N-(3-(4-Hydroxyphenyl)-propenoyl)-amino acid tryptamides as SIRT2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.023

A series of N-(3-(4-hydroxyphenyl)-propenoyl)-amino acid tryptamides was based on a previously reported new SIRT2 inhibitor from our group, and it was designed to study if the molecular size of the compound could be reduced. The most potent compounds, N-(3-(4-hydroxyphenyl)-propenoyl)-2-aminoisobutyric acid tryptamide and N-(3-(4-hydroxyphenyl)-propenoyl)-l-alanine tryptamide, were equipotent, 30% smaller in molecular weight, and slightly more selective (SIRT2/SIRT1) than the parent compound.

Full text of DOI:10.1016/j.bmcl.2007.02.023

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2448–2451
N-(3-(4-Hydroxyphenyl)-propenoyl)-amino acid tryptamides
as SIRT2 inhibitors
Pa¨ivi H. Kiviranta,a,* Jukka Leppa¨nen,a Valtteri M. Rinne,a Tiina Suuronen,b
Olga Kyrylenko,b Sergiy Kyrylenko,b,ꢀ Erkki Kuusisto,b Anu J. Tervo,a,ꢁ
a
a,§
Tomi Ja¨rvinen,a Antero Salminen,b,c Antti Poso and Erik A. A. Wallen
´
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
bDepartment of Neuroscience and Neurology, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
cDepartment of Neurology, Kuopio University Hospital, PO Box 1777, 70211 Kuopio, Finland
Received 24 January 2007; accepted 9 February 2007
Available online 13 February 2007
Abstract—A series of N-(3-(4-hydroxyphenyl)-propenoyl)-amino acid tryptamides was based on a previously reported new SIRT2
inhibitor from our group, and it was designed to study if the molecular size of the compound could be reduced. The most potent
compounds, N-(3-(4-hydroxyphenyl)-propenoyl)-2-aminoisobutyric acid tryptamide and N-(3-(4-hydroxyphenyl)-propenoyl)-L-ala-
nine tryptamide, were equipotent, 30% smaller in molecular weight, and slightly more selective (SIRT2/SIRT1) than the parent
compound.
Ó 2007 Elsevier Ltd. All rights reserved.
Silent information regulator human type 2 (SIRT2) en-
zyme belongs to the class III histone deacetylase
(HDAC) protein family.1,2 SIRT2, which is located prin-
cipally in the cytoplasm of brain and muscle cells,3 is one
of the seven human sirtuin type homologues.1,4 The
class III proteins are nicotinamide adenine dinucleotide
(NAD+) dependent proteins, from which SIRT2 has
been shown to catalyze the deacetylation of an acetylat-
ed lysine-40 of a-tubulin, resulting in free nicotinamide
and 20- and 30-O-acetyl-ADP-ribose.1,5–7 The Sir2 pro-
tein family have been connected with several cellular
functions, for example, chromatin silencing, cell cycle,
metabolism, and life span.1,8–10 It has been shown that
SIRT2 is a mitotic checkpoint protein11 and it interacts
with the homeobox transcription factor, HOXA10.12 It
has been postulated that inhibitors of SIRT2 might be
beneficial in the treatment of cancer and neurodegener-
ative diseases.13,14
The NAD+-dependent deacetylase activity is inhibited
by nicotinamide. A few other SIRT2 inhibitors have
also been reported. A3 and sirtinol were the first potent
SIRT2 inhibitors.15 1,4-Bis-[2-(4-hydroxy-phenyl)-eth-
ylamino]-anthraquinone,16 para-sirtinol,17 EX-527,18
cambinol,13 and Ro 31-822019 have been reported
recently.
Based on our previous results16,20 and examinations of
favorable interactions between the known SIRT2 inhib-
itors and the putative binding site, a receptor-based vir-
tual screening was carried out. Compound 1 (Tripos
360702) was the most potent compound that was found
Keywords: SIRT2; HDAC; Inhibitor.
*
H
N
Corresponding author. Tel.: +358 17 163714; fax: +358 17 162456;
e-mail: paivi.kiviranta@uku.fi
O
H
N
ꢀ
N
H
Present address: Department of Biochemistry, University of Kuopio,
PO Box 1627, 70211 Kuopio, Finland.
NH
O
ꢁ
Present address: AstraZeneca R&D Mo¨lndal, SC2, Pepparedsleden
1, 43183 Mo¨lndal, Sweden.
O
F
F
§
Present address: Division of Pharmaceutical Chemistry, Faculty of
Pharmacy, PO Box 56, 00014, University of Helsinki, Finland.
Figure 1. Compound 1 found by modeling and virtual screening.21
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.023

P. H. Kiviranta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2448–2451
2449
in this study and it had an IC50 of 51 lM (Fig. 1).21
Compound 1 had a structural scaffold that was new
for SIRT2 inhibitors. The desire to reduce the molecular
size of compound 1 was the main focus for the present
study. The design of the new series of compounds was
based on simplifying the structure and finding the essen-
tial parts of 1 for the inhibitory activity.
(6) was synthesized by protecting the phenolic hydrox-
yl group with acetic anhydride. Compound 7a was
synthesized from 5 with ethyl chloroformate activation
followed by reaction with 4a.23 The removal of the Boc
group yielded compound 7a. To form compound 7b,
compound 5 was reacted with oxalyl chloride to form
the acid chloride which was reacted with 2-amino-
isobutyric acid tryptamide (4b) in DCM. Compounds
7d, [S]-7e, [R]-7e, and 7g were also synthesized from
6 with ethyl chloroformate activation followed by reac-
tion with the appropriate amino acid tryptamide.23
Compound 7f was synthesized from compound 6 with
DCC activation followed by reaction with glycine try-
ptamide (4f). The acetyl protection group was hydro-
lyzed with K2CO3 in water and methanol or
CH3ONa in methanol, yielding compounds 7d–7g. In
most amide formation reactions ethyl chloroformate
activation was found to be the most successful method.
The yields of the amide bond formations varied be-
tween 14% and 93%.
The synthetic routes are presented in Scheme 1. The
starting material 4-amino-1-Boc-piperidine-4-carboxyl-
ic acid was synthesized as described in the literature.22
The amino groups of the amino acids 2a, 2b, and 2f
were protected with benzyl chloroformate. The carbox-
ylic acid was activated with ethyl chloroformate23 or
DCC24 and reacted with tryptamine. N-Cbz-L-alanine
N-hydroxysuccinimide ester and N-Cbz-D-alanine
N-hydroxysuccinimide ester were reacted with trypt-
amine in THF.25 The Cbz group was removed with
palladium (10%) on activated charcoal and either
ammoniumformate or hydrogen gas in methanol. 3-
(4-Benzyloxyphenyl)-propenoic acid (5) was synthe-
sized from 3-(4-hydroxyphenyl)-propenoic acid and
benzyl bromide. 3-(4-Acetoxyphenyl)-propenoic acid
Another synthetic procedure was used for compound
7c. 3-(4-Methoxyphenyl)-propenoic acid (8) was syn-
R2 R3
H
R2 R3
O
H
N
N
H2N
R2
R3
OH
O
O
NH
O
N
H
b
NH
O
a
O
O
N
H
R4
R4
2a, 2b, and 2f
4a, 4b, [S]- and [R]-4e, 4f, and 4g
3a, 3b, [S]- and [R]-3e, 3f, and 3g
R2 R3
O
O
H
N
5: R1 = CH2-Ph
6: R1 = CO-CH3
c
OH
N
H
4b or [S]- and [R]-4e or 4f or 4g
NH
f
O
O
R1
HO
R4
5, 6
7d, [S]- and [R]-7e, 7f, and 7g
e
4b
O
c
4a
H
N
N
H
NH
O
O
7b
d
H
N
2a-4a: R2, R3= (CH2)2-NBoc-(CH2)2, and R4= H
O
H
2b-4b: R2, and R3= CH3, and R4= H
N
N
H
7d: R2, and R3= CH3, and R4= H
NH
O
[S]-3e, [S]-4e, [S]-7e: R2= CH3, R3= H, and R4= H
[R]-3e, [R]-4e, [R]-7e: R2= H, R3= CH3, and R4= H
O
7a
2f-4f, 7f: R2, R3, and R4= H
3g, 4g, 7g: R2, and R3= CH3, and R4= F
O
O
O
H
N
g
h, i
O
OH
N
H
N
H
NH
O
O
9
7c
8
O
O
O
Scheme 1. Reagents and conditions: (a) 1—Et3N, ethyl chloroformate, DCM or THF, -20 or 0 °C, 2—Et3N, tryptamine; or DMAP, tryptamine,
DCC, CHCl3, 0 °C to reflux (N-Cbz-L-alanine N-hydroxysuccinimide ester and N-Cbz-D-alanine N-hydroxysuccinimide ester were reacted with
tryptamine in THF, yielding compounds [S]- and [R]-3e.); (b) ammoniumformate, 10% Pd/C, MeOH; or H2, 10% Pd/C, MeOH; (c) 1—Et3N, ethyl
chloroformate, THF or DCM, -20 or 0 °C, 2—Et3N, 4a or 4b or [S]-4e or [R]-4e or 4g; or DMAP, 4f, DCC, DCM, and 1,4-dioxane, 0 °C; (d) TFA,
anisole, sodium thiophenolate, DCM, 0 °C; (e) 1—oxalyl chloride, DMF, DCM; 2—4b, Et3N, DCM; (f) K2CO3, H2O, MeOH, 0 °C; or 1M CH3ONa
in MeOH, DCM, 0 °C; (g) 1—Et3N, pivaloyl chloride, DCM, 0–25 °C, 2—Et3N, 2-amino-isobutyric acid methyl ester; (h) LiOHÆH2O, H2O, MeOH;
(i) 1—Et3N, ethyl chloroformate, DCM, -20 °C, 2—Et3N, tryptamine.

2450
P. H. Kiviranta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2448–2451
thesized as described in the literature.26 Compound 8
was activated with pivaloyl chloride and reacted with
2-amino-isobutyric acid methyl ester. The methyl ester
of the obtained product 9 was hydrolyzed with LiO-
HÆH2O in water and methanol. The obtained free car-
boxylic acid was activated with ethyl chloroformate
and reacted with tryptamine to yield compound 7c.
This synthetic procedure was not useful when 3-(4-
hydroxyphenyl)-propenoic acid was used because the
removal of the methyl ester would also have removed
the acetyl protection group of the phenolic hydroxyl
group. EX-527 was used as a reference compound.
EX-527 was synthesized as described in the
literature.18
resulting in 7d. Compound 7d was equipotent with 1
and it showed that the chemical structure could be re-
duced in size without affecting the inhibitory activity
for SIRT2. The molecular weight of 1 was reduced
about 30%. Replacing the aminoisobutyric acid group
by an L-alanine group gave [S]-7e, which had an inhi-
bition of 88.6% at 200 lM and an IC50 of 47 lM. Com-
pound [S]-7e showed also selectivity for SIRT2.
However, replacing the aminoisobutyric acid group
by a D-alanine group gave [R]-7e, with an inhibition
of 8.5% at 200 lM. The structure was further simpli-
fied by replacing the alanine group by a glycine group
resulting in 7f, which was less potent than a L-alanine
group but more potent than a D-alanine group. Final-
ly, the effect of the fluorine atom on the indole ring was
studied with 7g. Compound 7g showed that the fluo-
rine atom does not have a positive effect on the inhib-
itory activity since 7g had a slightly lower inhibitory
activity compared to 7d.
The compounds and their inhibitory activities are pre-
sented in Table 1. The inhibitory activities were tested
in a Fluor de Lys fluorescence-based assay. Poor water
solubility of some compounds was observed when
determining the inhibition at higher concentrations.
This was the main reason not to determine the IC50
values of the compounds with less than 50% inhibition
at 200 lM. The importance of the fluorine atoms for
the inhibitory activity of 1 was investigated with 7a.
Removal of the fluorine atoms did not have a signifi-
cant effect on the inhibitory activity. Opening the
piperidine ring and removing the amine functionality
in the side-chain by using a 2-aminoisobutyric acid
group in the middle of the structure resulted in 7b, with
a significantly lower inhibitory activity, inhibition of
32.6% at 200 lM. Furthermore, removal of the phenyl
group resulted in 7c, which was slightly more potent,
with an inhibition of 55.4% at 200 lM and an IC50
of 99 lM. The structure was further simplified by
removing the methyl group from the methoxy group,
In addition, the selectivity for SIRT2 was studied (Table
1). As earlier reported, SIRT2 inhibitors are also good
SIRT1 inhibitors. Interestingly, compound 7d showed
a slightly higher selectivity for SIRT2 than 1. At the con-
centration of 200 lM compound 1 inhibits SIRT1 94.1%
but 7d only 6.6%. Compound [S]-7e is almost as selec-
tive as 7d.
In conclusion, the essential parts of compound 1 for the
inhibitory activity were identified, and the study showed
that the molecular weight of compound 1 could be re-
duced 30% while maintaining the inhibitory activity.
In addition, the most potent compounds 7d and [S]-7e
were slightly more selective for SIRT2 (SIRT2/SIRT1)
than compound 1.
Table 1. Compounds and their inhibitory activities for SIRT2 and SIRT1 (95% confidence intervals for IC50 given in parentheses)
R2
R3
O
O
H
N
N
H
NH
R1
O
R4
7
Compound
R1
R2
R3
R4 Inhibition at
Inhibition at
IC50 (lmol/L) IC50
200 lM SD,a (%) 200 lM SD,a (%) SIRT2b
(lmol/L)
SIRT2
SIRT1
SIRT1b
1
7a
CH2-Ph-F (CH2)2–NH–(CH2)2
(CH2)2–NH–(CH2)2
F
77.3 4.8
79.1 1.4
32.6 18.2
55.4 3.3
83.3 3.7
88.6 0.8
8.5 6.0
94.1 3.5
97.0 1.9
10.2 6.4
10.3 4.3
6.6 1.8
17.7 1.0
3.2 5.5
9.9 1.2
7.0 4.0
98.9 0.3
51 (27–75)
63 (41–96)
—
73 (47–114)
52 (38–70)
CH2–Ph
CH2–Ph
H
H
H
H
H
H
H
F
7b
7c
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
H
—
—
CH3
H
99 (66–150)
50 (23–109)
47 (28–79)
—
7d
[S]-7e
[R]-7e
—
—
H
H
CH3
H
—
—
7f
7g
H
H
CH3
44.3 10.6
67.5 1.1
89.4 2.8
—
80 (53–120)
14 (8–25)
H
CH3
—
0.28 (0.23–0.34)
1018 EX-527
Cl
N
NH2
H
O
a SD, standard deviation.
b IC50 were determined for compounds which had over 50% inhibition at 200 lM for SIRT2 or SIRT1.

P. H. Kiviranta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2448–2451
2451
11. Dryden, S. C.; Nahhas, F. A.; Nowak, J. E.; Goustin, A.
S.; Tainsky, M. A. Mol. Cell Biol. 2003, 23, 3173.
12. Bae, N. S.; Swanson, M. J.; Vassilev, A.; Howard, B. H.
J. Biochem. (Tokyo) 2004, 135, 695.
13. Heltweg, B.; Gatbonton, T.; Schuler, A. D.; Posak-
ony, J.; Li, H.; Goehle, S.; Kollipara, R.; DePinho,
R. A.; Gu, Y.; Simon, J. A.; Bedalov, A. Cancer
Res. 2006, 66, 4368.
14. Grubisha, O.; Rafty, L. A.; Takanishi, C. L.; Xu, X.;
Tong, L.; Perraud, A. L.; Scharenberg, A. M.; Denu, J. M.
J. Biol. Chem. 2006, 281, 14057.
15. Grozinger, C. M.; Chao, E. D.; Blackwell, H. E.; Moazed,
D.; Schreiber, S. L. J. Biol. Chem. 2001, 276, 38837.
16. Tervo, A. J.; Kyrylenko, S.; Niskanen, P.; Salminen, A.;
Leppanen, J.; Nyronen, T. H.; Jarvinen, T.; Poso, A.
J. Med. Chem. 2004, 47, 6292.
Acknowledgments
We thank Prof. Jouko Vepsa¨la¨inen for guidance in
NMR, and Tiina Koivunen, and Ulrich Bergmann for
their skilful technical assistance, and we thank the Grad-
uate School of Drug Discovery, Finnish Funding Agency
for Technology and Innovation, the Academy of Finland
(Grant No. 108649 to E.A.A.W., Grant No. 106313 to
J.L., and Grant No. 111453 to A.P.), Association of
Finnish Chemical Societies, and the Gustav Komppa
Fund of the Kordelin Foundation for financial support.
Supplementary data
17. Mai, A.; Massa, S.; Lavu, S.; Pezzi, R.; Simeoni, S.;
Ragno, R.; Mariotti, F. R.; Chiani, F.; Camilloni, G.;
Sinclair, D. A. J. Med. Chem. 2005, 48, 7789.
18. Napper, A. D.; Hixon, J.; McDonagh, T.; Keavey,
K.; Pons, J. F.; Barker, J.; Yau, W. T.; Amouzegh,
P.; Flegg, A.; Hamelin, E.; Thomas, R. J.; Kates,
M.; Jones, S.; Navia, M. A.; Saunders, J. O.;
DiStefano, P. S.; Curtis, R. J. Med. Chem. 2005,
48, 8045.
Detailed experimental procedures for the syntheses,
NMR spectra, ESI-MS results, and elemental analysis
data for the new compounds, and procedures obtaining
human SIRT1 and SIRT2 recombinant proteins, and
in vitro assay for SIRT2 activity are available. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2007.02.023.
19. Trapp, J.; Jochum, A.; Meier, R.; Saunders, L.; Marshall,
B.; Kunick, C.; Verdin, E.; Goekjian, P.; Sippl, W.; Jung,
M. J. Med. Chem. 2006, 49, 7307.
References and notes
20. Kiviranta, P. H.; Leppanen, J.; Kyrylenko, S.; Salo, H.
S.; Lahtela-Kakkonen, M.; Tervo, A. J.; Wittekindt, C.;
Suuronen, T.; Kuusisto, E.; Jarvinen, T.; Salminen, A.;
Poso, A.; Wallen, E. A. A. J. Med. Chem. 2006, 49,
7907.
21. Tervo, A. J.; Suuronen, T.; Kyrylenko, S.; Kuusisto, E.;
Kiviranta, P. H.; Salminen, A.; Leppanen, J.; Poso, A.
J. Med. Chem. 2006, 49, 7239.
22. Wysong, C. L.; Yokum, T. S.; Morales, G. A.; Gundry, R.
L.; McLaughlin, M. L.; Hammer, R. P. J. Org. Chem.
1996, 61, 7650.
23. Sam, J.; Minor, W. F.; Perron, Y. G. J. Am. Chem. Soc.
1959, 81, 710.
24. Jung, M. E.; Gervay, J. J. Am. Chem. Soc. 1991, 113, 224.
25. Garcia-Lopez, M. T.; Gonzalez-Muniz, R.; Molinero, M.
T.; Naranjo, J. R.; Del Rio, J. J. Med. Chem. 1987, 30,
1658.
26. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.;
Tatchell, A. R. Vogel’s Textbook of Practical Organic
Chemistry; Longman Scientific & Technical: New York,
1989, pp 1040–1041.
1. Sauve, A. A.; Wolberger, C.; Schramm, V. L.; Boeke, J. D.
Annu. Rev. Biochem. 2006, 75, 435.
2. Smith, J. S.; Brachmann, C. B.; Celic, I.; Kenna, M. A.;
Muhammad, S.; Starai, V. J.; Avalos, J. L.; Escalante-
Semerena, J. C.; Grubmeyer, C.; Wolberger, C.; Boeke, J.
D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6658.
3. Afshar, G.; Murnane, J. P. Gene 1999, 234, 161.
4. Frye, R. A. Biochem. Biophys. Res. Commun. 2000, 273,
793.
5. Tanner, K. G.; Landry, J.; Sternglanz, R.; Denu, J. M.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14178.
6. Smith, B. C.; Denu, J. M. Biochemistry 2006, 45, 272.
7. North, B. J.; Marshall, B. L.; Borra, M. T.; Denu, J. M.;
Verdin, E. Mol. Cell 2003, 11, 437.
8. Brachmann, C. B.; Sherman, J. M.; Devine, S. E.;
Cameron, E. E.; Pillus, L.; Boeke, J. D. Genes Dev.
1995, 9, 2888.
9. Guarente, L. Genes Dev. 2000, 14, 1021.
10. Lin, S. J.; Ford, E.; Haigis, M.; Liszt, G.; Guarente, L.
Genes Dev. 2004, 18, 12.