Structure-based design of human immuno- and constitutive proteasomes inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.01.013

Starting from the X-ray structure of our previous tripeptidic linear mimics of TMC-95A in complex with yeast 20S proteasome, we introduced new structural features to induce a differential inhibition between human constitutive and immunoproteasome 20S part

Full text of DOI:10.1016/j.ejmech.2018.01.013

Accepted Manuscript
Structure-based design of human immuno- and constitutive proteasomes inhibitors
Nicolas Richy, Daad Sarraf, Xavier Maréchal, Naëla Janmamode, Rémy Le Guével,
Emilie Genin, Michèle Reboud-Ravaux, Joëlle Vidal
PII:
S0223-5234(18)30013-8
10.1016/j.ejmech.2018.01.013
EJMECH 10090
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2017
Revised Date: 4 January 2018
Accepted Date: 4 January 2018
Please cite this article as: N. Richy, D. Sarraf, X. Maréchal, Naë. Janmamode, Ré. Le Guével,
E. Genin, Michè. Reboud-Ravaux, Joë. Vidal, Structure-based design of human immuno- and
constitutive proteasomes inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.01.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-based design of human immuno- and constitutive proteasomes inhibitors
a
a
b
a
c
Nicolas Richy , Daad Sarraf , Xavier Maréchal , Naëla Janmamode , Rémy Le Guével ,
a,1
b,*
a,*
Emilie Genin, Michèle Reboud-Ravaux , Joëlle Vidal
a
Université Rennes 1, Institut des Sciences Chimiques de Rennes, CNRS UMR 6226,
Bâtiment 10A , Campus de Beaulieu, 35042 Rennes Cedex, France.
b
Sorbonne Universités, UPMC Univ Paris 06-CNRS, IBPS, UMR 8256, Inserm ERL1164,
B2A, 7 Quai Saint Bernard, F75005 Paris, France.
c
Université Rennes 1, Technology platform ImPACcell, SFR UMS CNRS 3480 - INSERM
0
18, Bâtiment 8, Campus de Villejean, 35043 Rennes Cedex, France.
1
Current adress: Univ. Bordeaux, Institut des Sciences Moléculaires, UMR CNRS 5255, 351
Cours de la Libération, F-33405, Talence Cedex, France
*
Corresponding authors
E-mail addresses: joelle.vidal@univ-rennes1.fr (J. Vidal), michele.reboud@upmc.fr (M.
Reboud-Ravaux)
Abstract
Starting from the X-ray structure of our previous tripeptidic linear mimics of TMC-95A in
complex with yeast 20S proteasome, we introduced new structural features to induce a
differential inhibition between human constitutive and immunoproteasome 20S particles.
Libraries of 24 tripeptidic and 6 dipeptidic derivatives were synthesized. The optimized
preparation of 3-hydroxyoxindolyl alanine residues from tryptophan and their incorporation in
peptides were described. Several potent inhibitors of human constitutive proteasome and
immunoproteasome acting at the nanomolar level (IC = 7.1 nM against the chymotrypsin-
5
0
like activity for the best inhibitor) were obtained. A cytotoxic effect at the submicromolar
level was observed against 6 human cancer cell lines.
Graphical abstract:
1

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Highlights
•
•
•
•
•
X-ray structures drive design of immuno- and constitutive proteasome inhibitors.
Di- and tripeptide inhibitors are tailored in order to gain subunit selectivity.
Enantiopure 3-hydroxyoxindolylalaninamide derivatives are efficiently synthesized.
One, 2 or 3 activities of the proteasomes are inhibited in the nanomolar range.
The most potent inhibitors are cytotoxic against cancer cell lines.
Keywords
Proteasome inhibitors
Immunoproteasome
Constitutive proteasome
3
-Hydroxyoxindolylalanine derivatives
Tryptophan oxidation
1
. Introduction
At the end of the ubiquitin-proteasome pathway, the proteasome is a central hub of non
lysosomal cellular proteolysis in the cytoplasm as well as in the nucleus of eukaryotic cells [1,
2
]. Its inhibition leads to a large variety of cellular responses such a cell cycle arrest and
increase of proapoptotic factors and tumor suppressors. The proteasome is an established drug
target in the treatment of hematologic malignancies with three approved drugs, bortezomib



(
Velcade ) [3], carfilzomib (Kyprolis ) [4] and the orally administered ixazomib (Ninlaro )
5] (Figure 1).
[
2

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Figure 1: Covalent inhibitors used in cancer therapy.
All these approved molecules target the 20S catalytic core with covalent modification of the
γ
Thr1O group of the catalytic subunits. Moreover, proteasome is a potential target for immune
diseases [2], tuberculosis [6] or malaria [7]. Devoid of a reactive function, noncovalent
inhibitors have also been developed and have some advantages over covalent ones, such as
better tissue penetration, inhibitor stability, lower reactivity and potentially higher selectivity
[
8]. Several peptides are noncovalent inhibitors [9], such as the natural TMC-95A and its
cyclic and linear mimics 1a and 2a [10-17], dipeptides (e.g. 3-4) [18-24], and pseudopeptides
25-27] (Figure 2).
[
Figure 2: Some noncovalent peptide inhibitors.
Nonpeptidic noncovalent inhibitors have also been described (e. g. sulfonamides [28-31],
hydroxyurea [32], 1,2,4-oxadiazoles [33], pyrazoles [34], phakellins [35], quinolines [36],
psoralene [37]). The three approved covalent drugs inhibit mainly the
3
β5 activity of the

ACCEPTED MANUSCRIPT
catalytic core of the constitutive proteasome (cCP) but also that of the inducible
immunoproteasome (iCP). This partly explain side-effects and resistance observed during
cancer treatments [38]. The cCP and iCP catalytic cores are both made of two outer rings of 7
α
-subunits and two inner rings of
β
-subunits. The 2
1i, 2 2i and 2
β
1c, 2
β
2c and 2
β
5c catalytic units of
1, 2 and β5
cCP are replaced by the inducible 2
β
β
β
3i subunits in iCP. The
β
β
catalytic units exhibit caspase-like or post-acid (C-L), trypsin-like (T-L) and chymotrypsin-
like (ChT-L) activities, respectively. The catalytic pockets of cCP and iCP show subtle
differences that can be exploited to discriminate between the inhibitors targeting proteasomes
[
[
39]. Our successes in developing linear mimics of TMC-95A either monovalent 1a and 2a
16, 17] or bivalent [40, 41] and their cellular penetration [42] prompted us to pursue their
structure-driven development. Based on previous co-crystals of compounds 1a and 2a
complexed with yeast cCP (Figure 1S, supporting information) [17], and using the reported
structural data obtained with mouse constitutive and immunoproteasome 20S particles [39],
we decided to explore potential structure-based improvements of the inhibitory effect of
tripeptidic (1, 2) and dipeptidic (5) linear mimics. The influence on the ChT-L, T-L and C-L
activities of both cCP and iCP of the structural variations of residues P1, P2, P3 and P4
present in the starting molecules 1a and 2a was systematically explored (Figure 3). We report
here the synthesis of molecules 1, 2 and 5 from the original enantiopure 3-
hydroxyoxindolylalaninamide residues 6 and 7, efficiently prepared from Z-tryptophan. After
the in vitro enzymatic evaluation, the best inhibitors were selected and assayed on seven
human tumor cell lines to evaluate their potential as cytotoxic tumor agents.
4

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Figure 3: Structures of the studied noncovalent inhibitors 1a-v, 2a-c and 5a-f and their 3-
hydroxyoxindolyl alanine chemical precursors 6 and 7. The occupancy of the S1-S4 binding
pockets by P1-P4 groups, respectively, is deduced from co-crystallization of compounds 1a
and 2a with yeast proteasome [17].
2
. Results and discussion
2
.1. Structure driven drug-design
The design started from the X-ray structure of epimeric tripeptides 1a and 2a in complex with
yeast cCP, that showed an antiparallel -sheet in the substrate binding channel implicating the
5/ 6 subunits. Their binding were identical, except for the epimeric oxindole moiety of the
β
β
β
side chain in P2 which was rotated (Figure 1S) [17]. The crystallographic knowledge gained
from the structures of mouse cCP and iCP and their covalent complexes with the tripeptide
epoxy ketone ONX-0914 (PR-957, Figure 2S) was also used [39], as well as the
crystallographic structure of human constitutive proteasome [43]. Structural features have
been identified in order to potentially target
ones [39]. In the 5i subunit, the subsite S1 appears significantly more spacious than that of
5c, and the subsite S3 is smaller and more hydrophilic. A defined and shallow S2 pocket is
also present in 5i. Regarding the 1i subunit, the S1 pocket is small and hydrophobic
whereas the corresponding one in
β1i or β5i subunits preferentially to β1c or β5c
β
β
β
β
β
1c accommodates P1 negatively charged residues. Lastly,
5

ACCEPTED MANUSCRIPT
the S3 subsite of β1i is smaller and more polar than that of β1c. As P1 and P3 residues play a
1
3
major role, the main structural variations in 1 were introduced in the R and R groups (Table
1
1
). For R , the methylene group mimicking the
α
-carbon of a decarboxylated peptide was
substituted by hydrogen (1b, 2b), aromatic groups with variable bulkiness (1a, 1c, 1f-i),
primary (1j-m), secondary (1n) and tertiary (1o-p) aliphatic groups. The Ala residue (P3) of
reference compound 1a, was replaced by the smaller Gly residue (1r), D-Ala (1q), the more
polar and hydrophilic Asn, Thr or Lys (1s-v) residues. Modifications of the oxindole group at
P2 (configuration of the C3-carbon: (R) for tripeptides 1 or (S) for tripeptides 2, N-substitution
1
c and 2c) were also introduced. The peptide sequence was also shortened by removing the
Tyr residue of tripeptides 1 (P4 modulation) in order to explore the activity of dipeptides 5
Table 2).
(
2
.2. Chemistry
Molecules 1, 2 and 5 were prepared starting from epimeric 3-hydroxy-2-oxindole derivatives
and 7 (Scheme 1). The key intermediates in the preparation of these non-proteogenic amino
6
acids 6 and 7 were respectively the epimeric N-Z spirolactones 9 and 10.
They were obtained in 64-82% total yield by oxidation of Z-L-tryptophan 8 with DMSO in
the presence of t-BuBr [44, 45], as mixtures with a moderate amount of the corresponding
2
brominated derivative (X = Br), when R = H, indicating that electrophilic bromine was
released in the medium. In order to avoid bromination of the aromatic ring, attempts using
DMSO activated with methanesulfonic anhydride [46] or oxalyl chloride [47], were
performed and led to very low yields (ca 10%) in 9a and 10a (X = H). As t-BuBr could
generate hydrogen bromide at 45°C in the presence of DMSO (Scheme 2), the charge-transfer
complex Br -Me S was formed rather than the electrophilic sulfonium [48]. It became the
2
2
source of electrophilic bromine [48, 49], that reacted with the nucleophilic enanime moiety of
the indole ring to give intermediate A (Scheme 2). After trapping of the iminium cation by
DMSO and bromo-lactonization, intermediate B might lead to spiro-oxindole C. The more
difficult electrophilic bromination of the benzene ring in C gave a small amount of D
(Scheme 2). The oxidative lactonization of indole propanoic acids mediated by oxygen
peroxide and catalytic iodide [50] was also applied to Z-tryptophan 8a and afforded the
expected pure 9a (X = H) and 10a (X = H) in 53% overall yield, which was lower than that
obtained using DMSO and t-BuBr (ca 82%, X = H or Br). We found that the epimeric
lactones 9 and 10 could be separated by column chromatography over silica gel on a 12 g
scale in respectively 22-37% and 42-45% yields. Opening of the lactone ring in 9 or 10 by
6

ACCEPTED MANUSCRIPT
diversely substituted primary amines was easy at room temperature. The presence of the small
amount of brominated derivative (X = Br) was not a problem, since further hydrogenation in
the presence of palladium on charcoal cleaved the Z group and also the carbon bromine bond.
The expected derivatives 6 or 7 were obtained as their stable hydrochloride or TFA salts in
1
5-39% overall yields from Z-tryptophan 8. The formation of the rigid lactams 11 and 12 by
the slow isomerization of the free amine compounds 6a and 7a [51], allowed the
1
configuration determination of the quaternary asymmetric carbon using H NMR NOESY
experiments in DMSO-D solvent (scheme 1).
6
Scheme 1: Synthesis of 3-hydroxyoxindolyl alanine precursors 6 and 7 and assignment of the
1
configuration at the created asymmetric carbon in 11 and 12 by H NMR NOESY experiment.
Reagents and conditions: i. DMSO, t-BuBr, 45 °C, 24 h, separation of 9 and 10 by
chromatography; ii. H O , catalytic KI (11%), CH CN, RT, 18 h, separation of 9a and 10a by
2
2
3
1
chromatography. iii. R NH , THF, RT, 1.5 to 18 h; iv. H , Pd/C, MeOH, RT, overnight, then
2
2
1
eq. anhydrous HCl or TFA; v. water, 72 h, RT, more than 85% conversion.
Scheme 2: Proposed mechanism for the oxidative lactonization of indole propanoic acid
mediated by DMSO/t-BuBr.
7

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A library of peptides 1, 2 and 5 was then prepared from the 3-hydroxyoxindolyl alanine
residues 6 and 7 using peptide conventional liquid-phase synthesis [52] (scheme 3 and tables
1
and 2). As the tertiary alcohol of the side chain in 6 or 7 was not protected, a mild activation
of the peptide coupling partner was required and was realized by the pre-formed succinimidyl
Su) esters. In the convergent synthesis, the succinimidyl esters of dipeptides 13 [16] were
coupled to the 3-hydroxyoxindolyl alanine residues 6 and 7 to give the expected tripeptides
a-c, 1f-i, 1o-r, 1u in 31-76% yields. As some epimerization of activated dipeptide 13 was
(
1
sometimes observed in large scale experiments, the linear synthesis of tripeptides 1-2 was also
performed. N-Z-dipeptides 5 were obtained in 56-88% yields from 6 and commercial
activated Z-amino acids 14. Hydrogenolysis of 5 followed by coupling with the tyrosine
residue afforded expected tripeptides 1 in 26-64% overall yields from the corresponding 6. In
the case of the reference compound 1a, the linear synthesis worked better than the convergent
one and was applied to a gram scale preparation. Cleavage of protecting groups in tripeptides
1
a, 1u or 1j was also realized. Hydrogenolysis of the O-benzyl residue in 1a led to 1d in
quantitative yield. Acidic cleavage of the Boc residue in 1a and 1u to give 1e and 1v was
complete in less than 30 min at 0°C and should be controlled, since higher temperature or
longer reaction time resulted in extensive decomposition of the compounds. Saponification of
the methyl ester in 1j gave quantitatively 1k. The N-terminus groups R encountered in peptide
3
[18] and an isomeric group [19] were also introduced in N-capped dipeptides 5d-f by
deprotection of dipeptides 5a or 5b followed by coupling with the corresponding activated
esters R-OSu.
8

ACCEPTED MANUSCRIPT
Scheme 3: Synthesis of non-covalent inhibitors 1a-v, 2a-c and 5a-g.
Reagents and conditions: i. DCC, HOSu, DME, overnight; ii. HCl, 6 or HCl, 7, Et N,
3
DMF/CH Cl , RT, overnight; iii. H , Pd/C, MeOH; iv. Boc-Tyr(Bn)-OSu, RT, overnight; v.
2
2
2
(
3-PhO)C H CH CO Su or (4-BnO)-C H CO Su; vi. anhydrous HCl, MeOH, 0 °C, 30 min;
6 4 2 2 6 4 2
vii: LiOH, H O/THF, then aqueous HCl.
2
2
.2 In vitro inhibition studies
The inhibitory activities of compounds 1a-v, 2a-c and 5a-f on the rate of hydrolysis of
5 substrate Suc-LLVY-AMC and the 2 substrate Boc-LLR-AMC catalyzed by cCP and
iCP were determined (Figure 4, Tables 1 and 2). The 1 substrates Z-LLE- NA and Ac-
PAL-AMC were used to detect the activities of cCP and iCP respectively.
the
β
β
β
β
1
00
100
80
60
40
20
0
100
80
8
6
4
2
0
0
0
0
0
6
4
2
0
0
0
0
ChT-L, iCP
PA, iCP
T-L, iCP
40 50
0
0,05 0,1 0,15 0,2 0,25 0,3
0
5
10 15 20 25 30 35 40
0
10
20
30
[
5f] (µM)
[1r] (µM)
[1v] (µM)
9

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1
00
100
80
100
80
60
40
20
0
80
60
40
20
0
60
40
2
0
0
ChT-L, cCP
4 5
PA, cCP
40
T-L, cCP
80 100
0
1
2
[
3
0
10
20
[1t] (µM)
30
50
0
20
40
60
1t] (µM)
[5e] (µM)
Figure 4: Inhibition profile of the ChT-L, C-L and T-L activities of cCP and iCP by
compounds 1r,t,v or 5e,f at pH 8 and 37 °C. [cCP] = [iCP] = 0.3 nM; [Suc-LLVY-AMC] =
0
2
0 µM (ChT-L activity); [Boc-LLR-AMC] = 50 µM (T-L activity); [Z-LLE-βNA] = 50 µM
0 0
(
C-L activity of cCP); [Ac-PAL-AMC] = 50 µM (C-L activity of iCP). Experimental points
0
were fitted to equation 1 or 2.
A similar efficiency of the starting compound 1a was observed against cCP and iCP
ChT-L and C-L activities whereas the inhibition of the iCP T-L activity was quasi-abolished.
Reduced inhibitory activity was observed when the P1 benzylic group was replaced by the
smaller methyl group (1a/1b) with a larger effect for iCP inhibition. This was expected since
based on the mouse crystallographic structures of cCP and iCP [39],
β5i seems able to
accommodate larger amino acid side chain at position P1 than 5c. The epimerization at
β
carbon 3 (2a/1a and 2b/1b) resulted in a significantly loss of activity whereas no significant
2
effect was observed for 1c/2c in which P2 substituent R = H present in both 1a and 2a was
5
replaced by a methyl group. The replacement of the larger R Boc group (1a) by H
(compound 1e) led to a similar decrease effect by a factor ≈ 3 on the ChT-L, C-L and T-L
4
inhibition of cCP and iCP. The removal of the R benzyl group (1d versus 1a) was very
unfavorable. For further potential optimization, we enlarged side chains at P1 using CH (1-
2
Naphthyl) (1f), CH(Ph) (1g), CH -(4-PhC H ) (1h) and CH -(4-iPrC H ) (1i) to be compared
2
2
6
4
2
6
4
to the side chain Bn (1a). Their efficiencies against ChT-L and C-L activities of both
proteasomes were quite similar, except for 1f and 1g which were more efficient (factors 1.8 to
5
.4) on the ChT-L and PA activities of cCP and iCP. Whereas compounds 1g-i were
inefficient to inhibit iCP and cCP T-L activity, 1f inhibited T-L activities as efficiently than
C-L one (IC of 2.7 µM). The adamantyl compound 1p was significantly less potent on iCP
5
0
1
0

ACCEPTED MANUSCRIPT
ChT-L and C-L activities than on the same cCP activities (factors of 4.7 and 2.2,
respectively). The ONX-0914 derivative bearing an adamantyl at P1 failed to inhibit both CPs
[
53].
Attempts to enhance efficiency against C-L activity by introducing Asp analogs (compounds
j and 1k) were unsuccessful. Conversely to that observed for epoxyketone peptides [53], a
cyclohexyl group at the P1 position did not enhance 5i selectivity (compounds 1m and 1n).
Whereas the efficiency against ChT-L activity of both CPs did not vary compared to that of
a, the efficiency against C-L activity was improved (factors of 1.3-3.7) and the effect on the
T-L activity was poorer or lost. We investigated the SAR of the P3 residues. The -Ala
residue (1q) was less favorable than the -Ala one (1a). The Asn residue also found in the
natural product TMC-95A favored inhibition of the ChT-L activity of both CPs (factors of ∼
). An enhanced ChT-L inhibitory activity was observed when the P3 methyl group
compound 1t) was replaced by the polar lateral chain of Thr (factors of 28 for cCP and 17 for
1
β
1
D
L
2
(
iCP). The inhibition of the C-L activity of both CPs was poorly affected whereas the iCP T-L
activity was noticeable (IC = 5.8 ± 0.3 µM). Finally, the Lys side chain (compound 1v) but
50
not its Boc form (compound 1u) were active against the T-L activity of both CPs at the
submicromolar level. The most important improvement was obtained with dipeptides 5. As
for tripeptide derivatives, a favorable effect of the Asn (5b) and Thr (5c) on the ChT-L
activity was observed in comparison with 5a, factors of 14 for cCP and 9.4 for iCP
(compound 5b) and of 5.8 for cCP and 7 for iCP (compound 5c). The T-L activity was not
affected whereas the C-L one was very poorly inhibited only for compound 5c. The CO(4-
BnO)C H N-terminal blocking group known to potentially occupy small accessory
6
4
hydrophobic pockets AS1 and AS2 [18, 25] led to efficient and selective ChT-L inhibition (5f
3
and 5e). This effect was increased by more than one order of magnitude when the R alanine
group (5e) was replaced with the threonine residue (5f). The IC values of compound 5f for
5
0
the ChT-L activity were of 7.1 ± 0.2 nM (cCP) and 10.2 ± 0.1 nM (iCP). Finally, after 15 min
treatment, compound 5f appeared to be more efficient than the covalent inhibitor ONX-0914
against ChT-L activity by a factor of 2 (cCP) and 5 (iCP). This compound is known to act
selectively on the ChT-L activity of immunoproteasome [54].
1
1

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Table 1: Inhibition by tripeptides 1a-v and 2a-c of purified human 20S constitutive
proteasome (values displayed in normal characters) and human 20S immunoproteasome
a
(values in bold) at pH 8 and 37 °C
1
2
3
4
5
R
R
R
R
R
IC50 (µM)
C-L
11.5 ± 0.5
9.9 ± 0.3
20%
ChT-L
3.2 ± 0.1
.53 ± 0.07
79±2
T-L
7.1 ± 0.2
28 ± 2
36%
b,c
c
1
a
Bn
Me
H
H
Me
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Bn
Bn
Bn
Bn
Bn
Bn
H
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
2
1
b
2
4%
13%
79 ± 3
19%
1
c
Bn
5.1 ± 0.1
.93 ± 0.08
7.0 ± 0.3
11.4 ± 0.3
10.32 ± 0.07
17.2 ± 0.4
10.6 ± 0.2
ni
3
27%
ni
c
2
2
a
Bn
7
.3 ± 0.2
34 ± 9
13%
c
b
Me
ni
8%
10%
19%
2
c
Bn
6.8 ± 0.2
13.4 ± 0.5
8.9 ± 0.2
ni
12%
30 ± 1
21.5 ± 0.6
2.14 ± 0.09
2.77 ± 0.08
2.2 ± 0.2
4 ± 1
17.6 ± 0.4
13 ± 1
37%
56%
6
.1 ± 0.1
18%
19%
c
1
d
Bn
32 ± 2
3
4 ± 2
10.7 ± 0.2
3 ± 1
1.40 ± 0.01
.90 ± 0.03
1.74 ± 0.03
.70 ± 0.06
28.7 ± 0.9
9.7 ± 0.7
10.0 ± 0.4
10%
c
1
e
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
24.8 ± 0.9
27.2 ± 0.8
2.9 ± 0.1
2.7 ± 0.1
30%
1
1
f
CH
2
(1-Naph)
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
1
1
g
h
CH(Ph)
-(4-PhC
-(4-iPrC
(CH CO Me
(CH CO
(CH Ph
(CH -C
CH -C
CH -tBu
CH -Ada
Bn
2
2
ni
1
CH
2
6
H
4
)
ni
x 1.5
d
1
1
i
CH
2
6
H
4
)
ni d
1
7.1 ± 0.9
46 ± 3
15%
17%
19%
x 1.3
1
j
2
)
2
2
64 ± 6
14%
3
1%
18%
4%
6.0 ± 0.1
.5 ± 0.1
2.8 ± 0.1
.7 ± 0.1
4.3 ± 0.1
.2 ± 0.1
11.3 ± 0.2
2.6 ± 0.3
2.13 ± 0.03
0.0 ± 0.2
9.2 ± 0.3
106 ± 7
37.6 ±0.7
22 ± 2
45%
1
k
2
)
2
2
H
1
17%
1
l
2
)
2
4.9 ± 0.2
7.31 ± 0.06
2.7 ± 0.2
3.5 ± 0.1
4.7 ± 0.3
4.8 ± 0.2
15.1 ± 0.3
21 ± 1
2.6 ± 0.2
5.7 ± 0.1
17.1 ±0.5
12.5± 0.9
12.4 ± 0.5
12.6 ± 0.5
20.1 ± 0.8
16.1 ± 0.9
6.08 ± 0.09
8.8 ± 0.7
15%
6
26%
1
m
2
)
2
6
H
11
36%
2
19%
1
n
o
p
q
2
6
H
11
19.3 ±0.4
16 ± 1
31 ± 2
30 ± 3
19%
5
1
2
1
1
1
2
1
ni
(
D
)Me
H
18.6 ± 0.2
21.4 ± 0.4
30 ± 1
8.9 ± 0.2
1
3.2 ± 0.3
1
r
Bn
Bn
Bn
Bn
Bn
6.2 ± 0.1
7
.8 ± 0.2
1.48 ± 0.04
.6 ± 0.1
0.125 ± 0.004
.20 ± 0.01
22.0 ± 0.4
5.1 ± 0.9
2.9 ± 0.1
.8 ± 0.1
d
1
s
t
CH
2
CONH
2
x 1.4d
1
x 1.3
13.5 ± 0.8
5.8 ± 0.3
ni
1
(R)-CH(OH)Me
0
1
u
v
(CH
2
)
4
NHBoc
NH
d
2
59 ± 5
13.0 ± 0.9
4.3 ± 0.6
x 1.3
1
(CH
2
)
4
2
0.52 ± 0.08
3
0.82 ± 0.07
a
The inhibition was evaluated after 15 min incubation of the enzyme with the respective
compound before adding the appropriate fluorogenic substrate (Suc-LLVY-AMC for ChT-L
1
2

ACCEPTED MANUSCRIPT
activities of cCP and iCP, Z-LLE-βNA for C-L activity of cCP, Ac-PAL-AMC for C-L
activity of iCP and Boc-LLR-AMC for T-L activities). ni: no inhibition. IC values were
5
0
determined when the % inhibition was higher than 85% at 50 µM. The % inhibition at 50 µM
b
c
is displayed in italicized text. Published results [42]. Inhibition of the ChT-L activity of
d
yeast proteasome was previously published [17]. Activation factor at 50 µM.
Table 2: Inhibition by dipeptides 5a-f of purified human 20 S constitutive proteasome (values
displayed in normal characters) and human 20S immunoproteasome (values in bold) at pH 8
a
and 37 °C
IC (µM)
ChT-L
5.1 ± 0.1
50
1
3
R
R
R
Z
C-L
T-L
5
5
5
5
5
5
a
b
c
d
e
f
Bn Me
Bn CH
ni
45 ± 2
ni
ni
20%
24.6 ± 0.7
40 ± 1
14 ± 1
35 ± 1
52 ± 2
ni
ni
ni
ni
ni
ni
3
.96 ± 0.09
0.36 ±0.02
.42 ± 0.01
0.88 ± 0.02
.572 ± 0.005
0.35 ± 0.01
.26 ± 0.01
0.113 ± 0.006
.170 ± 0.004
CONH
Z
2
2
0
Bn
(
R
)-CH(OH)Me
Z
0
Bn Me
Bn Me
COCH
(3-PhO)C H
6 4
16.2 ± 0.7
14.3 ± 0.6
26.0 ± 0.3
28.3 ± 0.7
13.2 ± 0.5
6.6 ± 0.6
4.7 ± 0.6
2
0
CO(4-BnO)C H
6 4
0
Bn
-
(
-
R
)-CH(OH)Me CO(4-BnO) C
H
0.0071 ± 0.0002 31.9 ± 0.6
.0102 ± 0.0001 23.8 ± 0.7
0.15 ± 0.02 22 ± 1
.052 ± 0.007
6
4
0
ONX-0914
-
0
0.59 ± 0.03 4.85 ± 0.07
a
The inhibition was evaluated after 15 min incubation of the enzyme with the respective
compound before adding the appropriate fluorogenic substrate (Suc-LLVY-AMC for ChT-L
activities of cCP and iCP, Z-LLE-βNA for C-L activity of cCP, Ac-PAL-AMC for C-L
activity of iCP and Boc-LLR-AMC for T-L activities). ni: no inhibition. IC values were
5
0
determined when the % inhibition was higher than 85% at 50 µM. The % inhibition at 50 µM
is displayed in italicized text.
2
.4 Tumor cells assays
The cell viability of the best inhibitors was evaluated on several cancer cell lines:
hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco2, colorectal carcinoma
HCT-116, breast carcinoma MDA-MB 231, breast carcinoma MCF7, prostate carcinoma PC3
1
3

ACCEPTED MANUSCRIPT
and lung carcinoid NCI-H727) (Table 3, Figure S3). Human skin fibroblasts were the
reference for non-tumor cells. DMSO was used as negative control and the proteasome
inhibitor ONX-0914 as positive control. After 48 h treatment with the inhibitors, cells were
fixed and the nuclei were stained with Hoechst 33342, and then counted. The survival
percentages were calculated as the number of cells after compound treatment over the number
of cells after DMSO treatment. According to dose-response curves, the EC values were
5
0
determined (Table 3). Dipeptides 5e,f were more cytotoxic than tripeptides 1a,s-t. Dipeptide
f displayed toxicities in the sub-micromolar range against 6 over 7 cancer cell lines. Its effect
5
was close to that obtained with ONX-0914 on MDA-MB 231, HCT-116, PC3, NCI-H727 and
MCF7. Noticeably, normal fibroblasts were not affected by treatment with our compounds.
Table 3. Effect of compounds 1a,s,t and 5e,f on the survival of human cancer cells and
normal human skin fibroblasts using Hoechst 3342 staining.
EC (µM)
50
HuH7 CaCo-2 MDA-MB-231 HCT-116 PC3 NCI-H727 MCF7 Fibroblasts
1
1
1
5
5
a
s
t
ne
ne
ne
ne
ne
ne
ne
32
ne
5
ne
32
ne
ne
ne
10
24
0.4
ne
ne
ne
ne
2
26
ne
ne
ne
ne
9
9
9
ne
e
f
23
0.7
0.27
10
0.2
0.11
21
0.7
0.26
ne
ne
ONX-0914 0.39
1.3
0.33 0.3
0.16
ne: no effect at 25 µM.
3
. Conclusion
We have designed and synthesized 22 new tripeptidic and 6 dipeptidic linear analogs of the
natural non covalent inhibitor TMC-95A and analyzed their inhibitory activity against human
cCP and iCP. We investigated analogs with various P1 and P3 residues for tripeptides, and
various P1, P3 residues and N-terminal groups for dipeptides reasoning that the interaction of
these residues with the enzyme subsites described in previous studies by us [17] or others [19-
2
1] would contribute to affinity and orientate the inhibitor selectivity towards iCP over cCP.
Conversely to that observed with covalent inhibitors such as epoxyketones [53, 55], bulky P1
groups such as naphtyl, 4-isopropyl, 4-biphenyl or cyclohexyl groups did not favor the
inhibition of iCP over cCP. The simultaneous introduction of the threonine residue at the P3
position and the CO(4-BnOC H ) as N-terminal group in dipeptides led to the inhibition of
6
4
both cCP and iCP at the nanomolar level. Discrepancies between covalent and noncovalent
1
4

ACCEPTED MANUSCRIPT
inhibitors may be correlated to differences in conformational changes upon their binding with
γ
the catalytic subsites and the formation of a covalent bond with Thr1O in the case of covalent
inhibitors [56, 57].
4
. Experimental section
4
.1. Chemistry
General chemistry methods are described in the supporting information.
4
4
.1.1. Synthesis of 3-hydroxyoxindolyl alanine residues 6 and 7
.1.1.1. General procedure for the preparation of spirolactones 9a and 10a
To a solution of Z-L-Trp-OH (12.00 g, 35.5 mmol) in DMSO (26.8 mL, 377 mmol), was
added slowly t-BuBr (41.5 mL, 357 mmol). The mixture was stirred at 45°C for 20 hours. The
excess of t-BuBr and DMSO was removed under reduced pressure. After dilution by CH Cl2
2
(150 mL), the mixture was washed by iced water twice (2 x 20 mL). The organic phase was
concentrated in vacuo and the residue was purified by chromatography over silica gel (835 g,
eluent 5, 10, 15, 20% of AcOEt in CH Cl ) to afford two fractions: the less polar 3(R)-
2
2
spirolactones 9a as a brown amorphous solid (4.90 g, 70/30 mixture of non brominated (X
H)/ brominated (X = Br) derivatives, ca 37% yield) and the more polar 3(S)-spirolactones
0a as a brown amorphous solid (6.00 g, 70/30 mixture of non brominated (X =H)/
brominated (X = Br) derivatives, ca 45% yield). An authentic sample of 9a (X = H) and 10a
X = H) was obtained by reaction at room temperature of Z- -Trp-OH (0.364 g, 1.07 mmol)
=
1
(
L
in acetonitrile (6 mL) with potassium iodide (20.3 mmg, 0.12 mmol) and 35 % hydrogene
peroxide (0.57 mL, 7.04 mmol). The brownish reaction mixture was stirred overnight at room
temperature, and then concentrated in vacuo. After chromatography over silica gel (16 g,
eluent AcOEt in CH Cl ), spirolactones 9a (X = H, 57 mg, 15% yield) and 10a (X = H, 142
2
2
mg, 38%) were afforded as solids.
(R)-spirolactone 9a (X = H). Yellowish solid. Mp (decomposition) 150 °C. H NMR (300
MHz, CDCl ) δ 2.49 (dd, J = 14.1, 5.4 Hz, 1H), 2.83 (dd, J = 14.1, 9.6 Hz, 1H), 5.11 (m, 1H),
1
3
3
5
7
6
1
1
.19 (s, 2H), 6.22 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 7.15 (t, J = 6.9 Hz, 1H), 7.35-
1
3
.40 (m, 7H), 7.94 (br s, 1H). C NMR (75.5 MHz, CD COCD ) δ 37.14 (CH ), 51.00 (CH),
3
3
2
7.31 (CH ), 81.80 (C), 111.67 (CH), 123.97 (CH), 125.09 (CH), 128.03 (C), 128.78 (CH),
2
28.82 (CH), 129.24 (CH), 132.05 (CH), 137.82 (C), 142.93 (C), 156.70 (C), 174.47 (C),
+
75.79 (C). HRMS (ESI) calcd for C H N O Na [(M+Na) ] 375.0953, found 375.0955. R
1
9
16
2
5
f
(
1/4 AcOEt/CH Cl ) 0.45.
2 2
1
5

ACCEPTED MANUSCRIPT
1
3
(S)-spirolactone 10a (X = H). White solid. Mp (decomposition) 164 °C. H NMR (300 MHz,
CDCl ) δ 2.76 (m, 1H), 2.92 (m, 1H), 5.00 (m, 1H), 5.14 (s, 1H), 5.68 (d, J = 6.0 Hz, 1H),
3
1
3
6
.91 (d, J = 7.8 Hz, 1H), 7.13 (m, 1H), 7.33-7.55 (m, 7H), 8,31 (br s, 1H). C NMR (75.5
MHz, CD COCD ) δ 36.79 (CH ), 51.12 (CH), 67.21 (CH ), 80.78 (C), 111.46 (CH), 123.87
3
3
2
2
(CH), 125.82 (CH), 127.40 (C), 128.74 (CH), 128.79 (CH), 129.24 (CH), 132.16 (CH),
1
37.75 (C), 143.49 (C), 156.81 (C), 174.50 (C), 176.43 (C). HRMS (ESI) calcd for
+
C H N O Na [(M+Na) ] 375.0953, found 375.0951. R (1/4 AcOEt/CH Cl ) 0.3.
19
16
2
5
f
2
2
4
.1.1.2. Spirolactones 9b and 10b
According to the same procedure starting from Z-L-methyltryprophan (4.84 g, 13.7 mmol),
DMSO (9.9 mL, 140 mmol) and tert-butyl bromide (15.6 mL, 139 mmol), the less polar
spirolactone 9b (1.10 g, 22 % yield) and the more polar spirolactone 10b (2.03 g, 42% yield)
were obtained as vitreous orange solids after purification by chromatography over silica gel
(
260 g, eluent 5, 10, 15, 20% of AcOEt in CH Cl ).
2 2
1
3
(R)-spirolactone 9b: Mp (decomposition) 67 °C. H NMR (300 MHz, CDCl ) δ 2.43 (dd, J
3
=
14.0, 5.0 Hz, 1H), 2.78 (dd, J = 14.0, 9.3 Hz, 1H), 3.20 (s, 3H), 5.08 (m, 1H), 5.15 (s, 2H),
6
.42 (d, J = 9.3 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 7.15 (t, J = 7.3 Hz, 1H), 7.43-7.47 (m, 7H).
C NMR (75.5 MHz, CDCl ) δ 26.56 (CH ), 37.35 (CH ), 50.07 (CH), 67.36 (CH ), 81.35
1
3
3
3
2
2
(C), 109.23 (CH), 123.96 (CH), 124.30 (CH), 125.44 (C), 128.14 (CH), 128.17 (CH), 128.53
(CH), 131.56 (CH), 135.97 (C), 143.68 (C), 155.97 (C), 173.58 (C), 173.84 (C). HRMS (ESI)
+
calcd for C H N O Na [(M+Na) ] 389.1108, found 389.1107. R = 0.49 (AcOEt/CH Cl
2
0
18
2
5
f
2
2
5
3
2
/95)
1
(S)-spirolactone 10b: Mp (decomposition) 140 °C. H NMR (300 MHz, CDCl ) δ 2.80 (m,
3
H), 3.18 (s, 3H), 4.94-5.03 (m, 1H), 5.14 (s, 2H), 5.65 (d, J = 6.1 Hz, 1H), 6.85 (d, J = 7.8
1
3
Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H,), 7.32-7.43 (m, 6H,), 7.52 (d, J = 7.2 Hz, 1H). C NMR
(75.5 MHz, CDCl ) δ 26.34 (CH ), 36.62 (CH ), 50.65 (CH), 67.30 (CH ), 81.13 (C), 108.80
3 3 2 2
(CH), 123.80 (CH), 125.03 (CH), 125.43 (C), 128.15 (CH), 128.30 (CH), 128.55 (CH),
1
31.44 (CH), 135.77 (C), 144.03 (C), 155.65 (C), 173.70 (C), 173.84 (C). HRMS (ESI) calcd
+
for C H N O Na [(M+Na) ] 389.1108, found 389.1105. R = 0.31 (AcOEt/ CH Cl 5/95)
2
0
18
2
5
f
2
2
4
.1.1.3. General procedure for the synthesis of (S)-2-amino-N-methyl-3-((R)-3-hydroxy-2-
oxoindolin-3-yl)propanamide 6a:
1
6

ACCEPTED MANUSCRIPT
Spirolactone 9a (200 mg, ca 0.53 mmol, based on a 70:30 ratio of H- and Br-oxindole starting
material, X = H or Br) was reacted with 2 M MeNH in THF (2 mL, 4 mmol) for 3 h at room
2
temperature. Evaporation of the solvent under reduced pressure and chromatography over
silica gel (2% MeOH in CH Cl ) afforded a 70:30 mixture of non-brominated and brominated
2
2
Z-protected 6a (170 mg, yield ca 79%) as a yellow amorphous solid. MPLC purification of a
small sample (38 mg) over reversed phase silica (C18, 5 g, 50% MeOH in water) afforded
after lyophilization, (S)-2-benzyloxycarbonylamino-N-methyl-3-((R)-3-hydroxy-2-oxoindolin-
1
3
-yl)propanamide (22.3 mg, H NMR (200 MHz, CDCl ) δ 2.36 (broad s, 2H, CH β), 1.54 (s,
3
2
3
H, NCH ), 4.26 (broad s, 1H, Hα), 4.88 (s, 2H, CH Ph), 5.56 (broad s, 1H, OH), 6.27-7.14
3 2
+
(
m, 9H, aromatic H), 8.80 (broad s, 1H, NH). HRMS (LSIMS) calcd for C H N O [M+H] :
20 22 3 5
3
84.1559, found 384.1550. R (RP18, 50% MeOH in water) 0.18.) and (S)-2-
f
benzyloxycarbonylamino-N-methyl-3-((R)-5-bromo-3-hydroxy-2-oxoindolin-3-
79
21
+
yl)propanamide (3.5 mg, HRMS (LSIMS) calcd for C H BrN O [M+H] : 462.0665,
2
0
3
5
found 462.0632. R (RP18, 50% MeOH in water) 0.05).
f
A 70:30 mixture of non-brominated and brominated Z-protected 6a (132 mg) was placed
overnight with 10% Pd on carbon (20 mg) in methanol (2 mL) under H atmosphere. The
2
mixture was filtered through celite and treated with 1 M anhydrous HCl in methanol (0.7 mL,
0
.7 mmol). After concentration in vacuo, product 6a, hydrochloride was obtained as a white
solid (96.4 mg, 30% from Z-Trp).
1
H NMR (300 MHz, D O) δ 2.38 (s, 3H, NCH ), 2.44 (d, J = 6.9 Hz, 2H, CH β), 4.02 (t, J =
2
3
2
1
3
6
.9 Hz, 1H, Hα), 7.02 (d, J = 7.5 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H,), 7.31-7.35 (m, 2H). C
NMR (75.5 MHz, D O) δ 26.6 (NCH ), 37.4 (CH ), 50.2 (CHα), 75.1 (C), 111.9 (CH), 124.4
2
3
2
(CH), 125.0 (CH), 129.5 (C), 131.5 (CH), 140.7 (C), 169.3 (C=O), 180.2 (C=O oxindole).
+
HRMS (ESI) calcd for C H N O [M+H] : 250.1192, found 250.1192.
1
2
16
3
3
4
.1.1.4. (S)-2-Amino-N-benzyl-3-((R)-3-hydroxy-2-oxoindolin-3-yl)propanamide 6b:
According to the general procedure described above, spirolactone 9a (314 mg, ca 0.83 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and benzylamine (110 µL,
0
.96 mmol) were reacted overnight. Purification by chromatography over silica gel (23.6 g,
eluent 40% of AcOEt in CH Cl then AcOEt) afforded an orange amorphous solid (419 mg)
2
2
that was hydrogenated according to the general procedure. Product 6b, hydrochloride was
obtained as a white solid (221 mg, 27% from Z-Trp).
1
7

ACCEPTED MANUSCRIPT
1
H NMR (300 MHz, DMSO-d ) δ 2.08 (dd, J = 13.5, 3.0 Hz, 1H, CHβ), 2.26 (dd, J = 13.5,
6
9
.0 Hz, 1H, CHβ), 4.15 and 4.27 (ABX system, JAB = 15.0 Hz, JAX = JBX = 5.7 Hz, 2H,
NCH ), 4.41 (t, J = 4.5 Hz, 1H, Hα), 6.87 (d, J = 6.0 Hz, 1H, H aromatic), 7.02 (td, J = 7.5,
2
+
1
8
2
4
.5 Hz, 1H, H aromatic), 7.2-7.46 (m, 7H, aromatics), 8.08-8.10 (d, J = 3.0 Hz, 3H, NH ),
3
1
.83 (t, J = 6.0 Hz, 1H, NH), 10.54 (s, 1H, NH oxindole). H NMR (300 MHz, D O + HCl) δ
2
.37 and 2.44 (ABX system, 2H, JAX = 6.7 Hz, JBX = 7 Hz, JAB = 14.7 Hz, CH β), 3.96 and
2
.11 (AB system, J = 14.7 Hz, 2H, CH Ph), 4.20 (t, J = 6.8 Hz, 1H, CHα), 6.95 (d, J = 8.0
AB
2
13
Hz, 1H), 7.06-7.15 (m, 3H), 7.23-7.33 (m, 5H). C NMR (75.5 MHz, D O + HCl) δ 37.5
2
(
CH ), 44.1 (CH ), 50.1 (CH), 75.1 (C), 111.9 (CH), 124.4 (CH), 124.9 (CH), 128.1 (CH),
2 2
1
28.3 (CH), 129.4 (CH), 129.9 (C), 131.5 (CH), 137.6 (C), 140.6 (C), 169.0 (C=O), 180.3
+
(
CO oxindole). HRMS (ESI) calcd for C H N O Na [M + Na] : 348.1324, found 348.1323.
1
8
19
3
3
4
.1.1.5.
(S)-2-Benzyloxyamino-N-benzyl-3-((R)-3-hydroxy-1-methyl-2-oxoindolin-3-yl)
propanamide:
According to the general procedure described above, spirolactone 9b (1.60 g, 4.75 mmol) and
benzylamine (560 µL, 5.12 mmol) were reacted overnight. The solid was filtered and washed
with CH Cl2 to afford (S)-2-benzyloxyamino-N-benzyl-3-((R)-3-hydroxy-1-methyl-2-
2
oxoindolin-3-yl)propanamide (N-Z-protected 6c) as a white solid (1.52g, 68%). Mp 156-158
1
°
C. H NMR (300 MHz, CDCl ) δ 2.25 (dd, J = 15.6, 4.5 Hz, 1H), 2.50 (dd, J = 15.6, 4.2 Hz,
3
1
1
7
5
1
H), 3.01 (s, 3H), 4.33 (dd, J = 14.8, 5.1 Hz, 1H), 4.48 (dd, J = 14.6, 6.3 Hz, 1H), 4.78 (br,
H), 5.13 (m, 2H), 6.54 (d, J = 6.5 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H),
13
.43-7.47 (m, 14H). C NMR (75.5 MHz, CDCl ) δ 26.09 (CH ), 42.23 (CH ), 43.55 (CH ),
3
3
2
2
0.13 (CH), 67.35 (CH ), 74.28 (C), 108.60 (CH), 123.64 (CH), 123.68 (CH), 127.47 (CH),
2
27.56 (CH), 128.21 (CH), 128.31 (CH), 128.57 (CH), 128.67 (CH), 129.88 (CH), 130.97
(C), 135.89 (C), 137.70 (C), 142.44 (C), 156.66 (C), 171.29 (C), 178.60 (C). HRMS (ESI)
+
calcd for C H N O Na [(M+Na) ] 496.1843, found 496.1844. R (2/3 AcOEt/CH Cl ) 0.31.
2
7
27
3
5
f
2
2
4
.1.1.6. (S)-2-Amino-N-benzyl-3-((R)-3-hydroxy-1-methyl-2-oxoindolin-3-yl)propanamide 6c:
Hydrogenation of (S)-2-benzyloxyamino-N-benzyl-3-((R)-3-hydroxy-1-methyl-2-oxoindolin-
3
-yl)propanamide (965 mg, 2.04 mmol) according to the general procedure afforded 6c as a
1
white solid (691 mg, quantitative, 15% from Z-
L
-methyl tryptophan). H NMR (300 MHz,
DMSO-d ) δ 1.88-2.04 (m, 2H), 3.09 (s, 3H), 3.64 (dd, J = 10.5, 3.90 Hz, 1H), 4.20 (d, J = 4.2
6
Hz, 2H), 6.95 (d, J = 7.5 Hz, 1H), 7.03 (t, J = 7.2 Hz, 1H), 7.17-7.32 (m, 1H), 8.38 (br s, 1H)
1
8

ACCEPTED MANUSCRIPT
4
.1.1.7.
(S)-2-Amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)-N-(naphthalen-1-ylmethyl)
propanamide 6d: According to the general procedure described above, spirolactone 9a (208
mg, ca 0.55 mmol based on a 70:30 ratio of H- and Br-oxindole starting material) and
naphtylmethylene amine (140 µL, 0.90 mmol) were reacted overnight. Purification by
chromatography over silica gel (8.1 g, eluent 10% of AcOEt in CH Cl then then 25% AcOEt
2
2
in CH Cl ) afforded an orange amorphous solid (291 mg) that was hydrogenated according to
2
2
the general procedure. Product 6d, hydrochloride was obtained as a white solid (191 mg, 31%
from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 2.14 (dd, J = 15.3, 3.0 Hz, 1H, CHβ), 2.63 (dd, J = 15.2, 4.2
3
Hz, 1H, CHβ), 4.75 ( m, 3H, Hα, NCH ), 6.89 (d, J = 7.8 Hz, 1H aromatic), 7.00 (td, J = 7.5,
2
0
7
5
4
1
.6 Hz, 1H aromatic), 7.21 (td, J = 7.8, 1.0 Hz, 1H aromatic), 7.29-7.43 (m, 5H aromatics) ,
+
3
.68-7.78 (m, 2H aromatics), 7.89-7.93 (m, 1H aromatic), 8.42 ( br s, NH , OH) , 8.85 (t, J =
1
3
.4 Hz, 1H, NH), 10.31 (s, 1H, NH oxindole). C NMR (75.5 MHz, CD OD) δ 39.08 (CH ),
3
2
2.48 (CH ), 50.86 (CH), 75.67 (C), 111.68 (CH), 124.04 (CH), 124.22 (CH), 124.67 (CH),
2
23.36 (CH), 126.86 (CH), 127.26 (CH), 127.40 (CH), 129.39 (CH), 129.73 (CH), 131.13
(CH), 132.45 (C), 132.70 (C), 134.11 (C), 135.17 (C), 141.86 (C), 170.21 (CO), 180.70 (CO).
+
HRMS (ESI) calcd for C H N O Na [M + Na] : 398.1475, found 398.1476.
2
2
21
3
3
4
.1.1.8. (S)-2-Amino-N-benzhydryl-3-((R)-3-hydroxy-2-oxoindolin-3-yl)propanamide 6e:
According to the general procedure described above, spirolactone 9a (233 mg, ca 0.62 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and benzhydrylamine (130 µL,
0
.73 mmol) were reacted overnight. Purification by chromatography over silica gel (8 g,
eluent 50% of AcOEt in CH Cl then 80% AcOEt in CH Cl ) afforded an orange amorphous
2
2
2
2
solid (189 mg) that was hydrogenated according to the general procedure. Product 6e,
hydrochloride was obtained as a white solid (148 mg, 19% from Z-Trp).
1
H NMR (300 MHz, DMSO-d ) δ 2.07 (dd, J = 13.5, 3.0 Hz, 1H, CHβ), 2.26 (dd, J = 9.0, 3.0
6
Hz, 1H, CHβ), 3.97 (s, 1H, OH), 4.22 (ddd, J = 18.0, 15.0, 1.5 Hz, 2H, NCH ), 4.55 (br s, 1H,
2
Hα), 6.07 (d, J = 9.0 Hz, 1H, CH(Ph) ), 6.87 (d, J = 6.0 Hz, 1H aromatic), 6.93 (m, 1H
2
aromatic), 7.02 (td, J = 7.5, 1.0 Hz, 1H aromatic), 7.17-7.37 (m, 11H aromatic) , 8.08 (br s,
+
13
3
H, NH ) , 9.3 (d, J = 9.0 Hz, 1H, NH), 10.58 (s, 1H, NH oxindole). C NMR (75.5 MHz,
3
CD OD) δ 38.97 (CH ), 50.86 (CH), 58.61 (CH), 75.66 (C), 111.68 (CH), 124.06 (CH),
3
2
1
24.67 (CH), 128.51 (CH), 128.59 (CH), 129.18 (CH), 129.56 (CH), 129.6 (CH), 129.89
(CH), 131.18 (CH), 132.83 (C), 141.98 (C), 142.32 (C), 142.42 (C), 169.09 (CO), 180.63
+
(
CO). HRMS (ESI) calcd for C H N O Na [M+Na] : 424.1632, found 424.1630.
2
4
23
3
3
1
9

ACCEPTED MANUSCRIPT
4
.1.1.9.
(S)-N-([1,1'-Biphenyl]-4-ylmethyl)-2-amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)
propanamide 6f:. According to the general procedure described above, spirolactone 9a (261
mg, ca 0.69 mmol based on a 70:30 ratio of H- and Br-oxindole starting material) and 4-
phenylbenzylamine (151 mg, 0.82 mmol) were reacted overnight. Purification by
chromatography over silica gel (20 g, eluent 30% of AcOEt in CH Cl ) afforded an orange
2
2
amorphous solid (300 mg) that was hydrogenated according to the general procedure. Product
6
f, trifluoroacetate salt was obtained as a white solid (291 mg, 30% from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 2.12 (dd, J = 15.0, 3.0 Hz, 1H, CHβ), 2.22 (dd, J = 15.3, 10.8
3
Hz, 1H, CHβ), 4.34-4.38 (m, 2H, CH ), 4.71 (dd, J = 10.8, 3.0 Hz, 1H, Hα), 6.93 (d, J = 7.8
2
Hz, 1H aromatic), 7.02 (td, J = 7.5, 3.0 Hz, 1H aromatic), 7.22-7.32 (m, 7H aromatics), 7.45-
+
7
.49 ( m, 4H aromatics), 8.32 (br s, 3H, NH ), 8.79 (t, J = 6.0 Hz, 1H, NHCO), 10.21 (s, NH
3
13
oxindole). C NMR (75.5 MHz, CD OD) δ 38.98 (CH ), 44.04 (CH ), 50.08 (CH), 75.59
3
2
2
(C), 111.66 (CH), 124.03 (CH), 124.72 (CH), 127.77 (CH), 128.04 (CH), 128.28 (CH),
1
1
4
29.14 (CH), 129.76 (CH), 131.14 (CH), 132.70 (C), 138.23 (C), 141.44 (C), 141.78 (C),
+
41.92 (C), 169.73 (CO), 180.56 (CO). HRMS (ESI) calcd for C H N O [M+H] :
2
4
24
3
3
02.1812, found 402.1802.
4
.1.1.10.
(S)-2-Amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)-N-(4-isopropylbenzyl)
propanamide 6g:
According to the general procedure described above, spirolactone 9a (284 mg, ca 0.75 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and 4-(isopropyl)benzylamine
(
132 mg, 0.89 mmol) were reacted overnight. Purification by chromatography over silica gel
11 g, eluent 40% of AcOEt in CH Cl ) afforded an orange amorphous solid (299 mg) that
(
2
2
was hydrogenated according to the general procedure. Product 6g, trifluoroacetate salt was
obtained as a white solid (271 mg, 27% from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 1.15 (s, 3H, CH ), 1.16 (s, 3H, CH ), 2.09 (dd, J = 15.0, 3.0
3
3
3
Hz, 1H, CHβ), 2.22 (dd, J = 15.3, 10.8 Hz, 1H, CHβ), 2.74-2.83 (m, 1H, CH), 4.22-4.33 (m,
H, CH -NHCO), 4.68 (dd, J = 8.3, 3.0 Hz, 1H, Hα), 6.92 (d, J = 7.5 Hz, 1H aromatic), 7.03
2
2
(td, J = 7.5, 1.0 Hz, 1H aromatic), 7.08-7.15 (m, 4H aromatics), 7.25 (td, J = 7.8, 1.2 Hz, 1H
+
aromatic), 7.31-7.36 (m, 1H, aromatic), 8.30 (br s, 4H, NH , OH), 8.70 (t, J = 6.0 Hz, 1H,
3
13
NHCO), 10.26 (s, NH oxindole1H). C NMR (75.5 MHz, CD OD) δ 24.36 (CH ), 34.93
3
3
(
CH), 39 (CH ), 44.2 (CH ), 50.84 (CH), 75.64 (C), 111.69 (CH), 124.04 (CH), 124.71 (CH),
2 2
1
27.50 (CH), 127.74 (CH), 131.14 (CH), 132.68 (C), 136.45 (C), 141.90 (C), 149.27 (C),
2
0

ACCEPTED MANUSCRIPT
+
1
3
69.65 (CO), 180.61 (CO). HRMS (ESI) calcd for C H N O [M+H] : 368.1969, found
21 26 3 3
68.1968.
4
.1.1.11.
Methyl
3-((S)-2-amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)propanamido)
propanoate 6h:
According to the general procedure described above, spirolactone 9a (284 mg, ca 0.75 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material), β-alanine methyl ester,
hydrochloride (134 mg, 0.96 mmol) and triethylamine (300 µL, 2.13 mmol) were reacted
overnight. Purification by chromatography over silica gel (8 g, eluent 15% of AcOEt in
CH Cl ) afforded an orange amorphous solid (213 mg) that was hydrogenated according to
2
2
the general procedure. Product 6h, trifluoroacetate salt was obtained as a white solid (191 mg,
2
1% from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 2.06 (dd, J = 15.3, 3.0 Hz, 1H), 2.45-2.55 (m, 3H), 3.37-3.51
3
(m, 2H), 3.61 (s, 3H), 4.59 (dd, J = 10.8, 3.0 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 7.08 (t, J = 7.5
13
Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H). C NMR (75.5 MHz, CD OD) δ
3
3
4.30 (CH ), 36.59 (CH ), 39.13 (CH ), 50.69 (CH), 52.21 (CH ), 75.60, 111.63 (CH), 124.05
2 2 2 2
(CH), 124.70 (CH), 131.18 (CH), 132.85 (C), 142.02 (C), 169.95 (C), 173.65 (C), 180.68 (C).
+
HRMS (ESI) calcd for C H N O Na [M+Na] : 344.1217, found 344.1212.
1
5
19
3
5
4
.1.1.12. (S)-2-amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)-N-phenethylpropanamide 6i:
According to the general procedure described above, spirolactone 9a (260 mg, ca 0.7 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and phenethyl amine (102 µL,
0
.81 mmol) were reacted overnight. Purification by chromatography over silica gel (15 g,
eluent 2% of MeOH in CH Cl ) afforded an orange amorphous solid (300 mg) that was
2
2
hydrogenated according to the general procedure. Product 6i, trifluoroacetate salt was
obtained as a white solid (241 mg, 28% yield from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 1.72 (dd, J = 15.5, 3.0 Hz, 1H), 2.30 (dd, J = 15.5, 11.4 Hz,
3
1
H), 2.68-2.87 (m, 2H), 3.28-3.37 (m, 1H), 3.50-3.60 (m, 1H), 4.50 (dd, J = 11.4, 3.0 Hz,
1
3
1
H), 6.94 (d, J = 7.5 Hz, 1H), 7.05-7.16 (m, 6H), 7.31-7.34 (m, 2H). C NMR (75.5 MHz,
CD OD) δ 34.64 (CH ), 38.03 (CH ), 40.44 (CH ), 49.35 (CH), 74.28 (C), 110.35 (CH),
3
2
2
2
1
22.70 (CH), 123.41 (CH), 126.04 (CH), 128.05 (CH), 128.51 (CH), 129.83 (CH), 131.42
(
[
C), 138.64 (C), 140.56 (C), 168.50 (C), 179.27 (C). HRMS (ESI) calcd for C H N O Na
1
9
21
3
3
+
M+Na] : 362.1475, found 362.1476.
2
1

ACCEPTED MANUSCRIPT
4
.1.1.13. (S)-2-amino-N-(2-cyclohexylethyl)-3-((R)-3-hydroxy-2-oxoindolin-3-yl)propanamide
6
j:
According to the general procedure described above, spirolactone 9a (260 mg, ca 0.7 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and 2-cyclohexylethyl amine
(
103 mg, 0.81 mmol) were reacted overnight. Purification by chromatography over silica gel
15 g, eluent 2% of MeOH in CH Cl ) afforded an orange amorphous solid (272 mg) that was
(
2
2
hydrogenated according to the general procedure. Product 6j, trifluoroacetate salt was
obtained as a white solid (208 mg, 24% yield from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 0.85-0.96 (m, 2H), 1.13-1.41 (m, 6H), 1.68 (m, 5H), 2.06 (dd,
3
J = 15.3, 3.3 Hz, 1H), 2.51 (dd, J = 15.3, 11.1 Hz, 1H), 3.21 (m, 2H), 4.59 (dd, J = 11.1, 3.3
Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.38 (d, J
1
3
=
7.5 Hz, 1H). C NMR (75.5 MHz, CD OD) δ 27.25 (CH ), 27.54 (CH ), 34.18 (CH), 36.41
3 2 2
(
(
(
CH ), 37.50 (CH ), 38.50 (CH ), 39.21 (CH ), 50.76 (CH), 75.63 (C), 111.64 (CH), 124.02
2 2 2 2
CH), 124.68 (CH), 131.15 (CH), 132.81 (C), 142.01 (C), 169.61 (C), 180.63 (C). HRMS
+
ESI) calcd for C H N O Na [M+Na] : 368.1945, found 368.1948.
1
9
27
3
3
4
.1.1.14. (S)-2-Amino)-N-cyclohexylmethyl-3-((R)-3-hydroxy-2-oxoindolin-3-yl)propanamide
6
k:
According to the general procedure described above, spirolactone 9a (151 mg, ca 0.40 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and cyclohexylmethylene
amine (113.2 mg, 0.4 mmol) were reacted overnight. Purification by chromatography over
silica gel (6 g, eluent 50% of AcOEt in CH Cl ) afforded an orange amorphous solid (163 mg)
2
2
that was hydrogenated according to the general procedure. Product 6k, trifluoroacetate salt
was obtained as a white solid (121 mg, 26% yield from Z-Trp).
1
H NMR (300 MHz, CD OD) δ 0.85-0.97 (m, 2H), 1.19-1.26 (m, 3H), 1.46 (br s, 1H), 1.67-
3
1
.71 (m, 5H), 2.06 (dd, J = 15.3, 3.0 Hz, 1H), 2.52 (dd, J = 15.3, 11.1 Hz, 1H), 3.03 (d, J =
.0 Hz, 2H), 4.62 (dd, J = 11.1, 2.7 Hz, 2H), 6.95 (d, J = 7.8 Hz, 1H), 7.10 (td, J = 7.8, 1.0
6
1
3
Hz, 1H), 7.32 (td, J = 7.8, 1.5 Hz, 1H), 7.38 (d, J = 6.0 Hz, 1H). C NMR (75.5 MHz,
CD OD) δ 26.9 (CH ), 27.5 (CH ), 31.9 (CH ), 32.0 (CH ), 39.0 (CH), 39.3 (CH ), 47.0
3
2
2
2
2
2
(
CH ), 50.8 (CH), 75.7 (C), 111.7 (CH), 123.1 (CH), 124.8 (CH), 131.3 (CH), 133.0 (C),
2
+
1
42.2 (C), 169.9 (CO), 180.8 (CO). HRMS (ESI) calcd for C H N O [M + H] : 332.1969,
1
8
26
3
3
found 332.1972.
4
.1.1.15. (S)-2-Amino-3-((R)-3-hydroxy-2-oxoindolin-3-yl)-N-neopentylpropanamide 6l:
2
2

ACCEPTED MANUSCRIPT
According to the general procedure described above, spirolactone 9a (250 mg, ca 0.66 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and tert-butylmethylene amine
(92 µL, 0.78 mmol) were reacted overnight. Purification by chromatography over silica gel (8
g, eluent 20% of AcOEt in CH Cl ) afforded an orange amorphous solid (250 mg) that was
2
2
hydrogenated according to the general procedure. Product 6l, hydrochloride was obtained as a
white solid (149 mg, 24% from Z-Trp).
1
H NMR (300 MHz, DMSO-d ) δ 0.80 (s, 9H, t-Bu), 2.03 (dd, J = 14.7, 3.9 Hz, 1H, CHβ),
6
2
.22 (dd, J = 14.7, 10.2 Hz, 1H, CHβ), 2.81 and 2.90 (ABX system, 2H, JAX = 6.3 Hz, JBX
=
6
.3 Hz, J = 12.9 Hz, 2H, CH -NHCO), 4.36 (dd, J = 10.2, 2.1 Hz, 1H, Hα), 6.88 (d, J = 7.8
AB
2
Hz, 1H aromatic), 7.02 (td, J = 7.5, 1.0 Hz, 1H aromatic), 7.24-7.38 (m, 2H aromatics), 7.77
+
13
(
br s, NH , OH, 4H), 8.23 (t, J = 6.0 Hz, 1H, NHCO), 10.56 (s, 1H, NH oxindole). C NMR
3
(
75.5 MHz, CD OD) δ 26.20 (CH ), 31.68 (C), 38.03 (CH ), 49.43 (CHα), 50.47 (CH ), 74.26
3
3
2
2
(C), 110.24 (CH), 122.70 (CH), 123.25 (CH), 129.81 (CH), 131.56 (C), 140.66 (C), 168.70
+
(
CO), 179.31 (CO). HRMS (ESI) calcd for C H N O Na [M+Na] : 328.1632, found
16
23
3
3
3
28.1632.
.1.1.16.
4
(2S)-N-(adamantan-1-yl)methyl)-2-amino-3-((R)-3-hydroxy-2-oxoindolin-3-
yl)propanamide 6m:
According to the general procedure described above, spirolactone 9a (208 mg, ca 0.55 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and adamantylmethylene
amine (140 µL, 0.90 mmol) were reacted overnight. Purification by chromatography over
silica gel (8 g, eluent 15% of AcOEt in CH Cl ) afforded an orange amorphous solid (240 mg)
2
2
that was hydrogenated according to the general procedure. Product 6m, trifluoroacetate salt
was obtained as a white solid (150 mg, 20% from Z-Trp).
1
H NMR (300 MHz, DMSO-d ) δ 1.37-1.89 (m, 15H, adamantyl), 2.02 (d, J = 3.9 Hz, 1H,
6
CHβ), 2.18-2.27 (m, 1H, CHβ), 2.70-2.77 (m, 2H, CH -NHCO), 4.38 (br s, 1H, Hα), 6.88 (d,
2
J = 7.8 Hz, 1H aromatic), 7.02 (td, J = 7.5, 1 Hz, 1H aromatic), 7.24-7.32 (m, 2H aromatics),
+
13
8
.02 (br s, NH , OH), 8.16 (t, J = 6 Hz, 1H, NH), 10.57 (s, 1H, NH oxindole). C NMR
3
(
(
(
75.5 MHz, CD OD) δ 29.61 (CH adamantyl), 35.05 (C), 37.88 (CH ), 39.36 (CH ), 41.16
3
2
2
CH ), 50.81 (CHα), 52.35 (CH ), 75.66 (C), 111.70 (CH), 124.06 (CH), 124.65 (CH), 131.16
2
2
CH), 132.85 (C), 141.98 (C), 160.61 (CO), 170.17 (CO), 180.65 (CO). HRMS (ESI) calcd
+
for C H N O Na [M+Na] : 406.2101, found 406.2105.
2
2
29
3
3
2
3

ACCEPTED MANUSCRIPT
4
.1.1.17.
(S)-2-Amino-N-methyl-3-((S)-3-hydroxy-2-oxoindolin-3-yl)propanamide
7a:
According to the general procedure described above, spirolactone 10a (243 mg, ca 0.64 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and 2 M methylamine in THF
(2 mL, 4 mmol) were reacted for 3h. Purification by chromatography over silica gel (8.1 g,
eluent 10% of AcOEt in CH Cl then then 25% AcOEt in CH Cl ) afforded an orange
2
2
2
2
amorphous solid (189 mg). MPLC purification of a small sample (20 mg) over reversed phase
silica (C18, 5 g, 50 % MeOH in water) afforded after lyophilization, (S)-2-
benzyloxycarbonylamino-N-methyl-3-((S)-3-hydroxy-2-oxoindolin-3-yl)propanamide (9 mg,
1
H NMR (300 MHz, CDCl ) δ 2.32-2.55 (m, 2H, CH β), 4.50 (m, 1H, Hα), 4.88 (broad s, 3H,
3
2
1
3
CH Ph + OH), 5.80 (broad, 1H, NHZ), 6.73-7.31 (m, 9H, aromatic H), 9.22 (s, 1H, NH). C
2
NMR (75.5 MHz, CDCl ) δ 26.2 (NCH ), 39.8 , 51.2, 66.9 (OCH Ph), 75.7 (C ), 110.5-140.6
3
3
2
γ
(
aromatic C), 155.9 (C=O), 172.4 (C=O), 179.9 (C=O). R = 0.27 (RP18, 50 % MeOH in
f
+
water), HRMS (LSIMS) calcd for C H N O [M+H] : 384.1559, found 384.1555) and (S)-2-
2
0
22
3
5
benzyloxycarbonylamino-N-methyl-3-(5-bromo-(S)-3-hydroxy-2-oxoindolin-3-
1
yl)propanamide (5.5 mg, H NMR (300 MHz, CDCl ) δ 2.16-2.59 (m, 2H, CH β), 2.72 (s,
3
2
3
,
H, NCH ), 4.48 (broad s, 1H, Hα), 4.90 (s, 2H, CH Ph), 5.84 (broad s, 1H, OH), 6.33 (broad
3 2
1H, NH), 6.59-7.60 (m, 8H, aromatic H), 9.20 (broad s, 1H, NH). R = 0.13 (RP18, 50 %
f
79
21
+
MeOH in water), HRMS (LSIMS) calcd for C H Br N O [M+H] : 462.0665, found
2
0
3
5
4
62.0669).
As above, hydrogenation of the mixture (169 mg) afforded 7a, HCl salt as a white solid (114
mg, 28 % from Z-Trp).
1
H NMR (200 MHz, D O + HCl) δ 2.39 (ABX system, 1H , J = 7 Hz, JAB = 14.7 Hz,
2
A
AX
CH β), 2.55 (ABX system, 1H , J = 7 Hz, J = 14.7 Hz, CH β), 2.69 (broad s, 3H, NCH ),
2
B
BX
AB
2
3
4
.13 (t, 1H, J = 7 Hz, Hα), 7.05 (d, 1H, J = 7.8 Hz ,H ), 7.21 (t, 1H, J = 7.8 Hz, H ), 7.33 (t,
7 5
1
3
1
H, J = 7.8 Hz, H ), 7.47 (d, 1H, J = 7.8 Hz, H ). C NMR (75.5 MHz, D O + HCl) δ 26.5
6
4
2
(
Me), 37.7 (CH ), 50.4 (CHα), 75.1 (C), 112.1 (CH), 124.5 (CH), 124.8 (CH), 129.6 (C),
2
1
31.5 (CH), 140.9 (C), 164.4 (C=O), 169.6 (C=O), 180.5 (C=O). HRMS (LSIMS) calcd for
+
C H N O [M+H] : 250.1192, found 250.1196.
12
16
3
3
4
.1.1.18.
(S)-2-Amino-N-benzyl-3-((S)-3-hydroxy-2-oxoindolin-3-yl)propanamide
7b:
According to the general procedure described above, spirolactone 10a (351 mg, ca 0.93 mmol
based on a 70:30 ratio of H- and Br-oxindole starting material) and benzylamine (120 µL,
1
.10 mmol) were reacted overnight. Purification by chromatography over silica gel (8 g,
2
4

ACCEPTED MANUSCRIPT
eluent 15% of AcOEt in CH Cl ) afforded an orange amorphous solid (385 mg) that was
2
2
hydrogenated according to the general procedure. Product 7b, HCl salt was obtained as a
white solid (290 mg, 39% from Z-Trp).
1
H NMR (300 MHz, D O) δ 2.32 and 2.46 (ABX system, 2H, J = 7.5 Hz, J = 6.2 Hz, J
AB
2
AX
BX
=
14.9 Hz, CH β), 4.23 (t, 1H, J = 6.3 Hz, CHα), 4.28 (broad s, 2H, CH Ph), 6.93 (d, 1H, J =
2 2
1
3
7
4
.8 Hz), 7.13 (t, 2H, J = 6.9 Hz), 7.23-7.42 (m, 6H). C NMR (75.5 MHz, D O) δ 37.8 (CH ),
2 2
4.1 (CH ), 50.4 (CHα), 75.1 (Cγ), 112.1 (CH), 124.4 (CH), 124.7 (CH), 128.3 (CH), 128.4
2
(CH), 129.5 (CH), 129.8 (C), 131.5 (CH), 137.7 (C), 140.8 (C), 158.5 (C=O), 169.1 (C=O),
+
1
80.6 (C=O). HRMS (ESI) calcd for C H N O [M+H] : 326.1505, found 326.1512.
1
8
20
3
3
4
.1.1.19.
(S)-2-Benzyloxyamino-N-benzyl-3-((S)-3-hydroxy-1-methyl-2-oxoindolin-3-
yl)propanamide:
According to the general procedure described above, spirolactone 10b (1.60 g, 4.75 mmol)
and benzylamine (560 µL, 5.12 mmol) were reacted overnight. Purification by
chromatography over silica gel (60 g, eluent 10% of AcOEt in CH Cl ) afforded (S)-2-
2
2
benzyloxyamino-N-benzyl-3-((S)-3-hydroxy-1-methyl-2-oxoindolin-3-yl)propanamide as a
1
beige solid (1.54 g, 69%). Mp 56-59 °C. H NMR (300 MHz, CDCl ) δ 2.18 (dd, J = 14.7, 7.2
3
Hz, 1H), 2.56 (dd, J = 14.8, 6.8 Hz, 1H), 3.11 (s, 3H), 4.40 (m, 2H), 4.69 (m, 1H), 5.01 (m,
2
H), 5.90 (br s, 1H), 6.77 (d, J = 7.7 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.17-7.36 (m, 14H).
C NMR (75.5 MHz, CDCl ) δ 26.01 (CH ), 39.70 (CH ), 43.40 (CH ), 51.23 (CH), 66.75
1
3
3
3
2
2
(
(
(
[
CH ), 74.91 (C), 108.47 (CH), 123.23 (CH), 123.77 (CH), 127.11 (CH), 127.40 (CH), 127.86
2
CH), 127.93 (CH), 128.28 (CH), 128.39 (CH), 129.46 (CH), 130.49 (C), 135.98 (C), 137.68
C), 142.43 (C), 155.89 (C), 171.93 (C), 177.47 (C). HRMS (ESI) calcd for C H N O Na
2
7
27
3
5
+
(M+Na) ] 496.1843, found 496.1842. R (2/3 AcOEt/CH Cl ) 0.28.
f
2
2
4
.1.1.20.
(S)-2-Amino-N-benzyl-3-((S)-3-hydroxy-1-methyl-2-oxoindolin-3-yl)propanamide
of (S)-2-benzyloxyamino-N-benzyl-3-((S)-3-hydroxy-1-methyl-2-
7
c: Hydrogenation
oxoindolin-3-yl)propanamide (972 mg, 2.05 mmol) according to the general procedure
1
afforded 7c as a white solid (697 mg, quantitative, 29% from Z-
L
-methyl tryptophan). H
NMR (300 MHz, DMSO-d ) δ 1.85 (m, 2H), 3.09 (s, 3H), 3.71 (dd, J = 8.1, 5.4 Hz, 1H), 4.24
6
(s, 2H), 6.99 (d, J = 7.8 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 7.22-7.30 (m, 6H), 7.42 (d, J = 7.5
Hz, 1H), 8.42 (br s, 1H).
2
5

ACCEPTED MANUSCRIPT
4
.1.2. Lactams 10 and 11
1
Lactam10: H NMR (400 MHz, DMSO-d ) δ 2.06 (dd, J = 8.8, 12.0 Hz, 1H, Hβ), 2.60 (d, J =
6
4
.6 Hz, 3H, Me), 2.77 (dd, J = 12.3, 6.1 Hz, 1Η, Hβ), 3.62 (dd, J = 8.8, 6.2 Hz, 1H, Hα), 5.25
(
s, 2H, NH ), 6.10 (s, 1H, OH), 6.50 (ddd, J = 8.6, 7.3, 1.3 Hz, 1H), 6.69 (dd, J = 8.0, 1.2 Hz),
2
6
8
.82 (dd, J = 7.7, 1.6 Hz, 1H), 7.00 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 8.01 (q, J = 4.6 Hz, 1H),
.31(br s, 1H, NH).
1
Lactam 11: H NMR (400 MHz, DMSO-d ) δ 2.31 (dd, J = 13.5, 5.7 Hz, 1H, Hβ trans to
6
Hα), 2.52 (m, 4H, Hβ cis to Hα and Me), 4.06 ( dd, J = 7.5, 5.7 Hz, 1H, Hα), 5.05 (s, 2H,
2
NH ), 6.10 (s, 1H, OH), 6.48 (ddd, J = 8.1, 7.3, 1.3 Hz, 1H, H ), 6.61 (dd, J = 8.0, 1.2 Hz, 1H,
2
4
3
1
H ), 6.94 (ddd, J = 8.8, 7.3, 1.6 Hz, 1H, H ), 7.13 (dd, J = 7.7, 1.6 Hz, 1H, H ), 7.94 (q, J =
1
3
4
.6 Hz, 1H, NH amide), 8.24 (br s, 1H, NH lactam). C NMR (DMSO, 100 MHz) δ 25.5
(
CH ), 39.4 (CH ), 52.64 (CH), 115.2 (CH), 115.8 (CH), 125.2(C), 126.7 (CH), 127.7 (CH),
3
2
1
46.0 (C), 171.9 (CO amide), 176.4 (CO lactam).
4
4
.1.3. Synthesis of tripeptides 1-2 and dipeptides 5
.1.3.1. General procedure for the convergent synthesis of tripeptide 1a: A mixture of Boc-
3
Tyr(Bn)-Ala-OSu (278.1 mg, 0.515 mmol prepared from 13 (R = Me) [16], DCC and HOSu
according to the literature [52]) and oxidized tryptophan 6b, HCl (152.3 mg, 0.421 mmol) in
dry DMF (0.7 mL), triethylamine (60 µl, 0.43 mmol) and dry CH Cl (1.3 mL) was stirred at
2
2
room temperature for two days. After evaporation of the solvent under high vacuum and
chromatography of the residue over silica gel (3% MeOH in CH Cl ), tripeptide 1a was
2
2
afforded as a white solid (155.7 mg, 49%). Characterization datas of 1a were previously
published [17]. R (5/95 MeOH/CH Cl ) 0.49. HPLC (column 1): t = 27.2 min, area percent
f
2
2
R
>99% at 254 nm.
4
.1.3.2. General procedure for the linear synthesis of tripeptide 1a: To a solution of oxidized
tryptophan HCl, 6b (1.38 g, 3.83 mmol) and commercial Z-Ala-OSu (1.23 g, 3.83 mmol) in
dry CH Cl (25 mL) was added triethylamine (840 µL, 6 mmol). The mixture was stirred
2
2
overnight at room temperature. The mixture was washed by water. The aqueous phase was
extracted three times with CH Cl . The combined organic phases were dried over sodium
2
2
sulfate and then concentrated under reduced pressure. After chromatography of the residue
over silica gel (60 g, 40% AcOEt in CH Cl ), Z-dipeptide 5a was afforded as a white solid
2
2
1
(
1.80 g, 88%). H NMR (500 MHz, DMSO-d ) δ 1.19 (d, J = 7.2 Hz, 3H), 2.18 (dd, J = 13.9,
6
2
6

ACCEPTED MANUSCRIPT
9
.0 Hz, 1H), 2.45-2.50 (m, 1H), 3.83-3.90 (m, 1H), 4.11 (dd, J = 15.3, 5.5 Hz, 1H), 4.16 (td, J
=
8.6, 3.6 Hz, 1H), 4.25 (dd, J = 15.3, 6.4 Hz, 1H), 4.87 (d, J = 12.6 Hz, 1H), 4.98 (d, J = 12.6
Hz, 1H), 6.01 (s, 1H), 6.75 (d, J = 7.7 Hz, 1H), 6.94 (t, J = 7.4 Hz, 1H), 7.16-7.37 (m, 12H),
1
3
7
1
1
.47 (d, J = 5.8 Hz, 1H), 7.91-7.96 (m, 2H), 10.08 (s, 1H). C NMR (125 MHz, DMSO-d ) δ
6
7.3, 38.6, 42.0, 49.1, 50.9, 65.5, 74.3, 109.5, 121.4, 124.4, 126.5, 126.8, 127.0, 127.1, 127.7,
28.0, 128.2, 128.9, 131.0, 136.7, 139.1, 141.8, 156.2, 171.0, 171.9, 178.9. A mixture of the
Z-dipeptide 5a (1.80 g, 3.40 mmol) and 10 % palladium on charcoal (542 mg) in methanol
20 mL) was stirred overnight at room temperature under H atmosphere. After filtration
(
2
through celite, the filtrate was concentrated under reduced pressure to afford the deprotected
1
dipeptide as a white solid (1.46 g, quantitative). H NMR (300 MHz, CD OD+HCl) δ 1.44 (d,
3
J = 7.2 Hz, 3H), 2.31 (dd, J = 14.4, 7.5 Hz, 1H), 2.50 (dd, J = 14.4, 5.1 Hz, 1H), 3.87 (q, J =
7
.2 Hz, 1H), 4.09 and 4.30 (AB system, JAB = 15.3 Hz, 2H), 4.43 (dd, J = 7.5, 5.1 Hz, 1H),
.91 (d, J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.21-7.37 (m, 7 H). A mixture of the
6
deprotected dipeptide (1.46 g, 3.68 mmol), commercial Boc-Tyr(Bn)-OSu (1.85 g, 3.96
mmol) and triethylamine (690 µL, 4.95 mmol) in dry CH Cl (25 mL) was stirred overnight at
2
2
room temperature. After dilution by CH Cl , the mixture was washed with water. The
2
2
aqueous phase was extracted three times with CH Cl . The combined organic phases were
2
2
dried over sodium sulfate and then concentrated under reduced pressure. After
chromatography of the residue over silica gel (88 g, 50% AcOEt in CH Cl ), tripeptide 1a was
2
2
afforded as a white solid (2.00 g, 73%). The overall yield from oxidized tryptophan 6b was
6
4%. Characterization datas of 1a were previously published [17].
4
.1.3.3. Tripeptide 1b: It was prepared by the convergent synthesis according to the general
procedure starting from Boc-Tyr(Bn)-Ala-OSu (159.3 mg, 0.295 mmol) and 6a (78.1 mg,
.313 mmol) in DMF (0.3 mL). Tripeptide 1b was afforded as a white solid (63 mg, 32%).
Characterization datas of 1b were previously published [17].
0
4
.1.3.4. Tripeptide 1c: It was prepared by the convergent synthesis according to the general
procedure starting from Boc-Tyr(Bn)-Ala-OSu (1.00 g, 1.85 mmol) and 6c (0.691 g, 2.04
mmol) in CH Cl (20 mL). Tripeptide 1c was afforded as a white solid (0.789 g, 56%).
2
2
1
H NMR (300 MHz, DMSO-d ) δ 1.13 (d, J = 7.2 Hz, 3H), 1.27 (s, 9H), 2.20 (m 1H), 2.60
6
(m, 1H), 2.90 (m, 1H), 2.98 (s, 3H), 3.93 (m, 1H), 4.09-4.30 (m, 4H), 5.05 (s, 2H), 6.07 (s,
1
3
1
H), 6.85-7.04 (m, 5H), 7.13-7.43 (m, 14H), 7.87-7.94 (m, 3H). C NMR (75.5 MHz,
DMSO-d ) δ 18.11 (CH ), 25.71 (CH ), 28.11 (CH ), 36.23 (CH ), 38.72 (CH ), 42.12 (CH ),
6
3
3
3
2
2
2
2
7

ACCEPTED MANUSCRIPT
4
8.45 (CH), 49.17 (CH), 55.87 (CH), 69.12 (CH ), 74.00 (C), 78.13 (C), 108.30 (CH), 114.33
2
(CH), 122.16 (CH), 123.90 (CH), 126.61 (CH), 126.91 (CH), 127.54 (CH), 127.69 (CH),
1
1
28.09 (CH), 128.35 (CH), 129.17 (CH), 130.15 (CH), 130.27 (C), 137.22 (C), 139.16 (C),
43.43 (C), 155.33 (C), 156.85 (C), 170.62 (C), 171.43 (C), 171.63 (C), 176.82 (C). HRMS
+
(
ESI) calcd for C H N O Na [(M+Na) ] 786.3473, found 786.3470. R (AcOEt) 0.44.
4
3
49
5
8
f
4
.1.3.5. Tripeptide 1d: A mixture of tripeptide 1a (26 mg, 0.035 mmol) and 10% palladium
on charcoal (5 mg) in MeOH (1 mL) was hydrogenated at atmospheric pressure overnight.
After filtration over a pad of celite, the filtrate was concentrated and the resulting residue was
washed with ether to afford 1d as a white solid (22.7 mg, 99%). Characterization datas of 1d
were previously published [17].
4
.1.3.6. Tripeptide 1e: Tripeptide 1a (34.29 mg, 0.0457 mmol) was dissolved in MeOH
(0.1 mL) and treated at 0 °C for 30 min with anhydrous 12 M HCl in MeOH (0.2 mL, 2.4
mmol). The resulting mixture was then concentrated in vacuo. Purification by MPLC over RP
silica (C18, MeOH/0.005 M aqueous HCl 40/60) afforded tripeptide 1e (13.0 mg, 41%).
Characterization datas of 1e were previously published [17].
4
.1.3.7. Tripeptide 1f: It was prepared by the convergent synthesis according to the general
procedure starting from Boc-Tyr(Bn)-Ala-OSu (231 mg, 0.428 mmol) and 6d, HCl salt (216.5
mg, 0.435 mmol) in CH Cl (1.5 mL). Tripeptide 1f was afforded as a white solid (123.5 mg,
2
2
3
6% yield). It was also prepared by the linear synthesis according to the general procedure.
Intermediate Z-dipeptide was obtained as a white solid (214 mg, 77%) from oxidized
1
tryptophan, HCl salt 6d (236 mg, 0.483 mmol) and Z-Ala-OSu (156 mg, 0.49 mmol). H NMR
(
300 MHz, CDCl ) δ 1.22 (d, J = 7.2 Hz, 3H), 2.36 (dd, J = 16.5, 4.2 Hz, 1H), 2.59 (dd, J =
3
1
6.5, 6.6 Hz, 1H), 4.21 (q, J = 7.2 Hz, 1H), 4.64 (m, 1H), 4.80-5.01 (m, 4H), 5.42 (s, 1H),
.45 (d, J = 7.2 Hz, 1H), 6.59 (d, J = 7.8 Hz, 1H), 7.02-7.49 (m, 13H), 7.69-7.94 (m, 5H).
5
Hydrogenation and then coupling with Boc-Tyr(Bn)-OSu (133 mg, 0.290 mmol) afforded
tripeptide 1f as a white solid (142 mg, 48%). The overall yield from oxidized tryptophan 6d
was 37%.
1
H NMR (500 MHz, DMSO-d ) δ 1.14 (d, J = 7.0 Hz, 3H, CH ), 1.29 (s, 9H, Boc), 2.08 (dd, J
6
3
=
14.5, 3.0 Hz, 1H, CHβ), 2.46 (dd, J = 14.5, 3.5 Hz, 1H, CHβ), 2.59 (dd, J = 14.5, 9.0 Hz,
1
H, CHβ), 2.93 (dd, J = 15.0, 4.5 Hz, 1H), 4.06-4.13 (m, 2H, NCH ), 4.23-4.28 (m, 1H,
2
CHα), 4.57 (dd, J = 15.0, 5.5 Hz, 1H), 4.71 (dd, J = 15.0, 6.0 Hz, 1H), 5.05 (s, 2H, OCH2),
2
8