Exploiting differences in caspase-2 and -3 S2 subsites for selectivity: Structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.08.020

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P₂ residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P₂ elements for their predicted ability to clash sterically with a residue of the caspase-3 S₂ pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ～200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.

Full text of DOI:10.1016/j.bmc.2011.08.020

Bioorganic & Medicinal Chemistry 19 (2011) 5833–5851
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploiting differences in caspase-2 and -3 S2 subsites for selectivity:
Structure-based design, solid-phase synthesis and in vitro activity of novel
substrate-based caspase-2 inhibitors
Michel C. Maillard a, , Frederick A. Brookfield b, Stephen M. Courtney b, Florence M. Eustache b,
⇑
Mark J. Gemkow c, Rebecca K. Handel b, Laura C. Johnson b, Peter D. Johnson b, Mark A. Kerry b,
Florian Krieger c, Mirco Meniconi b, Ignacio Muñoz-Sanjuán a, Jordan J. Palfrey b, Hyunsun Park a,
Sabine Schaertl c, Malcolm G. Taylor b, Derek Weddell b, Celia Dominguez a
a CHDI Management, Inc., 6080 Center Drive Suite 100, Los Angeles, CA 90045, USA
b Evotec (UK) Ltd, 114 Milton Park, Abingdon, OX14 4SA, UK
c Evotec AG, 114 Schnackenburgalee, Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Several caspases have been implicated in the pathogenesis of Huntington’s disease (HD); however, exist-
ing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual
caspases in the neuronal cell death associated with HD. In order to explore the potential role played
by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally
modified at the P2 residue in an attempt to decrease its activity against caspase-3. With the aid of struc-
Received 16 June 2011
Revised 6 August 2011
Accepted 9 August 2011
Available online 16 August 2011
tural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-L-pro-
Keywords:
line along with four additional scaffold variants were selected as P2 elements for their predicted ability to
clash sterically with a residue of the caspase-3 S2 pocket. These elements were then incorporated by
solid-phase synthesis into pentapeptide aldehydes 33a–v. Proline-based compound 33h bearing a bulky
3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to
60-fold increased selectivity for caspase-2 and ꢀ200-fold decreased caspase-3 potency compared to
the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery
of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates
to HD.
Caspase inhibitors
Huntington’s disease
Proline-derived P2 scaffolds
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
the understanding of Huntington’s disease (HD) as it relates to
expediting the development of therapies.
The human caspases (Csp) are a family of intracellular cysteine
aspartyl-specific proteases with at least 12 members that play
essential roles in inflammation, apoptosis and a variety of other
physiological and pathological processes.1 The promise of pro-
inflammatory Csp-1 as a drug target for rheumatoid arthritis has
encouraged intensive research efforts culminating with two Csp-
1 inhibitors reaching clinical stage evaluation.2 Additionally, in
apoptotic-driven indications, especially neurodegenerative dis-
eases, efficacy in proof-of-concept in vivo experiments has resulted
in an increased focus on several members of the family.3 Conse-
quently, caspases represent a key drug target family of particular
interest to our research organization whose mission is to improve
HD is a fatal genetic disorder characterized pathologically by
the selective loss of neurons, predominantly in the striatum,
and clinically by uncontrollable movements, progressive cogni-
tive deficit, and psychiatric disturbance.4 The underlying genetic
deficit of this disease is a CAG trinucleotide repeat expansion
encoding an abnormally long polyglutamine tract in the hunting-
tin (HTT) protein. Early reports suggested that polyglutamine
expansion in HTT mediates apoptosis through caspase activation,
in particular Csp-1, -3, -8 and -9,5 while more recently Csp-2 and
-7 activation has been reported in HD post-mortem tissue.6
Moreover, as many as five caspases (Csp-1, -2, -3, -6 and -7)6,7
have been implicated in mutant HTT (mHTT) cleavage resulting
in the production of potentially more toxic polyglutamine-
expanded HTT fragments.8 In testing this toxic fragment hypoth-
esis, YAC mice expressing Csp-6 cleavage-resistant mHTT did not
develop striatal neurodegeneration.9 Thus, whether it is through
the induction of apoptosis or through the production of toxic
⇑
Corresponding author. Tel.: +1 310 342 5506; fax: +1 310 342 5519.
E-mail addresses: michel.maillard@chdifoundation.org, michel.maillard@
earthlink.net (M.C. Maillard).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.020

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M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
HTT fragments and their downstream consequences, there is
sufficient in vitro and in vivo experimental evidence suggesting
Due to the absence of small-molecule leads, a substrate-based
approach represented, in our opinion, the best option for the rapid
development of selective and cell permeable Csp-2 inhibitors.
Relatively recently, a PSCL approach was used to attempt to ad-
dress this issue of cross reactivity of peptide-based caspase inhib-
itors.19 Unfortunately, despite the use of both natural and
unnatural amino acids, the selectivity against Csp-3 of the de-
scribed Csp-8 and Csp-9 inhibitors remained insufficient. In this
context, we favored a rational design approach based on structural
information of the targeted caspases of interest. At the time of this
work, crystal structures were available for Csp-220 and Csp-321 but
not for Csp-6,22 therefore we decided to target Csp-2 with the goal
to design potent inhibitor with >100-fold selectivity against Csp-3.
Our design strategy commenced with a detailed comparative
structural analysis of the S1 through S5 Csp-2 and Csp-3 subsites
(Table 2) searching for residue differences in the active site cleft
from which selectivity against Csp-3 could be derived. The S4 pock-
et was examined first as this subsite is the most divergent in the
caspase family and it had been successfully exploited to develop
potent Csp-1 tetrapeptidic inhibitors with >100-fold selectivity
against Csp-3.12 In the case of Csp-2, the triad Asn-Trp-Tyr of the
S4 subsite is unfortunately almost completely conserved in Csp-3
(Asn-Trp-Phe) which is reflected in the nearly exclusive preference
for an Asp in S4 for both caspases.14b,17 Our analysis continued with
the key recognition S1 pocket which was quickly discounted be-
cause S1 is completely conserved.23 Similarly, Csp-2 and -3 share
structurally equivalent hydrophobic solvent exposed S5 pockets24
which are not amenable to our selectivity design. The same conclu-
sion was also reached for the S3 pockets which in Csp-2 and -3 are
both flat polar surfaces providing no opportunity to drive
selectivity.14b,25
Finally, important differences in terms of shape and size were
observed in the S2 subsites of Csp-2 and -3. The most distinguish-
able residues are Ala-228 in Csp-2 which is replaced in Csp-3 by
Tyr-204 and Phe-279 in Csp-2 which is moved by about 3 Å inside
the pocket in Csp-3 (Phe-256). In part as a result of this amino acid
change, the bowl-shaped lipophilic S2 subsite of Csp-3 is changed
into a groove, with a narrow opening perpendicular to the ac-
tive-site cleft (Fig. 1A and B). Thus, these S2 subsite differences rep-
resented the best opportunity on the unprime side for the design of
selective Csp-2 inhibitors as we hypothesized that a bulky P2
accommodated in Csp-2 would be likely to clash sterically with
Tyr-204 of Csp-3. Molecular modeling also predicted limited
mobility for Tyr-204 which further reinforced our idea. The second
stage of the design was to ‘engineer’ a vector from the peptidic
backbone to provide an adequate trajectory for the selected bulky
P2 to clash with Tyr-204. Modeling experiments indicated that di-
a
role for caspase inhibition in HD to warrant further
investigation.
Further substantiating these findings is the efficacy of broad-
spectrum peptide-based caspase inhibitors in preventing HTT
cleavage and reducing toxicity in vitro7b,10 and in HD animal
models.11 However, the lack of selective caspase inhibitors has
hampered further progress in understanding the specific involve-
ment of individual caspases in mHTT-induced apoptosis or mHTT
cleavage; consequently, we set out to develop these selective
inhibitors. Selective Csp-1,12 Csp-312a,13 and Csp-713a inhibitors
have been disclosed; however, for Csp-2 and Csp-6, the two lead-
ing caspase targets for HD, there are no reports to our knowledge
of selective inhibitors. In order to be able to rapidly test our
inhibitor design strategy, we decided to focus our effort initially
on the development of Csp-2 inhibitors with reduced activity
for Csp-3.
We report here the successful application of structure-based
design to the development of potent inhibitors of Csp-2 with im-
proved selectivity. We discuss the rationale for targeting the S2
pocket and choosing proline-based templates as the P2 element.
Synthesis of these key P2 scaffolds and their incorporation into
pentapeptide aldehydes via solid-phase chemistry are presented
along with a detailed analysis of the biochemical and cellular selec-
tivity of these new inhibitors.
2. Inhibitor design
Ideally, non-peptide small-molecule inhibitors would represent
the most desirable starting point to achieve our goal of developing
a selective and cell permeable Csp-2 inhibitor. Unfortunately, al-
most twenty years after the cloning of the first caspase,14a the
identification of non-covalent small-molecule caspase inhibitors
remains the ‘Holy Grail’ in the field because of the extreme chal-
lenges in transforming an electrophilic warhead-based tetra- or
pentapeptide with an aspartic acid into a non-peptide mimetic.
Not surprisingly, screening campaigns have had very limited suc-
cess with only a few reports of validated hits15 including isatins.13
However, we recently disclosed unsuccessful attempts to prepare
potent Csp-2 isatin inhibitors and to reproduce the activity of sev-
eral published non-peptidic Csp-3 and -6 inhibitors identified by
screening.16
In the peptide class, potent inhibitors have been derived from
substrate sequences obtained by positional scanning combinato-
rial libraries (PSCL) or peptide sequence variants employed in
the seminal work of the Thornberry and Talanian groups.14b,17
It is sobering to note that over a decade later, the vast majority
of the numerous substrate-derived caspase inhibitors including
the commercial ones lack sufficient specificity to inhibit individ-
ual caspase pathways. In particular for Csp-2, the canonical inhib-
itor Ac-VDVAD-CHO (1, Table 1) is more potent against Csp-3
which casts some doubt about the validity of the data generated
with this reagent when employed in a cellular context as a ‘selec-
tive’ Csp-2 inhibitor. Other groups have already expressed the
same specificity concerns regarding existing reagents and
stressed the urgent need for more selective inhibitors in the cas-
pase field.18 In particular, selectivity against Csp-3 is critically
important because of the high concentration of active Csp-3 rel-
ative to other caspases in almost all tissues and of its promiscu-
ous nature.18c Furthermore, the neuroprotective effects of a Csp-3
inhibitor has already been reported in a HD model.11b For these
reasons, it was important to develop a Csp-2 tool with no or little
Csp-3 activity in order to be able to specifically evaluate the role
of Casp-2 in HD.
rect substitution on the peptidic backbone
a-carbon often led to
the large P2 side chains to point away from the Tyr-204 into sol-
vent as a result of movement at the P1–P2 junction. This led us to
consider L-proline analogues since we envisioned that a rigid tem-
plate could lock a substituent in the required trajectory and reduce
the possibility of movement from the peptidic backbone (Fig. 1C).
Another attractive feature of L-proline is that it has been frequently
used as a key building block in peptidomimetic design including
for Csp-1.2b,26
To our knowledge, there is no report of P2 proline Csp-2 inhibitors;
however, there are P2 Pro-containing inhibitors for Csp-3,27 -728
and the caspase-like granzyme B.29 Crystal co-complexes of these
three proteins with P2 proline substrate-like inhibitors are also
available.27–29 Examination of these three co-structures revealed
that a common feature is the presence of a Phe residue nestled
against the P2 proline. These three P2 Phe residues overlay well
with Phe-279 of Csp-2 which supports the notion that a proline
residue should also be well tolerated as a P2 in Csp-2. Furthermore,
PSCL results corroborated our structure-based hypothesis that

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5835
Table 1
Biological activity of pentapeptide aldehydes
O
O
OH
O
O
O
O
H
N
H
N
N
H
N
H
P2
OH
O
O
Casp-3/
Casp-2
ratio
Enzyme assay
Casp-2 IC50 (nM)
Enzyme assay
Casp-3 IC50 (nM)
Cell assay Casp-
2 IC50 (nM)
Cell assaya Casp- Casp-2 cell/
P2
3 IC50 (nM)
enzyme ratio
1
2
(S)-Ala
46
41
15
29
0.3
0.7
1334
1690
346
437
29
41
(S)-O-tBu-serine
33a
33b
X = H
X = OCH3
41
108
12
59
0.3
0.5
635
1940
92
965
15
18
X
33c
33d
33e
33f
33g
33h
33i
X = OCH(CH3)2
X = OCH(CH2CH3)CH3 413
246
5533
5125
9300
22,500
9920
2925
10,400
2765
4075
22
12
20
29
45
60
24
1.7
30
12,600
>50,000
32,000
35,667
17,400
2360
50,000
>65,000
13,607
>100,000
>25,000
nd
51
>121
71
46
79
N
X = OCH2CH(CH3)2
X = O-cyclopentyl
X = O-(2-pyrimidyl)
X = CH2C(CH3)3
X = phenyl
453
772
219
50
441
1600
136
nd
>100,000
78,350
nd
>50,000
42,160
65
113
>41
100
33j
33k
X = 4-CH3-phenyl
X = cyclohexyl
33l
150
289
2
781
nd
5
N
33m
33n
X = H
X = OCH(CH3)2
86
52,800
26
81,500
0.3
1.5
1120
>100,000
24
>100,000
13
>2
X
N
33o
33p
33q
33r
X = H, Y = H, ⁄ = S
22
233
1410
3000
7970
11
25
60
4
2260
5533
6670
nd
>35,000
>60,000
>100,000
103
92
130
>45
Y
X = CH3, Y = H, ⁄ = R/S 60
X = H, Y = CH3, ⁄ = R/S 50
X
X = OCH3, Y = OCH3,
2190
>100,000
⁄ = R/S
33s
33t
33u
33v
X = H, Y = OH, ⁄ = R/S
X = OH, Y = H, ⁄ = R/S
X = H, Y = Br, ⁄ = R/S
X = Br, Y = H, ⁄ = R/S
1980
459
163
573
58,600
23,000
907
30
50
6
12,823
23,567
7470
nd
nd
nd
39,170
6
*
50
46
5
N
1075
1.8
3110
a
nd, not determined.
Table 2
have the required trajectory and size to clash with Tyr-204 of
Csp-3 and would be tolerated in the S2 groove of Csp-2. Finally, tak-
ing a variable degree of synthetic accessibility into account, we
designed a set of analogs based on five different proline-based mo-
tifs as P2 scaffolds (i)–(v) (Fig. 1D) which would assess the design
principles described above.
Generalized structure of pentapeptide aldehyde inhibitors: (P5–P4–P3–P2–P1–CHO)
and key residues of S1–S5 subsites in Csp-2 and Csp-3
3. Chemistry
Csp-2
Csp-3
Subsite
Tyr-273
Ala-274
Pro-275
Asn-232
Trp-238
Tyr-273
Ser-55
Thr-233
Arg-231
Ala-228
Met-230
Phe-279
Arg-54
Arg-231
Gln-153
3.1. P2 Amino acid synthesis
Phe-250
Ser-251
Phe-252
Asn-208
Trp-214
Phe-250
Ser-65
Ser-209
Arg-207
Tyr-204
Trp-206
Phe-256
Arg-64
Arg-207
Gln-161
The synthetic routes to the P2 amino acids are outlined in
Schemes 1–4. The synthesis of 3-oxyproline analogues 6a–f is de-
scribed in Scheme 1. Reaction of N-Fmoc-3-(S)-hydroxy-L
-proline30
S5
S4
S3
S2
S1
3 with methanol under acidic conditions afforded the methyl ester
4 which was then alkylated using a series of alkyl iodides in a Ag2O
mediated etherification to give compounds 5a–e; final acid
mediated hydrolysis of the ester afforded compounds 6a–e. The
2-pyrimidyl analogue 6f was obtained via a SNAr reaction of
N-Boc-3-(S)-hydroxy-L-proline 7 with 2-chloropyrimidine and sub-
sequent protective group interconversion. Synthesis of the 3-(S)-
alkyl and aryl proline analogues 15a–d were performed using the
proline would be accepted in the S2 subsite of Csp-2.14b The final
step in the design was the identification of the position, orientation
and size of the substituent on the proline ring. Additional modeling
experiments indicated that from the 3-position large alkyl or alk-
oxy groups (isobutyl, isopropoxy) in the (S)-configuration would

5836
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
A
B
Figure 1. Shape of S2 binding subsites with surface colored in grey for (A) Csp-3 (PDB code 2DKO21b) and (B) Csp-2 (PDB code 1PYO20). (C) Putative steric clash of 3(S)-proline
substituent (-R) in S2 pocket of Csp-3. (D) Selected scaffold variants (i)–(v). Parts (A) and (B) were generated using PyMOL.39
R
R
OH
OH
O
O
i
ii
iii
OH
O
O
OH
N
N
N
N
O
O
fmoc
fmoc
O
O
fmoc
fmoc
3
4
5a-e
6a-f
N
a
b
c
d
R = CH3
R = CH(CH3)2
R = CH(CH3)CH2CH3
R = CH2CH(CH3)2
R = cyclopentyl
R = 2-pyrimidyl
N
OH
O
iv
OH
v, vi
OH
N
e
N
boc
O
boc
f
O
7
8
Scheme 1. Synthetic procedure for 3-oxyproline amino acid analogues 6a–f. Reagents and conditions: (i) 4N HCl in dioxane, MeOH, rt, 24 h, 68%; (ii) RI, Ag2O, DCE, rt or
reflux, 24 h, 3–63%; (iii) 6N HCl (aq), dioxane, reflux, 15 h, 42–95%; (iv) 2-chloropyrimidine, KOtBu, tBuOH, 1 h, 35%; (v) 4 N HCl in dioxane, rt, 1 h; (vi) NaHCO3, FmocCl,
dioxane, water, 0 °C to rt, 48 h, 65%.
chemistry described in Scheme 2. Compound 9 was synthesized
following the procedure of Herdeis and Hubmann31 and subse-
quent Michael addition of organocuprates afforded 3-(S)-substi-
tuted pyrrolidinones 10a–c in good yield. Reduction of the
pyrrolidine amide using borane–dimethylsulfide complex gener-
ated 3-(S)-substituted pyrrolidines 11a–c and TBAF deprotection
of the silyl protecting group gave hydroxymethylpyrrolidines
12a–c which were subjected to Jones oxidation to give acids
13a–c. Switching of the carbamate protecting group from Boc to
Fmoc was performed using standard deprotection and protection
conditions to give Fmoc-3-(S)-substituted prolines 15a–c. Com-
pound 15d was synthesized from the commercially available
3-(R)-phenyl-L-proline 14 via Fmoc protection.
Scheme 3 describes the synthesis of N-Fmoc-3-(S)-isopropoxy-
pipecolic acid analogue 24. The PMB protected hydroxyl azide 16
was synthesized following the procedure described by Kumar et
al.32 and alkylation of this compound was successfully performed
using isopropyl iodide and Ag2O to give compound 17. Deprotec-
tion of the PMB group was performed using DDQ resulting in
hydroxy azide 18 which was converted to 19 using a one pot

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5837
R
R
ii
OTBDPS
i
OTBDPS
OTBDPS
O
N
O
iv
N
N
Boc
9
Boc
Boc
10a-c
11a-c
R
R
R
v, vi
iii
OH
OH
OH
N
N
N
O
O
Boc
Boc
Fmoc
13a-c
12a-c
15a-d
a R = CH2CH(CH3)3
b R = 4-CH3-Ph
vii
OH
c R = cyclohexyl
N
H
d R = Ph
O
14
Scheme 2. Synthetic procedure for Fmoc-3-(S)-alkyl proline 15a–d. Reagents and conditions: (i) RLiCu or RMgBrCu, TMSCl, Et2O, ꢁ78 °C to rt, 2 h, 70–80%; (ii) BH3ꢂDMS, THF,
reflux, 1 h, 82–100%; (iii) TBAF, THF, rt, 24 h, 65–91%; (iv) Jones reagent, acetone, rt, 5 h, 61–100%; (v) TFA/DCM (1:1), 0 °C, 1 h; (vi) FmocCl, NaHCO3, dioxane, rt, 18 h, 55–
100%; (vii) FmocCl, NaHCO3, dioxane, water, 0 °C to rt, 18 h, 78%.
OH
O
OR1
O
O
O
i
iii
O
O
O
OPMB
N3
OR2
ii
N3
OH
NHBoc
17 R1= iPr, R2= PMB
16
19
18 R1= iPr, R2= H
O
O
O
O
v
vii
iv
O
OH
OR
N
N
R
O
NHBoc
S
O
boc
O
O
O
21 R = Et
22 R = H
23 R = H
20
vi
viii
24 R = Fmoc
Scheme 3. Synthetic procedure for 3-isopropoxypiperidine amino acid analogue 24. Reagents and conditions: (i) iPrI, Ag2O, DCE, 70 °C, 17 h, 18%; (ii) DDQ, DCM, H2O, rt, 1 h
30, 73%; (iii) H2, 10%Pd/C, Boc2O, EtOAc, rt, 15 h, 80%; (iv) MsCl, TEA, DCM, 0 °C to rt, 2 h, 76%; (v) NaH, DMF, 0 °C to rt, 4 h, 59%; (vi) LiOH, EtOH/H2O, rt, 3 h, 50%; (vii) TFA,
DCM, 0 °C, 2 h, 90%; (viii) FmocCl, dioxane, 0 °C to rt, 16 h, 50%.
reduction and N-protection strategy. Mesylation of 19 with
methanesulfonyl chloride afforded 20 which was successfully
cyclized using NaH to give the pipecolic ester 21. Transformation
of 21 into the Fmoc pipecolic acid 24 was performed following
standard hydrolysis and Boc deprotection/Fmoc protection
strategies.
Scheme 4 describes the synthesis of N-Fmoc-6-, 7-mono substi-
tuted and 6,7-disubstitued tetrahydroisoquinoline-1-carboxylic
acids 29a–g. The N-Fmoc-6-hydroxy tetrahydroisoquinoline-1-car-
boxylic acid 29a was prepared by reaction of the commercially
available 3-(2-amino-ethyl)-phenol 25a with glyoxylic acid fol-
lowed by Fmoc-protection. Compounds 29b–d were prepared by
Fmoc protection of the commercially available tetrahydroisoquin-
oline-1-carboxylic acids 26b–d. Fmoc protection of the substituted
phenethylamines 27e–g followed by a Pictet–Spengler cyclisation
using glyoxylic acid afforded compounds 29e–g.
3.2. Solid-phase synthesis
Synthesized Fmoc amino acids 6a–f, 15a–d, 24 and 29a–g and
commercially available, Fmoc amino acids ((S)-Fmoc-O-tert-butyl-
serine, (S)-Fmoc-proline, Fmoc-2,3-dihydro-1H-isoindole-1-car-
boxylic acid, (S)-Fmoc-1,2,3,4-tetrahydroisoquinoline-1-carboxylic
acid, (S)-Fmoc pipecolic acid) were loaded onto aspartic acid func-
tionalized semi-carbazide resin 30 as described by Grimm et al.33
(Scheme 5). Fmoc deprotection of resins 31 using 20% piperidine
in DMF followed by coupling of the resin immobilized P2 amino
acids with AcVD(OtBu)V-OH (synthesized using classic linear

5838
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
R1
R2
`
R1
R2
NH2
i
ii
OH
N
H
O
R1
25a
26a
26b-d*
R2
H
N
iii
R1
R2
R1
R2
NH2
iv
fmoc
OH
N
fmoc
O
29a-g
27e-g
28e-g
a R1 = OH, R2 = H; b R1 = CH3 R2 = H; c R1 = H R2 = CH3; d R1 = R2 = OCH3
e R1 = H, R2 = OH; f R1 = H, R2 = Br; g R1 = Br, R2 = H
⁄
Scheme 4. Synthetic procedure for Fmoc-tetrahydroisoquinoline-1-carboxylic acid analogues 29a–g. denotes commercial compounds. Reagents and conditions: (i)
Glyoxylic acid, TEA, EtOH, 5 °C to rt, 1 h, 70%; (ii) FmocCl, NaHCO3, dioxane, water, 0 °C to rt, 18 h, 33–83%; (iii) Fmoc, DIPEA, DCM, 0 °C to rt, 20 h, 23–55%; (iv) Glyoxylic acid,
H2SO4, AcOH, rt, 24 h, 21–36%.
peptide elongation)34 using HATU gave resin bound peptides 32.
The final peptide aldehydes 2 and 33a–v were obtained using a
one step acid mediated deprotection and cleavage from resin using
TFA/water.
commercially available 3-(S)-hydroxy-L-proline would allow rapid
access to a range of analogs to study the influence of substituent
size on selectivity. We started to probe the steric requirements
with two analogs: the methoxy derivatives 33b which, as predicted
by modeling, showed no improvement in selectivity because the
3-(S)-OMe was too small to clash with Tyr-204 and the 3-(S)-iso-
propoxy analogs 33c which had ꢀ20-fold selectivity against
Csp-3. Encouraged by this result, compounds 33d–f bearing larger
substituents were prepared in an attempt to further improve this
selectivity ratio. Unfortunately, we obtained only modest improve-
ments (Table 1) with the O-(2-pyrimidyl) derivatives 33g having
the best selectivity (45-fold) and the activity in Csp-2 for this com-
pound decreased in the triple-digit nanomolar range as in 33c. A
crystal structure of 33c bound to Csp-2 was obtained and it re-
vealed that the 3-iPro substituent is in a pseudo axial orientation
(Fig. 2). On the basis of this structural information and docking
studies, we decided to introduce the 3-iPro substituent on the
piperidine scaffold (ii) because this substituent would then be
locked in the putative bioactive axial conformation and would
have a favorable trajectory to clash with the Tyr-204 of Csp-3. As
intended, compound 33n had only residual Csp-3 activity but very
disappointingly Csp-2 activity was also almost totally abolished.
This result was surprising because molecular modeling had pre-
dicted the axial conformation to be the lowest energy conforma-
tion for 33n while the equatorial diastereoisomer (3R) could not
be docked in Csp-2. Therefore, for this analogue, other factors as
yet unknown must be exerting an effect.
In a further attempt to achieve higher selectivity against Csp-3
in the proline series, it was sought to increase the steric bulk in the
vicinity of the 3-(S)-hydroxy by introducing a tert-butyl group. A
tert-butoxy was however not compatible with the final deprotec-
tion step of the solid-phase route and instead the neopentyl analog
was prepared 33h. This compound offered no significant improve-
ment in selectivity (60-fold) compared to our previously prepared
analogs, however unlike in the 3-(S)-alkoxyproline series, the dou-
gle-digit nanomolar Csp-2 activity was maintained. The oxygen to
carbon replacement could explain this difference because a carbon
atom in the 3-position is likely to make a better contact with Phe-
279 than an oxygen in the same position. Interestingly, the flexible
(S)-O-(tert-butyl)-serine P2 analog 2 corresponding to 33h is a
4. Results and discussion
4.1. Unsubstituted P2 scaffolds
We set out to synthesize first the unsubstituted compounds
corresponding to the five scaffolds (i–v) represented in Figure 1D.
It was gratifying to see our P2 proline hypothesis proven correct
as analog Ac-VDVPD-CHO 33a (Table 1) had the same activity in
Casp-2 as the reference inhibitor Ac-VDVAD-CHO 1. As expected,
the homologous piperidine analog 33m also had similar activity
to proline 33a, and hence, both scaffolds (i) and (ii) were validated
for the next step of substitution at the 3-position. According to
molecular docking, the isoindole 33l and isoquinoline 33o pre-
sented a reduced steric clash with the Tyr-204 of Csp-3 when com-
pared to the large 3(S)-substituent on the proline, however these
two analogs were easily accessible and prepared to test the robust-
ness of the docking model. It was interesting to observe that, unlike
the proline 33a and piperidine 33m, these homologous bicyclic
systems had different activity profile: isoquinoline 33o showed a
10-fold selectivity against Csp-3 whilst maintaining high Casp-2
activity but the isoindoline 33l had no selectivity against Csp-3
and showed a decrease in Csp-2 and Csp-3 activity compared to
the proline 33a. The resolution of the modeling was insufficient
to explain this difference in activity. Finally, we found the 3-spiro
series (iii) to be chemically intractable, probably due to steric hin-
drance caused by the 2-position substituent on the proline; how-
ever, having already identified three scaffolds (i), (ii) and (v)
which were tolerated in the S2 pocket of Csp-2, we elected to depri-
oritize scaffold (iii).
4.2. (S)-Substituted proline and piperidine P2 scaffolds
The next step in the proline series was the introduction of the
3(S)-substituent designed to clash with Tyr-204 of Csp-3. We chose
to introduce alkoxy groups because functionalization of

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5839
O
O
O
O
O
N
O
N
H
N
H
Fmoc
Fmoc
N
OH
O
2
N
H2N
2
H
O
O
OH
O
O
O
N
O
O
H
N
O
H
H
O
O
O
O
N
H
N
H
N
H
N
N
2
N
N
H
H
N
H
2
O
O
OH
O
O
O
2
N
N
HN
N
N
2
=
=
O
NH2
H
N
H
Merrifield resin
N
O
Scheme 5. General synthetic procedure for solid phase synthesis of pentapeptides 2, 33a–v. ⁄ denotes (2R/S)-configuration except for 33o (2S), \ denotes commercial Fmoc
protected P2 building block. Reagents and conditions: (i) HATU, DIPEA, DMF, rt, 18 h, (ii) 20% piperidine in DMF, rt, 1 h; (iii) AcValAsp(OtBu)Val-OH, HATU, DMF, rt, 18 h;
(iv) trifluoroacetic acid/water (9:1), rt, 1 h.
double-digit nanomolar inhibitor of both Csp-2 and Csp-3. This
absence of selectivity against Csp-3 of the flexible analog of 33h
is a vindication of our choice of rigid templates. Finally, three ana-
logs 33i–k with a ring directly attached to the 3-proline position
were prepared, however they did not display better selectivity
than the lead compound 33h.
easily accessible 6,7-diOMe 33r which unfortunately suffered a
ꢀ40-fold loss of Csp-2 activity compared to the Me analogs. Simi-
larly, the 6- and 7-OH analogs 33s–t lost Csp-2 activity which indi-
cated that creating a substantial interaction with the upper S2
pocket of Csp-2 from these two positions will be difficult. Taken to-
gether, these results suggest that despite the encouraging profile of
6-Me analog 33q, it will probably be more challenging to improve
the selectivity and potency in Csp-2 in the isoquinoline series than
in the 3(S)-substituted proline series which remains the most
promising series with the neopentyl analog 33h as the lead
compound.
4.3. Isoquinoline P2 scaffold
In this series, attempts were made to improve on the 10-fold
selectivity observed with the unsubstituted isoquinoline 33o. The
6- and 7-positions of the isoquinoline nucleus were selected based
on modeling considerations, that is, the possibility of creating a
contact with the upper part of the S2 pocket of Csp-2 (6-, 7-OH ana-
logs 33s–t) and the introduction of a bulky group in order to try to
take advantage of the different steric requirements of Csp-2 and
Csp-3 (6-, 7-Me and 6-, 7-Br; 33p–q, u–v). The results for these
six analogues are summarized in Table 1 and showed that the 6-
Me analog 33q had coincidently the same profile has our best
3(S)-substituted analog 33h (60-fold selectivity and 50nM in Csp-
2). The other five analogs had lower selectivity and weaker activity
in both Csp-2 and Csp-3. The two bulky 6- and 7-bromo substitu-
ents 33u–v were too large but the improved selectivity of both the
6-Me 33p (60-fold) and 7-Me 33q (25-fold) led us to prepare the
4.4. Cellular activity and future direction
Csp-2 and Csp-3 cell activities and cell-to-enzyme (C/E) potency
ratio in Csp-2 are reported in Table 1. With the exception of VDVPD-
CHO 33a and isoindoline 33l which have respectively an IC50 of 630
nM and 780 nM in the Csp-2 cell assay, all other compounds display
micromolar activity with C/E ratios ranging from 5 to 130. In the 3-
(S)-substituted proline series, the C/E ratio generally increases with
the size of the 3-substituent and the most Csp-2 selective analogs
33h had a C/E of 65. In the isoquinoline series, the Csp-2 C/E ratio
does not correlate with size as the bromo analog 33v had surpris-
ingly one of the best C/E ratios (C/E = 5) whereas the unsubstituted

5840
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
selectivity against Csp-3. Replacement of the neopentyl group in
the most selective inhibitor 33h by an alternative large substituent,
from which additional Csp-2 potency could be derived may ulti-
mately produce the pharmacological tool compound needed to un-
cover the precise role of Csp-2 in particular pathways or disease
mechanisms.
6. Experimental
6.1. Biochemical assays for Csp-2 and Csp-3
6.1.1. Materials
Coumarin-120-based Csp-2, and -3 substrates Ac-VDVAD-AMC
(AMC: 7-amino-4-methyl-coumarin, Coumarin-120) and Ac-
DEVD-AMC were purchased from PeptaNova GmbH (Sandhausen,
Germany) or Peptide Institute (Osaka, Japan). Csp-2 (170–452aa
containing large and small subunits) and Csp-3 (1–277aa contain-
ing full length pro-enzyme) fused with a C- terminal histidine-tag
were cloned into a pET-23b vector (Novagen). The plasmids were
transformed into E. Coli BL21 (DE3) pLysS cell and expression was
carried out by overnight induction with 0.2 mM IPTG at 16 °C. The
protein was purified in two steps using Ni-NTA affinity and anion
exchange chromatography. 2-(N-Morpholino)ethanesulfonic acid
(MES), piperazine-N,N0-bis(2-ethanesulfonic acid) (PIPES), ethy-
lenediamine-tetra acetic acid (EDTA), dimethyl sulfoxide (DMSO),
Pluronic F127 (PF127), and glutathione (GSH) were ordered from
SIGMA-Aldrich (St. Louis, MO, USA). 4-(2-Hydroxyethyl)-1-pipera-
zine-ethane-sulfonic acid (HEPES) was obtained from Merck
(Darmstadt, Germany). Substrate-based Csp-2 (Ac-VDVAD-CHO,
1) and Csp-3 (Ac-DEVD-CHO) inhibitors were purchased from BA-
CHEM (Bubendorf, Switzerland).
Figure 2. X-ray co-crystal structure at 2.6 Å of compound 33c bound to Csp-2 (PDB
code 3RJM). The surface of Csp-2 active site is colored in grey and compound 33c by
atom type. The Fo ꢁ Fc composite omit electron density map contoured at 2.5
r is
displayed in blue mesh around 33c. Figure generated by PyMOL.39
isoquinoline 33o had a C/E of 100. The most Csp-2 selective isoquin-
oline analog 33q also had a poor C/E of 130. In the end, the com-
pound that emerged from this effort with the most interesting
profile is 3(S)-neopentyl-proline 33h which has a number of
attractive characteristics compared to the reference inhibitor
Ac-VDVAD-CHO (1). Compounds 33h and 1 are equipotent both in
Csp-2 biochemical and cell assays with IC50 values of respectively
6.1.2. Fluorescence-based enzyme inhibition assays
The assay is based on the cleavage of a fluorogenic substrate
comprised of the recognition sequence of the individual caspase
(see above) and coumarin-120. Cleavage of the substrate converts
the quenched fluorophore into a fluorescent product. All assays
were performed in a 384 well format (Matrix 384 well plate, black,
polystyrene; cat.-no. 4318; Thermo Fisher Scientific, Hudson, NH,
ꢀ50 nM & ꢀ2
60-fold selectivity against Csp-3 and is 200-fold less potent in
Csp-3 than 1 (ꢀ3 M vs 15 nM, Table 1). In the cell assays, com-
pound 33h is 20-fold selective for Casp-2 and also very weakly
active in Csp-3 (78 M) whereas compound 1 is quite potent
lM. However, in the biochemical assays, 33h has a
l
l
(346 nM). All together, these data make 33h a better tool compound
than other VDVAD-based inhibitors including compound 1 and
demonstrates the validity of the structured-based approach. These
results are particularly encouraging for an exploratory study and
we expect that additional progress can be made in the 3(S)-substi-
tuted proline series. The current limitation with this series comes
from the fact that the neopentyl group in 33h does not improve
Csp-2 activity compared to the unsubstituted proline 33a. Based
on the analysis of the S2 pocket of Csp-2, we hypothesize that it
should be possible to identify an alternative bulky substituent
capable of not only clashing sterically with a residue of the Csp-3
S2 pocket but also engaging in interactions with residue(s) of the
S2 pocket of Csp-2. These new interactions should increase Csp-2
potency which could allow deletion of the P5 group. The resulting
tetrapeptide would be expected to be potent with an improved
C/E ratio compared to 33h, thus making it a valuable pharmacolog-
ical tool compound.
USA) in a total volume of 20 ll per well. Standard reaction condi-
tions for Csp-2 consisted of 50 mM MES (pH 6.5), 150 mM NaCl,
1.5% sucrose, 5 mM GSH and 0.03% Pluronic F127. Standard reac-
tion conditions for Csp-3 consisted of 50 mM HEPES (pH 7.4),
100 mM NaCl, 5 mM GSH and 0.03% Pluronic F127. The chosen
concentration of fluorogenic substrates corresponded to the
respective KM-values (see below for details). The peptidic inhibi-
tors Ac-VDVAD-CHO (Csp-2) and Ac-DEVD-CHO (Csp-3) were used
as reference inhibitors on each individual assay plate. The activity
measured for the reference inhibitors was as expected from the lit-
erature.14b,24a,35 Reference inhibitors and test compounds were
dissolved in DMSO, threefold serial dilutions with eleven concen-
trations were prepared in 100% DMSO at 100-fold final concentra-
tion and 0.2
ll of the compound dilutions added to each well
containing 10
l
l of freshly prepared solutions of either 20 nM
Csp-2 or 0.6nM Csp-3 in the respective reaction buffers using a
CyBiÒ—Well vario manipulator (CyBio AG, Jena, Germany). Plates
were incubated at 37 °C for 5 min before the enzymatic reaction
5. Conclusion
was started by adding 10
final concentration in the individual reaction buffer) resulting in
20 M Ac-VDVAD-AMC for Csp-2 and 10 M Ac-DEVD-AMC for
Csp-3, respectively. The final concentration of DMSO was 1%. Plates
were incubated for 20 min at 37 °C and reaction was stopped by
ll of the substrate solution (twofold of
The paucity of selective caspase inhibitors is an obstacle to elu-
cidating the precise role that caspases may play in HD pathogene-
sis. We demonstrate here that a structure-based design strategy is
effective for the identification of pentapeptide-based Csp-2 inhibi-
tors with a superior Csp-3 selectivity profile in enzyme and cellular
assays compared to existing VDVAD-based reagents. The proline
scaffold introduced as the P2 element along with the bulkiness of
its 3-(S)-substituent have proven to be crucial features to improve
l
l
addition of 3 ll of 5 M aqueous acetic acid solution followed by a
short centrifugation step to remove air-bubbles. Fluorescence
was quantified using a Tecan Safire 2 microplate fluorometer
(Tecan Austria GmbH, Grödig, Austria) with the following

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5841
wavelength settings: excitation at 380 nm and emission at 440 nm
for coumarin-120, respectively.
determined with the A+-software using a four parameter Hill equa-
tion. Normalized percent activity was calculated using the median
values of the controls containing 1% DMSO (no inhibition) and
6.2. Cellular assays for Csp-2 and Csp-3
1 lM of the peptide inhibitors Ac-VDVAD-CHO (Csp-2) and Ac-
DEVD-CHO (Csp-3) (full inhibition). Final IC50 values are the aver-
Determination of cellular potency of the compounds was per-
formed using HEK293 T17 cells that were transiently transfected
with the full-length cDNAs of Csp-2 or Csp-3. The cDNAs used were
human Myc-DDK tagged Csp-2 in pCMV6 (RC200669, Origene,
Rockville, USA) and full-length cDNA of Csp-3 in pcDNA 3.1 with
in-frame fusion and FLAG tag at the C-terminus. Cells were grown
at 8% CO2 and 37 °C in culture medium consisting of DMEM (cat.-
no. D5796, Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal calf
serum and 1% (v/v) penicillin/streptomycin-solution (cat.-no.
P11-013, PAA Laboratories, Cölbe, Germany).
age of at least two independent experiments in triplicates.
6.4. Structural biology
Csp-2 used for crystallography was produced by co-expression
of the P12 subunit (amino acids 345–452) with a C-terminal
hexa-histidine affinity tag, and the untagged P19 subunit (amino
acids 167–333) in E. coli overnight at 16 °C. The soluble complex
was purified by Ni affinity chromatography followed by heparin-
sepharose chromatography. The protein was dialyzed into 20 mM
HEPES, pH 7.5, 150 mM NaCl, 10% sucrose, 8 mM DTT and concen-
trated to 2 mg/ml for crystallization. Complexes of Csp-2 and 33c
were prepared by incubating Csp-2 with a 5ꢃ molar excess of
33c at 4 °C. Crystallization trials were carried out using sitting drop
vapor diffusion at 16 °C. Crystals grew from 20% PEG3000, NH4Cit-
rate, pH 5.5. Crystals display a symmetry of P212121 with cell
dimensions a = 63.77 Å, b = 97.38 Å, c = 97.98 Å. Atomic coordi-
nates for the Csp-2 complex with 33c have been deposited in the
Protein Data Bank under PDB ID code 3RJM and statistics for the
X-ray data sets are shown in the Supplementary data.
6.2.1. Csp-2 cellular assay
The assay was performed in poly-L-lysine coated 96-well plates
(CellCoat, cat.-no. 655,936, Greiner bio-one, Frickenhausen, Ger-
many). For one 96-well plate 20 ml of cell suspension was pre-
pared as follows: 1,100,000 cells/ml in culture medium in a total
volume of 18 ml were harvested. A solution containing 20
mid mixture (2 g Csp-2 and 18 g empty vector pcDNA3.1neo)
and 60
l Fugene HD (FuGENEÒ HD Transfection Reagent, Roche,
lg plas-
l
l
l
Switzerland) in OptiMEM (cat.-no. 31,985, Invitrogen, Darmstadt,
Germany) was prepared, vortexed and kept at room temperature
for 15 min. Cells were then transfected by adding transfection
solution to cells. Cells were then seeded into the 96-well plate at
6.5. Computational chemistry—Docking of peptides using low-
mode MD simulations
a density of 20,000 cells/well (200 ll of transfection suspension).
After 4 h of incubation at 37 °C and 8% CO2 in a humidified incuba-
tor the standard inhibitor Ac-VDVAD-CHO (final concentration
10 lM) was added and cells were incubated another 20 h before
culture medium and inhibitor were replaced. 48 h after transfec-
tion the assay was performed by removing the medium and replac-
ing it with 40 ll of phenol red-free medium (DMEM High Glucose,
cat.-no. E15-870, PAA Laboratories) per well. Compounds were di-
luted from a DMSO stock solution using the phenol red-free med-
The crystal structures of Csp-2 and Csp-3 used in the in silico
docking experiments were obtained from the Brookhaven protein
data bank (PDB codes: 1PYO20 and 2DKO respectively21b). These
structures were prepared for docking by removing all ligands and
water molecules followed by adding hydrogen atoms using the
Protonate-3D tool within MOE.36 Tetrapeptides were utilized for
docking purposes rather than pentapeptides to conserve the com-
putation time for the docking of these peptides. It has been shown
that the P1, P3 and P4 groups were sufficient to stabilize and cor-
rectly orient the P2 group in the S2 pocket and that there is no ben-
efit to using the pentapeptides for docking when considering the P2
substituent orientation (unpublished results). Low-mode MD sim-
ulations were chosen as a method to dock the tetrapeptides due to
their ability to effectively sample the conformational space of both
the ligand and the side chains of the protein residues involved in
the binding.37
The tetrapeptides were manually placed in the binding pocket
of Csp-2. The starting position and conformation of the tetrapep-
tides for the docking were chosen by optimizing the overlap with
the Ac-LDESD peptide in Csp-2 crystal structure (1PYO). A covalent
bond was set between the sulphur atom of the catalytic Cys-155
and the reactive carbon atom of the P1 aldehyde of the tetrapep-
tide. In order to take into account the protein flexibility in the S2
pocket, the side chains of Cys-155, Ala-228, Thr-160, Met-230
and Phe-279 were left unconstrained in the calculations. The
geometry of the ligand–protein complex was then optimized with-
in MOE using the MMFF94x force-field.38 Covalent docking was
carried out using the low-mode MD simulations tool under default
settings as implemented in MOE, using the MMFF94x force-field.
All the binding hypotheses output were individually analyzed
and the most representative poses among the ones with the lowest
energy were chosen. The model was initially validated by compar-
ing the binding hypotheses generated for Ac-DESD with the crystal
structure of Ac-LDESD in Csp-2 (1PYO).
ium to yield
a concentration 10-fold higher than final test
concentration in 10% DMSO. Five microliters of this compound
dilution was subsequently added per well and after incubation
for 2 h 5
ll of Ac-VDVAD-AMC substrate (Peptide Institute, Osaka,
Japan) were added to yield a final concentration of 200
l
M. Fluo-
rescence was measured every hour for 5 h to monitor the reaction
using a Tecan Safire 2 plate-reader and settings for the AMC sub-
strate as described above. Typically, the fluorescence values deter-
mined after 5 h were used to calculate the IC50 values.
6.2.2. Csp-3 cellular assay
The assay was performed in poly-L-lysine coated 384 well plates
(CellCoat, cat.-no. 78,1936, Greiner bio-one, Frickenhausen, Ger-
many). For one 384 well plate 20 ml of cell suspension was pre-
pared entirely as described above and using 2
encoding Csp-3 instead of Csp-2. Transfected cells were seeded
into the 384 well plate at a density of 10,000 cells/well (50 l of
transfection suspension). 48 h after transfection, the assay was
performed by removing the medium and replacing it with 40
lg of plasmid
l
l
l
of phenol red-free medium per well. Compounds were diluted
and added to cells entirely as described above and reaction started
by addition of 5 ll of Ac-DEVD-AMC substrate (Peptide Institute
Osaka, Japan) at a final concentration of 200
lM. Readout was
again performed as described above.
6.3. Data analysis
The docking of the tetrapeptides in Csp-3 was completed fol-
lowing the same protocol as described above except that the P4
group of the tetrapeptide was constrained. This was necessary as
Data from the various biochemical and cellular assays were cor-
rected, normalized and concentration–response relationships

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M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
the P4 group had a tendency to exit the subsite S4, thus destabiliz-
ing the low-mode MD simulation. A covalent bond was set be-
tween the sulphur atom of the catalytic Cys-163 of Csp-3 and the
reactive carbon atom of the P1 aldehyde of the tetrapeptide. The
side chains of Cys-163, Leu-168, Tyr-204, Trp-206 and Phe-256
were left unconstrained in the calculations. The model was vali-
dated by comparing the binding hypotheses generated for Ac-
DEVD with the crystal structure of Ac-DEVD in Csp-3 (2DKO).
sulfate), NaHCO3 (sodium bicarbonate), Na2SO4 (sodium sulfate),
TBME (tert-butyl methyl ether), THF (tetrahydrofuran).
6.6.2. Synthetic procedures
6.6.2.1. (2S,3S)-3-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-
(9H-fluoren-9-ylmethyl) ester 2-methyl ester (4).
A 4 N
hydrochloric acid solution in dioxane (50 mL) was added to a stir-
red solution of (2S,3S)-3-hydroxy-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester 3 (19.97 g, 56.51 mmol) in MeOH
(150 mL). The reaction mixture was stirred at room temperature
for 24 h, then concentrated under vaccum and the resulting oil
was purified by flash column chromatography (heptane/EtOAc
1:0 to 1:1) to provide the title compound (14.2 g, 68% yield) as a
white solid. MS (ES+) m/z (M+1) 368.0. 1H NMR (500 MHz, CDCl3)
6.6. Chemistry
6.6.1. Material and general methods
All reagents including (S)-Fmoc-O-tert-butyl-serine, (S)-Fmoc-
proline, Fmoc-2,3-dihydro-1H-isoindole-1-carboxylic acid, (S)-
Fmoc-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, (S)-Fmoc
pipecolic acid were used as supplied from commercial sources
and all solvents were HPLC grade.
d
ppm 7.77 (t, J = 6.78 Hz, 2H) 7.60–7.66 (m, 1H) 7.56 (t,
J = 7.41 Hz, 1H) 7.37–7.44 (m, 2H) 7.29–7.36 (m, 2H) 4.34–4.55
(m, 3.6H) 4.26–4.33 (m, 0.5H) 4.25 (s, 0.4H) 4.19 (t, J = 6.62 Hz,
0.5H) 3.65–3.81 (m, 5H) 1.90–2.28 (m, 3H). 13C NMR (126 MHz,
CDCl3) d 171.09, 171.06, 155.31, 154.81, 144.10, 143.93, 143.85,
141.39, 127.84, 127.82, 127.77, 127.19, 127.15, 125.30, 125.23,
125.06, 125.03, 120.10, 120.06, 75.34, 74.18, 68.31, 67.88, 67.82,
67.63, 52.68, 52.64, 47.33, 47.26, 45.00, 44.69, 32.74, 31.99. Mix-
ture of rotamers. HPLC purity 85%.
1H NMR spectra were recorded on a Bruker DRX 500 MHz spec-
trometer or Bruker DPX 250 MHz spectrometer in deuterated sol-
vents. Chemical shifts (d) are in parts per million. Thin-layer
chromatography (TLC) analysis was performed with Kieselgel 60
F254 (Merck) plates and visualized using UV light.
Analytical HPLC-MS was performed on Shimadzu LCMS-2010EV
systems using reverse phase Atlantis dC18 columns (3 lm,
2.1 ꢃ 50 mm), gradient 5–100% B (A = water/0.1% formic acid,
6.6.2.2. (2S,3S)-3-Methoxy-pyrrolidine-1,2-dicarboxylic acid 1-
B = acetonitrile/0.1% formic acid) over 3 min, injection volume
(9H-fluoren-9-ylmethyl) ester 2-methyl ester (5a).
Silver
3
l
l, flow = 1.0 ml/min. UV spectra were recorded at 215 nm using
oxide (4.48 g, 16.33 mmol) and methyl iodide (1.02 mL, 2.32 g,
16.33 mmol) were added to a solution of (2S,3S)-3-hydroxy-
pyrrolidine-1,2-dicarboxylic acid 1-(9H-fluoren-9-ylmethyl) ester
2-methyl ester 4 (2.00 g, 5.44 mmol) in DCE (20 mL) at room tem-
perature under nitrogen. The reaction mixture was stirred in the
dark at room temperature for 24 h, then the whole was filtered
through Keiselguhr and the filtrate was concentrated under vac-
uum to provide a crude residue which was purified by flash column
chromatography (heptane/EtOAc 1:0 to 4:1) to afford the title com-
pound (1.30 g, 63% yield) as a colorless oil. MS (ES+) m/z (M+23)
404.4. 1H NMR (500 MHz, CDCl3) d ppm 7.77 (dd, J = 7.4, 4.6 Hz,
2H) 7.63 (dd, J = 11.5, 7.6 Hz, 1H) 7.56 (t, J = 8.0 Hz, 1H) 7.38–
7.44 (m, 2H) 7.29–7.36 (m, 2H) 4.53 (s, 0.5H) 4.33–4.49 (m,
2.5H) 4.26–4.31 (m, 0.5H) 4.18–4.23 (m, 0.5H) 3.94–3.99 (m, 1H)
3.75–3.81 (m, 2H) 3.58–3.74 (m, 3H) 3.40 (d, J = 17.50 Hz, 3H)
2.00–2.19 (m, 2H). Mixture of rotamers. HPLC purity 100%.
a Waters 2788 dual wavelength UV detector. Mass spectra were
obtained over the range m/z 150–850 at a sampling rate of 2 scans
per second using Waters LCT or analytical HPLC-MS on Shimadzu
LCMS-2010EV systems using reverse phase Water Atlantis dC18
columns (3
0.1% formic acid, B = acetonitrile/0.1% formic acid) over 7 min,
injection volume 3 l, flow = 0.6 ml/min. UV spectra were recorded
lm, 2.1 ꢃ 100 mm), gradient 5–100% B (A = water/
l
at 215 nm using a Waters 2996 photo diode array. Data were inte-
grated and reported using Shimadzu psiport software.
Preparative-HPLC for intermediate compounds was performed
using a Gilson 215 Liquid Handler and Gilson 321 Pump, utilising a
reverse phase Waters Sunfire C18 column (5
100 mm), gradient 5–100% B (A = water, B = acetonitrile) over
18 min, injection volume 1000 l, flow = 20 ml/min. UV spectra
were recorded at 215 nm using a Gilson 151 UV/vis Detector system.
Data was processed using Gilson Unipoint V5.11 software. Prepara-
tive-HPLCfor final compounds (2, 33a–v) was performed using a Gil-
son 215 Liquid Handler and 2 ꢃ Gilson 306 Pumps with Gilson 805
Manometric Module, utilising a Waters Atlantis T3 OBD column
lm, 19 mm ꢃ
l
6.6.2.3. (2S,3S)-3-Isopropoxy-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester 2-methyl ester (5b).
Silver
oxide (7.88 g, 34.00 mmol) and isopropyl iodide (27.76 mL,
34.70 g, 204.14 mmol) were added to a solution of (2S,3S)-3-hy-
droxy-pyrrolidine-1,2-dicarboxylic acid 1-(9H-fluoren-9-ylmethyl)
ester 2-methyl ester 4 (2.50 g, 6.80 mmol) in DCE (50 mL) at room
temperature under nitrogen. The reaction mixture was stirred in
the dark at reflux for 15 h, then cooled to room temperature and
filtered through Keiselguhr. The filtrate was concentrated under
vacuum to provide a crude residue which was purified successively
by flash column chromatography (heptane/EtOAc 1:0 to 7:3) and
preparative HPLC to provide the title compound (0.077 g, 3% yield)
as a colorless oil. MS (ES+) m/z (M+1) 410.0. 1H NMR (250 MHz,
CDCl3) d ppm 7.77 (dd, J = 7.2, 2.5 Hz, 2H) 7.52–7.68 (m, 2H)
7.27–7.46 (m, 4H) 4.12–4.49 (m, 5H) 3.61–3.83 (m, 6H) 1.92–
2.22 (m, 2H) 1.20 (dd, J = 6.2, 2.3 Hz, 6H). Mixture of rotamers.
HPLC purity 100%.
(5
l
m, 19 mm ꢃ 100 mm), gradient 0–100% B (A = water, B = meth-
anol) over 18 min, injection volume 1000
ll, flow = 20 ml/min. UV
spectra were recorded at 215 nm using a Gilson 119 UV/vis Detector
system. Data was processed using Gilson Unipoint V5.11 software.
High Resolution Mass Spectra (HRMS) were collected using a
Waters Micromass LCT Premier orthogonal acceleration Time-of-
Flight Mass Spectrometer 4 GHz TDC with LockSpray™ under elec-
trospray positive detection and operating MassLynx v4.1 SCN 704
software. All compounds prepared for biochemical screening dis-
played purities determined by HPLC as P95% unless otherwise
stated.
The following abbreviations were used in the text: CDCl3 (deuter-
ated chloroform), D2O (deuterium oxide), DCE (1,2-dichloroethane),
DCM (dichloromethane), DDQ (2,3-dichloro-5,6-dicyanobenzoqui-
none), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformam-
ide), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), Et2O
(diethyl ether), EtOAc (ethyl acetate), EtOH (ethanol), FmocCl (9-flu-
orenylmethyl chloroformate), HATU (2-(7-aza-1H-benzotriazole-1-
6.6.2.4. (2S,3S)-3-sec-Butoxy-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester 2-methyl ester (5c).
The ti-
tle compound was prepared following the procedure used for com-
pound 5b. The crude residue was purified by preparative HPLC to
provide the title compound (0.204 g, 9% yield) as a viscous
yl)-1,1,3,3-tetramethyluronium
(1-hydroxybenzotriazole), MeOH (methanol), MgSO4 (magnesium
hexafluorophosphate),
HOBt

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5843
colorless oil. MS (ES+) m/z (M+23) 446.2. 1H NMR (250 MHz, CDCl3)
d ppm 7.74–7.81 (m, 2H) 7.52–7.67 (m, 2H) 7.29–7.45 (m, 4H)
4.12–4.46 (m, 5H) 3.66–3.80 (m, 5H) 3.40–3.56 (m, 1H) 1.95–
2.17 (m, 2H) 1.46–1.55 (m, 2H) 1.12–1.22 (m, 3H) 0.84–0.98 (m,
3H). Mixture of rotamers. HPLC purity 89%.
m/z (M+1) 410.0. 1H NMR (500 MHz, CDCl3) d ppm 7.78 (d,
J = 7.6 Hz, 1.3H) 7.73 (d, J = 7.5 Hz, 0.7H) 7.61 (t, J = 8.1 Hz, 1.3H)
7.55 (t, J = 7.0 Hz, 0.7H) 7.26–7.44 (m, 4H) 5.06 (br s, 1H) 4.16–
4.50 (m, 5H) 3.59–3.74 (m, 2H) 3.39–3.53 (m, 1H) 1.92–2.19 (m,
2H) 1.41–1.59 (m, 2H) 1.13–1.19 (m, 3H) 0.84–0.94 (m, 3H). Mix-
ture of rotamers. HPLC purity 100%.
6.6.2.5. (2S,3S)-3-Isobutoxy-pyrrolidine-1,2-dicarboxylic acid 1-
(9H-fluoren-9-ylmethyl) ester 2-methyl ester (5d).
The title
6.6.2.10. (2S,3S)-3-Isobutoxy-pyrrolidine-1,2-dicarboxylic acid
compound was prepared following the procedure used for com-
pound 5b. The crude residue was purified successively by flash col-
umn chromatography and preparative HPLC to provide the title
compound (0.555 g, 19% yield) as a colorless oil. MS (ES+) m/z
(M+1) 424.1. 1H NMR (250 MHz, CDCl3) d ppm 7.77 (m, 2H)
7.50–7.68 (m, 2H) 7.27–7.46 (m, 4H) 4.15–4.53 (m, 4H) 3.98–
4.08 (m, 1H) 3.57–3.86 (m, 5H) 3.17–3.38 (m, 2H) 1.95–2.17 (m,
2H) 1.76–1.95 (m, 1H) 0.92 (dd, J = 6.6, 2.1 Hz, 6H). Mixture of rota-
mers. HPLC purity 100%.
1-(9H-fluoren-9-ylmethyl) ester (6d).
The title compound
was prepared following the procedure used for compound 6a.
The crude residue was purified by flash column chromatography
(heptane/EtOAc 1:0 to 1:1) to afford the title compound (0.403 g,
42% yield) as a colorless oil. MS (ES+) m/z (M+1) 410.1. 1H NMR
(500 MHz, CDCl3) d ppm 7.72–7.81 (m, 2H) 7.52–7.64 (m, 2H)
7.28–7.44 (m, 4H) 4.04–4.51 (m, 6H) 3.58–3.72 (m, 2H) 3.17–
3.33 (m, 2H) 1.98–2.15 (m, 2H) 1.80–1.91 (m, 1H) 0.87–0.94 (m,
6H). Mixture of rotamers. HPLC purity 100%.
6.6.2.6. (2S,3S)-3-Cyclopentyloxy-pyrrolidine-1,2-dicarboxylic
6.6.2.11. (2S,3S)-3-Cyclopentyloxy-pyrrolidine-1,2-dicarboxylic
acid
(5e).
1-(9H-fluoren-9-ylmethyl)
The title compound was prepared following the proce-
ester
2-methyl
ester
acid 1-(9H-fluoren-9-ylmethyl) ester (6e).
The title com-
pound was prepared following the procedure used for compound
6a. The crude residue was purified by flash column chromatogra-
phy (heptane/EtOAc 1:0 to 8:2) to afford the title compound
(0.085 g, 54% yield) as a colorless oil. MS (ES+) m/z (M+1) 422.0.
1H NMR (500 MHz, CDCl3) d ppm 7.70–7.82 (m, 2H) 7.52–7.65
(m, 2H) 7.25–7.45 (m, 4H) 4.35–4.52 (m, 2.5H) 4.25–4.34 (m,
1.5H) 4.13–4.22 (m, 0.7H) 3.97–4.09 (m, 1H) 3.59–3.72 (m, 2.3H)
1.93–2.15 (m, 2H) 1.49–1.84 (m, 7H) 1.23–1.36 (m, 2H). Mixture
of rotamers. HPLC purity 99%.
dure used for compound 5b. The crude residue was purified suc-
cessively by flash column chromatography and preparative HPLC
to provide the title compound (0.161 g, 5% yield) as a colorless
oil. MS (ES+) m/z (M+1) 436.4. 1H NMR (500 MHz, CDCl3) d ppm
7.77 (t, J = 6.5 Hz, 2H) 7.63 (dd, J = 12.6, 7.4 Hz, 1H) 7.56 (t,
J = 8.3 Hz, 1H) 7.41 (q, J = 6.7 Hz, 2H) 7.29–7.36 (m, 2H) 4.26–
4.50 (m, 3.6H) 4.21 (t, J = 6.7 Hz, 0.4H) 3.98–4.14 (m, 2H) 3.59–
3.81 (m, 5H) 1.94–2.17 (m, 2H) 1.50–1.85 (m, 8H). Mixture of rota-
mers. HPLC purity 98%.
6.6.2.12.
boxylic acid 1-tert-butyl ester (8).
(1.81 g, 16.23 mmol) was added to a stirred solution of (2S,3S)-3-
hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester
(2S,3S)-3-(Pyrimidin-2-yloxy)-pyrrolidine-1,2-dicar-
6.6.2.7. (2S,3S)-3-Methoxy-pyrrolidine-1,2-dicarboxylic acid 1-
Potassium tert-butoxide
(9H-fluoren-9-ylmethyl) ester (6a).
A 6 N hydrochloric acid
aqueous solution (5 mL) was added to a stirred solution of
(2S,3S)-3-methoxy-pyrrolidine-1,2-dicarboxylic acid 1-(9H-fluo-
ren-9-ylmethyl) ester 2-methyl ester 5a (1.30 g, 3.40 mmol) in
dioxane (5 mL) at room temperature. The reaction mixture was
stirred at reflux for 18 h. The mixture was cooled to room temper-
ature, dioxane was removed under vacuum and the aqueous resi-
due was extracted with EtOAc (3 ꢃ 30 mL). The organic extracts
were combined, dried over Na2SO4, filtered and concentrated un-
der vacuum. The crude residue was purified by flash column chro-
matography (heptane/EtOAc 1:0 to 7:3) to provide the title
compound (0.70 g, 56% yield) as a white solid. MS (ES+) m/z
(M+1) 368.0. 1H NMR (500 MHz, CDCl3) d ppm 7.70–7.82 (m, 2H)
7.52–7.64 (m, 2H) 7.35–7.45 (m, 2H) 7.28–7.35 (m, 2H) 4.24–
4.54 (m, 4H) 4.14–4.22 (m, 1H) 4.00 (br s, 0.3H) 3.76–3.81 (m,
0.3H) 3.57–3.72 (m, 2.4H) 3.35–3.43 (m, 3H) 1.99–2.16 (m, 2H).
Mixture of rotamers. HPLC purity 100%.
7
(0.50 g, 2.16 mmol) in tert-butanol (20 mL) at room temperature
and the mixture was stirred for 15 min. 2-Chloropyrimidine
(1.24 g, 10.82 mmol) was added at room temperature and the reac-
tion mixture was stirred for 1 hour. The mixture was quenched
with a 10% w/w aqueous citric acid solution (ꢀ2 mL), and parti-
tioned between EtOAc (100 mL) and brine (100 mL). The layers
were separated and the aqueous phase was extracted with EtOAc
(3 ꢃ 100 mL). The organic extracts were combined, washed with
brine (1 ꢃ 50 mL), dried over MgSO4, filtered and concentrated un-
der vacuum. The resulting crude residue was purified by flash col-
umn chromatography (DCM/MeOH 1:0 to 4:1) to provide the title
compound (0.237 g, 35% yield) as a yellow gum. MS (ES+) m/z
(M+23) 332.1. 1H NMR (500 MHz, DMSO-d6) d ppm 13.07 (br s,
1H), 8.65 (d, J = 4.7 Hz, 2H), 7.20 (t, J = 4.8 Hz, 1H), 5.55 (d,
J = 4.3 Hz, 0.6H), 5.51 (d, J = 4.0 Hz, 0.4H), 4.25 (s, 0.4H), 4.20 (s,
0.6H), 3.50–3.60 (m, 1H), 3.36–3.49 (m, 1H), 1.98–2.28 (m, 2H),
1.41 (s, 4H), 1.34 (s, 5H). Mixture of rotamers. HPLC purity 100%.
6.6.2.8. (2S,3S)-3-Isopropoxy-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester (6b).
The title compound
was prepared following the procedure used for compound 6a.
The crude residue was purified by flash column chromatography
(heptane/EtOAc 1:0 to 8:2) to provide the title compound
(0.148 g, 95% yield) as a colorless oil. MS (ES+) m/z (M+1) 396.1.
1H NMR (500 MHz, CDCl3) d ppm 7.69–7.80 (m, 2H) 7.52–7.65
(m, 2H) 7.25–7.45 (m, 4H) 4.15–4.51 (m, 5H) 3.61–3.81 (m, 4H)
1.91–2.17 (m, 2H) 1.20 (d, J = 6.0 Hz, 6H). Mixture of rotamers.
HPLC purity 99%.
6.6.2.13.
(2S,3S)-3-(Pyrimidin-2-yloxy)-pyrrolidine-1,2-dicar
(2S,3S)-
boxylic acid 1-(9H-fluoren-9-ylmethyl) ester (6f).
3-(Pyrimidin-2-yloxy)-pyrrolidine-1,2-dicarboxylic acid 1-tert-bu-
tyl ester 8 (0.220 g, 0.71 mol) was dissolved in a 4 N hydrochloric
acid solution in dioxane (4 mL) and the whole was stirred at room
temperature for 1 hour before being concentrated to dryness under
vacuum. The resulting orange solid was dissolved in dioxane
(5 mL) and a solution of NaHCO3 (0.226 mg, 2.13 mmol) in water
(2 mL) was added, the mixture was cooled to 0 °C and a solution
of FmocCl (0.202 g, 0.78 mmol) in dioxane (1 mL) was added drop
wise. The mixture was stirred at 0 °C for 30 min and room temper-
ature for a further 48 h. Dioxane was removed under vacuum and
the resulting aqueous residue was diluted with water (5 mL),
washed with TBME (3 ꢃ 3 mL), acidified to pH 2 using a 2 N
6.6.2.9. (2S,3S)-3-sec-Butoxy-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester (6c).
The title compound
was prepared following the procedure used for compound 6a.
The crude residue was purified by preparative HPLC to afford the
title compound (0.173 g, 50% yield) as a colorless glass. MS (ES+)

5844
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
hydrochloric acid aqueous solution and extracted with EtOAc
(3 ꢃ 5 mL). The organic extracts were combined, dried over MgSO4,
filtered and concentrated under vacuum to provide the title com-
pound (0.201 g, 65% yield) as a colorless glass. MS (ES+) m/z
(M+1) 432.0. 1H NMR (500 MHz, DMSO-d6) d ppm 8.67 (dd,
J = 16.4, 4.7 Hz, 2H) 7.82–7.94 (m, 2H) 7.68 (dd, J = 7.2, 2.0 Hz,
1H) 7.57 (d, J = 7.6 Hz, 1H) 7.32–7.5 (m, 3H) 7.19–7.31 (m, 2H)
5.55–5.66 (m, 1H) 4.50 (s, 0.4H) 4.23–4.37 (m, 2.6H) 4.14–4.23
(m, 1H) 3.52–3.73 (m, 3H) 2.14–2.31 (m, 2H). Mixture of rotamers.
HPLC purity 97%.
THF) (2.00 mL, 4.00 mmol) was added to a stirred solution of
(2S,3S)-2-(tert-butyl-diphenyl-silanyloxymethyl)-3-(2,2-dimethyl-
propyl)-5-oxo-pyrrolidine-1-carboxylic acid tert-butyl ester 10a
(1.05 g, 2.00 mmol) in THF (5 mL) at room temperature. The result-
ing solution was stirred at reflux for 1 hour, then cooled to room
temperature, quenched with MeOH (20 mL) and concentrated un-
der vacuum. The resulting white residue was dissolved in water
(50 mL) and the solution was extracted with EtOAc (3 ꢃ 50 mL);
the organic extracts were combined, dried, filtered over MgSO4, fil-
tered and concentrated under vacuum. The title compound (1.02 g,
88% yield) was obtained as a white gum and used in the subse-
quent step without any further purification. MS (ES+) m/z (M+23)
532.2. 1H NMR (250 MHz, CDCl3) d ppm 7.54–7.79 (m, 4H) 7.30–
7.51 (m, 6H) 3.40–4.05 (m, 4H) 3.26 (dt, J = 10.7, 7.0 Hz, 1H) 2.54
(br s, 1H) 1.97–2.16 (m, 3H) 1.61–1.76 (m, 1H) 1.22–1.44 (m, 9H)
1.06 (s, 9H) 0.81–1.01 (m, 9H). HPLC purity 72%.
6.6.2.14.
(2,2-dimethyl-propyl)-5-oxo-pyrrolidine-1-carboxylic acid tert-
butyl ester (10a). solution of neopentylmagnesium
(2S,3S)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-
A
bromide (0.5 M in THF) (57.57 mL, 28.78 mmol) was added to a
stirred solution of (methylsulfanyl)methane–bromocopper (1:1)
(0.592 g, 2.88 mmol) in THF (5 mL) at ꢁ35 °C and the mixture
was stirred at this temperature for 20 min. The mixture was cooled
to ꢁ78 °C and a solution of (S)-2-(tert-butyl-diphenyl-silanyloxym-
ethyl)-5-oxo-2,5-dihydro-pyrrole-1-carboxylic acid tert-butyl es-
ter 9 (1.30 g, 2.88 mmol) in THF (5 mL) and chlorotrimethylsilane
(0.73 mL, 0.625 mg, 5.76 mmol) was added. The reaction mixture
was slowly warmed to room temperature and stirred for 2 h before
being cooled to 0 °C, quenched with a saturated ammonium chlo-
ride aqueous solution (3 mL) and warmed to room temperature.
The mixture was extracted with EtOAc (3 ꢃ 5 mL), the organic ex-
tracts were combined, washed with a saturated ammonium chlo-
ride aqueous solution (3 ꢃ 3 mL), dried over Na2SO4, filtered and
concentrated under vacuum. The resulting residue was purified
by flash column chromatography (heptane/EtOAc 1:0 to 3:1) to af-
ford the title compound (1.05 g, 70% yield) as a colorless oil. MS
(ES+) m/z (M+23) 546.4. 1H NMR (250 MHz, CDCl3) d ppm 7.54–
7.71 (m, 4H) 7.30–7.51 (m, 6H) 3.76–3.97 (m, 2H) 3.58–3.77 (m,
1H) 2.93 (dd, J = 17.6, 8.9 Hz, 1H) 2.28–2.51 (m, 1H) 2.19 (dd,
J = 17.6, 2.7 Hz, 1H) 1.44 (s, 9H) 1.27 (s, 2H) 1.06 (s, 9H) 0.93 (s,
9H). HPLC purity 74%.
6.6.2.18.
(2S,3R)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-
3-p-tolyl-pyrrolidine-1-carboxylic acid tert-butyl ester
(11b). The title compound was prepared according to the proce-
dure used for compound 11a. The crude residue was not purified
and the title compound (82% yield) was obtained as a colorless oil.
(ES+) m/z (M+23) 552.1. HPLC purity 84%.
6.6.2.19.
cyclohexyl-pyrrolidine-1-carboxylic acid tert-butyl ester
(11c). The title compound was prepared according to the pro-
(2S,3R)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-
cedure used for compound 11a. The crude residue was not purified
and the title compound (100% yield) was obtained as a colorless oil.
(ES+) m/z (M+23) 544.2. 1H NMR (500 MHz, CDCl3) d ppm 7.58–
7.70 (m, 4H) 7.31–7.49 (m, 6H) 3.43–4.04 (m, 4H) 3.27 (dt,
J = 10.6, 7.4 Hz, 1H) 2.08–2.26 (m, 1H) 1.86–1.99 (m, 1H) 1.59–
1.80 (m, 6H) 1.33–1.50 (m, 9H) 1.12–1.24 (m, 3H) 1.05 (s, 9H)
0.84–1.01 (m, 3H). HPLC purity 92%,
6.6.2.20. (2S,3S)-3-(2,2-Dimethyl-propyl)-2-hydroxymethyl-pyr-
rolidine-1-carboxylic acid tert-butyl ester (12a).
A 1 M
6.6.2.15.
oxo-3-p-tolyl-pyrrolidine-1-carboxylic acid tert-butyl ester
(10b). The title compound was prepared according to the pro-
(2S,3R)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-5-
solution of tetrabutyl ammonium fluoride in THF (3.54 mL,
3.54 mmol) was added to a stirred solution of (2S,3S)-2-(tert-
butyl-diphenyl-silanyloxymethyl)-3-(2,2-dimethyl-propyl)-pyr-
rolidine-1-carboxylic acid tert-butyl ester 11a (0.723 g,
1.42 mmol) in THF (15 mL) at room temperature. The reaction
mixture was stirred at room temperature for 24 h before being
concentrated under vacuum. The resulting residue was dissolved
in EtOAc (10 mL) and the solution was washed with a saturated
ammonium chloride aqueous solution (2 ꢃ 10 mL) and water
(2 ꢃ 10 mL), dried over MgSO4, filtered and concentrated under
vacuum. The crude residue was purified by flash column chroma-
tography (heptane/EtOAc 1:0 to 9:1) to afford the title compound
(0.251 g, 65% yield) as a colorless crystalline solid. MS (ES+) m/z
cedure used for compound 10a. The crude residue was purified by
flash column chromatography (heptane/EtOAc 1:0 to 98.5:1.5) to
afford the title compound (0.971 g, 68% yield) as a white gum.
MS (ES+) m/z (M+23) 566.0. 1H NMR (500 MHz, CDCl3) d ppm
7.60–7.70 (m, 4H) 7.34–7.49 (m, 6H) 7.14 (d, J = 7.9 Hz, 2H) 7.06
(d, J = 8.1 Hz, 2H) 4.09 (dt, J = 4.2, 2.1 Hz, 1H) 3.97 (dd, J = 10.5,
4.3 Hz, 1H) 3.81 (dd, J = 10.5, 2.3 Hz, 1H) 3.40–3.54 (m, 1H) 3.19
(dd, J = 18.0, 9.5 Hz, 1H) 2.57 (dd, J = 17.8, 2.8 Hz, 1H) 2.34 (s, 3H)
1.42 (s, 9H) 1.09 (s, 9H). HPLC purity 91%.
6.6.2.16.
cyclohexyl-5-oxo-pyrrolidine-1-carboxylic acid tert-butyl ester
(10c). The title compound was prepared according to the pro-
(2S,3R)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-
(M+23) 294.1. 1H NMR (500 MHz, CDCl3)
d ppm 5.22 (d,
J = 8.9 Hz, 1H) 3.71 (d, J = 9.8 Hz, 1H) 3.49–3.64 (m, 2H) 3.44 (t,
J = 7.9 Hz, 1H) 3.21 (d, J = 10.5, 6.5 Hz, 1H) 1.98–2.11 (m, 1H)
1.62–1.77 (m, 1H) 1.48 (s, 11H) 1.28 (dd, J = 14.1, 8.6 Hz, 1H)
0.92 (s, 9H). HPLC purity 100%.
cedure used for compound 10a. The crude residue was purified by
flash column chromatography (heptane/EtOAc 1:0 to 95:5) to af-
ford the title compound (0.693 g, 80% yield) as a pale yellow oil.
(ES+) m/z (M+23) 558.0. 1H NMR (500 MHz, CDCl3) d ppm 7.56–
7.68 (m, 4H) 7.32–7.51 (m, 6H) 3.94–4.01 (m, 1H) 3.89 (dd,
J = 10.3, 4.3 Hz, 1H) 3.65 (dd, J = 10.3, 2.3 Hz, 1H) 2.82 (dd,
J = 18.0, 9.8 Hz, 1H) 2.31 (dd, J = 18.0, 2.0 Hz, 1H) 2.05–2.19 (m,
1H) 1.76 (dd, J = 12.6, 2.2 Hz, 2H) 1.57–1.72 (m, 2H) 1.46 (s, 9H)
1.11–1.38 (m, 5H) 1.05 (s, 9H) 0.86–1.03 (m, 2H). HPLC purity 95%.
6.6.2.21. (2S,3R)-2-Hydroxymethyl-3-p-tolyl-pyrrolidine-1-car-
boxylic acid tert-butyl ester (12b).
The title compound was
prepared according to the procedure used for compound 12a. The
crude residue was purified by flash column chromatography (hep-
tane/EtOAc 1:0 to 4:1) to afford the title compound (91% yield) as a
colorless oil. (ES+) m/z (M+23) 314.1. 1H NMR (500 MHz, CDCl3) d
ppm 7.14 (s, 4H) 5.03 (d, J = 7.9 Hz, 1H) 3.67–4.01 (m, 3H) 3.58–
3.65 (m, 1H) 3.35 (d, J = 10.5, 6.5 Hz, 1H) 2.77–2.96 (m, 1H) 2.34
(s, 3H) 2.07–2.20 (m, 1H) 1.95 (m, J = 12.3, 10.3, 10.3, 8.1 Hz, 1H)
1.51 (s, 9H). HPLC purity 96%.
6.6.2.17.
(2,2-dimethyl-propyl)-pyrrolidine-1-carboxylic acid tert-butyl
ester (11a). A solution of borane-dimethyl sulfide (2 M in
(2S,3S)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5845
6.6.2.22. (2S,3R)-3-Cyclohexyl-2-hydroxymethyl-pyrrolidine-1-
carboxylic acid tert-butyl ester (12c). The title compound
2.11–2.25 (m, 1H) 1.52–1.68 (m, 2H) 1.29 (dd, J = 14.0, 7.3 Hz, 1H)
0.88–0.99 (m, 9H). Mixture of rotamers. HPLC purity 93%.
was prepared according to the procedure used for compound
12a. The crude residue was purified by flash column chromatogra-
phy (heptane/EtOAc 1:0 to 9:1) to afford the title compound (66%
yield) as a colorless oil. (ES+) m/z (M+23) 306.1. 1H NMR (500 MHz,
CDCl3) d ppm 4.82 (d, J = 7.3 Hz, 1H) 3.44–3.86 (m, 4H) 3.06–3.24
(m, 1H) 1.51–1.97 (m, 9H) 1.48 (s, 8H) 0.84–1.37 (m, 6H). HPLC
purity 75%.
6.6.2.27. (2S,3R)-3-para-Tolyl-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-yl methyl) ester (15b).
The title compound
was prepared according to the procedure used for compound
15a. The crude residue was purified by flash column chromatogra-
phy (DCM/MeOH 1:0 to 99:1) to afford the title compound
(0.317 g) in quantitative yield as a pale pink gum. (ES+) m/z
(M+23) 450.2. 1H NMR (500 MHz, CDCl3) d ppm 7.68–7.85 (m,
2H) 7.49–7.65 (m, 2H) 7.30–7.46 (m, 4H) 7.04–7.19 (m, 4H) 4.55
(dd, J = 10.7, 6.9 Hz,1H) 4.48 (dd, J = 11.1, 6.4 Hz, 1.3H) 4.22–4.32
(m, 0.6H) 4.17 (t, J = 5.8 Hz, 0.7H) 3.67–3.80 (m, 2H) 3.54–3.67
(m, 1H) 3.48 (q, J = 6.5 Hz, 0.4H) 2.26–2.41 (m, 4H) 1.89–2.12 (m,
1H). Mixture of rotamers. HPLC purity 100%.
6.6.2.23. (2S,3S)-3-(2,2-Dimethyl-propyl)-pyrrolidine-1,2-dicar-
boxylic acid 1-tert-butyl ester (13a).
trioxide (0.180 mg, 1.801 mmol) in sulfuric acid (98
A solution of chromium
l, 1.84 mmol)
l
and water (0.5 ml) was added to a stirred solution of (2S,3S)-3-
(2,2-dimethyl-propyl)-2-hydroxymethyl-pyrrolidine-1-carboxylic
acid tert-butyl ester 12a (0.251 g, 0.901 mmol) in acetone (5 mL) at
room temperature. The reaction mixture was stirred at room tem-
perature for 5 h before being quenched with a saturated ammo-
nium chloride aqueous solution (10 mL). The mixture was
extracted with EtOAc (3 ꢃ 10 mL) and the combined organics were
washed with water (2 ꢃ 20 mL) and extracted with a 1 M sodium
hydroxide aqueous solution (3 ꢃ 40 mL). The aqueous extracts
were combined, acidified to pH 4 with 1 M hydrochloric acid aque-
ous solution and extracted with EtOAc (3 ꢃ 40 mL). These organic
extracts were combined, dried over MgSO4, filtered, and concen-
trated to afford the title compound (0.187 g, 73% yield) as a color-
less solid. MS (ES+) m/z (M+23) 308.0. HPLC purity 88%.
6.6.2.28. (2S,3R)-3-Cyclohexyl-pyrrolidine-1,2-dicarboxylic acid
1-(9H-fluoren-9-ylmethyl) ester (15c).
The title compound
was prepared according to the procedure used for compound
15a. The crude residue was purified by flash column chromatogra-
phy (DCM/MeOH 1:0 to 97:3) to afford the title compound
(0.214 g, 55% yield) as a colorless oil. (ES+) m/z (M+1) 420.2. 1H
NMR (500 MHz, CDCl3) d ppm 7.68–7.82 (m, 2H) 7.51–7.64 (m,
2H) 7.35–7.46 (m, 2H) 7.28–7.35 (m, 2H) 4.44–4.54 (m, 1H)
4.33–4.44 (m, 1H) 4.04–4.31 (m, 2H) 3.53–3.68 (m, 1H) 3.47 (dt,
J = 10.4, 7.4 Hz, 1H) 2.06 (s, 2H) 1.57–1.85 (m, 6H) 1.08–1.42 (m,
4H) 0.90–1.08 (m, 2H). HPLC purity 98%.
6.6.2.24. (2S,3R)-3-p-Tolyl-pyrrolidine-1,2-dicarboxylic acid 1-
6.6.2.29. (2S,3R)-3-Phenyl-pyrrolidine-1,2-dicarboxylic acid 1-
tert-butyl ester (13b).
The title compound was prepared
(9H-fluoren-9-ylmethyl) ester (15d).
(2S,3R)-3-Phenyl-pyr-
according to the procedure used for compound 13a. The crude res-
idue was not purified and the title compound (61% yield) was ob-
tained as a white solid. (ESꢁ) m/z (Mꢁ1) 304.2. 1H NMR (500 MHz,
CDCl3) d ppm 7.05–7.21 (m, 4H) 4.18–4.53 (m, 1H) 3.71–3.82 (m,
1H) 3.54–3.70 (m, 1H) 3.42–3.54 (m, 1H) 2.25–2.37 (m, 4H)
1.96–2.09 (m, 1H) 1.37–1.58 (m, 9H). HPLC purity 96%.
rolidine-2-carboxylic acid 14 (0.096 g, 0.502 mmol) was suspended
in dioxane (3 mL) and the suspension was cooled to 0 °C. A solution
of NaHCO3 (0.088 g, 1.054 mmol) in water (5 mL) was added to the
mixture and the stirring was continued at 0 °C for 5 min. A solution
of FmocCl (0.130 g, 0.502 mmol) in dioxane (2 mL) was added
slowly and the reaction mixture was stirred for 18 h with gradual
warming to room temperature. The reaction mixture was concen-
trated under vacuum to remove the dioxane; the resulting aqueous
residue was diluted with water (10 mL), washed with TBME
(2 ꢃ 5 mL), acidified to pH 2 using 2 N hydrochloric acid aqueous
solution and extracted with EtOAc (3 ꢃ 10 mL). The organic ex-
tracts were combined, dried over Na2SO4, filtered and concentrated
under vacuum. The crude material was purified by flash column
chromatography (heptane/EtOAc 1:2 to 2:3) to provide the title
compound (0.163 g, 78% yield) as a white solid. (ES+) m/z (M+1)
414.0. 1H NMR (250 MHz, CDCl3) d ppm 7.15–7.85 (m, 13H)
4.42–4.60 (m, 2.4H) 4.26–4.36 (m, 1H) 4.08–4.22 (m, 0.6H) 3.41–
3.75 (m, 3H) 2.28–2.49 (m, 1H) 1.97–2.15 (m, 1H) 1.22–1.32 (m,
0.7H) 0.85–0.93 (m, 0.3H). Mixture of rotamers. HPLC purity 100%.
6.6.2.25. (2S,3R)-3-Cyclohexyl-2-hydroxymethyl-pyrrolidine-1-
carboxylic acid tert-butyl ester (13c).
The title compound
was prepared according to the procedure used for compound
13a. The crude residue was not purified and the title compound
(100% yield) was obtained as a colorless oil. (ES+) m/z (M+23)
320.1. 1H NMR (500 MHz, CDCl3) d ppm 3.96–4.17 (m, 1H) 3.27–
3.64 (m, 2H) 2.13–2.43 (m, 1H) 1.93–2.03 (m, 1H) 1.61–1.87 (m,
6H) 1.37–1.54 (m, 9H) 0.90–1.31 (m, 6H). HPLC purity 85%.
6.6.2.26. (2S,3S)-3-(2,2-Dimethyl-propyl)-pyrrolidine-1,2-dicar-
boxylic acid 1-(9H-fluoren-9-ylmethyl) ester (15a).
(2S,3S)-3-
(2,2-dimethyl-propyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 13a (0.187 g, 0.66 mmol) was dissolved in a mixture of DCM/
trifluoroacetic acid (1:1) (2 mL) at 0 °C and stirred for 45 min at this
temperature. The reaction mixture was concentrated under vacuum
and the resulting residue was dissolved in dioxane (1.5 mL); a 10%
w/w sodium carbonate aqueous solution (1 mL) and a solution of
FmocCl (0.170 g, 0.66 mmol) in dioxane (2 mL) were added and
the mixture was stirred at room temperature for 18 h. The reaction
mixture was diluted with water (5 mL), acidified to pH 1 with 1 N
hydrochloric acid aqueous solution and extracted with EtOAc
(2 ꢃ 5 mL). The organic extracts were combined, dried over MgSO4,
filtered and concentrated under vacuum. The crude residue was
purified by flash column chromatography (DCM/MeOH 1:0 to
98:2) to afford the title compound (0.168 g, 63% yield) as a yellow
brittle foam. MS (ES+) m/z (M+23) 430.1. 1H NMR (500 MHz, CDCl3)
d ppm 7.69–7.83 (m, 2H) 7.50–7.65 (m, 2H) 7.27–7.45 (m, 4H) 4.46–
4.55 (m, 1H) 4.33–4.44 (m, 1H) 4.12–4.33 (m, 1H) 3.76–4.08 (m, 1H)
3.55–3.69 (m, 1H) 3.51 (dt, J = 10.4, 7.0 Hz, 1H) 2.27–2.62 (m, 1H)
6.6.2.30.
(2S,3S)-2-Azido-3-isopropoxy-6-(4-methoxy-benzyl-
oxy)-hexanoic acid ethyl ester (17). 2-iodopropane (16.5 mL,
28.1 g, 165.3 mmol) and silver oxide (38.3 g, 165.3 mmol) were
added to a solution of (2S,3S)-2-azido-3-hydroxy-6-(4-methoxy-
benzyloxy)-hexanoic acid ethyl ester 16 (4.60 g, 13.8 mmol) in
1,2-dichloroethane (190 mL) under nitrogen at room temperature.
The mixture was heated at 70 °C for 17 h. The reaction mixture
was cooled to room temperature, filtered over glass fiber paper
and the filtrate was concentrated under vacuum. The crude residue
was purified by flash column chromatography (heptane/EtOAc 1:0
to 6:4) to afford the title compound (0.92 g, 18% yield) as a color-
less oil. MS (ES+) m/z (M+23) 402.5. 1H NMR (500 MHz, CDCl3) d
ppm 7.26 (d, J = 8.6 Hz, 2H) 6.88 (d, J = 8.6 Hz, 2H) 4.43 (s, 2H)
4.25 (q, J = 7.1 Hz, 2H) 4.11 (d, J = 4.4 Hz, 1H) 3.78–3.83 (m, 4H)
3.72 (dt, J = 12.2, 6.1 Hz, 1H) 3.40–3.50 (m, 2H) 1.59–1.82 (m,

5846
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
3H) 1.49–1.57 (m, 1H) 1.29–1.33 (m, 3H) 1.16 (dd, J = 9.7, 6.1 Hz,
6H). HPLC purity 100%.
diluted with water (10 mL), pH was adjusted to 6 using a 10% w/
w citric acid aqueous solution and the mixture was extracted with
EtOAc (3 ꢃ 50 mL). The organic extracts were combined, washed
with brine (2 ꢃ 30 mL), dried over MgSO4, filtered and concen-
trated under vacuum. The crude residue was purified by flash col-
umn chromatography (heptane/EtOAc 1:0 to 9:1) to afford the title
compound (0.225 g, 59% yield) as a colorless oil. MS (ES+) m/z
(M+23) 338.4. 1H NMR (500 MHz, CDCl3) d ppm 4.71–5.03 (m,
1H) 4.20 (q, J = 7.1 Hz, 2H) 3.88–4.11 (m, 2H) 3.65–3.76 (m, 1H)
2.83–3.10 (m, 1H) 1.75–1.94 (m, 2H) 1.31–1.52 (m, 11H) 1.28 (t,
J = 7.1 Hz, 3H) 1.16 (d, J = 6.0 Hz, 6H). Mixture of rotamers. HPLC
purity 86%.
6.6.2.31. (2S,3S)-2-Azido-6-hydroxy-3-isopropoxy-hexanoic acid
ethyl ester (18).
To a solution of (2S,3S)-2-azido-3-isopro-
poxy-6-(4-methoxy-benzyloxy)-hexanoic acid ethyl ester 17
(0.92 g, 2.42 mmol) in DCM (20 mL) at 0 °C was added water
(1.5 mL) and DDQ (0.82 g, 3.61 mmol) portion wise. The reaction
mixture was stirred at 0 °C for 10 min and room temperature for
a further 90 min. The reaction mixture was filtered through glass
fiber paper and the residue was washed with DCM (4 ꢃ 5 mL).
The filtrate phases were separated and the aqueous phase was ex-
tracted with DCM (1 ꢃ 10 mL). The organic extracts were com-
bined, dried over MgSO4, filtered and concentrated under
vacuum. The crude residue was purified by flash column chroma-
tography (heptane/EtOAc 1:0 to 4:6) to afford the title compound
(0.46 g, 73% yield) as a colorless oil. MS (ES+) m/z (M+23) 282.0.
1H NMR (500 MHz, CDCl3) d ppm 4.27 (q, J = 7.1 Hz, 2H) 4.15 (d,
J = 4.7 Hz, 1H) 3.84 (dt, J = 8.0, 4.02 Hz, 1H) 3.76 (dt, J = 12.2,
6.0 Hz, 1H) 3.67 (m, 2H) 1.68–1.79 (m, 2H) 1.59–1.68 (m, 2H)
1.52–1.59 (m, 1H) 1.33 (t, J = 7.2 Hz, 3H) 1.19 (d, J = 6.1 Hz, 6H).
HPLC purity 100%.
6.6.2.35. (2S,3S)-3-Isopropoxy-piperidine-1,2-dicarboxylic acid
1-tert-butyl ester (22).
A solution of lithium hydroxide
(0.431 g, 17.980 mmol) in water (2 mL) was added to a stirred
solution of (2S,3S)-3-isopropoxy-piperidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-ethyl ester 21 (0.567 g, 1.798 mmol) in THF
(5 mL) at room temperature. The reaction mixture was stirred at
room temperature for 3 h. THF was removed under vacuum and
the resulting aqueous residue was diluted with water (5 mL),
washed with TBME (2 ꢃ 5 mL), acidified to pH 4 using a 1 N hydro-
chloric acid aqueous solution, extracted with EtOAc (3 ꢃ 20 mL).
The organic extracts were combined, washed with brine
(1 ꢃ 10 mL), dried over MgSO4, filtered and concentrated to pro-
vide the title compound (0.256 g, 50% yield) as a white solid. MS
(ES+) m/z (M+23) 310.0. 1H NMR (500 MHz, CDCl3) d ppm 4.83–
5.09 (m, 1H) 3.92–4.15 (m, 2H) 3.66–3.78 (m, 1H) 2.85–3.09 (m,
1H) 1.78–1.94 (m, 2H) 1.33–1.54 (m, 11H) 1.17 (d, J = 6.0 Hz, 6H).
Mixture of rotamers. HPLC purity 100%.
6.6.2.32. (2S,3S)-2-tert-butoxycarbonylamino-6-hydroxy-3-iso-
propoxy-hexanoic acid ethyl ester (19).
Boc anhydride
(0.584 g, 2.661 mmol) was added to a stirred solution of (2S,3S)-
2-azido-6-hydroxy-3-isopropoxy-hexanoic acid ethyl ester 18
(0.460 g, 1.774 mmol) in EtOAc (20 mL). The solution was degassed
using nitrogen and palladium on carbon (10% w/w, 50% wet with
water) (0.156 mg, 0.701 mmol) was added. The reaction vessel
was purged and put under hydrogen. The reaction mixture was
stirred for 15 h at room temperature. The mixture was filtered
through celite and the residue was washed with EtOAc (10 mL).
The filtrate was concentrated under vacuum and the resulting res-
idue was purified by flash column chromatography (heptane/
EtOAc 1:0 to 0:1) to afford the title compound (0.591 g, 80% yield)
as a colorless oil. MS (ES+) m/z (M+23) 356.0. 1H NMR (250 MHz,
CDCl3) d ppm 5.20 (d, J = 9.0 Hz, 1H) 4.55 (dd, J = 8.7, 4.0 Hz, 1H)
4.22 (q, J = 7.1 Hz, 2H) 3.59–3.82 (m, 4H) 1.41–1.87 (m, 14H)
1.29 (t, J = 7.1 Hz, 3H) 1.14 (dd, J = 6.02, 2.4 Hz, 6H). HPLC purity
100%.
6.6.2.36. (2S,3S)-3-Isopropoxy-piperidine-2-carboxylic acid tri-
fluoroacetic acid salt (23).
Trifluoro acetic acid (50% v/v
solution in DCM) (2 mL) was added to a stirred solution of
(2S,3S)-3-isopropoxy-piperidine-1,2-dicarboxylic acid 1-tert-butyl
ester 22 (0.130 g, 0.452 mmol) in DCM (1 mL) at 0 °C. The reaction
mixture was stirred for 2 h at 0 °C and concentrated under vacuum
to produce the title compound (0.123 g, 90% yield) as a viscous pale
yellow oil. MS (ES+) m/z (M+1) 188.0. 1H NMR (250 MHz, MeOD) d
ppm 4.04–4.16 (m, 2H) 3.80 (dt, J = 12.2, 6.1 Hz, 1H) 3.25–3.41 (m,
1H) 3.07–3.21 (m, 1H) 1.93–2.16 (m, 1H) 1.62–1.93 (m, 3H) 1.20
(d, J = 7.3 Hz, 6H). HPLC purity 91%.
6.6.2.33. (2S,3S)-2-tert-butoxycarbonylamino-3-isopropoxy-6-
methanesulfonyloxy-hexanoic acid ethyl ester (20).
Trieth-
6.6.2.37. (2S,3S)-3-Isopropoxy-piperidine-1,2-dicarboxylic acid
ylamine (0.39 mL, 0.28 g, 2.80 mmol) and mesyl chloride (0.13 mL,
0.19 g, 1.68 mmol) were added to a solution of (2S,3S)-2-tert-but-
oxycarbonylamino-6-hydroxy-3-isopropoxy-hexanoic acid ethyl
ester 19 (0.46 g, 1.38 mmol) in DCM (26 mL) at 0 °C under nitro-
gen. The mixture was stirred at 0 °C for 1 hour and room temper-
ature for 1 hour before being concentrated under vacuum. The
resulting residue was purified by flash column chromatography
(heptane/EtOAc 1:0 to 2:1) to provide the title compound (0.47 g,
76% yield) as a colorless oil. MS (ES+) m/z (M+23) 434.1. 1H NMR
(500 MHz, CDCl3) d ppm 5.13–5.25 (m, 1H) 4.46–4.56 (m, 1H)
4.19–4.31 (m, 4H) 3.74 (s, 2H) 3.01 (s, 3H) 1.90–2.01 (m, 1H)
1.77–1.87 (m, 1H) 1.59–1.66 (m, 1H) 1.49–1.56 (m, 1H) 1.46 (s,
9H) 1.30 (t, J = 7.09 Hz, 3H) 1.14 (d, J = 5.99 Hz, 6H). HPLC purity
100%.
1-(9H-fluoren-9-ylmethyl) ester (24).
A sodium carbonate
aqueous solution (10% w/w) (0.791 mL, 0.950 mmol) was added
to a stirred solution of (2S,3S)-3-isopropoxy-piperidine-2-carbox-
ylic acid trifluoroacetic acid salt 23 (0.123 mg, 0.408 mmol) in
dioxane (1 mL) at room temperature and the solution was cooled
to 0 °C. A solution of FmocCl (0.117 g, 0.452 mmol) in dioxane
(1 mL) was added drop wise at 0 °C and the reaction mixture was
allowed to warm to room temperature and stirred for 16 h. Diox-
ane was removed under reduced pressure and the resulting basic
solution was diluted with water (3 mL), washed with TBME
(2 ꢃ 2 mL) and acidified to pH 2 using 2 N hydrochloric acid aque-
ous solution. The acidic phase was extracted with EtOAc
(3 ꢃ 5 mL), the organic extracts were combined, dried over MgSO4,
filtered and concentrated under vacuum. The crude residue was
purified by flash column chromatography (heptane/EtOAc 1:0 to
1:1) to provide the title compound (0.092 g, 50% yield) as a color-
less glass. MS (ES+) m/z (M+23) 432.4. 1H NMR (500 MHz, CDCl3) d
ppm 7.70–7.83 (m, 2H) 7.53–7.66 (m, 2H) 7.28–7.44 (m, 4H) 5.10
(br s, 0.6H) 4.72 (br s, 0.4H) 4.39–4.53 (m, 2H) 4.22–4.37 (m, 1H)
3.97–4.17 (m, 2H) 3.74 (dt, J = 11.9, 5.7 Hz, 0.6H) 3.45–3.55 (m,
0.4H) 3.14–3.24 (m, 0.6H) 2.96 (t, J = 12.4 Hz, 0.4H) 1.78–2.04 (m,
6.6.2.34. (2S,3S)-3-Isopropoxy-piperidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-ethyl ester (21).
mineral oil) (0.049 g, 1.215 mmol) was added to a stirred solution
of (2S,3S)-2-tert-butoxycarbonylamino-3-isopropoxy-6-metha-
Sodium hydride (60% in
nesulfonyloxy-hexanoic acid ethyl ester 20 (0.500 g, 1.215 mmol)
in DMF (7 mL) at 0 °C under nitrogen. The mixture was allowed
to warm to room temperature over 4 h. The reaction mixture was

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5847
2H) 1.36–1.59 (m, 2H) 1.22–1.35 (m, 1H) 1.04–1.22 (m, 6H). HPLC
purity 99%.
(15 mL):2 N HCl (aq) (15 mL). The phases were separated and the
aqueous phase was extracted with EtOAc (3 ꢃ 15 mL). The organic
extracts were combined, dried over Na2SO4, filtered and concen-
trated under vacuum to provide a sticky off-white solid which
was triturated in Et2O to provide the title compound (0.90 g, 83%
yield) as an off-white solid. (ES+) m/z (M+1) 438.1. 1H NMR
(500 MHz, DMSO-d6) d ppm 9.50 (s, 0.4H) 9.49 (s, 0.6H) 7.83–7.95
(m, 2H) 7.66 (q, J = 6.7 Hz, 2H) 7.38–7.48 (m, 2H) 7.21–7.37 (m,
3H) 6.62–6.70 (m, 1H) 6.59 (s, 1H) 5.34 (s, 0.4H) 5.27 (s, 0.6H)
4.22–4.41 (m, 3H) 3.63–3.72 (m, 1H) 3.51–3.63 (m, 1H) 2.75–2.85
(m, 1H) 2.61–2.75 (m, 1H). Mixture of rotamers. HPLC purity 83%.
6.6.2.38. 6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-1-carbox-
ylic acid (26a).
Triethylamine (0.76 mL, 0.55 g, 5.47 mmol)
was added to a stirred solution of 3-(2-aminoethyl)phenol hydro-
chloride (1:1) 25a (1.00 g, 5.76 mmol) in EtOH (26 mL). The mix-
ture was cooled to 5 °C and a solution of glyoxylic acid (0.53 g,
5.76 mmol) in EtOH (6 mL) was added drop wise. The resulting
mixture was stirred for 1 hour with gradual warming to room tem-
perature. The resulting solid was filtered and washed with EtOH to
provide the title compound (0.78 g, 70% yield) as an off-white solid.
(ES+) m/z (M+1) 193.9. 1H NMR (500 MHz, DMSO-d6) d ppm 9.35
(br s, 1H) 7.50 (d, J = 8.6 Hz, 1H) 6.60 (dd, J = 8.6, 2.0 Hz, 1H) 6.49
(d, J = 2.0 Hz, 1H) 4.33 (s, 1H) 3.42–3.47 (m, 1H) 3.27–3.34 (m,
1H) 3.06–3.14 (m, 1H) 2.81–2.89 (m, 1H) 2.69–2.78 (m, 1H).
6.6.2.43. 6-Methyl-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29b).
The title
compound was prepared according to the procedure used for com-
pound 29a The crude residue did not require purification and the
title compound (0.454 g, 63% yield) was obtained as a white pow-
der. (ES+) m/z (M+1) 414.0. 1H NMR (250 MHz, CDCl3) d ppm
6.95–7.83 (m, 12H) 5.62 (s, 0.6H) 5.33 (s, 0.4H) 4.38–4.61 (m,
2H) 4.16–4.36 (m, 1H) 3.66–3.97 (m, 2H) 2.73–3.06 (m, 2H)
2.29–2.37 (m, 3H). Mixture of rotamers. HPLC purity 100%.
6.6.2.39. [2-(3-Hydroxy-phenyl)-ethyl]-carbamic acid 9H-fluo-
ren-9-ylmethyl ester (28e).
A solution of FmocCl (4.3 g,
16.8 mmol) in DCM (20 mL) was added to a stirred solution of 3-
(2-amino-ethyl)-phenol 27e (2.0 g, 14.6 mmol) and DIPEA
(7.2 mL, 5.6 g, 43.7 mmol) in DCM (30 mL) at 0 °C. After 15 min
at 0 °C, the mixture was stirred for 20 h at room temperature.
The reaction mixture was diluted with DCM (50 mL), washed suc-
cessively with a saturated aqueous NaHCO3 solution (1 ꢃ 20 mL)
and brine (1 ꢃ 20 mL). The organic phase was dried over Na2SO4,
filtered and concentrated under vacuum. The crude residue was
purified by flash column chromatography (DCM/MeOH 1:0 to
95:5) to provide the title compound (1.2 g, 23% yield) as a white
solid. (ES+) m/z (M+23) 382.3. HPLC purity 95%.
6.6.2.44. 7-Methyl-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29c).
The title
compound was prepared according to the procedure used for com-
pound 29a. The crude residue did not require purification and the
title compound (0.402 g, 55% yield) was obtained as a white
powder. (ES+) m/z (M+1) 414.0. 1H NMR (250 MHz, CDCl3) d ppm
7.01–7.83 (m, 12H) 5.61 (s, 0.6H) 5.32 (s, 0.4H) 4.36–4.63 (m,
2H) 4.07–4.35 (m, 1H) 3.65–3.98 (m, 2H) 2.84 (d, J = 6.4 Hz, 2H)
2.35 (s, 3H). Mixture of rotamers. HPLC purity 89%.
6.6.2.40. [2-(4-Bromo-phenyl)-ethyl]-carbamic acid 9H-fluoren-
9-ylmethyl ester (28f).
The title compound was prepared
6.6.2.45. 6,7-Dimethoxy-3,4-dihydro-1H-isoquinoline-1,2-dicar-
according to the procedure used for compound 28e. The crude res-
idue was purified by trituration in TBME to provide the title com-
pound (1.24 g, 50% yield) as a white powder. (ES+) m/z (M+23)
444.1/445.9. 1H NMR (500 MHz, CDCl3) d ppm 7.78 (d, J = 7.6 Hz,
2H) 7.57 (d, J = 7.4 Hz, 2H) 7.39–7.46 (m, 4H) 7.29–7.36 (m, 2H)
7.05 (d, J = 8.2 Hz, 1.7H) 6.90 (br s, 0.3H) 4.74 (d, J = 4.7 Hz, 1H)
4.55 (br s, 0.3H) 4.43 (d, J = 6.6 Hz, 1.7H) 4.19–4.26 (m, 1H) 3.43
(q, J = 6.4 Hz, 1.7H) 3.22 (br s, 0.3H) 2.78 (t, J = 6.7 Hz, 1.7H) 2.53
(br s, 0.3H). Mixture of rotamers. HPLC purity 100%.
boxylic acid 2-(9H-fluoren-9-ylmethyl) ester (29d).
The title
compound was prepared according to the procedure used for com-
pound 29a. The crude residue was purified by flash column chro-
matography (heptane/EtOAc 1:0 to 1:1) to afford the title
compound (0.219 g, 33% yield) as an off-white powder. (ES+) m/z
(M+23) 482.5. 1H NMR (500 MHz, CDCl3) d ppm 7.52–7.83 (m,
4H) 7.18–7.45 (m, 5H) 7.01 (s, 0.6H) 6.93 (s, 0.4H) 6.61–6.69 (m,
1H) 5.58 (s, 0.6H) 5.33 (s, 0.4H) 4.42–4.59 (m, 2H) 4.21–4.35 (m,
1H) 3.78–4.00 (m, 7.4H) 3.62–3.76 (m, 0.6H) 2.77–2.96 (m, 2H).
Mixture of rotamers. HPLC purity 98%.
6.6.2.41. [2-(3-Bromo-phenyl)-ethyl]-carbamic acid 9H-fluoren-
9-ylmethyl ester (28g).
The title compound was prepared
6.6.2.46. 7-Hydroxy-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
according to the procedure used for compound 28e. The crude res-
idue was purified by trituration in Et2O to provide the title com-
pound (2.45 g, 55% yield) as a white powder. (ES+) m/z (M+23)
444.2/446.2. 1H NMR (500 MHz, CDCl3) d ppm 7.78 (d, J = 7.6 Hz,
2H) 7.58 (d, J = 7.4 Hz, 2H) 7.30–7.45 (m, 6H) 7.08–7.22 (m, 2H)
4.78 (br s, 1H) 4.57 (br s, 0.3H) 4.43 (d, J = 6.8 Hz, 1.7H) 4.20–
4.28 (m, 1H) 3.45 (q, J = 6.5 Hz, 1.7H) 3.21 (br s, 0.3H) 2.80 (t,
J = 6.7 Hz, 1.7H) 2.52 (br s, 0.3H). Mixture of rotamers. HPLC purity
100%.
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29e).
acid (0.28 g, 3.74 mmol) was added to a stirred solution of 9H-flu-
oren-9-ylmethyl N-[2-(4-bromophenyl)ethyl]carbamate 28e
Glyoxylic
(1.21 g, 3.37 mmol) in a AcOH/H2SO4 mixture (3:1, 30 mL) at room
temperature. The reaction mixture was stirred at room tempera-
ture for 24 h, wherupon the reaction mixture was poured carefully
onto ice/water (ꢀ100 mL). The resulting mixture was left to warm
to room temperature and then extracted with DCM (3 ꢃ 50 mL);
the organic extracts were combined, dried over Na2SO4, filtered
and concentrated under vacuum. The crude residue was purified
by flash column chromatography (DCM/MeOH 1:0 to 97.5:5) to
provide the title compound (0.31 g, 21% yield) as a sticky foam.
(ES+) m/z (M+1) 416.1. 1H NMR (500 MHz, CDCl3) d ppm 6.65–
7.79 (m, 13H) 5.57 (br s, 1H) 4.06–4.53 (m, 3H) 3.55–3.89 (m,
2H) 2.63–2.89 (m, 2H). HPLC purity 94%.
6.6.2.42. 6-Hydroxy-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29a).
6-Hydroxy-
1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid 26a (0.50 g,
2.59 mmol) was suspended in dioxane (7 mL) and the suspension
was cooled to 0 °C. A solution of NaHCO3 (0.24 g, 2.85 mmol) in
water (4 mL) was added to the mixture and the stirring was contin-
ued at 0 °C for 5 min. A solution of FmocCl (0.67 g, 2.59 mmol) in
dioxane (3 mL) was added slowly and the reaction mixture was stir-
red for 18 h with gradual warming to room temperature. The reac-
tion mixture was concentrated under vacuum to remove the
dioxane, the resulting residue was dissolved in a mixture EtOAc
6.6.2.47. 7-Bromo-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29f).
The title
compound was prepared according to the procedure used for com-
pound 29e. The crude residue was purified by flash column chro-
matography (DCM/MeOH 1:0 to 97.5:2.5) to provide a mixture of

5848
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
the 6- and 8-bromo isomers which were separated by preparative
HPLC and the title compound (0.506 g, 36% yield) was obtained as a
sticky oil. (ES+) m/z (M+23) 500.1/501.8. 1H NMR (250 MHz, CDCl3)
d ppm 7.49–7.83 (m, 5H) 7.18–7.47 (m, 5H) 6.97–7.11 (m, 1H) 5.63
(s, 0.5H) 5.24 (s, 0.5H) 4.38–4.61 (m, 2H) 4.16–4.34 (m, 1H) 3.76–
4.00 (m, 1.5H) 3.50–3.70 (m, 0.5H) 2.71–2.99 (m, 2H). Mixture of
rotamers. HPLC purity 89%.
preparative HPLC (MeOH/water mobile phase) to afford the final
pentapeptide aldehyde product.
6.7.3.1.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(S)-2-tert-butoxy-1-((S)-2-hydroxy-5-oxo-tetrahydro-
furan-3-ylcarbamoyl)-ethylcarbamoyl]-2-methyl-propyl}-suc-
cinamic acid (2).
The title compound was prepared following
the procedure used for peptide synthesis described above (use of
(S)-O-tert-butyl serine in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in
stage 2). The crude residue was purified by preparative HPLC to af-
ford the title compound (5 mg) as a white solid. MS (ES+) m/z
(M+1) 616.2. 1H NMR (500 MHz, D2O) d ppm 4.97–5.07 (m, 1H)
4.70–4.71 (m, 1H) 4.40–4.54 (m, 1H) 4.18–4.28 (m, 1H) 4.02–
4.16 (m, 2H) 3.61–3.75 (m, 2H) 2.45–3.14 (m, 4H) 1.96–2.18 (m,
5H) 1.17 (s, 9H) 0.85–0.95 (m, 12H). Mixture of isomers. HRMS
(ESI) calcd for C27H45N5O11 [M+H]+ m/z 616.3194, found m/z
616.3179. HPLC purity 100%.
6.6.2.48. 6-Bromo-3,4-dihydro-1H-isoquinoline-1,2-dicarbox-
ylic acid 2-(9H-fluoren-9-ylmethyl) ester (29g).
The title
compound was prepared according to the procedure used for com-
pound 29e. The crude residue was purified by flash column chro-
matography (DCM/MeOH 1:0 to 97.5:2.5) to provide a mixture of
the 6- and 8-bromo isomers which were separated by preparative
HPLC and the title compound (0.826 g, 30% yield) was obtained as a
white solid.(ES+) m/z (M+23) 499.9/501.9. 1H NMR (500 MHz,
CDCl3) d ppm 7.78 (d, J = 7.6 Hz, 1H) 7.70 (t, J = 8.2 Hz, 1H) 7.50–
7.63 (m, 2H) 7.28–7.44 (m, 6H) 7.22 (dd, J = 12.9, 7.1 Hz, 1H)
5.61 (s, 0.6H) 5.26 (s, 0.4H) 4.42–4.61 (m, 2H) 4.29 (t, J = 6.9 Hz,
0.6H) 4.21 (t, J = 5.7 Hz, 0.4H) 3.85–3.93 (m, 0.6H) 3.73–3.85 (m,
1H) 3.64 (ddd, J = 12.8, 8.0, 4.9 Hz, 0.4H) 2.76–2.99 (m, 2H). Mix-
ture of rotamers. HPLC purity 99%.
6.7.3.2.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-succinamic
acid (33a).
The title compound was prepared following the
procedure used for peptide synthesis described above (use of
Fmoc-(S)-proline in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage
2). The crude residue was purified by preparative HPLC to afford
the title compound (15 mg) as a white solid. MS (ES+) m/z (M+1)
570.1. 1H NMR (500 MHz, D2O) d ppm 4.88–5.04 (m, 1H), 4.64
(dd, J = 7.7, 6.1 Hz, 1H), 4.24–4.45 (m, 2H), 4.08–4.24 (m, 1H),
3.89–4.04 (m, 1H), 3.30–3.87 (m, 2H), 2.32–3.15 (m, 4H), 2.32–
3.15 (m, 4H), 2.09–2.31 (m, 1H), 1.65–2.09 (m, 8H), 0.66–0.97
(m, 12H). Mixture of isomers. HRMS (ESI) calcd for C25H39N5O10
[M+H]+ m/z 570.2775, found m/z 570.2776. HPLC purity 100%.
6.7. General method for synthesis of peptides
6.7.1. Stage 1—Loading of P2 amino acid to Webb semi-car
bazide resin (31)
Asp semi-carbazide Merrifield resin 30 (0.625 mmol/g,
1.0 equiv, typically 600–700 mg of resin) was added to a 20 ml frit-
ted plastic tube followed by P2 amino acid (2.0 equiv) and HATU
(2.2 equiv). To this mixture was added DMF (10 mL) and DIPEA
(5.0 equiv) and the mixture was agitated at room temperature
for 20 h. The resin was filtered and washed with 3 ꢃ 10 mL aliquots
of solvent in the following order: DMF, DCM, DMF, DCM, MeOH,
DCM, MeOH and Et2O to afford the functionalized resin which
was dried to constant weight under vacuum. Ninhydrin and IR
analysis of the resin was performed to confirm that there were
no free amine sites on the resin.
6.7.3.3.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylc-
arbamoyl)-3-methoxy-pyrrolidine-1-carbonyl]-2-methyl-pro-
pyl}-succinamic acid (33b).
The title compound was
prepared following the procedure used for peptide synthesis de-
scribed above (use of 6a in stage 1 and Ac-Val-Asp(OtBu)-Val-OH
in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (12 mg) as an off-white solid. MS
(ES+) m/z (M+1) 600.5. 1H NMR (500 MHz, D2O) d ppm 4.95–5.07
(m, 1H) 4.69–4.71 (m, 1H) 3.47–4.53 (m, 7H) 3.32–3.44 (m, 3H)
2.45–3.10 (m, 4H) 1.89–2.27 (m, 7H) 0.74–1.02 (m, 12H). Mixture
6.7.2. Stage 2—Resin deprotection followed by loading of Fmoc
Val-Asp-Val-OH onto resin (32)
To the stage 1 resin 31 in a 20 ml fritted plastic tube was added
10 ml of a 20% v/v solution of piperidine in DMF and the reaction
mixture was agitated for 1 h at room temperature. The resin was
then filtered and washed with DMF, DCM, DMF, DCM, MeOH,
DCM, MeOH and Et2O to afford the deprotected resin which was
dried to constant weight under vacuum. Ninhydrin and IR analysis
of the resin was performed to confirm that the coupling reaction
was completely successful.
of isomers. HRMS (ESI) calcd for
600.2881, found m/z 600.2900. HPLC purity 100%.
C
26H41N5O11 [M+H]+ m/z
6.7.3.4.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylc-
To the deprotected resin (1.0 equiv) in a 20 ml fritted plastic
tube was added Ac-Val-Asp(OtBu)-Val-OH and HATU (2.2 equiv)
followed by DMF (10 mL) and DIPEA (5.0 equiv). This mixture
was agitated at room temperature for 20 h. The resin was then fil-
tered and washed with 3 ꢃ 10 mL aliquots of solvent in the follow-
ing order: DMF, DCM, DMF, DCM, MeOH, DCM, MeOH and Et2O to
afford the functionalized resin which was dried to constant weight
under vacuum. Ninhydrin and IR analysis of the resin was per-
formed to confirm that there were no free amine sites on the resin.
arbamoyl)-3-isopropoxy-pyrrolidine-1-carbonyl]-2-methyl-
propyl}-succinamic acid (33c).
The title compound was pre-
pared following the procedure used for peptide synthesis described
above (use of 6b in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage
2). The crude residue was purified by preparative HPLC to afford
the title compound (13 mg) as a white powder. MS (ES+) m/z
(M+1) 628.1. 1H NMR (500 MHz, D2O) d ppm 4.96–5.09 (m, 1H)
4.69 (m, 1H) 4.16–4.52 (m, 4H) 3.52–4.10 (m, 4H) 2.43–3.16 (m,
4H) 1.90 -2.32 (m, 7H) 1.07–1.30 (m, 6H) 0.73–1.02 (m, 12H). Mix-
ture of isomers. HRMS (ESI) calcd for C28H45N5O11 [M+H]+ m/z
628.3194, found m/z 628.3195. HPLC purity 100%.
6.7.3. Stage 3—Cleavage of final compound from resin (2, 33a–v)
To the stage 2 resin (32) in a 20 ml fritted plastic tube was
added 5 ml of a solution of TFA/water (9:1). The resin was then agi-
tated at room temperature for 90 min after which the resin was fil-
tered, the resin was washed with two further aliquots of mixture
TFA/water (9:1, 2 ml) and the filtrate was concentrated under vac-
uum to dryness to give the crude product which was purified using
6.7.3.5.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3S)-3-se-butoxy-2-((S)-2-hydroxy-5-oxo-tetrahydro-
furan-3-ylcarbamoyl)-pyrrolidine-1-carbonyl]-2-methyl-pro-
pyl}-succinamic acid (33d).
The title compound was pre-
pared following the procedure used for peptide synthesis

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5849
described above (use of 6c in stage 1 and Ac-Val-Asp(OtBu)-Val-OH
in stage 2). The crude residue was purified by preparative HPLC to
afford the title compound (17 mg) as a yellow powder. MS (ES+) m/
z (M+1) 642.3. 1H NMR (500 MHz, D2O) d ppm 4.96–5.06 (m, 0.5H)
4.66–4.70 (m, 0.5H) 4.15–4.51 (m, 4H) 3.54–4.10 (m, 5H) 2.51–
3.12 (m, 4H) 1.96–2.31 (m, 7H) 1.37–1.57 (m, 2H) 1.07–1.22 (m,
3H) 0.74–0.99 (m, 15H). Mixture of isomers. HRMS (ESI) calcd for
2.06–2.09 (m, 2H) 2.05 (s, 3H) 1.63–1.76 (m, 1H) 1.49–1.61 (m,
1H) 1.29–1.43 (m, 1H) 0.85–0.99 (m, 21H). Mixture of isomers.
HRMS (ESI) calcd for C30H49N5O10 [M+H]+ m/z 640.3558, found
m/z 640.3546. HPLC purity 100%.
6.7.3.10. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3R)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylc-
arbamoyl)-3-phenyl-pyrrolidine-1-carbonyl]-2-methyl-pro-
C
29H47N5O11 [M+H]+ m/z 642.3350, found m/z 642.3347. HPLC pur-
ity 94%.
pyl}-succinamic acid (33i).
The title compound was
prepared following the procedure used for peptide synthesis de-
scribed above (use of 15d in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (8 mg) as a white solid. MS (ES+) m/z
(M+1) 646.2. 1H NMR (500 MHz, D2O) d ppm 7.23–7.47 (m, 5H)
4.87–5.00 (m, 1H) 4.66–4.71 (m, 1H) 3.90–4.55 (m, 5H) 3.74–
3.89 (m, 1H) 3.32–3.54 (m, 1H) 2.44–3.09 (m, 4H) 2.17–2.42 (m,
2H) 1.97–2.14 (m, 5H) 0.70–1.16 (m, 12H). Mixture of isomers.
HRMS (ESI) calcd for C31H43N5O10 [M+H]+ m/z 646.3088, found
m/z 646.3093. HPLC purity 99%.
6.7.3.6.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylc-
arbamoyl)-3-isobutoxy-pyrrolidine-1-carbonyl]-2-methyl-pro-
pyl}-succinamic acid (33e).
The title compound was
prepared following the procedure used for peptide synthesis de-
scribed above (use of 6d in stage 1 and Ac-Val-Asp(OtBu)-Val-OH
in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (7 mg) as a white powder. MS (ES+)
m/z (M+1) 642.3. 1H NMR (500 MHz, D2O) d ppm 4.95–5.08 (m,
1H) 4.68 (m, 1H) 4.06–4.54 (m, 5H) 3.66–4.01 (m, 2H) 3.25–3.44
(m, 2H) 2.43–3.12 (m, 4H) 1.94–2.31 (m, 7H) 1.79 (m, 1H) 0.80–
1.02 (m, 18H). Mixture of isomers. HRMS (ESI) calcd for
6.7.3.11. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3R)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-lcar-
bamoyl)-3-p-tolyl-pyrrolidine-1-carbonyl]-2-methyl-propyl}-
C
29H47N5O11 [M+H]+ m/z 642.3350, found m/z 642.3351. HPLC pur-
ity 95%.
succinamic acid (33j).
The title compound was prepared fol-
lowing the procedure used for peptide synthesis described above
(use of 15b in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage 2).
The crude residue was purified by preparative HPLC to afford the
title compound (23 mg) as a white solid. (ES+) m/z (M+1) 660.1.
1H NMR (500 MHz, D2O) d ppm 7.19–7.27 (m, 4H) 4.85–4.99 (m,
1H) 4.68–4.72 (m, 1H) 4.43 (d, J = 6.1 Hz, 1H) 4.03–4.30 (m, 4H)
3.68–3.84 (m, 1H) 3.23–3.43 (m, 1H) 2.43–3.07 (m, 4H) 2.15–
2.38 (m, 5H) 1.98–2.13 (m, 5H) 0.85–1.04 (m, 12H). Mixture of iso-
mers. HRMS (ESI) calcd for C32H45N5O10 [M+H]+ m/z 660.3245,
found m/z 660.3251. HPLC purity 100%.
6.7.3.7.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3S)-3-cyclopentyloxy-2-((S)-2-hydroxy-5-oxo-tetra-
hydro-furan-3-ylcarbamoyl)-pyrrolidine-1-carbonyl]-2-
methyl-propyl}-succinamic acid (33f).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 6e in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (7 mg) as a white powder. MS (ES+) m/
z (M+1) 654.2. 1H NMR (500 MHz, D2O) d ppm 4.92–5.09 (m, 1H)
4.67–4.70 (m, 1H) 3.53–4.46 (m, 8H) 2.46–3.12 (m, 4H) 1.96–
2.27 (m, 7H) 1.43–1.87 (m, 8H) 0.74–1.00 (m, 12H). Mixture of iso-
mers. HPLC purity 100%.
6.7.3.12. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(2S,3R)-3-cyclohexyl-2-((S)-2-hydroxy-5-oxo-tetrahy-
dro-furan-3-ylcarbamoyl)-pyrrolidine-1-carbonyl]-2-methyl-
6.7.3.8.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
propyl}-succinamic acid (33k).
The title compound was pre-
{(S)-1-[(2S,3S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylc-
arbamoyl)-3-(pyrimidin-2-yloxy)-pyrrolidine-1-carbonyl]-2-
pared following the procedure used for peptide synthesis described
above (use of 15c in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage
2). The crude residue was purified by preparative HPLC to afford
the title compound (15 mg) as a white solid. (ES+) m/z (M+1)
652.5. 1H NMR (500 MHz, D2O) d ppm 4.99–5.06 (m, 1H) 4.66–
4.69 (m, 1H) 4.24–4.46 (m, 2H) 4.04–4.20 (m, 2H) 3.85–4.00 (m,
1H) 3.54–3.68 (m, 1H) 2.45–3.14 (m, 4H) 1.95–2.21 (m, 7H)
1.55–1.86 (m, 6H) 0.96–1.48 (m, 6H) 0.84–0.95 (m, 12H). Mixture
methyl-propyl}-succinamic acid (33g).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 6f in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (18 mg) as a white powder. MS (ES+)
m/z (M+1) 664.2. 1H NMR (500 MHz, D2O) d ppm 8.52–8.69 (m, 2H)
7.14–7.28 (m, 1H) 5.50 (m, 1H) 5.03–5.19 (m, 1H) 4.82–4.86 (m,
1H) 4.68 (m, 1H) 3.61–4.50 (m, 5H) 2.46–3.15 (m, 4H) 2.25–2.43
(m, 2H) 1.90–2.17 (m, 5H) 0.62–1.04 (m, 12H). Mixture of isomers.
HRMS (ESI) calcd for C29H41N7O11 [M+H]+ m/z 664.2942, found m/z
664.2960. HPLC purity 100%.
of isomers. HRMS (ESI) calcd for
652.3558, found m/z 652.3549. HPLC purity 100%.
C
31H49N5O10 [M+H]+ m/z
6.7.3.13. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-1,3-dihydro-isoindole-2-carbonyl]-2-methyl-propyl}-
6.7.3.9.
(S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
succinamic acid (33l).
The title compound was prepared
{(S)-1-[(2S,3S)-3-(2,2-dimethyl-propyl)-2-((S)-2-hydroxy-5-oxo-
tetrahydro-furan-3-ylcarbamoyl)-pyrrolidine-1-carbonyl]-2-
following the procedure used for peptide synthesis described
above (use of Fmoc-1,3-dihydro-isoindole-1,2-dicarboxylic acid
in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage 2). The crude
residue was purified by preparative HPLC to afford the title com-
pound (36 mg) as an off-white solid. (ES+) m/z (M+1) 618.0. 1H
NMR (500 MHz, D2O) d ppm 7.28–7.55 (m, 4H) 5.52–5.68 (m,
1H) 5.11–5.23 (m, 2H) 5.01–5.04 (m, 1H) 4.65–4.70 (m, 1H)
3.87–4.59 (m, 3H) 2.48–3.13 (m, 4H) 1.74–2.22 (m, 5H) 0.54–
1.16 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
methyl-propyl}-succinamic acid (33h).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 15a in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (41 mg) as a colorless oil. MS (ES+) m/
z (M+1) 640.3. 1H NMR (500 MHz, D2O) d ppm 5.05–5.11 (m, 1H)
4.70–4.72 (m, 1H) 4.37–4.44 (m, 1H) 4.15–4.35 (m, 1H) 4.08–
4.13 (m, 1H) 3.96–4.06 (m, 1H) 3.73–3.86 (m, 1H) 3.59–3.69 (m,
1H) 2.53–2.98 (m, 4H) 2.31–2.42 (m, 1H) 2.17–2.18 (m, 1H)
C
29H39N5O10 [M+H]+ m/z 618.2775, found m/z 618.2790. HPLC
purity 100%.

5850
M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
6.7.3.14. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-piperidine-1-carbonyl]-2-methyl-propyl}-succinamic
Val-OH in stage 2). The crude residue was purified by preparative
HPLC to afford the title compound (39 mg) as a white solid. (ES+)
m/z (M+23) 668.2. 1H NMR (500 MHz, D2O) d ppm 7.05–7.37 (m,
3H) 5.59–6.10 (m, 1H) 4.95–5.05 (m, 1H) 4.39–4.67 (m, 2H)
3.64–4.26 (m, 4H) 2.43–3.20 (m, 6H) 2.29 (m, 3H) 1.70–2.18 (m,
5H) 0.56–0.99 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
acid (33m).
The title compound was prepared following the
procedure used for peptide synthesis described above (use of (S)-
N-Fmoc-piperidine-2-carboxylic acid in stage 1 and Ac-Val-Asp
(OtBu)-Val-OH in stage 2). The crude residue was purified by pre-
parative HPLC to afford the title compound (9 mg) as a white solid.
ES+) m/z (M+23) 606.1. 1H NMR (500 MHz, D2O) d ppm 4.86–5.05
(m, 2H) 3.97–4.68 (m, 4H) 3.88–3.96 (m, 1H) 3.12–3.35 (m, 1H)
2.37–3.05 (m, 4H) 1.89–2.30 (m, 6H) 1.52–1.77 (m, 3H) 1.18–
1.49 (m, 2H) 0.74–0.93 (m, 12H). Mixture of isomers. HRMS (ESI)
calcd for C26H41N5O10 [M+H]+ m/z 584.2932, found m/z 584.2927.
HPLC purity 100%.
C
31H43N5O10 [M+H]+ m/z 646.3088, found m/z 646.3097. HPLC pur-
ity 97%.
6.7.3.19. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-car-
bonyl]-2-methyl-propyl}-succinamic acid (33r).
The title
compound was prepared following the procedure used for peptide
synthesis described above (use of 29d in stage 1 and Ac-Val-As-
p(OtBu)-Val-OH in stage 2). The crude residue was purified by pre-
parative HPLC to afford the title compound (21 mg) as an off-white
solid. (ES+) m/z (M+1) 692.1. 1H NMR (500 MHz, D2O) d ppm 6.74–
7.07 (m, 2H) 5.46–5.99 (m, 1H) 4.90–4.98 (m, 1H) 4.54–4.68 (m,
2H) 3.61–4.27 (m, 10H) 2.32–3.05 (m, 6H) 1.69–2.11 (m, 5H)
0.42–0.98 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
6.7.3.15. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(S)-2-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-piperidine-1-carbonyl]-2-methyl-propyl}-succinamic
acid (33n).
The title compound was prepared following the
procedure used for peptide synthesis described above (use of 24
in stage 1 and Ac-Val-Asp(OtBu)-Val-OH in stage 2). The crude res-
idue was purified by preparative HPLC to afford the title compound
(38 mg) as a white solid. (ES+) m/z (M+1) 642.3. 1H NMR (500 MHz,
D2O) d ppm 5.06–5.45 (m, 1H) 4.92–5.04 (m, 1H) 4.50–4.69 (m, 2H)
3.93–4.37 (m, 4H) 3.36–3.82 (m, 2H) 2.44–3.09 (m, 4H) 1.97–2.16
(m, 5H) 1.44–1.87 (m, 4H) 0.72–1.24 (m, 18H). Mixture of isomers.
HRMS (ESI) calcd for C29H47N5O11 [M+H]+ m/z 642.3350, found m/z
642.3343. HPLC purity 96%.
C
32H45N5O12 [M+H]+ m/z 692.3143, found m/z 692.3124. HPLC pur-
ity 100%.
6.7.3.20. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[6-hydroxy-1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-
ylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
methyl-propyl}-succinamic acid (33s).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29a in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (7 mg) as an off-white solid. (ES+) m/z
(M+23) 670.4. 1H NMR (500 MHz, D2O) d ppm 7.18–7.33 (m, 1H)
6.69–6.88 (m, 2H) 5.43–6.08 (m, 1H) 4.91–5.07 (m, 1H) 4.33–
4.70 (m, 2H) 3.24–4.23 (m, 4H) 2.35–3.15 (m, 6H) 1.89–2.21 (m,
5H) 0.55–1.00 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
6.7.3.16. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[(S)-1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-methyl-pro-
pyl}-succinamic acid (33o).
The title compound was
prepared following the procedure used for peptide synthesis de-
scribed above (use of (1S)-2-[(9H-fluoren-9-ylmethoxy)carbonyl]-
1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in stage 1 and
Ac-Val-Asp(OtBu)-Val-OH in stage 2). The crude residue was puri-
fied by preparative HPLC to afford the title compound (22 mg) as a
white solid. (ES+) m/z (M+1) 632.3. 1H NMR (500 MHz, D2O) d ppm
7.14–7.38 (m, 4H) 5.52–6.06 (m, 1H) 4.91–4.97 (m, 1H) 4.59–4.68
(m, 2H) 3.64–4.22 (m, 4H) 2.34–3.18 (m, 6H) 1.82–2.14 (m, 5H)
0.48–0.98 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
C
30H41N5O11 [M+H]+ m/z 648.2881, found m/z 648.2886. HRMS
(ESI) calcd for C30H41N5O11 [M+H]+ m/z 648.2881, found m/z
648.2863. HPLC purity 95%.
6.7.3.21. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[7-hydroxy-1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-
ylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
C
30H41N5O10 [M+H]+ m/z 632.2932, found m/z 632.2948. HPLC pur-
ity 100%.
methyl-propyl}-succinamic acid (33t).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29e in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (15 mg) as an off-white solid. (ES+) m/
z (M+1) 648.0. 1H NMR (500 MHz, D2O) d ppm 7.09–7.19 (m, 1H)
6.73–6.94 (m, 2H) 5.55–6.07 (m, 1H) 4.93–5.08 (m, 1H) 4.42–
4.68 (m, 2H) 3.29–4.24 (m, 4H) 2.44–3.11 (m, 6H) 1.82–2.21 (m,
5H) 0.55–1.04 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
6.7.3.17. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-7-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
methyl-propyl}-succinamic acid (33p).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29c in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (28 mg) as a white solid. (ES+) m/z
(M+23) 668.2. 1H NMR (500 MHz, D2O) d ppm 7.09–7.26 (m, 3H)
5.53–6.06 (m, 1H) 4.95–5.10 (m, 1H) 4.42–4.66 (m, 2H) 3.70–
4.33 (m, 4H) 2.47–3.14 (m, 6H) 2.28–2.38 (m, 3H) 2.01–2.21 (m,
5H) 0.61–1.06 (m, 12H). Mixture of isomers. HRMS (ESI) calcd for
C
30H41N5O11 [M+H]+ m/z 648.2881, found m/z 648.2886. HPLC pur-
ity 89%.
6.7.3.22. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[6-bromo-1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-
ylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
C
31H43N5O10 [M+H]+ m/z 646.3088, found m/z 646.3073. HPLC pur-
ity 96%.
methyl-propyl}-succinamic acid (33u).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29g in stage 1 and Ac-Val-Asp(OtBu)-
Val-OH in stage 2). The crude residue was purified by preparative
HPLC to afford the title compound (18 mg) as an off-white solid.
(ES+) m/z (M+1) 709.9/711.9. 1H NMR (500 MHz, D2O) d ppm
7.41–7.56 (m, 2H) 7.17–7.34 (m, 1H) 5.61–6.08 (m, 1H) 4.95–
5.05 (m, 1H) 4.40–4.66 (m, 2H) 3.39–4.34 (m, 4H) 2.41–3.18 (m,
6.7.3.18. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-ylcarba-
moyl)-6-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
methyl-propyl}-succinamic acid (33q).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29b in stage 1 and Ac-Val-Asp(OtBu)-

M. C. Maillard et al. / Bioorg. Med. Chem. 19 (2011) 5833–5851
5851
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Giroux, A.; Zhu, Y.; Boucher, M.; Pikounis, B.; Xanthoudakis, S.; Roy, S.; Rigby,
M.; Zamboni, R.; Robertson, G. S.; Ng, G. Y.; Nicholson, D. W.; Flückiger, J. P. Br.
J. Pharmacol. 2004, 141, 689.
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W. E., Jr.; Keller, P. M.; Tomaszek, T.; Head, M. S.; Ryan, M. D.; Haltiwanger, R.
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6H) 1.84–2.17 (m, 5H) 0.57–1.03 (m, 12H). Mixture of isomers.
HRMS (ESI) calcd for C30H40BrN5O10 [M+H]+ m/z 710.2037, found
m/z 710.2047. HPLC purity 97%.
6.7.3.23. (S)-3-((S)-2-Acetylamino-3-methyl-butyrylamino)-N-
{(S)-1-[7-bromo-1-((S)-2-hydroxy-5-oxo-tetrahydro-furan-3-
ylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-
methyl-propyl}-succinamic acid (33v).
The title compound
was prepared following the procedure used for peptide synthesis
described above (use of 29f in stage 1 and Ac-Val-Asp(OtBu)-Val-
OH in stage 2). The crude residue was purified by preparative HPLC
to afford the title compound (2 mg) as an off-white solid. (ES+) m/z
(M+23) 709.9/712.0. 1H NMR (500 MHz, D2O) d ppm 7.43–7.74 (m,
2H) 7.12–7.31 (m, 1H) 5.71–6.12 (m, 1H) 4.93–5.01 (m, 1H) 3.42–
4.69 (m, 6H) 2.29–3.18 (m, 6H) 1.93–2.24 (m, 5H) 0.54–1.08 (m,
12H). Mixture of isomers. HRMS (ESI) calcd for C30H40BrN5O10
[M+H]+ m/z 710.2037, found m/z 710.2028. HPLC purity 100%.
Acknowledgments
15. Ivachtchenko, A. V.; Okun, I.; Tkachenko, S. E.; Kiselyov, A. S.; Ivanenkov, Y. A.;
Balakin, K. V. In Design of Caspase Inhibitors as Potential Clinical Agents; O’Brien,
T., Linton, S. T., Eds.; CRC Press, 2009. Chapter 5.
16. Ignacio Muñoz-Sanjuán, Hyusun Park, et al. In preparation for J. Biomol.
Screening.
We gratefully acknowledge the supply of recombinant human
Csp-2 and Csp-3 by Dr. James Wells and Dr. Michelle Arkin from
the Small Molecule Discovery Center at the University of California,
San Francisco. We also thank Dr. Donald Lorimer, Dr. Jan Abend-
roth, and Kateri Atkins of Emerald BioSystems for X-ray structure
determination.
17. Talanian, R. V.; Quinlan, C.; Trautz, S.; Hackett, M. C.; Mankovich, J. A.; Banach,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.020.
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