl3) δ 15.2, 37.6, 50.8, 56.6, 67.9, 73.2, 74.7, 76.2,
126.5, 127.6, 127.9, 128.4, 128.9, 131.2, 133.3, 136.5, 137.2,
141.9, 157.6, 166.7. Anal. Calcd for C30H31NO6 (501.57): C.
71.84; H, 6.23; N, 2.79. Found: C. 71.75; H, 6.20; N, 2.81.
5-(9H-Flu or en -9-ylm eth oxycar bon ylam in o)-4-h ydr oxy-
2-isobu tyl-6-p h en yl-h ex-2-en oic Acid Meth yl Ester 52. 1H
NMR (CDCl3) δ 1.10 (d, J ) 7 Hz, 3H), 1.16 (d, J ) 7 Hz, 3H),
1.80 (m, 1H), 1.96 (m, 2H), 2.76 (m, 1H), 2.86 (m, 1H), 3.01
(bs, 1H), 3.60 (s, 3H), 4.00 (m, 1H), 4.30-4.70 (m, 5H), 6.82
(d, J ) 8 Hz, 1H), 7.20-7.80 (m, 13H). 13C NMR (CDCl3) δ
22.6, 23.5, 25.8, 35.7, 36.9, 37.9, 50.8, 57.2, 71.2, 73.0, 125.8,
126.5, 127.9, 128.6, 131.4, 134.4, 135.2, 138.8, 141.6, 157.2,
165.0. Anal. Calcd for C32H35NO5 (513.62): C. 74.83; H, 6.87;
N, 2.73. Found: C. 74.76; H, 6.90; N, 2.70.
was added and the solvent evaporated. The residue was
dissolved in MeOH and passed through a short column filled
with Amberlite X. The solvent was evaporated and product
57 isolated by crystallization from ether: 58 mg, 70% yield.
1H NMR (DMSO-d6, 45 °C) δ 1.26 (d, J ) 7 Hz, 3H), 1.29 (s,
3H), 1.32 (s, 3H), 1.43 (d, J ) 6 Hz, 3H), 2.63 (d, J ) 2 Hz,
1H), 3.04 (dd, J ) 8, 2 Hz, 1H), 4.08 (m, 1H), 4.45-4.75 (m,
4H), 7.20-7.80 (m, 9H), 8.2 (bs, 2H). 13C NMR (DMSO-d6, 45
°C) δ 15.3, 17.6, 18.9, 19.8, 37.9, 42.0, 44.9, 54.6, 60.7, 63.6,
73.0, 126.5, 127.9, 128.9, 136.2, 141.9, 157.2, 177.0, 184.6.
2-(2-{3-[1-(2-Am in o-3-m eth yl-bu tyr yla m in o)-eth yl]-ox-
ir a n yl}-2-m et h yl-p r op ion yla m in o)-p r op ion ic Acid 59.
Resin 56 (250 mg) was submitted to an additional series of
deprotection (piperidine 15% in DMF) and coupling with Fmoc-
Val-OH, DMTMM and DIPEA in NMP (check with Kaiser
test). Finally the resin was deprotected with piperidine in DMF
and cleaved as previously described to give, after crystalliza-
1
tion from acetone, compound 59: 38 mg, 61% yield. H NMR
(DMSO-d6, 45 °C) δ 1.10 (d, J ) 7 Hz, 3H), 1.15 (d, J ) 7 Hz,
3H), 1.25 (s, 3H), 1.30 (s, 3H), 1.35 (d, J ) 6 Hz, 3H), 1.53 (d,
J ) 7 Hz, 3H), 2.05 (m, 1H), 2.60 (d, J ) 2 Hz, 1H), 3.10 (dd,
J ) 8, 2 Hz, 1H), 3.56 (m, 1H), 4.06 (m, 1H), 4.60 (m, 1H),
7.6-8.0 (series of broad signals, approximately 5 exchangeable
H). ES-MS (m/z) 344 (M+ + 1).
2-[3-(1-Am in o-et h yl)-oxir a n yl]-isob u t yr a m id e 64.
A
thiol resin (derived from a Merrifield resin as described by
Kobajashi, 250 mg of an approximately 1 mmol/g, 0.25 mmol)
was dispersed in CH2Cl2 and isobutyryl chloride (80 mg, 0.75
mmol) was added followed by DIPEA (1 mL). The mixture was
stirred (bubbling with N2) for 2 h (until negative Elman test).
The resin was rinsed several time with CH2Cl2, DMF, CH2Cl2
and THF. Finally the resin was dispersed in THF and cooled
to -40°. A solution of LDA (50 mL of a 1 M solution in THF)
was added followed by Me3SiCl (80 mg, 0.75 mmol). The
mixture was warmed to room temperature and then rinsed
with THF, NMP, pyridine and CH2Cl2. The mixture was cooled
again to -40 °C and aldehyde 9 (0.23 g, 0.5 mmol) was added
followed by TMSOTf (0.111 gr, 0.5 mmol). The mixture was
stirred (bubbling N2) for 5 h at -40° and 2 h at 0 °C (until the
TCT-Ali-R test resulted positive). The resin was rinsed with
CH2Cl2 and DMF and then warmed to room temperature. After
additional treatments with CH2Cl2, pyridine and CH2Cl2, the
resin was dried and transferred in a flask having a small
mechanical stirrer. MeOH (2 mL) was added, the flask was
cooled to 0 °C, and under stirring, Na2CO3 (0.05 g) was added
followed by MCPBA (85 mg of a 60% active sample, 0.3 mmol).
The mixture was stirred at 0 °C for 3 h (until the TCT-AliR
test was negative). The mixture was transferred again in the
reactor equipped with the sinter and the resin was rinsed with
MeOH, DMF, Pyridine and DMF. The Fmoc was deprotected
under standard conditions (Kaiser test positive) and the resin
was transferred in a sealed flask containing MeOH and NH3
(obtained bubbling dry NH3 in MeOH). The flask was gently
heated fro 1 h at 50°. The solution was analyzed at the HPLC
that showed the presence of several products. After filtration
of the residue resin, the solvent was evaporated and the
product was dissolved in MeCN and precipitated with diethyl
ether. The solid (26 mg, 61% yield overall) was analyzed at
the HPLC showing at least 3 peaks where one of the three
was about 80%. ES-MS showed that this peak had the expected
mass (m/z 173 (M+ + 1)). Product 64 was finally purified by
5-(9H-F lu or en -9-ylm eth oxyca r bon yla m in o)-2-isobu tyl-
4-(3,3,3-tr iflu or o-2-m eth oxy-2-p h en yl-p r op ion yloxy)-h ex-
2-en oic Acid Meth yl Ester 53. Gen er a l P r oced u r e. Prod-
uct 50 (20 mg, 0.04 mmol) was dissolved in benzene (1 mL)
and (R)-MTPA-Cl (11 mg, 0.04 mmol) was added followed by
pyridine (0.5 mL). The mixture was stirred for 12 h and then
filtered through a small pad of silica gel. The solvent was
evaporated and the residue submitted to high vacuum (0.01
mmHg) for 3 h. The residue was taken up in CDCl3 and
submitted to NMR analysis. The attribution of compounds 53-
1
55 was carried out by inspection of the H NMR signal of the
methoxy (δ ) 3.24), the 13C signal of CF3 (δ ) 120.3), and the
19F resonance (δ ) 11.0).
1
PTLC (eluent EtOH/CHCl3 1/7). H NMR (DMSO-d6, 45 °C) δ
2-(2-{3-[1-(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)-
et h yl]-oxir a n yl}-2-m et h yl-p r op ion yla m in o)-p r op ion ic
Acid 57. To Fmoc-Ala loaded onto a SASRIN-type resin (250
mg of a 0.75 mmol/g loaded resin, 0.18 mmol) previously
swelled in DMF was added piperidine in DMF (5 mL of a 15%
solution) and the mixture was bubbled gently with N2 until a
positive Kaiser test. The resin was rinsed several times with
DMF and NMP (N-methyl pyrrolidinone) and then compound
38 (168 mg, 0.36 mmol) was added followed by DMTMM (87
mg, 0.36 mmol) and DIPEA (0.2 mL). The mixture was stirred
(bubbling N2) for 1 h (Kaiser test negative) and the resin rinsed
several time with NMP. The resin was washed with CH2Cl2
and treated with a 1% solution of TFA in CH2Cl2 (5 mL) for
15 min at 0 °C. After filtration, to the solution Et3N (50 µL)
1.10 (d, J ) 7 Hz, 3H), 1.30 (bs, 6H), 2.12 (d, J ) 2 Hz, 1H),2.96
(dd, J ) 6, 2 Hz, 1H), 3.21 (m, 1H), 5.2-5.8 (large signal
containg the exchangeble protons). 13C NMR (DMSO-d6, 45 °C)
δ 18.3, 18.9, 21.1, 42.2, 48.5, 62.2, 64.6, 188.8. MS m/z 172
(M+).
Libr a r y. ES-MS analysis of the library prepared following
the procedures described for compound 64: m/z 158, 172, 200,
212, 228, 234, 248, 264, 324.
E xp er im en t s for In h ibit on of P a p a in e. Papain assay
was carried out at 25 °C on a spectrophotometer equipped with
the software UV-Visions 1.0 (Hitachi instruments Inc.), ac-
cording to a previous procedure9 slightly modified as follows.
Epoxide solutions were prepared as 40, 4, and 0.4 mM in

706 J . Org. Chem., Vol. 66, No. 3, 2001
Demarcus et al.
obtained by Dixon plots according to the equation
DMSO and directly added to the cuvette. Papain solutions
were activated before the kinetic measurements in 50 mM
phosphate buffer, pH 6.8, 2 mM EDTA, 2 mM 1,4-dithio-DL-
threitol at 25 °C for 30 min (papain concentration, 1.2 mg/
mL). The assay mixtures prepared in the same activation
buffer contained 1 mM L-BAPA, activated papain 40 mL/mL,
0-0.5 mM epoxides. In all cases the DMSO final concentration
was lower than 5%. The papain activity expressed as hydro-
lyzed L-BAPA was in all cases lower than 10 nmol/min/mL.
The absorbance increase at 405 nm was followed and the data
were analyzed with the software Microcal Origin 5.0 (Microcal
Software Inc.). The inhibition constant values (Ki) were
Rcont/Rx ) 1 + [x]/Kiapp
corrected to zero substrate concentration with the following
formula:
Ki ) Kiapp/(1 + [S]/Km)
The Km value of papain for L-BAPA, determined in our assay
conditions, was 2.5 mM.
J O000961W