Screen of pseudopeptidic inhibitors of human sirtuins 1-3: Two lead compounds with antiproliferative effects in cancer cells

Article abstract of DOI:10.1021/jm400438k

In the past few years sirtuins have gained growing attention for their involvement in many biological processes such as cellular metabolism, apoptosis, aging and inflammation. In this contribution, we report the synthesis of a library of thioacetylated ps

Full text of DOI:10.1021/jm400438k

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Article
Screen of Pseudopeptidic Inhibitors of Human Sirtuins 1-3: Two
Lead Compounds with Antiproliferative Effects in Cancer Cells
Paolo Mellini, Tarja Kokkola, Tiina Suuronen, Heikki S. Salo, Laura
Tolvanen, Antonello Mai, Maija Lahtela-Kakkonen, and Elina M. Jarho
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400438k • Publication Date (Web): 08 Aug 2013
Downloaded from http://pubs.acs.org on August 14, 2013
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Screen of Pseudopeptidic Inhibitors of Human
Sirtuins 1ꢀ3: Two Lead Compounds with
Antiproliferative Effects in Cancer Cells
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Paolo Mellini,†,§ Tarja Kokkola,† Tiina Suuronen,║ Heikki S. Salo,† Laura Tolvanen,† Antonello
Mai,§ Maija Lahtela-Kakkonen,† and Elina M. Jarho*,†
║
†School of Pharmacy and Department of Neurology, Institute of Clinical Medicine, University
of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland
§Istituto Pasteur ꢀ Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del
Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy
KEYWORDS
Histone deacetylases, Sirtuins, Sirtuin inhibitors, Pseudopeptides, Antiproliferative
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ABSTRACT
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In the past few years sirtuins have gained growing attention for their involvement in many
biological processes such as cellular metabolism, apoptosis, aging and inflammation. In this
paper, we report the synthesis of a library of thioacetylated pseudopeptides that were screened
against human sirtuins 1ꢀ3 to reveal their in vitro inhibition activities. Molecular modeling
studies were performed to acquire data about the binding modes of the inhibitors. Three sirtuin
inhibitors were subjected to cellular studies and all of them showed an increase in acetylation of
Lys382 of p53 after DNA damage. Furthermore, two of the compounds were able to inhibit both
A549 lung carcinoma and MCFꢀ7 breast carcinoma cell growth in micromolar concentration
with the ability to arrest cancer cell cycle in the G1 phase.
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INTRODUCTION
The reversible protein acetylation status plays an important role in the regulatory mechanisms
that control the function of histone and nonꢀhistone proteins. The class III histone deacetylases,
also called sirtuins (SIRT1ꢀ7),1 characterized by a conserved 270 amino acid catalytic core
domain, are NAD+ dependent enzymes able to catalyze substrateꢀspecific deacetylase (SIRT1ꢀ3,
and 6) and ADP ribosyltransferase2 (SIRT4 and 6) reactions. Furthermore, it has recently been
found that SIRT5 has potent demalonylase and desuccinylase enzymatic activity.3,4 Sirtuins are
able to deacetylate a variety of histone (H1, H3, H4) and nonꢀhistone targets such as PGCꢀ1α,
LXR, FOXO, NFꢀκB, p53, p73, p300, αꢀtubulin, AceCS1, and AceCS2.5,6 They also show
different cellular localizations, SIRT1, SIRT6, and SIRT7 being nuclear, SIRT2 cytoplasmic and
SIRT3ꢀ5 mitochondrial. Due to this it has been widely shown that the activity of sirtuins is
involved in the cellular metabolism regulation, neuroprotection, apoptosis, inflammation, cell
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survival/aging, telomere maintenance etc.7ꢀ10 SIRT1 has gained considerable attention for its
debated activity in tumor progression,7 in which it can act as a potential tumor suppressor or as
an oncogene. Indeed, it has been reported that SIRT1 can be upregulated in many cancer types
such as human prostate cancer,11,12 acute myeloid leukemia,13 adult Tꢀcell lymphoma,14 colon
cancer,15 breast cancer16 and lung cancer,17 in which SIRT1 can probably promote the cancer
development via the inactivation of a number of proteins, such as p53 and FOXO, associated
with tumor suppression.18 SIRT2 has also been suggested to play a role in tumorigenesis through
its inhibitory effects on p53.19 Furthermore, recently Alhazzazi and coꢀworkers showed that
SIRT3 is overexpressed in OSCC (oral squamous cell carcinoma) in vitro and in vivo and its
downꢀregulation reduced tumor burden in vivo.20 Lately, it has been suggested that targeting
both SIRT1 and SIRT2 could be a powerful approach in cancer therapy.21ꢀ24
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The thioacetylated peptidic inhibitors have represented the first successful approach to develop
potent competitive SIRT1/2 inhibitors.25 The inhibition mechanism (Figure 1), elucidated by
Smith and Denu, shows that after a fast nicotinamide cleavage, the thioacetyl inhibitor is readily
converted to a stalled intermediate 1′ꢀSꢀalkylamidate with a consequent retardation of the overall
turnover rate.26ꢀ28
Figure 1. The mechanism of sirtuin inhibition of thioacetylated peptides.
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In the scenario in which the peptidic scaffold gave compounds with potent inhibition activity29,30
but negligible drugꢀlike properties, Suzuki et al.31 and few years after, Huhtiniemi and coꢀ
workers,32 developed novel classes of pseudopeptides with an Nεꢀthioacetyl lysine residue
(Chart 1) in order to improve the cell permeability.
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Chart 1. Reference compound A by Suzuki et al.31 and reference compound B by Huhtiniemi et
al.32
Pursuing the versatility of the peudopeptidic scaffold, we focused our efforts on the preparation
and biological evaluation of a novel fragment based library (Chart 2, Tables 1ꢀ3). The library
was generated considering our previously reported binding hypothesis that was developed with
SIRT3 as the target structure.32 In this hypothesis, we suggested that a potential sirtuin inhibitor
should create an Hꢀbond network with Val292, Gly295, Glu296 and Glu325 (Figure 2). In
addition, in the Nꢀterminal area of a pseudopeptidic compound the binding affinity could be
improved by formation of a hydrogen bond to Leu298 and filling the cavity surrounded by
residues Pro297, Leu298, Phe302 and Leu303. On the other hand, in the Cꢀterminal a compound
with aromatic interactions with Phe294 or a hydrogen bond with Glu325 might have good
inhibition potency.
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Collection 1:
O
R1 =
or
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S
HN
R2 = miscellaneous
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Collection 2:
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OH
R1 = miscellaneous
O
R2 =
H
N
N
O
or
Cbz
R2
O
N
R1
H
Collection 3:
O
R1 = miscellaneous
R2 = miscellaneous
Chart 2. The general structure and the compound collections of the pseudopeptide library.
Figure 2. A scheme of the interaction pattern reported for a pseudopeptidic inhibitor in SIRT3
that fulfills the binding hypothesis. In cartoon, Hꢀbonds and aromatic interactions are represented
by dashed lines correspondingly in green and blue.
To improve the knowledge of pseudopeptidic interactions in various sirtuins and to get structureꢀ
activity relationship (SAR) data, a set of novel pseudopeptides was designed. A small library of
30 compounds was synthesized (Chart 2, Tables 1ꢀ3) and screened in vitro against SIRT1ꢀ3 with
the aim to understand how heterogeneous Nꢀ and Cꢀterminal modifications might induce changes
in the activity. Three compounds were selected for cellular studies. All three compounds were
cellꢀpermeable and nonꢀtoxic. Furthermore, two of the compounds showed antiproliferative
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effects on two different cancer cell lines (A549 and MCFꢀ7) causing cell cycle arrest at the G1
phase.
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CHEMISTRY
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The synthetic pathway to compounds 1ꢀ40 is depicted in Scheme 1. The Nεꢀthioacetylated
derivatives 1 and 24 were synthesized using ethyl dithioacetate and EtOH/10% (w/v) Na2CO3(aq)
at rt for 12 h according to the literature.25,32 Compounds 2ꢀ6 and 25ꢀ34 were obtained with a
coupling reaction between the activated acid 1 or 24 and an appropriate amine using the coupling
agent TBTU (Oꢀ(benzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀtetramethyluronium tetrafluoroborate) in
dimethylformamide (DMF)/pyridine under argon flow. The hydrochlorides 7ꢀ11 were gained via
Boc (tertꢀbutoxycarbonyl) deprotection33 performed at 60 °C using 15 eq of HCl(aq)12 N, 2ꢀ
propanol and 1,2ꢀethanedithiol as a scavenger for trapping the tertꢀbutyl cation generated in situ.
Initially, Boc deprotection was attempted in ethyl acetate under HClꢀgas flow in the absence of a
scavenger in order to precipitate the product as the HCl salt from the reaction mixture. However,
the 1Hꢀspectrum revealed the disappearance of the thioacetyl methyl singlet (2.4 ppm in DMSOꢀ
d6) but showed instead a methyl singlet at 1.8 ppm, a shift characteristic to an acetyl methyl
(unpublished data). Thus, the required amount of HCl was optimized; 15 eq of HCl divided in
three portions gave complete consumption of the starting material. This, together with the added
scavenger, protected the thioacetyl group during the deprotection step. Compounds 12ꢀ23 and
35ꢀ40 were synthesized according to the above mentioned coupling procedure; in this case the
appropriate acid was activated with TBTU to react with the corresponding unblocked amine. The
hydrochloride release of the starting material 7 was a critical step that reduced the yields of the
acetophenone (i.e. 2ꢀoxoꢀ2ꢀphenyl ethyl) derivatives 12ꢀ18. As a free amine, compound 7
underwent spontaneous sideꢀreactions; most likely an intramolecular cyclization between the free
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amino group and the ketone function responsible for generating several byproducts during the
coupling step.
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Scheme 1.a
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aReagents and conditions: (a) Ethyl dithioacetate, EtOH/10% (w/v) Na2CO3(aq), rt, 12 h; (b)
appropriate amine or carboxylic acid, TBTU, DMF/Pyridine (1:1), argon flow, rt, 2ꢀ3 h; (c) 2ꢀ
propanol, 1,2ꢀethanedithiol, HCl(aq) 12 N, 15 eq, 60°C, 2ꢀ3 h.
RESULTS AND DISCUSSION
StructureꢀActivity Relationship. A compound library of 30 pseudopeptides was synthesized.
One collection explored modifications in the Nꢀterminus (Table 1), the other collection explored
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the modifications in the Cꢀterminus (Table 2) and, in the third collection miscellaneous
modifications at both termini were studied (Table 3). All compounds were screened in vitro
against SIRT1ꢀ3 at 50 ꢁM concentration (Tables 1ꢀ3). However, the numbering of the target
protein amino acids is adopted from SIRT3 in the structureꢀactivity discussion unless otherwise
noted. The main differences between SIRT1ꢀ3 substrate binding sites are labeled in Figure 3.
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Figure 3. The overall structure of SIRT3 (PDB code: 3glr) is represented in blue ribbon (left).
The superimposition of the substrate binding sites (right) of SIRT1 (green) and SIRT2 (orange)
homology models and SIRT3 crystal structure (blue). The acetylated substrate is colored in
black, the Nꢀ and Cꢀterminal sides are represented by spheres V1 (pink) and V2 (yellow),
respectively.
The first set of compounds with modifications in the Nꢀterminus shared a 2ꢀoxoꢀ2ꢀphenylethyl
moiety in the Cꢀterminus (2, 12ꢀ18 in Table 1). These compounds fulfill the hydrogen bonding
network and the Cꢀterminal πꢀπ interaction requirements of the binding hypothesis (Figure 2).
For SIRT1, the fulfillment of these requirements is enough to give highly potent compounds;
despite the structural changes in the Nꢀterminus compounds 12ꢀ18 gave ≥ 95% inhibition.
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Compound 2 with the Nꢀterminal Bocꢀgroup gave 91% SIRT1 inhibition, although weakest
within this set it was still highly active. For SIRT2 and SIRT3, more variation was seen in the
inhibitory activities suggesting that the optimization of the Nꢀterminus is more critical for these
enzymes. However, the Cꢀterminal 2ꢀoxoꢀ2ꢀphenylethyl moiety seems to be very good also for
SIRT2 and SIRT3 because ≥ 90% inhibition was achieved in several examples.
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Compounds 12ꢀ14 show the same trend for all the three enzymes; elongation of the chain
improved inhibitory activity, the best compound being 14 with five methylene groups between
the phenyl and carbonyl functions. Based on the modeling studies, compound 14 seems to be
better complementary with the hydrophobic cavity compared to compounds 12 and 13.
Compound 2 with the Nꢀterminal Bocꢀgroup gave SIRT2 inhibition comparable to compound 14
but against SIRT1 and SIRT3 it showed decreased activity. Introduction of the pyridyl group did
not improve the inhibition of any of the enzymes (15 vs. 12). Compounds 16ꢀ18 were designed
to improve the Nꢀterminal interactions in the lipophilic cavity. Modeling studies also show that
the hydroxyl moiety of the 4ꢀhydroxyꢀLꢀprolyl group in compound 16 can form an additional Hꢀ
bond with Gly295 (Figure 4d). Interestingly, compound 16 was the only compound within the
whole compound library that achieved ≥ 90% inhibition of all three studied sirtuins and thus,
seems to possess a wellꢀoptimized structure.
The second set of compounds with modifications in the Nꢀterminus shared a phenyl moiety in
the Cꢀterminus (19ꢀ23 in Table 1). These compounds are missing one Hꢀbond of the hydrogen
bonding network due to the lack of the Cꢀterminal Hꢀbond acceptor. Furthermore, the phenyl
ring is not in the optimal position for the Cꢀterminal πꢀπ interaction. It is obvious that the Cꢀ
terminal phenyl is not as good a structure as the above studied 2ꢀoxoꢀ2ꢀphenylethyl moiety (19,
21, 23 vs. 15, 17, 16, respectively).
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Table 1. Screening of the Nꢀterminal modifications.
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Inhibitionꢀ% @ 50 ꢁMa
SIRT1 SIRT2 SIRT3
No.
R2
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HN
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O
H
N
R2
N
H
O
91 ± 0.6 87 ± 0.5 63 ± 1.2
96 ± 0.1 65 ± 1.5 68 ± 3.1
98 ± 0.1 76 ± 2.3 72 ± 0.2
98 ± 0.1 83 ± 0.4 82 ± 0.3
95 ± 0.3 58 ± 0.6 62 ± 1.3
2
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15
99 ± 0.0 96 ± 0.3 90 ± 0.2
97 ± 0.4 95 ± 0.5 76 ± 1.2
96 ± 0.2 73 ± 1.2 61 ± 0.3
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HN
S
H
N
R2
N
H
O
82 ± 0.2 39 ± 2.6 44 ± 0.3
62 ± 2.2 18 ± 1.1 16 ± 1.1
80 ± 0.5 44 ± 0.5 45 ± 2.2
86 ± 0.1 13 ± 3.1 19 ± 0.8
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22
90 ± 0.5 54 ± 1.4 69 ± 1.3
23
aFluor de Lys assay, values are means ± SD of at least three experiments.
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Figure 4. Docking poses for 16 (green), 23 (blue), 35 (yellow) in A) SIRT1, B) SIRT2, C)
SIRT3. On the protein surface the light green represents the hydrophilic and the light blue the
lipophilic areas. D) Compound 16 docked in SIRT3 highlighting the additional hydrogen bond
between the hydroxyl group and Gly295.
Again, the Nꢀterminal Cbzꢀ4ꢀhydroxyꢀ
(Figure 4). Bocꢀ ꢀprolyl (21) instead, shows only comparable activity to the 3ꢀ(3ꢀ
pyridyl)propanoyl fragment (19) in this series. Boc deprotection of 21 gave compound 22 with
an ꢀprolyl moiety and further simplification gave compound 20 with an N,Nꢀdimethylꢀglycine.
Against SIRT1, compound 22 was able to maintain inhibition (86%), possibly due to an Hꢀbond
Lꢀprolyl moiety (23) gave the most potent compound
L
L
from the NHꢀproton of the
L
ꢀproline to Gly223 (SIRT1 numbering). Compound 20 lacks this
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proton and shows decreased activity (62%). However, against SIRT2 and SIRT3 both 20 and 22
show only weak inhibition. This is in line with our previous finding with peptidic inhibitors29
that SIRT1 can be inhibited with smaller compounds. Small pseudopeptides provide an approach
to gain SIRT1 selectivity; compound 22 shows highest SIRT1 selectivity among the
pseudopeptide library.
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The first set of compounds with modifications in the Cꢀterminus shared a Cbz group in the Nꢀ
terminus (25ꢀ34 in Table 2). Although the Cꢀterminal 2ꢀoxoꢀ2ꢀphenylethyl moiety had proven to
be a very good structure (Table 1), it was causing sideꢀreactions during the synthesis reducing
the yields. Thus, it was decided to explore whether a more stable group with similar activity
could be found. The reduction (28) or deletion (25) of the carbonyl function (Figure 5) induced a
uniform loss in potency, probably because of the lost Hꢀbond to the carbonyl function.
Compound 28 was tested as a mixture of diastereomers but modeling did not suggest favorable
interactions for one over the other. Next, the phenyl function was positioned closer to the
thioacetylated lysine giving the benzyl derivative 26. For SIRT1 and SIRT3, this optimization of
the Cꢀterminal πꢀπ interaction could compensate for the lost Hꢀbond and high inhibition was
observed. SIRT2 instead showed different behavior; the Hꢀbond seems to be more crucial for the
SIRT2 inhibition. These results were verified also by another set of compounds (Figure 4, Table
2); compared to compound 16, compound 35 shows similar SIRT1 and SIRT3 inhibition but
decreased SIRT2 inhibition. However, both of these compounds show higher inhibition
compared to compound 23 with the Cꢀterminal phenyl group.
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Table 2. Screening of the Cꢀterminal modifications.
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Inhibitionꢀ% @ 50 ꢁMa
SIRT1 SIRT2 SIRT3
No.
R1
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HN
S
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15
O
H
N
O
N
R1
H
O
83 ± 0.5 81 ± 1.1 62 ± 1.5
97 ± 0.3 94 ± 0.3 74 ± 0.9
72 ± 3.5 52 ± 1.6 41 ± 1.9
91 ± 0.1 67 ± 0.6 71 ± 2.8
97 ± 0.1 97 ± 0.2 82 ± 1.3
77 ± 0.2 54 ± 3.1 45 ± 2.7
Ref.A
Ref.B
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28
85 ± 0.7 61 ± 2.3 46 ± 3.8
88 ± 0.3 66 ± 1.1 63 ± 1.2
87 ± 0.4 79 ± 0.6 68 ± 2.8
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31
84 ± 1.0 70 ± 1.3 59 ± 1.7
91 ± 0.3 72 ± 0.5 77 ± 0.1
96 ± 0.2 86 ± 0.2 85 ± 0.9
32
33
34
HN
S
O
H
N
HO
N
R1
H
N
O
Cbz
99 ± 0.0 96 ± 0.3 90 ± 0.2
90 ± 0.5 54 ± 1.4 69 ± 1.3
97 ± 0.2 87 ± 0.7 93 ± 0.1
16
23
35
aFluor de Lys assay, values are means ± SD of at least three experiments.
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Larger Cꢀterminal aromatic groups were studied by compounds 27 and 29ꢀ31. The tryptamine
moiety (29) does not show an optimal interaction with the Phe294. However, the tryptamine can
have other hydrophobic interactions with surrounding hydrophobic residues. Also, in SIRT1
there is a possibility for the tryptamine nitrogen to form an additional hydrogen bond with
Lys252 (SIRT1 numbering, compound 30 in Figure 5). Further bulkiness was gained by the
methoxy (30) and benzyloxy (31) groups. These hydrophobic groups increase Cꢀterminal
hydrophobic interactions and improve potency. Also, the methoxy group in compound 27 can
increase the hydrophobic interactions (Figure 5).
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Three smaller, more polar Cꢀterminal fragments were also studied. The pyridyl group in
compound 32 can have a πꢀπ interaction to the protein but surrounding water provides competing
interactions to the polar moiety. Compounds 33 and 34 lack an aromatic ring and cannot form πꢀ
π interactions. However, these compounds are able to retain a good inhibition potency which
may be due to an additional Hꢀbond to the terminal amide nitrogen. The stereochemistry of the
chiral compound 34 apparently orientates the hydrogen bonding more properly.
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Figure 5. Docking poses for 25 (yellow), 27 (green), 30 (blue) in A) SIRT1, B) SIRT2, C)
SIRT3. On the protein surface the light green represents the hydrophilic and the light blue the
lipophilic areas.
A pyridyl group is often used to improve water solubility of compounds. Therefore, Cꢀterminal
pyridyl moieties were combined to miscellaneous Nꢀterminal fragments (Table 3). Compounds 6
and 40 with the Cꢀterminal 2ꢀ(3ꢀpyridyl)ethyl fragment show weak to moderate sirtuin
inhibition. Compounds 38 and 39 possess the Cꢀterminal 3ꢀpyridyl moiety. In compound 38, the
Nꢀterminal Cbz group of compound 32 was replaced by a phenylethyl group giving decreased
SIRT2 and SIRT3 inhibition. However, compound 39 with an orthoꢀfluoro substituent on the
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phenyl ring shows improved affinity towards all studied sirtuins compared to compounds 32 and
38. The fluorine provides an Hꢀbond acceptor that may form an additional Hꢀbond to the enzyme
backbone (Leu298 in SIRT3) but the GlideScore scoring function does not reward fluorine as a
hydrogen bond acceptor. Finally, two compounds with Cꢀterminal benzyl groups were studied
(36 and 37). These compounds have an Nꢀterminal phenyl moiety at the end of a long, flexible
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chain. The length of these Nꢀterminal moieties is similar to Cbzꢀ4ꢀhydroxyꢀLꢀprolyl group. The
flexibility of the chains of 36 and 37 allows the phenyl groups be buried deep in the lipophilic
cavity. This gives excellent SIRT1 inhibition and good SIRT2 and SIRT3 inhibition.
Table 3. Screening of the miscellaneous modifications.
Inhibitionꢀ% @ 50 ꢁMa
Structure
No.
6
R2
R1
SIRT1
SIRT2
SIRT3
41 ± 1.9
28 ± 3.8 24. ± 0.3
71 ± 0.6 84 ± 0.2
74 ± 1.2 87 ± 0.3
21 ± 3.4 39 ± 1.2
74 ± 0.2 72 ± 0.6
100 ± 0.1
99 ± 0.2
83 ± 1.0
94 ± 0.4
66 ± 0.5
36
37
38
39
HN
S
H
N
R2
N
R1
H
O
49 ± 1.1 68 ± 3.0
40
aFluor de Lys assay, values are means ± SD of at least three experiments.
Three compounds with slightly different inhibition profiles, 16, 27 and 30, were chosen for
IC50 profile evaluation and cellular studies. Compound 16 was chosen because it is the only
compound giving ≥ 90% inhibition of all studied sirtuins. Compound 27 gives excellent SIRT1
and SIRT2 inhibition (97%) but slightly decreased SIRT3 inhibition (82%). Compound 30 has
the highest selectivity towards SIRT1 among these three compounds; it shows 88% SIRT1
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inhibition but 66% SIRT2 and 63% SIRT3 inhibition. Compounds 16 and 27 fulfill completely
the binding hypothesis while compound 30 with its Cꢀterminal tryptamine shows an alternative
Hꢀbond. The IC50 values of the three chosen compounds (Table 4, Figure 6) follow nicely the
preliminary screening results showing slightly different inhibition profiles. Also our previous
experience with the thioacetylated compounds has indicated that the preliminary screening
correlates well with the IC50 values. This may be due to the reaction mechanism and formation of
the stable 1’ꢀSꢀalkylamidate intermediate.
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Table 4. IC50 values, cLogP, PSA and MW for compounds 16, 27 and 30.
IC50 ꢁM (95% confidence interval)a
No.
SIRT1
SIRT2
SIRT3
cLogPb
PSAc
MW
0.24 (0.22ꢀ0.25) 1.80 (1.44ꢀ2.26)
0.89 (0.83ꢀ0.96) 2.53 (2.37ꢀ2.70)
5.98 (5.25ꢀ6.81) 25.8 (22.7ꢀ29.3)
3.89 (3.47ꢀ4.37)
2.61
287.85
568.69
16
27
30
8.35 (7.37ꢀ9.46)
3.66
262.74
485.60
29.4 (23.1ꢀ37.4)
4.50
257.70
510.65
aFluor de Lys assay, values are calculated from three independent determinations giving altogether 27
b
data points. Calculated logarithm of octanol/water partition coefficient, calculated with MOE 2011.10.34
cTotal Polar Surface Area (Å2), calculated with MOE 2011.10.34
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Figure 6. The IC50 curves of compounds 16, 27 and 30 for A) SIRT1, B) SIRT2 and C) SIRT3.
The effects of compounds 16, 27 and 30 in human cell lines. To get the first glimpse of the
cellꢀpenetration of the compounds, they were studied in two different cellꢀlines that we have
previously used for this purpose; ARPEꢀ19 retinal pigment epithelium cells and SHꢀSY5Y
neuroblastoma cells.32 In the literature, ARPEꢀ19 cells have been used to investigate ageꢀrelated
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macular degeneration, where SIRT1 is expected to have a therapeutic role35 and in SHꢀSY5Y
cells SIRT1 has been shown to have neuroprotective effects36. Because SIRT1 is known to
deacetylate Lys382 of p53,31,37 Western blot analysis was performed in order to detect changes
in acetylation level of p53 after etoposideꢀinduced DNA damage. Each of the compounds
showed increased p53 acetylation in ARPE cells while the effect in SHꢀSY5Y cells was not as
evident (Figure 7A). The low levels of lactate dehydrogenase (LDH) leakage from the treated
cells indicate that the tested compounds were not toxic to the cells at the used concentration
(Figure 7B).
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Figure 7. Inhibition of SIRT1 activity increases p53 acetylation after DNA damage. (A) Effect
of 20 ꢂM compounds 16, 27, and 30 on p53 acetylation after 100 ꢂM etoposide (eto) treatment
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for 5 h in ARPEꢀ19 and SHꢀSY5Y cell lines. The results are shown as mean ± SEM of 2
independent experiments. The representative Western blots are shown below. (B) Effect of
compounds 16, 27 and 30 on LDH leakage from ARPEꢀ19 and SHꢀSY5Y cells.
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As sirtuin inhibition has been suggested as a potential therapy for breast and lung cancer,16, 17, 38
the effects of SIRT inhibitors 16, 27, and 30 on the proliferation of two tumor cell lines, A549
lung carcinoma and MCFꢀ7 breast carcinoma, were measured with sulforhodamine B assay. Both
A549 and MCFꢀ7 cell growth was inhibited with ≥ 50 ꢁM of 27 and 30 (Figure 8). The other
tested SIRT inhibitor 16 did not show any substantial inhibitory effect on A549 or MCFꢀ7 cell
growth with concentrations up to 200 ꢁM (data not shown).
Figure 8. Effects of SIRT inhibitors on A549 and MCFꢀ7 cell proliferation. The cells were
treated with 0 to 100 ꢂM of 27 (left) or 30 (right). Cell proliferation was determined by
sulforhodamine B assay. The results are shown as mean ± SEM of 2ꢀ3 independent experiments.
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In order to understand the different inhibitory behavior of the three tested inhibitors on cell
proliferation, the cellular p53 and αꢀtubulin acetylation levels in the MCFꢀ7 cells were examined
and physicochemical parameters for the inhibitors were calculated (Table 4). The study showed
that all the three compounds were able to increase the acetylation level of p53 but compound 16
was less effective than 27 and 30 (Figure 9A). Instead, no significant effect was observed on the
αꢀtubulin acetylation status by any of the compounds at 50 ꢁM concentration. Despite being a
potent SIRT1ꢀ3 inhibitor, compound 16 did not inhibit MCFꢀ7 or A549 cell proliferation. This
could be explained by cell type specific differences in membrane permeability, together with
different physicoꢀchemical properties of the compounds (Table 4). As these compounds are
rather large, it can be speculated that compound 16 with the lowest cLogP and the highest polar
surface area and molecular weight may have difficulties in cellꢀpermeation. The compounds did
not show any toxic effect when tested with LDH assay (Figure 9B).
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Figure 9. Inhibition of SIRT1 activity increases p53 acetylation after DNA damage in MCFꢀ7
cells. (A) Effect of 50 ꢂM compounds 16, 27, and 30 on p53 acetylation in MCFꢀ7 cells after 100
ꢂM etoposide (eto) treatment for 5 h. The test was performed once for each compound. (B)
Effect of compounds 16, 27, and 30 on LDH leakage from MCFꢀ7 cells.
Propidium iodide staining and flow cytometric DNA content analysis of MCFꢀ7 and A549 cells
treated with 27 and 30 was performed in order to detect the effect of these compounds on cell
cycle progression. Cell cycle analysis revealed that both 27 and 30 arrested cancer cells in the
G1 phase, with > 50% reduction in DNA synthesis (S phase population), and no cell death was
observed (Figure 10).
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Control
Compd. 30
Compd. 27
%G1 = 62.3
%G2 = 19.4
%S = 17.8
%G1 = 85.7
%G2 = 9.9
%S = 3.0
%G1 = 82.2
%G2 = 9.4
%S = 7.7
A549
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Control
Compd. 27
Compd. 30
%G1 = 67.6
%G2 = 14.7
%S = 12.6
%G1 = 80.5
%G2 = 8.7
%S = 5.8
%G1 = 77.4
%G2 = 13.2
%S = 5.7
MCFꢀ7
Cellular DNA content
Figure 10. Effects of SIRT inhibitors on A549 and MCFꢀ7 cell cycle. The cells were subjected
to control treatment (0.5% DMSO) or treatment with 100 ꢂM of 27 or 30 for 18 h. Flow
cytometric analysis of DNA content was done after propidium iodide staining. Percentage of
cells in each phase of the cell cycle (G1, G2, and S) is indicated. Representative data from at least
two independent experiments are shown.
CONCLUSIONS
A library of 30 novel compounds around thioacetylated lysine scaffold was synthesized. The Cꢀ
terminal 2ꢀoxoꢀ2ꢀphenylethyl and benzyl moieties were found to be fragments that give excellent
SIRT1 inhibitions (2, 12ꢀ18 and 26, 35ꢀ37). The Nꢀterminal Cbzꢀ4ꢀhydroxyꢀLꢀprolyl or a long
flexible chain attached to a phenyl group (16, 23, 35 and 14, 36, 37) also gave a positive
contribution to the binding hypothesis hinting that the Hꢀbond with Gly295 and groups that can
be buried deep in the lipophilic cavity can improve inhibition potency.
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Three compounds, 16, 27 and 30, with slightly different inhibition profiles were chosen for more
detailed studies. Their effect on tumor cell proliferation was studied in A549 lung carcinoma and
MCFꢀ7 breast carcinoma cell lines. Interestingly, the in vitro inhibition potency did not correlate
with the cellular effect; the most potent compound 16 did not show any antiproliferative effect
while the two other compounds inhibited cell growth at ≥ 50 ꢁM concentration. The difference
may arise from different cell permeation of the compounds. Due to the observed induction of p53
acetylation in ARPEꢀ19 and MCFꢀ7 cells, SIRT1 is likely to be one of the cellular targets of
compounds 27 and 30.
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Compounds 27 and 30 were shown to be cellꢀpermeable and nonꢀtoxic sirtuin inhibitors. They
both inhibited cell growth by the arrest at the G1 phase in two tumor cellꢀlines, although the
effect of compound 30 was more pronounced. These two inhibitors are our new lead compounds
that will be taken to further studies.
EXPERIMENTAL SECTION
Chemistry. Chemical reagents and solvents used in this study were commercial high purity
quality. Organic solutions were dried over anhydrous sodium sulfate. Pyridine and
dimethylformamide (DMF) were dried over molecular sieves. Compounds 1, 24 were prepared
using the same amounts and following the procedure reported by Huhtiniemi and coworkers.25,32
Yields of all reactions refer to the purified products. NMR spectra were acquired on a Bruker
1
Avance 500 AV (Bruker Biospin, Swizerland) spectrometer operating at 500.1 MHz for H and
125.8 MHz for 13C. Chemical shift values are reported as δ (ppm) relatively to TMS
(tetramethylsilane) as an internal reference; coupling constant are given in Hz. Positive ion mass
spectra were recorded with a quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose,
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CA) equipped with an electrospray ionization source (ESIꢀMS). The purity of the compounds
was derermined by Elemental analysis obtained by an Thermo Quest CE Instruments EA 1110
CHNSꢀO elemental analyzer and the Analytical results are within ± 0.40% of the theoretical
values (see Supporting Information). Purity of tested compounds was ≥ 95%. The purity of
compounds 27 and 28 was also determined by HPLC (Agilent 1100 series) on a Cꢀ18 column
(Zorbax Eclipse XDBꢀC18, 4.6*50 mm, 1.8 ꢁm) using 45% acetate buffer (20 mM, pH 5) and
55% MeOH as eluent, flow rate 1 mL/min, column temperature 50°C and detection at 265 nm.
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General procedure for the coupling reactions of the end products 2, 6, 12ꢀ21, 23, 25ꢀ40:
(2S)ꢀ2ꢀtertꢀButyl
6ꢀethanethioamidoꢀ1ꢀoxoꢀ1ꢀ(2ꢀoxoꢀ2ꢀphenylethylamino)hexanꢀ2ꢀ
ylcarbamate (2). To a solution of the acid 1 (1.17 g, 1.0 eq, 3.84 mmol) in DMF (17.0 mL)
under Argon flow were added TBTU (1.36 g, 1.1 eq, 4.2 mmol), pyridine (17.0 mL) and
maintained under stirring for 10 min at rt. After this time 2ꢀaminoacetophenone hydrochloride
(0.66 g, 3.84 mmol) was added and the solution mixed 2 h 30 min at rt under Argon flow.
Solvents were evaporated and the obtained oil was dissolved in EtOAc (180 mL), washed with
HCl 0.5M, brine (two times), saturated NaHCO3 and finally dried with Na2SO4 and evaporated
under vacuo. The crude product obtained was purified by column chromatography using silica
Kieselgel for flash and EtOAc : MeOH (9.5 : 0.5 v/v) as eluent phase to give the pure product as
1
a sticky solid (1.34 g, 3.17 mmol, 83 %). HꢀNMR (DMSOꢀd6) : δ = 9.95 (s br, 1H), 8.13 (t, J =
5.36 Hz, 1H), 7.99 (d, J = 7.57 Hz, 2H), 7.67 (t, J = 7.57 Hz, 1H), 7.54 (t, J = 7.57 Hz, 2H), 6.90
(d, J = 8.20 Hz, 1H), 4.70ꢀ4.54 (m, 2H), 4.01ꢀ3.97 (m, 1H), 3.50ꢀ3.39 (m, 2H), 2.39 (s, 3H),
13
1.71ꢀ1.49 (m, 4H), 1.39ꢀ1.30 (m, 11H). CꢀNMR (CDCl3): δ = 201.06, 194.12, 172.33, 155.96,
134.43, 134.39, 129.13, 128.07, 54.11, 46.42, 46.12, 34.22, 32.67, 29.82, 28.46, 27.16, 22.88.
ESIꢀMS (m/z): 422.0 [M + H]+ , 444.20 [M + Na]+. Anal. (C21H31N3O4S) C, H, N.
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(2S)ꢀ2ꢀtertꢀButyl
6ꢀethanethioamidoꢀ1ꢀoxoꢀ1ꢀ[2ꢀ(pyridinꢀ3ꢀyl)ethylamino]hexanꢀ2ꢀ
5
6
7
8
1
ylcarbamate (6). HꢀNMR (DMSOꢀd6): δ = 9.93 (s br, 1H), 8.42 (m, 2H), 7.87 (t, J = 5.36 Hz,
1H), 7.62 (dt, J = 1.60, 7.57 Hz, 1H), 7.30 (dd, J = 4.73, 7.57Hz, 1H), 6.75 (d, J = 8.20 Hz, 1H),
3.84ꢀ3.79 (m, 1H), 3.44ꢀ3.24 (m, 4H), 2.74ꢀ2.71 (m, 2H), 2.37 (s, 3H), 1.52ꢀ1.34 (m, 13H), 1.25ꢀ
1.16 (m, 2H). 13CꢀNMR (DMSOꢀd6) : δ = 198.77, 172.10, 155.29, 149.59, 147.09, 136.66,
135.07, 123.47, 77.98, 54.25, 45.28, 32.81, 32.07, 31.74, 28.21, 26.93, 23.04, (ꢀNHꢀCH2ꢀCH2ꢀ
9
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pyr
under
DMSOꢀ
d6).
ESIꢀMS
(m/z):
409.15
[M
+
H]+.
Anal.
(C20H32N4O3Sꢃ0.03DMFꢃ0.03hexane) C, H, N.
(2S)ꢀ2ꢀ(3ꢀPhenylpropanoylamino)ꢀ6ꢀethanethioylaminoꢀNꢀ(2ꢀoxoꢀ2ꢀ
1
phenylethyl)hexanamide (12). HꢀNMR (DMSOꢀd6): δ = 9.95 (s br, 1H), 8.24 (t, J = 5.36 Hz,
1H), 8.03 (d, J = 8.20 Hz,1H), 7.99 (d, J = 7.57 Hz, 2H), 7.67 (t, J = 7.57 Hz, 1H), 7.54 (t, J =
7.57 Hz, 2H), 7.28ꢀ7.15 (ꢀAr, 5H), 4.65ꢀ4.55 (m, 2H), 4.37ꢀ4.32 (m, 1H), 3.46ꢀ3.38 (m, 2H),
2.84ꢀ2.79 (m, 2H), 2.47ꢀ2.43 (m, 2H), 2.38 (s, 3H), 1.70ꢀ1.62 (m, 1H), 1.55ꢀ 1.47 (m, 3H), 1.29ꢀ
1.22 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ = 198.74, 195.13, 172.11, 171.35, 141.27, 134.95,
133.50, 128.77, 128.20, 128.17, 127.79, 125.80, 52.16, 45.83, 45.33, 36.72, 32.78, 31.83, 31.04,
26.90, 22.82. ESIꢀMS (m/z): 454.14 [M + H]+. Anal. (C25H31N3O3S) C, H, N.
(2S)ꢀ2ꢀ(4ꢀPhenylbutyrylamino)ꢀ6ꢀethanethioylaminoꢀNꢀ(2ꢀoxoꢀ2ꢀphenylethyl)hexanamide
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(13). HꢀNMR (DMSOꢀd6): δ = 9.93 (s br, 1H), 8.21 (t, J = 5.36 Hz, 1H), 7.98ꢀ7.96 (m, 3H),
7.65 (t, J = 7.25 Hz, 1H), 7.54 (t, J = 7.25 Hz, 2H), 7.29ꢀ7.16 (ꢀAr, 5H), 4.66ꢀ4.54 (m, 2H), 4.37ꢀ
4.32 (m, 1H), 3.47ꢀ3.42 (m, 2H), 2.58ꢀ2.52 (m, 2H), 2.37 (s, 3H), 2.18ꢀ2.15 (m, 2H), 1.83ꢀ1.77
(m, 2H), 1.74ꢀ1.67 (m, 1H), 1.60ꢀ1.50 (m, 3H), 1.37ꢀ1.28 (m, 2H). 13CꢀNMR (DMSOꢀd6):
198.69, 195.10, 172.17, 171.86, 141.82, 134.95, 133.50, 128.76, 128.31, 128.23, 127.79, 125.69,
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52.19, 45.85, 45.32, 34.66, 34.66, 32.78, 31.72, 27.08, 26.89, 22.97. ESIꢀMS (m/z): 468.20 [M +
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H]+. Anal. (C26H33N3O3S) C, H, N.
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(2S)ꢀ2ꢀ(6ꢀPhenyhexanoylamino)ꢀ6ꢀethanethioylaminoꢀNꢀ(2ꢀoxoꢀ2ꢀphenylethyl)hexanamide
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(14). HꢀNMR (DMSOꢀd6): δ = 9.93 (s br, 1H), 8.20 (t, J = 5.67 Hz, 1H), 7.97 (d, J = 7.25 Hz,
2H), 7.92 (d, J = 8.20 Hz, 1H), 7.66 (t, J = 7.25 Hz, 1H), 7.54 (t, J = 7.25 Hz, 2H), 7.27ꢀ7.14 (ꢀ
Ar, 5H), 4.65ꢀ4.54 (m, 2H), 4.36ꢀ4.31 (m, 1H), 3.46ꢀ3.42 (m, 2H), 2.57ꢀ2.54 (m, 2H), 2.38 (s,
3H), 2.18ꢀ2.10 (m, 2H), 1.73ꢀ1.66 (m, 1H), 1.59ꢀ1.49 (m, 7H), 1.36ꢀ1.24 (m, 4H). 13CꢀNMR
(DMSOꢀd6): 198.75, 195.13, 172.21, 172.15, 142.25, 134.95, 133.51, 128.77, 128.22, 128.17,
127.79, 125.55, 52.10, 45.84, 45.33, 35.08, 35.05, 32.78, 31.77, 30.75, 28.27, 26.89, 25.07,
22.92. ESIꢀMS (m/z): 496.22 [M + H]+. Anal. (C28H37N3O3S) C, H, N.
(2S)ꢀ2ꢀ[3ꢀ(Pyridineꢀ3ꢀyl)propanoylamino]ꢀ6ꢀethanethioylaminoꢀNꢀ(2ꢀoxoꢀ2ꢀphenylethyl)
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hexanamide (15). HꢀNMR (DMSOꢀd6): δ = 9.97 (s br, 1H), 8.43 (s, 1H), 8.38 (d, J = 4.41 Hz,
1H), 8.27 (t, J = 5.36 Hz, 1H), 8.06 (d, J = 8.20 Hz, 1H), 7.99 (d, J = 7.57 Hz, 2H), 7.67 (t, J =
7.57 Hz, 1H), 7.62 (d, J = 7.47 Hz, 1H), 7.54 (t, J = 7.57 Hz, 2H), 7.29 (dd, J = 4.41, 7.47 Hz,
1H), 4.65ꢀ4.55 (m, 2H), 4.36ꢀ4.32 (m, 1H), 3.44ꢀ3.38 (m, 2H), 2.85ꢀ2.82 (m, 2H), 2.55ꢀ2.42 (m,
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2H), 2.38 (s, 3H), 1.69ꢀ1.62 (m, 1H), 1.55ꢀ1.44 (m, 3H), 1.27ꢀ1.18 (m, 2H). CꢀNMR (DMSOꢀ
d6): δ = 199.21, 195.61, 172.57, 171.48, 150.03, 147.67, 137.09, 136.28, 135.40, 134.03, 129.27,
128.30, 123.81, 52.61, 46.31, 45.81, 36.63, 33.27, 32.37, 28.58, 27.37, 23.29. ESIꢀMS (m/z):
455.19 [M+ H]+. Anal. (C24H30N4O3S. 0.5 H2O) C, H, N.
(2S,4R)ꢀNꢀ[(2S)ꢀ6ꢀEthanethioylaminoꢀ1ꢀ(2ꢀoxoꢀ2ꢀphenylethylamino)ꢀ1ꢀoxohexanꢀ2ꢀyl]ꢀ4ꢀ
hydroxypyrrolidineꢀ1ꢀbenzyloxycarbonylꢀ2ꢀcarboxamide (16). 1HꢀNMR (DMSOꢀd6):
rotamers A+B δ = 9.91ꢀ9.88 (m br, 1H), 8.19ꢀ8.13 (m, 2H), 7.99 (t, J = 7.57 Hz, 2H), 7.67 (t, J
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= 7.57 Hz,1H), 7.54 (t, J = 7.57 Hz, 2H), 7.37ꢀ7.28 (ꢀAr, 5H), 5.10ꢀ4.97 (m, 3H), 4.57ꢀ4.53 (m,
2H), 4.43ꢀ4.41 (m, 0.5H), 4.35ꢀ4.26 (m, 2.5H), 3.52ꢀ3.44 (m, 2H), 3.39ꢀ3.35 (m, 2H), 2.38 ( s,
1.5H), 2.37 (s, 1.5H) 2.14ꢀ2.05 (m, 1H), 1.90ꢀ1.86 (m, 1H), 1.78ꢀ1.72 (m, 0.5H), 1.65ꢀ1.52 (m,
1.5H), 1.48ꢀ1.34 (m, 2.5H), 1.29ꢀ1.19 (m, 1.5H).13CꢀNMR (DMSOꢀd6): rotamers A+(B) δ =
198.72, 195.11, 172.01 (171.90), 171.85 (171.71), 154.29 (154.01), 136.90, 134.92, 133.54,
128.78, 128.37 (128.17), 127.80, 127.46, 126.89, 68.48 (67.77), 65.93 (65.79), 58.80 (58.09),
55.34 (54.87), 52.39 (52.24), 45.85, 45.39 (45.29), 38.51, 32.77, 31.77 (31.57), 26.90 (26.82),
22.78. ESIꢀMS (m/z): 569.15 [M + H]+. Anal. (C29H36N4O6S) C, H, N.
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(2S)ꢀNꢀ[(2S)ꢀ6ꢀEthanethioylaminoꢀ1ꢀ(2ꢀoxoꢀ2ꢀphenylethylamino)ꢀ1ꢀoxohexanꢀ2ꢀyl]ꢀ
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pyrrolidineꢀ1ꢀtertꢀbutoxycarbonylꢀ2ꢀcarboxamide (17). HꢀNMR (DMSOꢀd6): rotamers A+B
δ = 9.91 (s br, 1H), 8.27 (t, J = 5.36 Hz, 0.6H), 8.15 (t, J = 5.36 Hz, 0.4H), 7.98ꢀ7.92 (m, 3H),
7.66 (t, J = 7.25 Hz, 1H), 7.54 (t, J = 7.25 Hz, 2H), 4.69ꢀ4.55 (m, 2H), 4.39ꢀ4.32 (m, 1H), 4.17ꢀ
4.15 (m, 1H), 3.47ꢀ3.37 (m, 3H), 3.28ꢀ3.25 (m, 1H), 2.38 (s, 3H), 2.14ꢀ2.00 (m, 1H), 1.86ꢀ1.67
(m, 4H), 1.62ꢀ1.52 (m, 3H), 1.41ꢀ1.30 (m, 11H). 13CꢀNMR (DMSOꢀd6): rotamers A+(B) δ =
198.77, 195.11 (195.03), 172.33, 171.97 (171.93), 153.76 (153.26), 134.92, 133.53, 128.78,
127.80, 78.64 (78.29), 59.28, 52.15, 46.74 (46.49), 45.85, 45.30, 32.77, 32.05 (31.80), 31.00
(29.58), 28.11 (28.00), 26.91, 23.98 (23.02), 22.85 (22.75). ESIꢀMS (m/z): 519.03 [M + H]+
Anal. (C26H38N4O5S) C, H, N.
(S)ꢀ2ꢀ(2ꢀ((2S,5S)ꢀ5ꢀBenzylꢀ3,6ꢀdioxopiperazinꢀ2ꢀyl)acetamido)ꢀ6ꢀethanethioamidoꢀNꢀ(2ꢀoxoꢀ
2ꢀphenylethyl)hexanamide (18). 1HꢀNMR (DMSOꢀd6): δ = 9.94 (s br, 1H), 8.28 (t, J = 5.67 Hz,
1H), 8.20 (s, 1H), 8.02 (d, J = 7.88 Hz, 1H), 7.97 (d, J = 7.57 Hz, 2H), 7.69ꢀ7.64 (m, 2H), 7.53
(t, J = 7.57 Hz, 2H), 7.29ꢀ7.19 (ꢀAr, 5H), 4.63ꢀ4.50 (m, 2H), 4.25ꢀ4.21 (m, 2H), 4.13ꢀ4.10 (m,
1H), 3.49ꢀ3.45 (m, 2H), 3.10 (m, 1H), 2.94 (m, 1H), 2.38ꢀ2.35 (m, 4H), 1.77ꢀ1.71 (m, 1H), 1.63ꢀ
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1.52 (m, 4H), 1.37ꢀ1.29 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ = 198.91, 195.09, 172.02, 169.43,
167.19, 166.51, 136.47, 134.81, 133.39, 130.21, 128.71, 128.01, 127.63, 126.75, 54.87, 52.73,
51.50, 45.72, 45.52, 38.57, 37.67, 32.97, 31.41, 26.75, 22.81. ESIꢀMS (m/z): 566.14 [M + H]+,
588.23 [M+ Na]+. Anal. (C29H35N5O5S) C, H, N.
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(2S)ꢀ6ꢀ(Ethanethioylamino)ꢀNꢀphenylꢀ2ꢀ[3ꢀ(pyridinꢀ3ꢀyl)propanoylamino]hexanamide (19).
1HꢀNMR (DMSOꢀd6): δ = 10.04 (s, 1H), 9.97 (s br, 1H), 8.44 (s, 1H), 8.38 (d, J = 4.88 Hz, 1H),
8.17 (d, J = 7.93 Hz, 1H), 7.63ꢀ7.58 (m, 3H), 7.32ꢀ7.27 (m, 3H), 7.05 (m, 1H), 4.40ꢀ4.36 (m,
1H), 3.44ꢀ3.41 (m, 2H), 2.85ꢀ2.82 (m, 2H), 2.54ꢀ2.46 (m, 2H), 2.36 (s, 3H), 1.69ꢀ1.62 (m, 1H),
1.57ꢀ1.48 (m, 3H), 1.32ꢀ1.18 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ = 198.80, 171.19, 170.76,
149.56, 147.18, 138.89, 136.57, 135.77, 128.68, 128.68, 123.29, 119.28, 53.22, 45.24, 36.07,
32.79, 31.81, 28.13, 26.92, 22.96. ESIꢀMS (m/z): 413.22 [M + H]+. Anal. (C22H28N4O2S) C, H,
N.
(2S)ꢀ6ꢀ(Ethanethioylamino)ꢀNꢀphenylꢀ2ꢀ[(dimethylamino)acetylamino]ꢀhexanamide (20).
1HꢀNMR (DMSOꢀd6): δ = 10.08 (s, 1H), 9.94 (s br, 1H) 7.82 (d, J = 8.20 Hz, 1H), 7.58 (d, J =
7.88 Hz, 2H), 7.30 (t, J = 7.88 Hz, 2H), 7.05 (t, J = 7.88 Hz, 1H), 4.50ꢀ4.45 (m, 1H), 3.46ꢀ3.43
(m, 2H), 2.95 (d, J = 15.26 Hz, 1H) 2.92 (d, J = 15.26 Hz, 1H), 2.35 (s, 3H), 2.24 (s, 6H), 1.78ꢀ
1.52 (m, 4H), 1.39ꢀ1.23 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ = 199.28, 170.93, 169.90, 139.22,
129.20, 123.91, 119.77, 62.94, 53.12, 45.89, 45.65, 33.24, 32.62, 27.38, 23.37. ESIꢀMS (m/z):
365.19 [M + H]+. Anal. (C18H28N4O2S) C, H, N.
(2S)ꢀNꢀ[(2S)ꢀ6ꢀEthanethioylaminoꢀ1ꢀphenylaminoꢀ1ꢀoxohexanꢀ2ꢀyl]ꢀpyrrolidineꢀ1ꢀtertꢀ
butoxycarbonylꢀ2ꢀcarboxamide (21). 1HꢀNMR (DMSOꢀd6): rotamers A+B δ = 10.06 (s, 0.6H),
9.93 (s br, 1H), 9.84 (s, 0.4H), 8.04 (d, J = 7.88 Hz, 1H), 7.59 (d, J = 7.57 Hz, 2H), 7.30 (t, J =
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