Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Article abstract of DOI:10.1016/j.bmc.2010.06.035

Sirtuins catalyze the NAD⁺ dependent deacetylation of N ^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP- ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N ^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε- alkanoyl, N^ε-α,β-unsaturated carbonyl and N ^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.

Full text of DOI:10.1016/j.bmc.2010.06.035

Bioorganic & Medicinal Chemistry 18 (2010) 5616–5625
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
e
N -Modified lysine containing inhibitors for SIRT1 and SIRT2
b
a
a
a
Tero Huhtiniemi a, , Tiina Suuronen , Maija Lahtela-Kakkonen , Tanja Bruijn , Sanna Jääskeläinen ,
*
Antti Poso a, Antero Salminen b,c, Jukka Leppänen a, Elina Jarho d,
a School of Pharmacy, University of Eastern Finland, Kuopio Campus, PO Box 1627, 70211 Kuopio, Finland
b Institute of Clinical Medicine, University of Eastern Finland, Kuopio Campus, PO Box 1627, 70211 Kuopio, Finland
c Department of Neurology, Kuopio University Hospital, PO Box 1777, 70211 Kuopio, Finland
d Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Göteborg, Sweden
a r t i c l e i n f o
a b s t r a c t
e
Sirtuins catalyze the NAD+ dependent deacetylation of N -acetyl lysine residues to nicotinamide, O0-
Article history:
e
Received 4 May 2010
Revised 10 June 2010
Accepted 11 June 2010
Available online 17 June 2010
acetyl-ADP-ribose (OAADPR) and N -deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala
sequence has been used as a probe for the screening of novel N -modified lysine containing inhibitors
against SIRT1 and SIRT2. N -Selenoacetyl and N -isothiovaleryl were the most potent moieties found in
this study, comparable to the widely studied N -thioacetyl group. The N -3,3-dimethylacryl and N -iso-
valeryl moieties gave significant inhibition in comparison to the N -acetyl group present in the sub-
strates. In addition, the studied N -alkanoyl, N -a,b-unsaturated carbonyl and N -aroyl moieties
showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small
changes in electronic and steric properties of the N -modification. These results are applicable for further
e
e
e
e
e
e
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Keywords:
e
e
e
Histone deacetylases
NAD+-dependent deacetylation
Sirtuin inhibitors
e
e
screening of N -acetyl analogues.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
acts as a nucleophile and forms an ADPR-peptidyl amidate (10-O-
alkylamidate),16,17 consequently cleaving the glycosidic bond and
The mammalian sirtuin family of class III deacetylases includes
seven enzymes, called SIRT1–SIRT7. Sirtuins are novel therapeutic
targets to treat medically important age-associated diseases, such
as cancer and metabolic, cardiovascular and neurodegenerative
diseases.1–4 Currently, there is intensive research to discover po-
tent activators and inhibitors of SIRT1 and SIRT2. The most relevant
therapeutic prospects for SIRT1 inhibitors are for the treatment of
cancer, either induce cell death or prevent angiogenesis.1,5–9 SIRT2
inhibitors have been demonstrated to rescue proteotoxicity in neu-
rodegenerative diseases.10,11 In addition to therapeutic studies,
small molecular activators and inhibitors of sirtuins have been
used as pharmacological tools in biological studies to reveal sirtuin
functions in cells.1–4 Thus, it is important to discover novel lead
molecules for SIRT1 and SIRT2 inhibition.
releasing nicotinamide. At this stage, the pyridyl-nitrogen of the
released nicotinamide can act as a competing nucleophile. This
reversibility of the transglycosidation makes nicotinamide a phys-
iological inhibitor for sirtuins.18,19 In the following steps, the active
site histidine acts as a base and activates the 20-hydroxyl-ribose in
the intermediate complex, which in turn attacks the 10-O-alkylam-
idate carbon and forms a cyclic intermediate. This is subsequently
cleaved by addition of water to form OAADPR and the deacetylated
protein as the end products.
e
N -Acetylated protein substrates provide a fresh starting point
for the development of novel sirtuin inhibitors as several inhibitors
binding putatively to NAD+ binding site has already been pub-
e
lished.20–26 Fatkins et al. were the first to report N -thioacetyl ly-
sine containing peptides, derived from human p53 tumor
e
Sirtuins catalyze deacetylation of N -acetyl lysine residues of
suppressor protein, human a-tubulin and human acetyl-coenzyme
various substrate proteins. This NAD+ dependent reaction has been
thoroughly defined.12 First binds an acetylated substrate,13 which
is followed by the binding of NAD+ into a conformation,14,15 where
the glycosidic bond between the nicotinamide and ribosyl moieties
of NAD+ becomes strained. The correctly positioned acetyl oxygen
A synthetase 2 proteins, as potent sirtuin inhibitors.27,28 Later, their
mechanism of inhibition has been fully elucidated.29–31 So far, a
e
limited number of different N -modifications at the lysine side
chain have been reported to inhibit sirtuins.27,32–36
e
We recently reported a series of N -thioacetyl lysine containing
tri-, tetra- and pentapeptides, based on alfa-tubulin and human tu-
mor suppressor protein p53 sequences, and demonstrated the ef-
fect of peptide length and selection of the side chains for the
SIRT1 and SIRT2 inhibitory activity.33 It transpired that the +2 res-
idue (the second residue towards the C-terminus calculating from
* Corresponding author. Tel.: +358 44 213 1886; fax: +358 17 162252.
E-mail address: Tero.Huhtiniemi@uef.fi (T. Huhtiniemi).
Present address: School of Pharmacy, University of Eastern Finland, Kuopio
Campus, PO Box 1627, 70211 Kuopio, Finland.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.035

T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
5617
order to develop new SIRT1 and SIRT2 inhibitors, a series of differ-
R1
R1
HN
HN
e
ent N -modifications was studied on the novel backbone and Cbz-
Lys-NH-Ph backbone, previously reported by Suzuki et al.32,35
(Fig. 1). Altogether, we report 36 novel compounds (Table 1), some
of them being very potent ones, and discuss their structure–activ-
ity relationships.
O
O
O
H
N
H
N
H
N
N
H
R2
O
N
H
O
O
O
2. Chemistry
e
e
Figure 1. Ac-Ala-Lys(N -R1)-Ala-R2 and Cbz-Lys(N -R1)-NH-Ph were used as
e
backbones for novel N -modified SIRT1 and SIRT2 inhibitors.
Compounds with Ac-Ala-Lys-Ala backbone were synthesized as
e
shown in Scheme 1. If an N -modification was introduced before
solid phase peptide synthesis (SPPS), the carboxyl terminal of N -
a
e
the N -thioacetyl lysine) was not crucial for the binding of the
Fmoc-Lys-OHꢁHCl was first protected as a methyl ester, then the
studied peptides to SIRT1 or SIRT2. However, the peptides lacking
e
N -site was modified, and in the end the carboxyl terminal was
the ꢀ2 residue (the second residue towards the N-terminus calcu-
e
a
e
deprotected (a–e in Scheme 1). Alternatively, the N -site of N -
Fmoc-Lys-OHꢁHCl was directly modified (f in Scheme 1). Treatment
lating from the N -thioacetyl lysine) could bind to SIRT1 but had
significantly decreased affinity towards SIRT2. Furthermore it
was shown that alanine replacements at the ꢀ1 and +1 positions
of the pentapeptide sequences were allowed by SIRT1 and SIRT2.
Based on the above findings, we here report a simple and easy-
to-synthesize Ac-Ala-Lys-Ala sequence which, with an appropriate
´
of compounds 2 and 8 with Woollin’s and Lawessons reagents,
respectively gave compounds 3 and 9 (c and d in Scheme 1). If
e
an N -modification was introduced after SPPS, the amino terminal
e
of N -Cbz-Lys-OH was protected with an Fmoc group prior to SPPS,
e
the Cbz group was removed by hydrogenation on palladium after
N -modification, has potential to interact with SIRT1 and SIRT2. In
Table 1
SIRT1 and SIRT2 activity assay results (95% confidence intervals for IC50 given in parentheses)
b
Compd
R1
R2
200
l
M
SDa (%)
SIRT2
IC50 (lM)
SIRT1
SIRT1
SIRT2
e
Ac-Ala-Lys(N -R1)-Ala-R2
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
64
65
66
67
68
69
70
71
72
73
74
75
76
78
79
80
81
82
CSCH3
CSeCH3
COCH3
COCH3
COCH3
COC(CH3)3
COC(CH3)3
OH
OH
OH
100
100
62
39
66
53
82
20
39
78
96
95
33
94
100
33
12
9
1
1
96
97
42
57
68
52
77
9
39
74
95
52
20
76
99
54
14
2
1
2
1
1
1
8
1
1
3
1
1
3
3
2
3
4
2
3
4
1
2
2
4
2
2
3
1
4
2
1
3
3
3
0.24 (0.20–0.28)
0.37 (0.28–0.50)
122 (105–142)
268 (160–448)
88 (77–101)
223 (151–330)
9.8 (8.7–11)
7.2 (5.2–9.9)
223 (152–328)
180 (122–266)
60 (47–77)
338 (270–422)
66 (59–74)
nd
1
3
4
2
1
2
1
1
1
1
4
1
3
3
1
2
4
4
2
2
2
2
4
1
2
2
1
2
4
6
1
OCH3
NHCH3
OCH3
NHCH3
OCH3
OCH3
OCH3
NHCH3
OH
40 (36–45)
(E)-COCH@CHCH3
(E)-COC(CH3)@CHCH3
COCH@C(CH3)2
COCH@C(CH3)2
COCH@C(CH3)2
(Z)-COCH@C(CH3CF3)
COCH2CH(CH3)2
CSCH2CH(CH3)2
CO-(cyclohex-1-ene)
COPh
CO(pyridin-3-yl)
CO(pyridin-2-yl)
COCH2(pyridin-3-yl)
COCH2(pyridin-2-yl)
CO(1H-pyrrol-2-yl)
CO(1-CH3–1H-pyrrol-2-yl)
CO-(p-N(CH3)2)Ph)
COCH2Cl
COCH2Br
COOCH3
COOBz
CON(CH3)2
SO2CH3
SO2CF3
SO2(p-CH3Ph)
SO2(p-NO2Ph)
ndc
nd
nd
46 (38–56)
6.1 (5.0–7.4)
11 (10–13)
nd
12 (10–14)
0.70 (0.53–0.91)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
69 (49–97)
23 (18–30)
77 (59–101)
nd
30 (23–40)
3.6 (2.5–5.2)
129 (90–184)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
NHCH3
NHCH3
NHCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
4
0
0
10
26
36
23
39
19
48
8
11
24
17
15
1
21
26
41
34
8
16
0
3
13
8
4
0
5
1767 (921–3392)
nd
nd
nd
nd
nd
nd
nd
0
e
Cbz-Lys(N -R1)-NH-Ph
8435
89
86
CSCH3
CSeCH3
PS(CH3)2
COOC(CH3)3
—
—
—
—
95
98
10
6
1
1
4
1
94
99
27
13
1
1
2
3
9.9 (8.3–12)
1.8 (1.2–2.8)
nd
nd
15 (13–17)
2.7 (2.2–3.3)
nd
83
nd
9027,28,33
9133
H-His-Lys-Lys(thioAc)-Leu-Met-OH
H-Lys-Lys(thioAc)-Leu-OH
100
100
1
1
98
47
1
1
0.33 (0.27–0.40)
0.57 (0.38–0.84)
6.4 (5.3–7.7)
151 (104–218)
a
b
c
Inhibition-% at 200
l
M
standard deviation with the Fluor de Lys based assay (n = 2–3).
IC50 was determined with the Fluor de Lys based assay if inhibition-% at 200
Not determined.
lM was over 50% (repeated at least three times).

5618
T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
e
SPPS, followed by N -derivatization by coupling chemistry (g, i, j in
Scheme 1). SPPS was performed on Wang-resin with Fmoc strategy
(h in Scheme 1), using TBTU and DIPEA in the coupling phase and
piperidine for the deprotection. Cleavage from the resin was
achieved either with 1 M NaOH (aq) in dioxane or with a mixture
of MeOH, DIPEA and DMF or with 8 M methylamine in EtOH giving
a free carboxylic group or a methyl ester or a methyl amide at the
C-terminus, respectively. Reference compounds 90 and 91 were
synthesized as described previously.33
the ꢀ1 and +1 positions are allowed by SIRT1 and SIRT2, encour-
aged us to synthesize tripeptide 48, with the sequence Ac-Ala-
Lys(thioAc)-Ala-OH. The resulting inhibitory activities were well
in agreement with the previous observations and show that the
ꢀ2 residue as such is not crucial for the recognition by SIRT2 be-
cause a plain acetyl group is sufficient to maintain affinity. Against
SIRT1, compound 48 (IC50 = 0.24
based pentapeptide 90 (IC50 = 0.31
l
l
M) was equipotent with p53-
M). Against SIRT2, compound
M) was only slightly less potent than pentapeptide
M) but 15–18 times more potent than the earlier re-
M) and
M).33 For this reason,
48 (IC50 = 9.8
90 (IC50 = 6.3
l
l
Compounds with Cbz-Lys-NH-Ph backbone were synthesized as
a
e
shown in Scheme 2. N -Cbz-N -Boc-Lys-OH was coupled with ani-
line using EDCꢁHCl and HOBt to give compound 83. It was selec-
ported tripeptides H-Asp-Lys(thioAc)-Thr-OH (IC50 = 175
H-Lys-Lys(thioAc)-Leu-OH (91) (IC50 = 151
l
l
e
tively Boc-deprotected in trifluoroacetic acid and the free N -site
Ac-Ala-Lys-Ala was chosen as a backbone for the acetyl analogues
was reacted with ethyl dithioacetate to gain compound 84. To gain
compound 86, the Boc protection group of compound 83 was re-
moved in 3.5 M HCl in EtOAc and the free N -site was then coupled
presented in this study.
Recently, Suzuki et al. presented a non-peptide sirtuin inhibitor
Cbz-Lys(thioAc)-NH-Ph (84).35 Although non-peptidic compounds
possess beneficial properties, like better cell permeability, over
peptidic ones, we wanted to compare these different types of
structures in terms of affinity. Against SIRT1, the inhibitory activity
of compound 84 was significantly lower in comparison to the pep-
tidic structures 48 and 90. However, against SIRT2 the inhibitory
activity of compound 84 was only slightly decreased. Previously,
we have shown that inhibition of SIRT2 requires longer sequences
than inhibition of SIRT1.33 Now obtained result indicates that it
may be rather the size of the substrate than the peptidic nature,
which is important.
e
with dimethyl thiophosphinic chloride. Synthesis of compound 89
a
started by protection of N -Cbz-Lys-OH as a methyl ester followed
e
by N -acetylation using acetic anhydride to get 87. Woollin’s re-
agent was then used to selectively change acetamide to selenoac-
etamide (88), while the carbonyl oxygen in the methyl ester
stays unchanged. The methyl ester was hydrolyzed in alkaline con-
ditions and the carboxylic group was coupled with aniline to gain
product 89.
3. Results and discussion
e
N -Thioacetyl lysine compounds have been reported to exploit
the deacetylation mechanism and form a stalled intermediate, 10-
Our previous observations concerning the importance of ꢀ2
S-alkylamidate instead of 10-O-alkylamidate in the normal deacet-
residue for SIRT2 binding and, that alanine replacements at
R1
+Cl-
NH3+Cl-
R1
NH3
Cbz
HN
HN
HN
a
b
e
g (42)
Fmoc
OH
Fmoc
O
Fmoc
O
Fmoc
OH
N
H
N
H
OH
N
H
N
H
H2N
O
O
O
O
O
1
2-22
23-47
f (23, 25, 26, 41)
R1 =
14, 37, 69: COCH2(pyridin-2-yl)
15, 38, 70: CO(1H-pyrrol-2-yl)
16, 39, 71: CO(1-CH3-1H-pyrrol-2-yl)
17, 40, 72: CO(p-(N(CH3)2)Ph)
18, 43, 78: CON(CH3)2
h
2, 25, 50, 51, 52: COCH3
3, 24, 49: CSeCH3
c (2 to 3)
d (8 to 9)
4, 27, 55: COCH=CHCH3
5, 28, 56: COC(CH3)=CHCH3
R1
HN
R2
=
6, 29, 57, 58, 59: COCH=C(CH3)2 19, 44, 79: SO2CH3
48-50, 59-62: OH
7, 30, 60: COCH=CH(CH3CF3)
8, 31, 61: COCH2CH(CH3)2
9, 32, 62: CSCH2CH(CH3)2
10, 33, 65: COPh
20, 45, 80: SO2CF3
21, 46, 81: SO2(p-CH3Ph)
22, 47, 82: SO2(p-NO2Ph)
23, 48: CSCH3
51, 53, 55-57, 65-69, 75-
76, 78-82: OCH3
52, 54, 58, 70-72: NHCH3
O
O
H
N
H
N
11, 34, 66: CO(pyridin-3-yl)
12, 35, 67: CO(pyridin-2-yl)
13, 36, 68: COCH2(pyridin-3-yl)
26, 53, 54: COC(CH3)3
41, 75: COOCH3
N
H
R2
O
O
42, 76: Cbz
48-62, 65-72, 75-76, 78-82
R1
Cbz
NH2
HN
HN
R1 =
j
i
64: CO(cyclohex-1-ene)
73: COCH2Cl
O
O
O
O
O
O
H
H
N
H
N
H
N
H
N
H
N
N
N
H
OCH3
N
H
OCH3 74: COCH2Br
N
H
OCH3
O
O
O
O
O
O
77
64, 73, 74
76
Scheme 1. Reagents and conditions: (a) SOCl2, MeOH, 0 °C to reflux, 45 min; (b) acid chloride or anhydride or TBTU activated carboxylic acid, pyridine or pyridine/DMF (1:1),
0 °C to rt, 2–16 h; (c) Woollin’s reagent, toluene/benzene (1:1), 70 °C, overnight; (d) Lawesson´ s reagent toluene, 70 °C, overnight; (e) NaOH, 0.8 M CaCl2 in i-PrOH/H20 (7:3), rt,
4–16 h; (f) 23: ethyl dithioacetate, EtOH/10% (w/v) Na2CO3 (aq) (3:1), rt, overnight; 25 and 26: appropriate anhydride, pyridine, 0 °C to rt, overnight; 41: methyl
chloroformate, NaHCO3, dioxane/H2O (1:1), 0 °C to rt, 3 h; (g) 9-fluorenylmethyl chloroformate, 0 °C to rt, 4 h; (h) solid phase peptide synthesis with Fmoc strategy:
(coupling) appropriate carboxylic acid, TBTU, DIPEA, DFM, rt, 1 h; (Fmoc-deprotection) 20% piperidine/DMF; (cleavage) R2 = OH: NaOH (1 M)/dioxane (2:6), rt, 1 h; R2 = OCH3:
DIPEA/MeOH/DMF (1:5:5), heat at reflux, overnight; R2 = NHCH3: 8 M methylamine in ethanol, rt, 2 h; (i) 10% Pd on charcoal, MeOH, rt, 2 h; (j) 64 and 73: TBTU activated
carboxylic acid, DMF/pyridine (1:1), 0 °C to rt, 3 h; 74: bromoacetyl bromide, DIPEA, 1 h at 0 °C, overnight at rt.

T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
5619
Boc
Boc
P
HN
HN
NH3+Cl-
HN
S
HN
S
b, c
a
e
H
N
H
N
H
N
H
N
Cbz
Cbz
OH
Cbz
Cbz
HN
Cbz
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
83
84
85
86
d
NH2
OH
Se
HN
O
HN
Se
f, g
h
i, j
H
N
Cbz
Cbz
N
Cbz
O
Cbz
O
N
H
H
N
H
N
H
O
O
O
O
87
88
89
Scheme 2. Reagents and conditions (a) EDCꢁHCl, HOBtꢁH2O, aniline, CH3CN, rt, overnight; (b) TFA/EtOAC (1:1), rt, 2 h; (c) ethyl dithioacetate, Na2CO3, EtOH/H2O (6:1), rt,
overnight; (d) 3.5 M HCl in EtOAc, 0 °C to rt, overnight; (e) dimethylthiophosphinic chloride, DIPEA, pyridine, 0 °C to rt, overnight; (f) SOCl2, MeOH, 0 °C to reflux, 45 min; (g)
acetic anhydride, pyridine, 0 °C to rt, overnight; (h) Woollin’s reagent, toluene, 70 °C overnight; (i) LiOH, MeOH/H2O (3:1), 0 °C to rt, 2.5 h; (j) TBTU, aniline, pyridine/DMF
(1:1), 0 °C to rt, 3 h.
e
ylation reaction.12,27,29,30,34 Therefore, we synthesized N -acety-
lated compounds 50–52 as reference compounds. The free car-
boxyl terminus of compound 50 was protected as a methyl ester
(51), and methyl amide (52). All three compounds inhibited SIRT1
and SIRT2 in vitro with little or moderate differences in their inhib-
itory activities, compound 52 with the methyl amide being the
(IC50 = 40 lM) and equipotent against SIRT2 (IC50 = 66 lM) in com-
e
parison to N -acetylated compound 52. These results suggest that
e
the binding cavity accepts also larger moieties than the N -acetyl
group.
Compounds 55–57 and 64 were synthesized to study different
e
e
N -
a
,b-unsaturated carbonyl groups. (E)-N -3-Methylacryl derivate
e
most favorable modification (IC50 = 88
l
M for SIRT1 and 60
lM
55 and (E)-N -2,3-dimethylacryl derivate 56 inhibited only weakly
e
e
for SIRT2). While N -thioacetyl compound 48 slows the reaction
kinetics, compounds 50–52 undergo a normal deacetylation reac-
tion and can be considered as competitive substrates in the assay.
if at all SIRT1 and SIRT2. The bulkier N -cyclohex-1-enecarbonyl
group (64) gave SIRT2 inhibition which was comparable to that
e
caused by the N -acetyl group (51). Surprisingly, the change to
e
e
The excellent potency of N -thioacetyl lysine possessing com-
the N -3,3-dimethylacryl group (57) increased inhibitory activity
pounds, first reported by Fatkins et al.,27 prompted us to synthesize
for both enzymes (IC50 = 46 lM for SIRT1 and 69 lM for SIRT2) in
e
e
the N -selenoacetylated derivative 49. The changes in the atomic
radius and electronegativity are smaller between sulfur and sele-
nium than between oxygen and sulfur. N -Thioacetylation (48)
and N -selenoacetylation (49) gave almost equipotent inhibitory
comparison to N -acetylated compound 51. There seems to be
space for bulkier groups but the shape of a group has crucial effect
on the inhibitory activity. To confirm this result, two more com-
e
e
e
pounds with the N -3,3-dimethylacryl group but with different
activities for SIRT1 and SIRT2. Encouraged by this result, we made
modifications at the C-terminus were synthesized (58 and 59).
They both were superior to their N -acetylated reference com-
pounds 52 and 50, respectively. In agreement with the previous re-
e
e
N -selenoacetylation on the Cbz-Lys-NH-Ph backbone also. Here,
e
e
the change from N -thioacetylation (84) to N -selenoacetylation
(89) increased the inhibitory activity 5.5-fold for both SIRT1 and
sults, the most potent SIRT1 (IC50 = 6.1
lM) and SIRT2
SIRT2. Compound 89 is one of the most potent reported SIRT2
(IC50 = 23 M) inhibition was achieved with compound 58, with
l
e
inhibitors (IC50 = 2.7
forms a stalled intermediate similar to N -thioacetyl lysine.
l
M). It is likely, that N -selenoacetyl lysine
the methyl amide at the C-terminus, while the free carboxylic
group (59) was slightly preferred over the methyl ester (57) by
SIRT1.
e
e
e
In addition to the N -thioacetylated and N -selenoacetylated
e
derivatives, several compounds were synthesized and tested to
In the N -3,3-dimethylacryl group, the carbonyl function is con-
e
study different functionalities as a substitute for the N -acetyl
jugated with a double bond which may increase its nucleophilicity.
In order to study if this electron donating property is important for
e
group and inhibit SIRT1 and SIRT2. In comparison to N -acetylated
e
reference compound 51, carbamates 75 and 76, dimethylurea 78,
and sulfonamides 79–82 were all significantly weaker inhibitors
the inhibitory activity, we synthesized (Z)-N -3-methyl-3-trifluo-
romethylacryl derivative 60 with electron withdrawing properties.
It had significantly decreased activity compared to compound 59.
We also prepared compounds 73 and 74 with the electron with-
e
of SIRT1 and SIRT2 or did not inhibit them at all. Also N -dim-
e
ethylphosphorothioylated derivative 86 and N -Boc protected
e
compound 83 failed to show any significant inhibitory activity.
These modifications are therefore considered to be mechanistically
inactive.
drawing groups attached on the N -acetyl group and they also
showed decreased activity compared to the reference compound
51. It seems that electron withdrawing groups near the nucleo-
philic oxygen abolish the inhibitory activity. Next we removed
The possible size restriction of the binding cavity was first stud-
e
e
ied with compounds 53 and 54, which introduce a N -pivaloyl
the acrylic double bond and synthesized N -isovaleryl derivative
group. Compound 53 showed equipotent SIRT1 inhibitory activity
with the corresponding N -acetylated compound 51, but slightly
61, with decreased nucleophilicity and increased flexibility. Com-
pared to compound 59, compound 61 was equipotent SIRT1 inhib-
e
decreased SIRT2 inhibition. However, compound 54 with methyl-
itor (IC50 = 12
lM) and over two times more potent SIRT2 inhibitor
amide at the C-terminus was two times more potent against SIRT1
(IC50 = 30 M). The good inhibitory activity of compound 61 indi-
l

5620
T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
cates that, although electron distribution may play some role, the
potency of compound 59 likely relies on complementary binding
and lipophilic interactions. Next we wanted to see if the beneficial
effects of a bulky lipophilic group and a thiocarbonyl group could
3.31 ppm and CHCl3 at 7.26 ppm. 13C NMR: (CD3)2SO at
39.52 ppm, CD3OD at 49.00 ppm and CDCl3 at 77.16 ppm). Positive
ion mass spectra (ESI-MS) were acquired with an LCQ quadrupole
ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped
with an electro spray ionization source. Combustion analysis for
CHN was measured on Thermo Quest CE Instruments EA 1110
CHNS–O elemental analyzer. The purity of the compounds 60
and 72 was determined using Agilent 1100 HPLC (Agilent Technol-
ogies Inc., Waldbronn, Karlsruhe, Germany) with diode array
detection, using reversed phase column (Zorbax Eclipse XDB C
e
be combined to the same molecule and synthesized N -isothiovale-
ryl derivative 62. It was clearly more potent SIRT1 (IC50 = 0.70
l
M)
e
and SIRT2 (IC50 = 3.6
lM) inhibitor than N -isovaleryl derivative
e
61. Compared to N -thioacetyl derivative 48, SIRT1 inhibition was
slightly decreased and SIRT2 inhibition slightly increased.
We studied also a series of bulky but flat five- and six-mem-
e
bered aromatic substituents at the N -site (65–72). Compared to
18, 4.6 ꢂ 50 mm, 1.8
lm, Agilent Technologies, Palo Alto, CA,
the aliphatic substituents, the aromatic structures differ in their
electronic or steric properties in the the acetyl binding pocket.
Compounds 65–67 and 70–72 have an aromatic ring directly at-
tached to the carbonyl carbon and all of them failed to inhibit
SIRT1 or SIRT2. Introduction of a methylene spacer between the
carbonyl and aromatic groups (compounds 68 and 69) or introduc-
tion of an electron donating substituent (70–72) didn’t improve the
inhibitory activity. Although nicotinamide is able to inhibit sirtuins
by attacking the 10-O-alkylimidate intermediate to regenerate
NAD+,18,19,30 pyridyl derivatives 66–69 did not show any significant
improvement in inhibitory activity compared to phenyl derivative
65.
USA), 5% Solvent B (0.05% AcOH in CH3CN) in solvent A (0.05%
AcOH in H2O) for 5 min, then linear gradient 5–80% B in A in
15 min with the flow rate 1 mL/min.
5.2. Manual solid phase peptide synthesis (SPPS)
Wang resin (polymer-bound p-alkoxy-benzyl alcohol) was used
as solid support for the peptide synthesis of compounds 48–62,
65–72, 75, 76 and 78–82. Solid phase synthesis was performed in a
10 mL syringe equipped with a frit. Fmoc-Ala-Wang resin (loading
0.4–0.8 mmol/g)wasswelled for 1 h in3 mL ofDMF. In the deprotec-
a
tion phase, the N -Fmoc protection group was removed with 5 mL of
20% (V/V) piperidine in DMF for 15 min and the resin was rinsed five
a
times with DMF. In the coupling phase, the following N -Fmoc-ami-
4. Conclusions
no acid or acetic acid (2–4 equiv) was preactivated (1–3 min) with
the coupling reagent TBTU (2–4 equiv) and DIPEA (5–10 equiv) in
3–5 mL of DMF. This solution was added on the resin and the syringe
was shaken at rt for 60 min. Then the resin was rinsed five times with
DMF. The cycle of Fmoc-deprotection and coupling was repeatedun-
til the desired resin-bound peptide was completed. Before cleavage
fromtheresin, theresin waswashedoncewithAcOH, fivetimeswith
DCM, and once with MeOH to remove the excess solvents and then
dried under vacuum.
e
SIRT2 inhibitory activity of N -thioacetylated tripeptides was
increased one order of magnitude by simple acetylation of the N-
terminus. This finding was combined with our previous observa-
tion that both SIRT1 and SIRT2 accept L-alanines at the positions
e
surrounding N -thioacetyllysine and, an Ac-Ala-Lys-Ala sequence
was developed. The sequence is easily applicable for screening of
e
different N -modifications for SIRT1 and SIRT2 inhibition, it is easy
to synthesize by solid phase peptide synthesis and, apart from the
lysine, it does not have reactive side chains. It can be cleaved from
resin as a free carboxylic acid, a methyl ester or a methyl amide
and can be purified on silica.
Several different N -modifications were studied with two differ-
ent backbones. N -Selenoacetylation gave equipotent or improved
SIRT1 and SIRT2 inhibition compared to N -thioacetylation.
Although the applications of seleno-compounds are limited to re-
search purposes, Cbz-Lys(selenoAc)-NH-Ph (89) was the most po-
tent SIRT2 inhibitor (2.7 lM) found in this study and is among
the most potent reported SIRT2 inhibitors. The studied bulky N -
alkanoyl and N -aroyl groups showed that both SIRT1 and SIRT2
can accept rather large groups at the active site. However, they
seem to be sensitive to even small changes in the electronic and
steric properties of the N -modification (see series 51–54, 55–57,
59–61 and 64–66). N -3,3-Dimethylacryl (57–59) and N -isovaleryl
(61) were the best aliphatic moieties found in this study. Further
modification gave N -isothiovaleryl derivative 62 which had
slightly decreased SIRT1 but slightly increased SIRT2 inhibition
when compared to N -thioacetylated compound 48.
5.3. Cleavage of peptide-resin as carboxylic acid
The dried peptide-resin was pre-swelled in dioxane for 15 min.
The excess of dioxane was removed and 8 mL of cool (0 °C) cleav-
age mixture of 1 M NaOH (aq)/dioxane (2 mL and 6 mL) was added
on the peptide-resin. The reaction mixture was shaken 1 h at rt.
The mixture was filtrated, filtrate was collected and the resin
was washed with H2O/dioxane (1:3), dioxane and H2O. The filtrate
was neutralized with 3 M HCl (aq) and the solvents were evapo-
rated. Acetone was added on the residue and the mixture was fil-
trated to remove inorganic salts. The solvent of the filtrate was
evaporated under reduced pressure to yield the crude product that
was purified by column chromatography or by crystallization.
e
e
e
e
e
e
e
e
5.4. Cleavage of peptide-resin as methyl ester
e
The dry peptide-resin was placed in a flask. Cleavage mixture
DIPEA/MeOH/DMF (1:5:5, 11–22 mL) was added and the reaction
mixture was refluxed under argon atmosphere overnight. The mix-
ture was filtrated, filtrate was collected and the resin was rinsed
five times with MeOH/DMF (1:1). Solvent was evaporated from
the combined filtrates under reduced pressure to yield the crude
product that was purified by column chromatography or by
crystallization.
e
5. Experimental section
5.1. General
All reagents and solvents were commercial high purity quality.
Purification of the products was performed by CombiFlashÒ col-
umn chromatography on silica column or by crystallization. NMR
spectra were recorded on a Bruker Avance 500 (Bruker Biospin,
Switzerland) 500.1 MHz for 1H and 125.8 MHz for 13C. The chemi-
cal shifts are expressed in ppm relative to the shift of used solvent
as an internal standard (1H NMR: DMSO at 2.50 ppm, CH3OH at
5.5. Cleavage of peptide-resin as methyl amide
5 mL of 8 M methylamine in EtOH was added to the dry pep-
tide-resin and the mixture was stirred for 2 h at rt. The resulting
white precipitate was dissolved in MeOH and the resin beads were
filtrated. The solvent of the filtrate was evaporated under reduced

T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
5621
pressure to yield the crude product that was purified by column
5.6.7. (S)-2-((S)-2-Acetamidopropanamido)-N-((S)-1-(methyl-
chromatography or by crystallization.
amino)-1-oxopropan-2-yl)-6-pivalamidohexanamide (54)
1H NMR ((CD3)2SO): d = 1.07 (s, 9H), 1.13–1.30 (m, 8H), 1.31–
1.42 (m, 2H), 1.44–1.71 (m, 2H),1.83 (s, 3H), 2.55–2.59 (m, 3H),
2.92–3.06 (m, 2H), 4.09–4.29 (m, 3H), 7.37–7.46 (m, 1H), 7.69–
7.78 (m, 1H), 7.78–7.86 (m, 1H), 7.89–7.97 (m, 1H) 8.04–8.13 (m,
1H). 13C NMR ((CD3)2SO): d = 18.02, 18.34, 22.49, 22.69, 25.54,
27.47, 28.87, 31.37, 37.94, 38.61, 48.08, 48.33, 52.54, 169.21,
171.09, 172.34, 172.59, 177.17. ESI-MS (m/z): 428.3 [M+H]+,
450.5 [M+Na]+. Anal. (C20H37N5O5ꢁ0.2H2O) C, H, N.
5.6. Analytical data for compounds 48–76, 78–84, 86 and 89
5.6.1. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-ethanethi-
oamidohexanamido)propanoic acid (48)
1H NMR (CD3OD): d = 1.35 (d, 3J = 7.2 Hz, 3H), 1.40 (d,
3J = 7.3 Hz, 3H), 1.43–1.52 (m, 2H), 1.60–1.93 (m, 4H), 1.98 (s,
3H), 2.45 (s, 3H), 3.51–3.63 (m, 2H), 4.26–4.40 (m, 3H). 13C NMR
(CD3OD): d = 17.82, 17.86, 22.40, 24.10, 28.21, 32.81, 33.12,
46.97, 49.67, 50.62, 54.21, 173.26, 173.68, 175.22, 176.27 (br),
201.52. ESI-MS (m/z): 389.24 [M+H]+. Anal. (C16H28N4O5Sꢁ1AcOH)
C, H, N.
5.6.8. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-((E)-
but-2-enamido)hexanamido)propanoate (55)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.1 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.21–1.44 (m, 4H), 1.45–1.71 (m, 2H), 1.77 (d,
3J = 6.7 Hz, 3H), 1.83 (s, 3H), 3.02–3.11 (m, 2H), 3.60 (s, 3H),
4.18–4.30 (m, 3H), 5.87 (d, 3J = 15.4 Hz, 1H), 6.58 (dddd,
3J = 15.4 Hz, 6.7 Hz, 1H), 7.76–7.89 (m, 2H), 7.99–8.08 (m, 1H),
8.20–8.30 (m, 1H). 13C NMR ((CD3)2SO): d = 16.79, 17.29, 18.04,
22.48, 22.57, 28.87, 31.72, 38.32, 47.52, 48.21, 51.82, 51.88,
126.02, 137.26, 164.74, 169.16, 171.45, 172.21, 172.92. ESI-MS
(m/z): 413.2 [M+H]+, 435.4 [M+Na]+. Anal. (C19H32N4O6ꢁ0.1H2O) C,
H, N.
5.6.2. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-ethanese-
lenoamidohexanamido)propanoic acid (49)
1H NMR (CD3OD): d = 1.34 (d, 3J = 7.2 Hz, 3H), 1.40 (d,
3J = 7.3 Hz, 3H), 1.44–1.54 (m, 2H), 1.64–1.94 (m, 4H), 1.98 (s,
3H), 2.54 (s, 3H), 3.58–3.69 (m, 2H), 4.26–4.43 (m, 3H). 13C NMR
(CD3OD): d = 17.62, 17.83, 22.39, 24.03, 27.99, 32.83, 37.18,
50.52, 50.62, 54.13, 173.25, 173.76, 175.20, 175.79, 204.95. ESI-
MS (m/z): 437.19 [M+H]+. Anal. (C16H28N4O5Seꢁ0.2H2Oꢁ0.8AcOH)
C, H, N.
5.6.9. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-((E)-
2-methylbut-2-enamido)hexanamido)propanoate (56)
5.6.3. (S)-2-((S)-6-Acetamido-2-((S)-2-acetamidopropanamido)
hexanamido)propanoic acid (50)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.1 Hz, 3H), 1.27 (d,
3J = 7.2 Hz, 3H), 1.21–1.45 (m, 4H), 1.45–1.71 (m, 2H), 1.67 (d,
3J = 6.8 Hz, 3H), 1.72 (s, 3H), 1.82 (s, 3H), 3.02–3.11 (m, 2H),
3.60 (s, 3H), 4.18–4.30 (m, 3H), 6.28 (ddd, 3J = 6.8 Hz, 1H),
7.64–7.73 (m, 1H), 7.78–7.87 (m, 1H), 7.98–8.08 (m, 1H), 8.20–
8.30 (m, 1H). 13C NMR ((CD3)2SO): d = 12.34, 13.56, 16.78,
18.04, 22.47, 22.59, 28.91, 31.77, 38.78, 47.51, 48.18, 51.79,
51.94, 128.75, 132.04, 168.25, 169.10, 171.45, 172.19, 172.89.
1H NMR ((CD3)2SO): d = 1.17 (d, 3J = 7.1 Hz, 3H), 1.26 (d,
3J = 7.3 Hz, 3H), 1.19–1.41 (m, 4H), 1.42–1.72 (m, 2H), 1.78 (s,
3H), 1,83 (s, 3H), 2.90–3.02 (m, 2H), 4.09–4.29 (m, 3H), 7.68–7.78
(m, 1H), 7.78–7.86 (m, 1H), 7.96–8.05 (m, 1H), 8.05–8.11 (m,
1H), 12.49 (br, 1H). 13C NMR ((CD3)2SO): d = 17.06, 18.04, 22.48,
22.56, 22.61, 28.81, 31.73, 38.41, 47.43, 48.23, 51.95, 168.93,
169.15, 171.27, 172.22, 173.95. ESI-MS (m/z): 373.1 [M+H]+. Anal.
(C16H28N4O6ꢁ0.7H2O) C, H, N.
ESI-MS
(m/z):
427.2
[M+H]+,
449.4
[M+Na]+.
Anal.
(C20H34N4O6ꢁ0.1H2O) C, H, N.
5.6.4. (S)-Methyl 2-((S)-6-acetamido-2-((S)-2-acetamidopropa-
namido)hexanamido)propanoate (51)
5.6.10. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(3-
methylbut-2-enamido)hexanamido)propanoate (57)
1H NMR (CD3OD): d = 1.33 (d, 3J = 7.2 Hz, 3H), 1.39 (d,
3J = 7.3 Hz, 3H), 1.38–1.57 (m, 4H), 1.62–1.88 (m, 2H), 1.93 (s,
3H), 1.97 (s, 3H), 3.11–3.22 (m, 2H), 3.71 (s, 3H), 4.26–4.43 (m,
3H). 13C NMR (CD3OD): d = 17.28, 17.84, 22.39, 22.58, 23.93,
29.88, 32.81, 40.21, 49.43, 50.58, 52.73, 54.16, 173.23, 173.23,
173.95, 174.52, 175.15. ESI-MS (m/z): 387.2 [M+H]+. Anal.
(C17H30N4O6) C, H, N.
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 6.9 Hz, 3H), 1.27 (d,
3J = 7.2 Hz, 3H), 1.21–1.43 (m, 4H), 1.45–1.71 (m, 2H), 1.76 (s,
3H), 1.83 (s, 3H), 2.06 (s, 3H), 2.97–3.08 (m, 2H), 3.60 (s, 3H),
4.19–4.30 (m, 3H), 5.62 (s, 1H), 7.65–7.72 (m, 1H), 7.80–7.87 (m,
1H), 8.00–8.07 (m, 1H), 8.22–8.29 (m, 1H). 13C NMR ((CD3)2SO):
d = 16.78, 18.03, 19.18, 22.47, 22.62, 26.72, 28.90, 31.76, 38.06,
47.51, 48.19, 51.81, 51.89, 119.22, 147.86, 165.85, 169.13, 171.45,
172.19, 172.91. ESI-MS (m/z): 427.2 [M+H]+, 449.4 [M+Na]+. Anal.
(C20H34N4O6) C, H, N.
5.6.5. (S)-6-Acetamido-2-((S)-2-acetamidopropanamido)-N-((S)-
1-(methylamino)-1-oxopropan-2-yl)hexanamide (52)
1H NMR ((CD3)2SO): d = 1.13–1.21 (m, 6H), 1.21–1.41 (m, 4H),
1.44–1.70 (m, 2H), 1.78 (s, 3H), 1.83 (s, 3H), 2.54–2.59 (m, 3H),
2.95–3.01 (m, 2H), 4.12–4.27 (m, 3H), 7.69–7.79 (m, 2H), 7.80–
7.86 (m, 1H), 7.89–7.95 (m, 1H), 8.05–8.09 (m, 1H). 13C NMR
((CD3)2SO): d = 17.95, 18.30, 22.48, 22.61, 22.69, 25.55, 28.76,
31.32, 38.34, 48.12, 48.37, 52.49, 168.96, 169.28, 171.08, 172.40,
172.60. ESI-MS (m/z): 386.5 [M+H]+. Anal. (C17H31N5O5ꢁ0.7H2Oꢁ
0.2hexane) C, H, N.
5.6.11. (S)-2-((S)-2-Acetamidopropanamido)-N-((S)-1-(methyl-
amino)-1-oxopropan-2-yl)-6-(3-methylbut-2-
enamido)hexanamide (58)
1H NMR ((CD3)2SO): d = 1.13–1.21 (m, 6H), 1.21–1.31 (m, 2H),
1.31–1.42 (m, 2H), 1.44–1.71 (m, 2H), 1.76 (s, 3H), 1.83 (s, 3H),
2.06 (s, 3H), 2.54–2.62 (m, 3H), 2.96–3.08 (m, 2H), 4.11–4.29 (m,
3H), 5.62 (s, 1H), 7.65–7.72 (m, 1H), 7.72–7.78 (m, 1H), 7.79–
7.87 (m, 1H), 7.88–7.96 (m, 1H) 8.04–8.12 (m, 1H). 13C NMR
((CD3)2SO): d = 17.97, 18.30, 19.19, 22.47, 22.77, 25.54, 26.73,
28.84, 31.38, 38.00, 48.09, 48.32, 52.46, 119.23, 147.87, 165.86,
169.23, 171.06, 172.38, 172.56. ESI-MS (m/z): 426.3 [M+H]+,
448.4 [M+Na]+. Anal. (C20H35N5O5ꢁ1H2Oꢁ0.1hexane) C, H, N.
5.6.6. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
pivalamidohexanamido)propanoate (53)
1H NMR (CD3OD): d = 1.17 (s, 9H), 1.34 (d, 3J = 7.2 Hz, 3H), 1.39
(d, 3J = 7.3 Hz, 3H), 1.35–1.57 (m, 4H), 1.62–1.89 (m, 2H), 1.97 (s,
3H), 3.12–3.22 (m, 2H), 3.71 (s, 3H), 4.27–4.43 (m, 3H). 13C NMR
(CD3OD): d = 17.30, 17.90, 22.40, 24.00, 27.89, 30.10, 32.85,
39.63, 40.37, 49.44, 50.57, 52.73, 54.25, 173.22, 173.99, 174.49,
175.14, 181.36. ESI-MS (m/z): 429.2 [M+H]+, 451.3 [M+Na]+. Anal.
(C20H36N4O6ꢁ0.1H2O) C, H, N.
5.6.12. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-(3-
methylbut-2-enamido)hexanamido)propanoic acid (59)
1H NMR ((CD3)2SO): d = 1.17 (d, 3J = 7.1 Hz, 3H), 1.26 (d,
3J = 7.3 Hz, 3H), 1.20–1.42 (m, 4H), 1.44–1.71 (m, 2H), 1.76 (s,

5622
T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
3H), 1,83 (s, 3H), 2.06 (s, 3H), 2.96–3.07 (m, 2H), 4.11–4.31 (m, 3H),
5.62 (s, 1H), 7.64–7.71 (m, 1H), 7.80–7.87 (m, 1H), 8.01–8.06 (m,
1H), 8.06–8.11 (m, 1H), 12.51 (br, 1H). 13C NMR ((CD3)2SO):
d = 17.07, 18.05, 19.19, 22.47, 22.66, 26.74, 28.89, 31.76, 38.08,
47.45, 48.21, 51.96, 119.21, 147.88, 165.85, 169.13, 171.27,
449.2 [M+H]+, 471.3 [M+Na]+. Anal. (C22H32N4O6ꢁ0.1hexane) C,
H, N.
5.6.18. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(nicotinamido)hexanamido)propanoate (66)
172.22,
173.96.
ESI-MS
(m/z):
411.2
[M+H]+.
Anal.
1H NMR (CD3OD): d = 1.32 (d, 3J = 7.2 Hz, 3H), 1.38 (d,
3J = 7.4 Hz, 3H), 1.44–1.56 (m, 2H), 1.60–1.93 (m, 4H), 1.95 (s,
3H), 3.36–3.48 (m, 2H), 3.68 (s, 3H), 4.25–4.42 (m, 3H), 7.49–
7.57 (m, 1H), 8.20–8.28 (m, 1H), 8.63–8.71 (m, 1H), 8.94–9.01
(m, 1H). 13C NMR (CD3OD): d = 17.26, 17.81, 22.37, 24.02, 29.91,
32.87, 40.84, 49.44, 50.62, 52.72, 54.17, 125.09, 132.21, 137.04,
149.15, 152.53, 167.79, 173.23, 173.95, 174.49, 175.16. ESI-MS
(m/z): 450.2 [M+H]+, 472.3 [M+Na]+. Anal. (C21H31N5O6ꢁ0.1hexane)
C, H, N.
(C19H32N4O6ꢁ0.5H2Oꢁ0.2hexane) C, H, N.
5.6.13. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-((Z)-4,4,4-
trifluoro-3-methylbut-2-enamido)hexanamido)propanoic acid
(60)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.1 Hz, 3H), 1.25 (d,
3J = 7.3 Hz, 3H), 1.21–1.34 (m, 2H), 1.35–1.45 (m, 2H), 1.45–1.72
(m, 2H), 1,82 (s, 3H), 2.16 (s, 3H), 3.04–3.14 (m, 2H), 4.09–4.29
(m, 3H), 6.47 (s, 1H), 7.84–7.91 (m, 1H), 8.01–8.11 (m, 2H), 8.35–
8.43 (m, 1H), 12.41 (br, 1H). 13C NMR ((CD3)2SO): d = 11.25,
17.12, 18.00, 22.43, 22.57, 28.44, 31.66, 38.44, 47.52, 48.24,
5.6.19. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(picolinamido)hexanamido)propanoate (67)
1
2
51.88, 123.77 (q, JCF 273.36 Hz), 134.37 (q, JCF 29.24 Hz), 125.44
1H NMR ((CD3)2SO): d = 1.15 (d, 3J = 7.7 Hz, 3H), 1.26 (d,
3J = 7.3 Hz, 3H), 1.22–1.37 (m, 2H), 1.47–1.73 (m, 4H), 1.82 (s,
3H), 3.24–3.34 (m, 2H), 3.59 (s, 3H), 4.20–4.29 (m, 3H), 7.56–
7.61 (m, 1H), 7.79–7.84 (m, 1H), 7.95–8.05 (m, 3H), 8.21–8.26
(m, 1H), 8.60–8.65 (m, 1H), 8.72–8.78 (m, 1H). 13C NMR
((CD3)2SO): d = 16.76, 18.04, 22.46, 22.57, 28.95, 31.76, 38.69,
47.49, 48.15, 51.76, 51.92, 121.76, 126.34, 137.71, 148.29,
150.11, 163.64, 169.07, 171.40, 172.16, 172.86. ESI-MS (m/z):
450.2 [M+H]+, 472.3 [M+Na]+. Anal. (C21H31N5O6ꢁ0.2H2O) C, H,
N.
3
(q, JCF 6.01 Hz), 163.34, 169.13, 171.18, 172.23, 173.92. ESI-MS
(m/z): 465.18, 466.18, 467.18, 468.18 [MꢀH]ꢀ. Anal. (C19H32N4O6ꢁ
1.5AcOHꢁ0.1hexane) C, H, N. HPLC: tR 9.31 min, area percent 97%
at 260 nm.
5.6.14. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-(3-
methylbutanamido)hexanamido)propanoic acid (61)
1H NMR ((CD3)2SO): d = 0.85 (d, 3J = 6.1 Hz, 6H), 1.17 (d,
3J = 7.1 Hz, 3H), 1.26 (d, 3J = 7.3 Hz, 3H), 1.21–1.42 (m, 4H), 1.43–
1.71 (m, 2H), 1.83 (s, 3H), 1.88–2.00 (m, 3H), 2.92–3.05 (m, 2H),
4.11–4.31 (m, 3H), 7.66–7.74 (m, 1H), 7.79–7.87 (m, 1H), 7.99–
8.08 (m, 1H), 8.05–8.10 (m, 1H), 12.48 (br, 1H). 13C NMR
((CD3)2SO): d = 17.07, 18.05, 22.29, 22.30, 22.47, 22.56, 25.45,
28.91, 31.69, 38.25, 44.75, 47.43, 48.20, 51.93, 169.08, 171.23,
171.24, 172.19, 173.93. ESI-MS (m/z): 413.4 [M+H]+. Anal.
(C19H34N4O6ꢁ0.5H2O) C, H, N.
5.6.20. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(2-
(pyridin-3-yl)acetamido)hexanamido)propanoate (68)
1H NMR ((CD3)2SO): d = 1.17 (d, 3J = 7.0 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.22–1.32 (m, 2H), 1.35–1.70 (m, 4H), 1.83 (s,
3H), 2.98–3.06 (m, 2H), 3.43 (s, 2H), 3.60 (s, 3H), 4.19–4.30 (m,
3H), 7.29–7.35 (m, 1H), 7.62–7.67 (m, 1H), 7.80–7.85 (m, 1H),
8.01–8.06 (m, 1H), 8.06–8.11 (m, 1H), 8.22–8.27 (m, 1H), 8.40–
8.46 (m, 2H). 13C NMR ((CD3)2SO): d = 16.78, 18.02, 22.47, 22.49,
28.71, 31.66, 38.62, 39.23, 47.49, 48.20, 51.79, 51.81, 123.28,
132.14, 136.46, 147.54, 149.94, 169.13, 169.25, 171.43, 172.19,
172.89. ESI-MS (m/z): 464.2 [M+H]+, 486.2 [M+Na]+. Anal.
(C22H33N5O6ꢁ0.6H2Oꢁ0.1hexane) C, H, N.
5.6.15. (S)-2-((S)-2-((S)-2-Acetamidopropanamido)-6-(3-
methylbutanethioamido)hexanamido)propanoic acid (62)
1H NMR (CD3OD): d = 0.89–0.95 (m, 6H), 1.32–1.41 (m, 6H),
1.41–1.56 (m, 2H), 1.62–1.94 (m, 4H), 1.92 (s, 3H), 2.17–2.28 (m,
1H), 2.45 (d, 3J = 7.4 Hz, 2H), 3.57–3.62 (m, 2H), 4.22–4.39 (m,
3H). 13C NMR (CD3OD): d = 17.92, 18.32, 22.25, 22.28, 22.42,
24.26, 28.24, 30.28, 32.72, 46.63, 50.37, 50.65, 54.36, 56.02,
173.26, 173.44, 175.27, 177.37, 205.37. ESI-MS (m/z): 431.25
[M+H]+, 453.30 [M+Na]+. Anal. (C19H34N4O5Sꢁ0.5H2Oꢁ0.1AcOH) C,
H, N.
5.6.21. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(2-
(pyridin-2-yl)acetamido)hexanamido)propanoate (69)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.0 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.22–1.32 (m, 2H), 1.35–1.70 (m, 4H), 1.83 (s,
3H), 2.99–3.07 (m, 2H), 3.58 (s, 2H), 3.60 (s, 3H), 4.19–4.30 (m,
3H), 7.20–7.25 (m, 1H), 7.28–7.34 (m, 1H), 7.68–7.74 (m, 1H),
7.79–7.85 (m, 1H), 7.99–8.09 (m, 2H), 8.21–8.27 (m, 1H), 8.44–
8.48 (m, 1H). 13C NMR ((CD3)2SO): d = 16.78, 18.03, 22.47, 22.52,
28.75, 31.68, 38.59, 44.91, 47.49, 48.19, 51.79, 51.84, 121.65,
123.67, 136.36, 148.81, 156.42, 168.87, 169.12, 171.43, 172.19,
172.89. ESI-MS (m/z): 464.3 [M+H]+, 486.2 [M+Na]+. Anal.
(C22H33N5O6ꢁ0.2H2O) C, H, N.
5.6.16. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(cyclohex-1-enecarboxamido)hexanamido)propanoate (64)
1H NMR (CD3OD): d = 1.33 (d, 3J = 7.2 Hz, 3H), 1.39 (d,
3J = 7.4 Hz, 3H), 1.40–1.91 (m, 10H), 1.97 (s, 3H), 2.11–2.27 (m,
4H), 3.13–3.27 (m, 2H), 3.71 (s, 3H), 4.27–4.43 (m, 3H), 6.54–
6.59 (m, 1H). 13C NMR (CD3OD): d = 17.29, 17.87, 22.39, 22.72,
23.33, 24.03, 25.29, 26.35, 30.08, 32.82, 40.27, 49.45, 50.59,
52.72, 54.25, 134.38, 134.62, 171.69, 173.22, 173.99, 174.49,
175.14. ESI-MS (m/z): 453.21 [M+H]+, 475.36 [M+Na]+. Anal.
(C22H36N4O6) C, H, N.
5.6.22. N-((S)-5-((S)-2-Acetamidopropanamido)-6-((S)-1-
(methylamino)-1-oxopropan-2-ylamino)-6-oxohexyl)-1H-
pyrrole-2-carboxamide (70)
5.6.17. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
benzamidohexanamido)propanoate (65)
1H NMR ((CD3)2SO): d = 1.13–1.22 (m, 6H), 1.22–1.38 (m, 2H),
1.39–1.74 (m, 4H), 1.83 (s, 3H), 2.55–2.59 (m, 3H), 3.12–3.22 (m,
2H), 4.12–4.29 (m, 3H), 6.02–6.09 (m, 1H), 6.69–6.77 (m, 1H),
6.78–6.85 (m, 1H),7.68–7.76 (m, 1H), 7.79–7.86 (m, 1H), 7.89–
7.98 (m, 2H), 8.04–8.12 (m, 1H) 11.32–11.42 (m, 1H). 13C NMR
((CD3)2SO): d = 17.98, 18.29, 22.47, 22.85, 25.53, 29.17, 31.39,
38.33, 48.09, 48.32, 52.56, 108.39, 109.59, 120.99, 126.44, 160.55,
169.26, 171.09, 172.36, 172.61. ESI-MS (m/z): 437.3 [M+H]+,
459.4 [M+Na]+. Anal. (C20H32N6O5ꢁ0.1H2O) C, H, N.
1H NMR (CD3OD): d = 1.32 (d, 3J = 7.2 Hz, 3H), 1.38 (d,
3J = 7.3 Hz, 3H), 1.43–1.56 (m, 2H), 1.60–1.93 (m, 4H), 1.95 (s,
3H), 3.36–3.43 (m, 2H), 3.68 (s, 3H), 4.27–4.42 (m, 3H), 7.41–
7.48 (m, 2H), 7.52 (t, 3J = 7.4, 1H), 7.78–7.85 (m, 2H). 13C
NMR (CD3OD): d = 17.27, 17.83, 22.37, 24.07, 30.05, 32.88,
40.76, 49.45, 50.61, 52.71, 54.23, 128.29, 129.50, 132.53,
135.90, 170.30, 173.25, 173.98, 174.49, 175.16. ESI-MS (m/z):

T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
5623
5.6.23. N-((S)-5-((S)-2-Acetamidopropanamido)-6-((S)-1-(meth-
ylamino)-1-oxopropan-2-ylamino)-6-oxohexyl)-1-methyl-1H-
pyrrole-2-carboxamide (71)
d = 16.79, 18.04, 22.39, 22.46, 29.14, 31.73, 40.23, 47.51, 48.19,
51.79, 51.90, 65.10, 127.72, 127.72, 128.33, 137.26, 156.05,
169.12, 171.42, 172.19, 172.90. ESI-MS (m/z): 479.1 [M+H]+,
501.3 [M+Na]+. Anal. (C23H34N4O7) C, H, N.
1H NMR ((CD3)2SO): d = 1.13–1.22 (m, 6H), 1.22–1.38 (m, 2H),
1.39–1.74 (m, 4H), 1.83 (s, 3H), 2.55–2.59 (m, 3H), 3.08–3.19 (m,
2H), 3.81 (s, 3H), 4.12–4.29 (m, 3H), 5.94–6.01 (m, 1H), 6.69–
6.76 (m, 1H), 6.83–6.88 (m, 1H), 7.69–7.76 (m, 1H), 7.80–7.87
(m, 1H), 7.89–7.98 (m, 2H), 8.05–8.11 (m, 1H). 13C NMR
((CD3)2SO): d = 17.99, 18.30, 22.47, 22.87, 25.54, 29.12, 31.44,
36.10, 38.29, 48.09, 48.33, 52.56, 106.45, 111.90, 125.74, 127.37,
161.24, 169.23, 171.09, 172.35, 172.59. ESI-MS (m/z): 451.3
[M+H]+, 473.4 [M+Na]+. Anal. (C21H34N6O5ꢁ0.9H2Oꢁ0.1hexane) C,
H, N.
5.6.29. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(3,3-dimethylureido)hexanamido)propanoate (78)
1H NMR (CD3OD): d = 1.33 (d, 3J = 7.2 Hz, 3H), 1.39 (d,
3J = 7.3 Hz, 3H), 1.37–1.56 (m, 4H), 1.63–1.87 (m, 2H), 1.97 (s,
3H), 2.88 (s, 6H), 3.14–3.17 (m, 2H), 3.71 (s, 3H), 4.30–4.41 (m,
3H). 13C NMR (CD3OD): d = 17.29, 17.90, 22.40, 23.94, 31.01,
32.89, 36.45, 36.45, 41.52, 49.51, 50.54, 52.71, 54.33, 161.20,
173.21, 174.04, 174.48, 175.13. ESI-MS (m/z): 416.1 [M+H]+. Anal.
(C18H33N5O6ꢁ1.0H20ꢁ1.0MeOH) C, H, N.
5.6.24. N-((S)-5-((S)-2-Acetamidopropanamido)-6-((S)-1-(meth-
ylamino)-1-oxopropan-2-ylamino)-6-oxohexyl)-4-(dimeth-
ylamino)benzamide (72)
5.6.30. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(methylsulfonamido)hexanamido)propanoate (79)
1H NMR ((CD3)2SO): d = 1.13–1.22 (m, 6H), 1.22–1.38 (m, 2H),
1.39–1.74 (m, 4H), 1.83 (s, 3H), 2.54–2.59 (m, 3H), 2.95 (s, 6H),
3.14–3.23 (m, 2H), 4.12–4.29 (m, 3H), 6.68 (d, 3J = 8.9 Hz, 2H),
7.70 (d, 3J = 8.9 Hz, 2H), 7.69–7.75 (m, 1H), 7.81–7.87 (m, 1H),
7.92–7.97 (m, 1H), 8.05–8.12 (m, 2H). 13C NMR ((CD3)2SO):
d = 17.99, 18.30, 22.49, 22.89, 25.55, 29.10, 31.42, 38.91, 39.75,
48.11, 48.35, 52.59, 110.75, 121.43, 128.45, 151.97, 165.99,
169.26, 171.12, 172.36, 172.61. ESI-MS (m/z): 491.3 [M+H]+,
513.4 [M+Na]+. Anal. (C24H38N6O5ꢁ1.5H2O) C, H, N. HPLC: tR
9.31 min, area percent 82% at 260 nm.
1H NMR ((CD3)2SO): d = 1.17 (d, 3J = 7.1 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.21–1.35 (m, 2H), 1.38–1.71 (m, 4H), 1.83 (s,
3H), 2.87 (s, 3H), 2.85–2.93 (m, 2H), 3.61 (s, 3H), 4.18–4.29 (m,
3H), 6.84–6.93 (m, 1H), 7.78–7.87 (m, 1H), 7.98–8.06 (m, 1H),
8.19–8.28 (m, 1H). 13C NMR ((CD3)2SO): d = 16.79, 18.03, 22.32,
22.47, 29.16, 31.17, 39.21, 42.45, 47.53, 48.21, 51.83, 51.83
169.16, 171.42, 172.22, 172.91. ESI-MS (m/z): 423.1 [M+H]+,
445.1 [M+Na]+. Anal. (C16H30N4O7S) C, H, N.
5.6.31. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(trifluoromethylsulfonamido)hexanamido)propanoate (80)
1H NMR ((CD3)2SO): d = 1.17 (d, 3J = 7.1 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.23–1.37 (m, 2H), 1.42–1.72 (m, 4H), 1.82 (s, 3H),
3.06–3.15 (m, 2H), 3.61 (s, 3H), 4.19–4.29 (m, 3H), 7.80–7.88 (m,
1H), 7.99–8.07 (m, 1H), 8.21–8.29 (m, 1H), 9.27–9.38 (m, 1H). 13C
NMR ((CD3)2SO): d = 17.28, 17.75, 22.35, 23.44, 30.91, 32.66, 44.75,
5.6.25. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(2-
chloroacetamido)hexanamido)propanoate (73)
1H NMR (CD3OD): d = 1.33 (d, 3J = 7.2 Hz, 3H), 1.39 (d,
3J = 7.3 Hz, 3H), 1.41–1.49 (m, 2H), 1.51–1.62 (m, 2H), 1.63–1.89
(m, 2H), 1.98 (s, 3H), 3.18–3.29 (m, 2H), 3.72 (s, 3H), 4.04 (s, 2H),
4.26–4.45 (m, 3H). 13C NMR (CD3OD): d = 17.28, 17.84, 22.39,
23.83, 29.77, 32.76, 40.51, 43.23, 49.62, 50.59, 52.74, 54.14,
169.29, 173.26, 173.94, 174.52, 175.16. ESI-MS (m/z):
421.07[M+H]+, 443.24 [M+Na]+. Anal. (C17H29ClN4O6) C, H, N.
1
49.45, 50.67, 52.76, 54.02, 121.50 (q, JCF = 321.05 Hz), 173.34,
173.85, 174.53, 175.19. ESI-MS (m/z): 477.06 [M+H]+, 499.22
[M+Na]+. Anal. (C16H27F3N4O7Sꢁ0.1hexane) C, H, N.
5.6.32. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(4-
methylphenylsulfonamido)hexanamido)propanoate (81)
1H NMR (CD3OD): d = 1.32 (d, 3J = 7.2 Hz, 3H), 1.38 (d,
3J = 7.3 Hz, 3H), 1.35–1.43 (m, 2H), 1.43–1.52 (m, 2H), 1.55–1.82
(m, 2H), 1.97 (s, 3H), 2.43 (s, 3H), 2.79–2.87 (m, 2H), 3.71 (s, 3H),
4.26–4.42 (m, 3H), 7.37 (d, 3J = 7.9 Hz, 2H), 7.72 (d, 3J = 8.2 Hz,
2H). 13C NMR (CD3OD): d = 17.28, 17.78, 21.42, 22.39, 23.39,
30.17, 32.65, 43.79, 49.43, 50.60, 52.54, 54.11, 128.05, 128.05,
130.71, 130.71, 138.95, 144.56, 173.29, 173.90, 174.53, 175.13.
ESI-MS (m/z): 499.08 [M+H]+, 521.27 [M+Na]+. Anal.
(C22H34N4O7Sꢁ0.1hexane) C, H, N.
5.6.26. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(2-
bromoacetamido)hexanamido)propanoate (74)
1H NMR (CD3OD): d = 1.34 (d, 3J = 7.2 Hz, 3H), 1.39 (d,
3J = 7.3 Hz, 3H), 1.41–1.49 (m, 2H), 1.50–1.61 (m, 2H), 1.62–1.89
(m, 2H), 1.98 (s, 3H), 3.15–3.27 (m, 2H), 3.71 (s, 3H), 3.83 (s, 2H),
4.26–4.45 (m, 3H). 13C NMR (CD3OD): d = 17.29, 17.85, 22.41,
23.82, 28.89, 29.66, 32.75, 40.65, 49.44, 50.61, 52.75, 54.13,
169.48, 173.25, 173.94, 174.54, 175.17. ESI-MS (m/z): 465.13
[M+H]+, 487.30 [M+Na]+. Anal. (C17H29BrN4O6) C, H, N.
5.6.27. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(methoxycarbonylamino)hexanamido)propanoate (75)
5.6.33. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(4-
nitrophenylsulfonamido)hexanamido)propanoate (82)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.1 Hz, 3H), 1.20–1.32 (m,
2H), 1.27 (d, 3J = 7.3 Hz, 3H), 1.33–1.42 (m, 2H), 1.46–1.68 (m,
2H), 1.83 (s, 3H), 2.92–2.95 (m, 2H), 3.50 (s, 3H), 3.60 (s, 3H),
4.20–4.28 (m, 3H), 7.01–7.09 (m, 1H), 7.79–7.85 (m, 1H), 7.99–
8.05 (m, 1H), 8.21–8.27 (m, 1H). 13C NMR ((CD3)2SO): d = 16.79,
18.04, 22.38, 22.47, 29.18, 31.74, 40.18, 47.53, 48.20, 51.11,
51.81, 51.92, 169.16, 171.44, 172.21, 172.21, 172.91. ESI-MS (m/
z): 425.3 [M+Na]+. Anal. (C17H30N4O7ꢁ0.1H2O) C, H, N.
1H NMR (CD3OD): d = 1.33 (d, 3J = 7.2 Hz, 3H), 1.38 (d,
3J = 7.3 Hz, 3H), 1.36–1.44 (m, 2H), 1.44–1.55 (m, 2H), 1.55–1.82
(m, 2H), 1.97 (s, 3H), 2.88–2.98 (m, 2H), 3.71 (s, 3H), 4.24–4.43
(m, 3H), 8.08 (d, 3J = 8.8 Hz, 2H), 8.41 (d, 3J = 8.7 Hz, 2H). 13C
NMR (CD3OD): d = 17.28, 17.77, 22.38, 23.57, 30.21, 32.63, 43.86,
49.51, 50.63, 52.78, 54.02, 125.43, 125.43, 129.39, 129.39, 147.95,
151.42, 173.32, 173.85, 174.54, 175.15. ESI-MS (m/z): 530.04
[M+H]+, 552.22 [M+Na]+. Anal. (C21H31N5O9Sꢁ0.5H2O) C, H, N.
5.6.28. (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-
(benzyloxycarbonylamino)hexanamido)propanoate (76)
1H NMR ((CD3)2SO): d = 1.16 (d, 3J = 7.1 Hz, 3H), 1.27 (d,
3J = 7.3 Hz, 3H), 1.21–1.33 (m, 2H), 1.34–1.70 (m, 4H), 1.82 (s,
3H), 2.91- 3.02 (m, 2H), 3.60 (s, 3H), 4.18–4.30 (m, 3H), 4.99 (s,
2H), 7.17–7.25 (m, 1H), 7.27–7.41 (m, 5H), 7.79–7.86 (m, 1H),
7.99–8.06 (m, 1H), 8.21–8.28 (m, 1H). 13C NMR ((CD3)2SO):
5.6.34. (S)-Benzyl 6-(tert-butoxycarbonylamino)-1-oxo-1-
(phenylamino)hexan-2-ylcarbamate (83)
1H NMR ((CD3)2SO): d = 1.19–1.42 (m, 4H), 1.35 (s, 9H), 1.62 (m,
2H), 2.89 (m, 2H), 4.11 (m, 1H), 5.03 (s, 2H), 6.78 (br, 1H), 7.04 (t,
3J = 7.3 Hz, 1H), 7.17–7.37 (m, 7H), 7.55 (d, 3J = 7.7 Hz, 1H), 7.60 (d,
3J = 8.1 Hz, 2H), 10.01 (s, 1H). 13C NMR ((CD3)2SO): d = 22.9, 28.3,

5624
T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
29.2, 31.5, 39.6, 55.4, 65.4, 77.3, 119.2, 123.2, 127.7, 127.8, 128.3,
128.7, 137.0, 139.0, 155.6, 156.1, 171.1. ESI-MS (m/z): 456.01
[M+H]+, 478.30 [M+Na]+. Anal. (C25H33N3O5) C, H, N.
SIRT2 assays differ in their active enzyme concentrations and
consequently, SIRT1 and SIRT2 IC50 values cannot be directly
compared.
5.6.35. (S)-Benzyl 6-ethanethioamido-1-oxo-1-
Acknowledgments
(phenylamino)hexan-2-ylcarbamate (84)
1H NMR ((CD3)2SO): d = 1.29–1.47 (m, 2H), 1.50–1.72 (m, 4H),
2.36 (s, 3H), 3.45 (m, 2H), 4.14 (m, 1H), 5.03 (s, 2H), 7.05 (m,
1H), 7.17–7.37 (m, 7H), 7.57–7.61 (m, 3H), 9.96 (br, 1H), 10.02 (s,
1H). 13C NMR ((CD3)2SO): d = 22.74, 27.10, 31.87, 34.28, 45.73,
55.28, 67.58, 120.24, 120.24, 124.91, 128.19, 128.19 128.53,
128.78, 128.78, 129.19, 129.19, 136.03, 137.44, 156.88, 170.02,
201.22. ESI-MS (m/z): 414.24 [M+H]+, 436.26 [M+Na]+. Anal.
(C22H27N3O3S) C, H, N.
We thank Professor Kristina Luthman for helpful discussions
during this work and M.Sc. Marko Lehtonen, M.Sc. Heikki Salo, Miia
Reponen, Tiina Koivunen and Pekka Savolainen for their skillful
assistance. We thank the Graduate School of Drug Discovery, Acad-
emy of Finland (Grant No. 127062), Orion Farmos Research Foun-
dation, Finnish Cultural Foundation, Emil Aaltonen Foundation
and the Saastamoinen Foundation for financial support. We thank
Biocenter Kuopio for the facilities for this study.
5.6.36. (S)-Benzyl 6-(dimethylphosphorothioylamino)-1-oxo-1-
(phenylamino)hexan-2-ylcarbamate (86)
Supplementary data
1H NMR ((CD3)2SO): d = 1.26–1.49 (m, 4H), 1.57–1.72 (m, 2H),
1.62 (s, 3H), 1.65 (s, 3H), 2.78 (m, 2H), 4.10–4.19 (m, 2H), 5.03 (s,
2H), 7.04 (t, 3J = 7.3 Hz, 1H), 7.18–7.37 (m, 7H), 7.57 (d,
3J = 8.1 Hz, 1H), 7.60 (d, 3J = 7.7 Hz, 2H), 10.02 (s, 1H). 13C NMR
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.035.
References and notes
1
1
((CD3)2SO): d = 22.7 (d, JCP = 2.9 Hz), 23.0, 23.4 (d, JCP = 3.1 Hz),
30.9, 31.0, 31.6, 55.5, 65.4, 119.2, 123.3, 127.7, 127.8, 128.3,
128.7, 137.0, 139.0, 156.1, 171.1. ESI-MS (m/z): 448.25 [M+H]+.
Anal. (C22H30N3O3PS) C, H, N.
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M SIRT1- or 138 M SIRT2-peptide substrate (concentrations
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M NAD+ (N6522, Sigma), recombi-
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M.33
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l
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0.01 M, 0.001
l
M, 1000
l
M, 100
lM, 10 lM, 1 lM, 0.1 lM,
l
l
M and 0.0001 l
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T. Huhtiniemi et al. / Bioorg. Med. Chem. 18 (2010) 5616–5625
5625
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