Novel cyclized Pifithrin-α p53 inactivators: Synthesis and biological studies

Article abstract of DOI:10.1016/j.bmcl.2005.01.075

Starting from various cyclic or bicyclic ketones, we have synthesized novel Pifithrin-α analogues bearing different methyl substituted phenyl ketone groups at the N₃-position of the 2-iminothiazole heterocycle. From stability studies in a biolo

Full text of DOI:10.1016/j.bmcl.2005.01.075

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1561–1564
Novel cyclized Pifithrin-a p53 inactivators:
synthesis and biological studies
a
Nicolas Pietrancosta, Flavio Maina, Rosanna Dono, Anice Moumen, C e´ drik Garino,
b
b
b
a
a
Younes Laras, St e´ phane Burlet, Gilles Qu e´ l e´ ver and Jean-Louis Kraus
a
a
a,
*
a
Unit e´ INSERM U-623, Laboratoire de Chimie Biomol e´ culaire, IBDM, Facult e´ des Sciences de Luminy,
Universit e´ de la M e´ diterran e´ e, 13288 Marseille Cedex 9, France
b
Unit e´ INSERM U-623, IBDM, Facult e´ des Sciences de Luminy, 13288 Marseille Cedex 9, France
Received 16 November 2004; revised 28 January 2005; accepted 31 January 2005
Abstract—Starting from various cyclic or bicyclic ketones, we have synthesized novel Pifithrin-a analogues bearing different methyl
substituted phenyl ketone groups at the N -position of the 2-iminothiazole heterocycle. From stability studies in a biological med-
3
ium as well as under specific chemical conditions, we have shown by NMR techniques that through a dehydration process, some
derivatives can generate their corresponding cyclized analogues. All of the new analogues, Pifithrin-like and polycyclic dehydrated
derivatives were assessed for their p53 inactivation potency by measuring survival of cortical neurons, whose death was induced by
the DNA-damaging agent etoposide. Pifithrin-a like 2f as well as the cyclic dehydrated 6b analogue were found to be one log more
potent p53 inactivators than reference compound Pft-a, with EC50 values ranging around 30 nM. These results support the finding
that p53 inactivation by Pft-a analogues could be also due to the presence of the cyclic dehydrated Pft-a forms, generated in situ in
the biological assay incubation medium.
Ó 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
S
N
S
N
(
CH )
NH
O
NH
O
2 m
During the last few years, p53 inactivators have received
increasing attention due to their possible application in
several major pathologies such as neurodegenerative dis-
orders (Alzheimerꢀs disease, Parkinsonꢀs disease and
stroke), cancer therapy and other pathologies related
to various signaling pathways. Pft-a 1 (Fig. 1) is the
leading compound of the known p53 inactivators. It
was isolated by screening of a chemical library in a
cell-based read-out system for its ability to reduce p53-
( )n
R1
1
2
3
R2
Pft-α
Pft-α analogues
1
2
2
Figure 1. General structure of new p53 inactivators.
dependent transactivation.
1
In a recent publication, 14 new Pft-a analogues bearing
various methylene substituted aromatic ketones at the
new derivatives were primarily synthesized to assess
the importance of the electrodonating and electrowith-
drawing substitution of the aromatic moiety of the sub-
N -position of the Pft-a scaffold have been described.
3
These analogues were found to be highly potent in pro-
tecting PC12 cells or primary hippocampal neurons
against DNA-damaging agent induced death. These
stituent at the N -position of the bicyclic backbone. In
3
this current study, we were interested in the design of
new Pft-a like derivatives in which, on the one hand,
the tetrahydrobenzo moiety was replaced by its octahy-
dronaphtho counterpart and, on the other hand, new
Keywords: Pft-a; Cortical neuron survival; p53 Inactivators; Dehydra-
tion reaction.
substituents were introduced at the N -position of the
3
*
Corresponding author. Tel.: +33 491 82 91 41; fax: +33 491 82 94
6; e-mail: kraus@luminy.univ-mrs.fr
heterocycle, such as a cyano group on the methylene
group on the acetophenone moiety (Fig. 1).
1
0
960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.075

1562
N. Pietrancosta et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1561–1564
In the course of our synthetic efforts to reach those Pft-a
derivatives, we discovered that some of the desired com-
pounds were dehydrated into their corresponding cyclic
imidazothiazole derivatives. We report the synthesis of
new Pft-a analogues, their chemical and biological sta-
bilities, as well as their p53 inhibitory activities.
after purification by crystallization. Their respective
structures were assigned through NMR and MS analysis
(Scheme 1).
5
Subsequently, the nitro Pft-a derivatives 2b and 2e were
submitted to reduction in order to isolate their aniline
analogues. This reduction process involved the use of
6
Fe/NH Cl as the reductant in refluxed protic solvents.
4
2
. Synthesis and chemical stability
We discovered that instead of the expected amino deriva-
tives 4b and 4e, we isolated mostly their dehydrated tri-
cyclic amino derivatives, 5b and 5e, and their corres-
ponding tricyclic nitro analogues 6b and 6e.
The synthesis of 2-aminothiazole hydroiodide salt
precursors was carried out according to the method
described by King and Hlavacek. The solvent free
4
reaction was performed by heating various cyclic ke-
tones (cyclohexanone, 2-decalone, cycloheptanone) with
thiourea in the presence of iodine. Basic treatment of the
hydroiodide salt allowed the isolation of the correspond-
ing free aminothiazoles 3a–c, which could be N-alkyl-
ated on the endocyclic N -position by the selected
3
a-bromoacetophenone at room temperature in toluene.
These unexpected results were confirmed by stability
study of the opened form of the nitro Pft-a analogues
2b and 2e in a protic solvent (EtOH, i-PrOH). The cor-
responding tricyclic nitro derivatives 6b and 6e resulting
from a dehydration process as suggested in Scheme 2
were isolated in quantitative yields and fully character-
ized. The ability of Pft-a analogues to undergo this
dehydration process depends on the electrodonating or
electrowithdrawing properties of the phenyl ring
The resulting N -substituted 2-iminothiazole derivatives
1
3
, 2a–g were isolated in yields ranging from 40% to 70%
(
i), (ii)
S
N
3
a: n = 1, m = 0
(
CH2)
(CH )
NH2
m
2 m
3b: n = 1, m = 4
3c: n = 2, m = 0
(
)n
( )n
O
(
iii)
1
2
Compound
n
m
R
R
S
N
(
CH2)
NH.HBr
O
m
1 (Pft-α)
1
1
1
1
1
1
1
2
0
0
0
4
4
4
0
0
H
CH
OCH
3
(
)n
2a
H
H
H
H
H
3
2
b
NO
CH
OCH
NO
2
R1
2c
3
2
d
3
3
2
e
2
2
f
CN OCH
CH
R2
2g
H
3
n = 1
(iv)
(
iv)
(v) or (vi)
X
S
N
S
N
S
N
(
CH2)m
NH
(CH )
(CH )
2 m
2 m
N
N
O
R1
R2
R2
2
1
2
5
6
b (m = 0, R = NH )
6 (Pft-β): m = 0, R = H, R = CH
6b: m = 0, R = H, R = NO
e: m = 4, R = H, R = NO
6f: m = 0, R = CN, R = OCH
2
3
2
1
2
b (m = 0, R = NO )
2
2
NH2
1
2
6
2
2
1
2
5
6
e (m = 4, R = NH )
2
3
4
4
b: m = 0
e: m = 4
2
e (m = 4, R = NO )
2
2
Scheme 1. Reagents and conditions: (i) thiourea, I
2
, 110 °C, 12 h; (ii) saturated Na
2
CO
3
, H
2
O; 3a (52%), 3b (47%), 3c (37%), for two steps; (iii) 4-R -
Cl, Fe, EtOH/H
5/3, v/v), reflux, 2 h; mixture of 5b and 6b (9/1); mixture of 5e and 6e (9/1); (v) MeOH, rt; quantitative for 6b and 6e; (vi) biological medium, 37 °C;
quantitative for 6, 6b, 6e, 6f.
1
C
6
H
4
-C(O)-CH(R )Br, toluene, rt, 48 h; 1 (54%), 2a (61%), 2b (55%), 2c (80%), 2d (41%), 2e (47%), 2f (55%), 2g (40%); (iv) NH
4
2
O
(

N. Pietrancosta et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1561–1564
1563
S
N
S
N
S
(
CH2)m
NH
(CH )
2 m
+ H
N
(
CH2)m
N
(
)n
( )n
R2
(
)n
N
R1
R1
R2
-
OH
R1
O
H
O
R2
H O
2
S
N
(
CH2)m
N
(
)n
R1
R2
Scheme 2.
substituents. Compounds with withdrawing substituents
such as a nitro group on the phenyl ring, 2b and 2e, were
found more sensitive to dehydration (few hours at room
temperature) than analogues bearing electrodonating
substituents, which needed a longer time to cyclize
ical medium used in the p53 inhibition assay (DMEM
1
serum, horse serum and glutamate). After extraction
from the biological medium, the residues were analyzed
by NMR.
(
one night in refluxed MeOH). These new cyclic 2-imid-
The NMR analysis showed that the signal correspond-
ing to the protons located at the a-position of the car-
bonyl function (d = 4.7 ppm) disappeared. At the same
time, a single proton signal corresponding to the newly
formed fused cyclic imidazole appeared at 7.3 ppm. In
the case of the a-cyano derivative, we noticed the com-
plete absence of the signal corresponding to the open
form.
azothiazoles represent new substrates suitable for fur-
ther new N-acylation or N-alkylation reactions.
The ability of Pft-a analogues to be dehydrated into
their corresponding cyclic forms led us to study their
biological stabilities.
1
The H NMR characteristic chemical shifts of the acy-
3
. Biological stability
clic and cyclic Pft-a derivatives are summarized in Table
1.
The ability of the new analogues to be dehydrated in
biological experimental conditions leading to the corre-
sponding cyclized analogues, questions about the possi-
bility for these Pft-a cyclic forms to be the bioactive
intermediates for p53 inactivation. We selected three
compounds, nitro derivatives 2b and 2e and the a-cyano
compound 2f as well as the reference compound Pft-a 1
to be incubated for several hours at 37 °C in the biolog-
The stability of the opened form in the assay could be
characterized by the values of the corresponding half-
reaction times. These values, reported in Table 2 varied
noticeably for the different compounds tested: 1, 2b, 2e
and 2f. Analogue 2f bearing a cyano group at the a-po-
sition of the keto function and a methoxy group on the
1
Table 1. H NMR characteristic shifts of Pft open and cyclic forms
S
S
(
CH2)m
NH
O
(CH2)m
N
N
N
Ha
R1
R1
R2
R2
open form
cyclic dehydrated form
1
R
2
R
1
Open form ! cyclic form
m
d(H
a
) ppm
d(R ) ppm
1
2
2
2
! 6
0
0
4
0
H
CH
3
4.7
4.8
4.8
5.7
7.3
7.3
7.4
—
b ! 6b
e ! 6e
f ! 6f
H
NO
NO
2
H
2
CN
OCH
3

1564
N. Pietrancosta et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1561–1564
1
Table 2. Stabilities of new Pifithrin-like derivatives in biological
a
medium
5. Open form analogues: 1 (Pft-a): H NMR (250 MHz,
DMSO-d ) d 9.60 (s, 1H), 7.95 (d, 2H, J = 8.2 Hz), 7.45 (d,
2H, J = 8.2 Hz), 4.70 (s, 2H), 2.51–2.31 (m, 5H), 1.83–1.79
6
1
R
2
R
Compound
m
t1/2 (h)
(
m, 2H), 1.36 (br s, 1H), 1.02 (d, 3H, J = 6.5 Hz). Anal.
Found: C, 52.33; H, 5.48; N, 7.91. C16 OSÆHBr
1
0
0
4
0
H
CH
3
4 ± 0.5
6 ± 0.5
5 ± 0.5
(
requires C, 52.32; H, 5.21; N, 7.63). ES/MS m/z 287
18 2
H N
2
2
2
b
e
f
H
NO
NO
2
2
H
+
1
(
M+H) . 2a: H NMR (250 MHz, DMSO-d ) d 9.47 (s,
6
CN
OCH
3
12 ± 0.5
1
(
4
H), 8.03 (d, 2H, J = 8.9 Hz), 7.16 (d, 2H, J = 8.9 Hz), 4.67
s, 2H), 3.89 (s, 3H), 2.55 (m, 2H), 2.32 (m, 2H), 1.73 (m,
H). Anal. (Found: C, 50.26; H, 4.83; N, 7.35.
SÆHBr requires C, 50.14; H, 5.00; N, 7.31).
ES/MS m/z 303 (M+H) . 2b: H NMR (250 MHz, DMSO-
d6) d 9.54 (s, 1H), 8.46 (d, 2H, J = 8.9 Hz), 8.28 (d, 2H,
J = 8.9 Hz), 4.80 (s, 2H), 2.56–2.37 (m, 4H), 1.74 (m, 4H).
a
DMEM, horse serum, glutamate.
16 18 2 2
C H N O
phenyl ring, appears to be the most stable analogue
t1/2 = 12 h) while 1 and analogues 2b and 2e have t1/2
+
1
(
values less than 6 h.
Anal. (Found: C, 45.52; H, 3.82; N, 10.52. C15
15 3 3-
H N O
SÆHBr requires C, 45.24; H, 4.05; N, 10.55). ES/MS m/z 318
+
1
(
1
(
6
M+H) . 2c: H NMR (250 MHz, DMSO-d
H), 8.34 (d, 2H, J = 8.9 Hz), 8.18 (d, 2H, J = 8.9 Hz), 4.80
s, 2H), 2.61–1.20 (m, 17H). Anal. (Found: C, 57.17; H,
.18; N, 6.42. C20 OSÆHBr requires C, 57.00; H, 5.98;
6
) d 9.71 (s,
4
. Biological activity: p53 inactivation
The newly synthesized compounds from both series, Pft-
a analogues as well as cyclic dehydrated analogues, were
assayed for their ability to protect mouse embryo corti-
24 2
H N
+
1
N, 6.65). ES/MS m/z 341 (M+H) . 2d: H NMR (250 MHz,
DMSO-d ) 9.47 (s, 1H), 8.03 (d, 2H, J = 8.9 Hz), 7.16 (d,
6
1
cal neurons against a DNA-damaging agent. Pft-a was
2
H, J = 8.9 Hz), 4.67 (s, 2H), 3.89 (s, 3H), 2.55–1.25 (m,
14H). Anal. (Found: C, 55.15; H, 5.46; N, 6.35.
C H N O SÆHBr requires C, 54.92; H, 5.76; N, 6.40).
used as external control (EC50 = 300 nM). In response
to etoposide used as death inducer, neuronal survival,
expressed in EC50 value, was determined for all the
tested compounds. Compounds 2f and 6b were found
to be the most active analogues, with EC50 values
around 30 nM.
2
0
24
2
2
+
1
ES/MS m/z 357 (M+H) . 2e: H NMR (250 MHz, DMSO-
) d 9.71 (s, 1H), 8.34 (d, 2H, J = 8.9 Hz), 8.18 (d, 2H,
J = 8.9 Hz), 4.80 (s, 2H), 2.61–1.20 (m, 14H). Anal.
Found: C, 50.52; H, 4.65; N, 9.19. C H N O SÆHBr
d
6
(
19 21
3
3
requires C, 50.45; H, 4.90; N, 9.29). ES/MS m/z 372
+
1
(
7
3
M+H) . 2f: H NMR (250 MHz, CDCl
3
) d 9.57 (s, 1H),
.91–8.05 (m, 2H), 6.88–7.00 (m, 2H), 5.65 (s, 1H), 3.84 (s,
H), 2.55 (m, 2H), 2.32 (m, 2H), 1.73 (m, 4H). Anal.
SÆHBr
requires C, 50.01; H, 4.44; N, 10.29). ES/MS m/z 328
In conclusion, as far as the two most potent p53 inacti-
vators are a Pft-a analogue (2f) and a cyclic dehydrated
analogue (6b), it could be suggested that p53 inactiva-
tion by Pft-a analogues could be due in part to the cyclic
dehydrated forms, which are generated in situ during the
incubation time in the biological medium. Additional
experiments are underway in order to verify this
hypothesis.
(
17 3 2
Found: C, 49.85; H, 4.46; N, 10.07. C17H N O
+
1
(M+H) . 2g: H NMR (250 MHz, DMSO-d ) d 9.60 (s,
1H), 7.95 (d, 2H, J = 8.2 Hz), 7.45 (d, 2H, J = 8.2 Hz), 4.22
(s, 2H), 2.65–2.50 (m, 7H), 1.74 (m, 6H). Anal. (Found: C,
6
5
5
3.71; H, 5.32; N, 7.52. C17
3.54; H, 5.55; N, 7.35). ES/MS m/z 301 (M+H) .
H N OSÆHBr requires C,
20 2
+
1
Cyclized form analogues: 6 (Pft-b): H NMR (250 MHz,
DMSO-d ) d 7.95 (d, 2H, J = 8.2 Hz), 7.45 (d, 2H,
J = 8.2 Hz), 7.32 (s, 1H), 2.51–2.31 (m, 5H), 1.83–1.79
Acknowledgements
6
(
(
7
m, 2H), 1.36 (br s, 1H), 1.02 (d, 3H, J = 6.5 Hz). Anal.
Found: C, 71.75; H, 5.82; N, 10.45. C16 S requires C,
1.60; H, 6.01; N, 10.44). ES/MS m/z 269 (M+H) . 6b: H
NMR (250 MHz, DMSO-d ) d 7.65 (d, 2H, J = 8.5 Hz),
INSERM and Conseil R e´ gional Provence Alpes C oˆ te
dꢀAzur are greatly acknowledged for financial support
(NP scholarship) and Innate Pharma (Marseille, France)
for helpful discussions.
16 2
H N
+
1
6
7
1
1
.45 (s, 1H), 7.15 (d, 2H, J = 8.5 Hz), 2.40–2.50 (m, 4H),
.80–1.95 (m, 4H). Anal. (Found: C, 60.38; H, 4.46; N,
4.28. C15H N O S requires C, 60.18; H, 4.38; N, 14.04).
13 3 2
+
1
References and notes
. Zhu, X. X.; Yu, Q. S.; Cutler, R. G.; Culmsee, C. W.;
Holloway, H. W.; Lahiri, D. K.; Mattson, M. P.; Greig, N.
H. J. Med. Chem. 2002, 45, 5090–5097.
ES/MS m/z 300 (M+H) . 6e: H NMR (250 MHz, DMSO-
d
6
) d 7.65 (d, 2H, J = 8.5 Hz), 7.45 (s, 1H), 7.15 (d, 2H,
1
J = 8.5 Hz), 2.55–1.25 (m, 14H). Anal. (Found: C, 64.64;
H, 5.32; N, 11.95. C H N O S requires C, 64.57; H, 5.42;
1
9
19
3
2
+
1
N, 11.89). ES/MS m/z 354 (M+H) . 6f: H NMR
2
. Komarov, P. G.; Komarova, E. A.; Kondratov, R. V.;
Christov-Tselkov, K.; Coon, J. S.; Chernov, M. V.;
Gudkov, A. V. Science 1999, 285, 1733–1737.
(250 MHz, CDCl ) d 7.91–8.05 (m, 2H), 6.88–7.00 (m,
3
2H), 3.84 (s, 3H), 2.55 (m, 2H), 2.32 (m, 2H), 1.73 (m, 4H).
Anal. (Found: C, 66.14; H, 5.12; N, 13.45. C H N OS
1
7
15
3
3
. Komarova, E. A.; Gudkov, A. V. Biochem.-Moscow 2000,
requires C, 66.00; H, 4.89; N, 13.58). ES/MS m/z 310
(M+H) .
6. Merlic, C.; Motamed, S.; Quinn, B. J. Org. Chem. 1995, 60,
3365–3369.
+
6
5, 41–48.
. King, L. C.; Hlavacek, R. J. J. Am. Chem. Soc. 1950, 72,
722–3725.
4
3