Synthesis, reactivity and biological evaluation of novel halogenated tripentones

Article abstract of DOI:10.1016/j.bmc.2009.09.027

We describe herein the synthesis and the biological evaluation of a novel series of a potent anticancer agents: the tripentones. For the first time, a halogen atom was introduced in high yields on the pyrrole ring of the tricycle. This synthesis and the r

Full text of DOI:10.1016/j.bmc.2009.09.027

Bioorganic & Medicinal Chemistry 17 (2009) 7783–7788
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, reactivity and biological evaluation of novel halogenated tripentones
a
Vittoria Perri a, Christophe Rochais a, Thierry Cresteil b, Patrick Dallemagne a, , Sylvain Rault
*
a Centre d’Etudes et de Recherche sur le Médicament de Normandie (UPRES EA 4258—FR CNRS 3038 INC3M), UFR des Sciences Pharmaceutiques,
Université de Caen Basse-Normandie, Boulevard Becquerel, 14032 Caen cedex, France
b ICSN—CNRS, UPR, 2301, Avenue de la Terrasse, 91198 Gif sur Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2009
Revised 10 September 2009
Accepted 16 September 2009
Available online 19 September 2009
We describe herein the synthesis and the biological evaluation of a novel series of a potent anticancer
agents: the tripentones. For the first time, a halogen atom was introduced in high yields on the pyrrole
ring of the tricycle. This synthesis and the reactivity of the novel halogenated tripentones in metallo-cat-
alysed cross-coupling reactions will be described in that article. Finally their influence on biological activ-
ity will be discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Tripentones
Metallo-catalysed cross coupling
Fused-ring systems
1. Introduction
Conserving the requirements for activity, our goal was first to
explore the chemical access to these structures but also to rational-
Anticancer researches at the laboratory have conduced to the
discovery of the thienopyrrolizinones family (Fig. 1) or tripen-
tones.1 Many works have been carried out in that series that gave
us crucial information and led to the discovery of MR22388 8b a
potent anticancer agent.2 However if 8b appeared to possess low
nanomolar activity against various cell lines its development has
been confronted to a lack of bioavailability. We then decided to ex-
plore novel chemical modulations of the tricycle. Different analogs
have been produced and evaluated replacing the thiophene ring by
a pyrrole, furane, pyrazole or benzene one.3
If the biological activity is closely related to the presence of the
thiophene and the 4-methoxy-3-hydroxyphenyl substituent very
little information are available concerning the influence of substi-
tution on the right hand part of the molecule and more precisely on
the pyrrole ring.4
ize their influence on activity.
Furthermore this substitution could be a good opportunity for
us to introduce groups that improve the hydrosolubility of that
particular series.
2. Chemistry
The general route towards the tripentone 8a, the benzylated
analog of MR22388 proceeded in an eight step synthesis from
the corresponding arylacetonitrile 1 (Scheme 1).1
Crucial intermediates could be identified as the thiophenic o-
aminoester 4a. It could be obtained in three steps from 1a, and in-
volved in a Clauson-Kaas reaction5 in order to introduce the pyr-
role ring. The ring closure procedure developed in that series is
using a Vilsmeier–Haack mechanism and therefore requests the
synthesis of carboxamide 6a.
O
S
This protected tripentone 8a will be one of our starting materi-
als for the novel pharmacomodulation. We will see that in many
cases, a final deprotection reaction will be needed to liberate the
phenolic group. Such a transformation will lead to novel analogs
of MR22388 whose biological activity could be assessed.
Various routes could be employed to substitute the pyrrole moi-
ety of the tripentone (Fig. 2). Indeed we could either introduce the
substituent from the cyclised tripentones and beneficiate from the
reactivity of the pyrrole ring to substitute the final tricyclic system
(route A) or introduce it during the preparation of the pyrrole ring
(route B) according to a modified Clauson-Kaas procedure. Results
exposed in that paper will be focused on route A.
N
R
8a R=pOMemOBn
8b R=pOMemOH
8c R=pOMe
Figure 1.
* Corresponding author. Tel.: +33 2 31 56 68 13; fax: +33 2 31 56 68 03.
E-mail address: patrick.dallemagne@unicaen.fr (P. Dallemagne).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.027

7784
V. Perri et al. / Bioorg. Med. Chem. 17 (2009) 7783–7788
ONa
CN
O
OSO2C6H5
ii
i
S
Ar
CN
Ar
Ar
CN
N
1a
2a
3a
Ar
iii
8a
O
CO2Me
N
N
N
v
CO2Me
NH2
S
S
iv
i
ii
iii
S
Ar
5a
Ar
6a
Ar
4a
O
O
O
S
vi
S
S
N
N
N
Br
I
Br
Br
Ar = C6H3(m-OBn,p-OMe)
Ar
O
Ar
Ar
N+
-
PO2Cl2
vii
S
9a
10a
11a
S
N
N
Ar
a: Ar = C6H3(m-OBn,p-OMe)
b : Ar = C6H3(m-OH,p-OMe)
iv
iv
Ar 7a
8a
Scheme 1. Reagents and conditions: (i) NaH, HCO2Et, THF; (ii) PhSO2Cl, NEt3,
toluene; (iii) HSCH2CO2Me, MeONa, MeOH; (iv) 2,5-dimethoxyTHF, 4-chloropyri-
dine HCl, dioxane, 100 °C; (v) pyrrolidine, 87 °C; (vi) POCl3, 70 °C; (vii) 10% NaOH,
50 °C.
8b
11b
10b
Scheme 2. Reagents and conditions: (i) Br2, CHCl3, rt; (ii) NBS, THF, ꢀ84 °C to rt,
80%; (iii) KOH, I2, DMF, rt, 90%; (iv) (a) 33% HBr in AcOH, 25 °C; (b) NaOH 1 N,
MeOH, 25 °C, 55%.
The first substituent we decided to introduce was a halogen
atom. It could be a source of multiple coupling reactions and there-
fore chemical diversity. Furthermore it should be easily introduce
beneficiating from the reactivity of the pyrrole ring towards elec-
trophilic aromatic substitution.
We decided to study the condition of electrophilic halogen-
ations of tripentone 8a. It appears that this type of compound is
really activated towards electrophilic bromination. The use of
one equivalent of bromine at room temperature conduced exclu-
sively to a mixture of mono- and di-bromo tripentone on position
2 and 6, both of the activated position of the tricycle. Looking for
milder conditions our best results were obtained when N-bromo-
succinimide was used at ꢀ84 °C. Compound 10a was exclusively
obtained in 80% yield. It appears that position 6 is the most reactive
towards electrophilic substitution (Scheme 2). The position of the
bromination was determined by a NMR study but also according
to the X-ray structure of 10a (Fig. 3).
We could compare this result with the reactivity of correspond-
ing substituted pyrrole ring. It is well established that when the 2
position of a pyrrole ring is occupied by an electron-withdrawing
group, electrophilic attack occurs more readily at the 4 position
than at the normally more reactive 5 position.6
We then tried to replace N-bromosuccinimide by N-iodosuccin-
imide in order to introduce an iodine atom. Unfortunately this
reaction conserved exclusively the starting material, even upon
heating. Iodination could however be easily realized with 90% yield
using a mixture of I2 and KOH to afford the isomer 11a.7 A solution
of hydrobromic acid was used to afford compound 10b in good
yields. Interestingly, under the same conditions 11a conduced in
high yields to the dehalogenated product 8b.
Figure 3.
The reactivity of these important intermediates was then eval-
uated in various metallo catalyzed cross-coupling reactions. Our
first attempts were to introduce alkyl chain using a Heck coupling
reaction.8 Starting from compound 11a and methyl acrylate the
novel compound 12a was obtained under microwave irradiation
(Scheme 3). The corresponding bromotripentone 10a remains inac-
tive under this condition. Compound 12a was deprotected accord-
ing to the standard reaction procedure.
The reactivity of the tripentone 10a was evaluated under Buch-
wald9 conditions in order to introduce an aromatic amine instead
of the halogen atom. Compound 13a was obtained when 10a was
reacted with p-aminoanisole. If unreactive towards the Heck cou-
pling, the bromo isomer 10a appears to be a good coupling partner
in the Suzuki–Miyaura cross-coupling reaction.10 The best condi-
O
O
CO2Me
S
S
S
R
N
N
N
R
Route A
Route B
Ar
Ar
Figure 2.
Ar

V. Perri et al. / Bioorg. Med. Chem. 17 (2009) 7783–7788
7785
Table 1
O
O
Cytotoxicity results for compounds 1–18b
i
ii
S
S
OMe
O
12b
N
N
I
S
O
Ar
Ar
N
a : Ar = C6H3(m-OBn,p-OMe)
R
11a
12a
b : Ar = C6H3(m-OH,p-OMe)
OH
Scheme 3. Reagents and conditions: (i) CH2CHCO2Me, PdOAc2, PPh3, TEA, dioxane,
50%; (ii) (a) 33% HBr in AcOH, 25 °C; (b) NaOH 1 N, MeOH, 25 °C, 40%.
O
Compds
R
% Inhib
% Inhib
IC50, nM
L1210
10ꢀ5
M
10ꢀ6
M
tions found in that particular series were the use of Pd(PPh3)4 as a
catalyst and K3PO4 as a base in DMF. Under these conditions a ser-
ies of novel tripentones arylated on position 6 14a–18a was syn-
thesized with good to excellent yields, before to be deprotected
(Scheme 4).
1a
H
Br
86
94
1
98
97
35
86
94
3
94
15
NTa
15
20
4.0
10b
12b
14b
15b
16b
17b
18b
17.0
NDb
113
NDb
NDb
NDb
NDb
(CH)2CO2Me
C6H4OMe
C6H3di-OMe
C6H2tri-OMe
C6H3pOMemOH 90
mC5NH4 64
3. Pharmacological results
a
NT: not tested.
ND: not determined.
The influence of these novel substitutions on the cytotoxic
activity of our tripentones was firstly evaluated in vitro on KB-cells
and reported in Table 1.
b
These results gave us some crucial information in order to de-
tails our SAR study and more particularly the influence of a novel
substituent on the pyrrole ring. It appears clearly that small substi-
tuent on that position, like bromo- and iodo- compounds con-
served an interesting cyctotoxicity. On the other hand its
replacement by a larger chain and an even bigger aromatic ring
caused an important decrease of the activity.
In conclusion, we have described here the synthesis and the
antiproliferative activities of novel pyrrolo[2,3-b]pyrrolizinones,
which have gave crucial information in the development of novel
tripentones compounds. Finally a novel and convergent synthesis
was reported through Suzuki cross-coupling and will be applied
to afford more diversity on these new structures.
4. Experimental
4.1. General
All commercial solvents and reagents were used as-received ex-
cept THF, which was distilled over Na/benzophenone under Argon.
Flash chromatography was realized on silica gel (SDS AAC 60, 70–
200) or on neutral alumina gel (Merck 90, 63–200). IR spectra were
recorded on KBr disks with a Perkin–Elmer BX FTIR apparatus. 1H
and 13C NMR spectra were recorded, respectively, at 400 and
100 MHz with a Jeol Lambda 400 NMR spectrometer. Chemical
shifts d are reported in parts per million with the solvent resonance
as the internal standard; coupling constants J are given in hertz.
Multiplicity is given as follows: s (singlet), d (doublet), t (triplet),
q (quartet), quint. (quintet), sept. (septet), m (multiplet). The
microwave reactions were performed using a Biotage Initiator
Microwave oven using 2–5 mL sealed vials. Temperature was mea-
sured with an IR-sensor and reaction times are given as hold times.
LC/MS (ESI) analyses were realized with a Waters alliance 2695 as
separating module using the following gradient: A (95%)/B (5%) to
A (5%)/B (95%) in 10 min. This ratio was hold during 3 min before
return to initial conditions in 1 min. Initial conditions were then
maintained for 5 min (A: H2O, B: CH3CN; each containing HCOOH:
0.1%; Column: C18 Xterra MSC118/2.1_50 mm). MS detection was
performed with a Micromass ZMD 2000 by positive ESI. EIMS and
HRMS (EI) were performed at 70 eV with a JEOL JMS GCMate. Melt-
ing points were determined on Kofler melting point apparatus.
O
S
N
Br
Ar
a : Ar = C6H3(
m-OBn,p-OMe)
10a
b : Ar = C6H3(
m-OH,
p-OMe)
ii
i
14
15
O
O
O
S
S
O
O
H
N
4.2. Halogenation procedure
N
Ar'
N
Ar
Ar
4.2.1. 3-(3-Benzyloxy-4-methoxyphenyl)-6-bromo-thieno[2,3-
b]pyrrolizin-8-one (10a)
O
13a
Ar' =
14a-18a
16
17
O
A solution of 100 mg (0.26 mmol) of the tripentone 8a in 5 mL
anhydrous THF was cooled to ꢀ84 °C. 46 mg (0.26 mmol, 1 equiv)
of N-bromosuccinimide was slowly added and the reaction mix-
ture was stirred at ꢀ85 °C for 1.5 h and 24 h at rt. The solvent
was removed under vacuum and the residue diluted in 150 mL
water. The solution was extracted with Et2O (2 ꢁ 100 mL). The
combined organic phases were washed with aqueous NaCl
(2 ꢁ 100 mL), dried (MgSO4) and evaporated. This residue was
purified by silica gel chromatography, eluting by cyclohexane/
ethyl acetate (1:2) to furnish the tripentone 10a as an orange solid
O
O
iii
iii
OR
O
13b
14b-18b
N
18
Scheme 4. Reagents and conditions: (i) CH3OC6H4NH2, Cs2CO3, BINAP, Pd2dba3,
dioxane, 70%; (ii) ArB(OH)2, Pd(PPh3)4, K3PO4, DMF, 60–80%; (iii) (a) 33% HBr in
AcOH, 25 °C; (b) NaOH 1 N, MeOH, 25 °C, 50–90%.

7786
V. Perri et al. / Bioorg. Med. Chem. 17 (2009) 7783–7788
(97 mg, 80%). Mp 110 °C. IR (KBr):
m
= 3429, 3119, 3065, 2924,
anhydrous 1,4-dioxane (1.5 mL). The tube was sealed and irradi-
ated at 160 °C for 40 min. After cooling to room temperature, the
reaction mixture was diluted with CH2Cl2, filtered and concen-
trated. This residue was purified by silica gel chromatography,
eluting by cyclohexane/ethyl acetate (2:1) to furnish 80 mg (36%)
of the desired product as a yellow solid. Mp 134 °C. IR (KBr):
1693(CO), 1524, 1503, 1439, 1413, 1377, 1275, 1255, 1155, 1139,
1023, 922, 771, 742, 697, 580 cmꢀ1 1H NMR (400 MHz, CDCl3):
d = 7.38 (m, 6H, Hphenyl + H2), 7.03 (dd, 1H, 4J = 1.9 Hz, 3J = 8.0 Hz,
.
3
4
0
0
0
H6 ), 7.01 (d, 1H, J = 8.0 Hz, H5 ), 6.96 (d, 1H, J = 1.9 Hz, H2 ), 6.65
(d, 1H, 4J = 0.9 Hz, H5), 6.63 (d, 1H, 4J = 0.9 Hz, H7), 5.19 (s, 2H,
OCH2Ph), 3.99 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3):
d = 173.31, 159.93, 150.42, 148.56, 136.50, 135.16, 119.70,
116.68, 114.00, 112.08, 100.34, 71.33, 56.14. MS (EI+) m/z: 466.9
(M+1, 31), 464.9 (Mꢀ1, 31).
m
= 3106 (NH), 2922, 2801, 2357, 1637 (CO), 1553, 1538, 1522,
1473, 1456, 1342, 1286, 1282, 1176, 766, 631 cmꢀ1.1H NMR
(400 MHz, CDCl3): d = 7.41d, 1H, J = 2.96 Hz, Harom), 7.36 (s, 1H,
H2), 7.35 (d, 2H, J = 1.96 Hz, Harom), 7.22 (d, 1H, J = 3.92 Hz, Harom),
00
00
7.10 (d, 2H, J = 8.79 Hz, H2 + H6 ), 6.95 (s, 1H, NH), 6.77 (d, 2H,
00
00
0
0
4.2.2. 3-(3-Benzyloxy-4-methoxyphenyl)-6-iodo-thieno[2,3-
b]pyrrolizin-8-one (11a)
J = 8.79 Hz, H3 + H5 ), 6.71 (m, 3H, H2 , H5 , Harom), 6.62 (dd, 1H,
4J = 1.96 Hz, J = 8.8 Hz, H6 ), 6.60 (d, 1H, J = 1.96 Hz, H5), 6.35 (d,
3
0
To a solution of 200 mg (0.52 mmol, 1 equiv) of the tripentone
(8a) in 10 mL DMF was added 87 mg (1.55 mmol, 3 equiv) of
KOH. The reaction mixture was cooled down to 0 °C and 262 mg
(1.03 mmol, 2 equiv) of iodine was added. The reaction mixture
was stirred at rt for 12 h before to be diluted with 50 mL of a sat-
urated solution of Na2S2O3. The combined organic phases were
washed with aqueous NaCl (2 ꢁ 100 mL), dried (MgSO4) and evap-
orated. This residue was purified by silica gel chromatography,
eluting by cyclohexane/ethyl acetate (1:2) to furnish the tripen-
tone 11a as an orange solid (180 mg, 90%). Mp 157 °C. IR (KBr):
1H, J = 1,96 Hz, H7), 4.80 (s, 2H, OCH2), 3.84 (s, 3H, OCH3), 3.72 (s,
3H, OCH3). 13C NMR (100 MHz, CDCl3): d = 157.34, 156.17,
148.96, 147.70, 139.90, 138.58, 136.88, 135.88, 130.41, 129.38,
128.76, 128.44, 127.70, 126.97, 126.75, 126.71, 126.42, 122.16,
121.55, 120.85, 120.71, 120.65, 114.88, 113.84, 112.15, 111.53,
96.79, 70.24, 55.84, 55.32. LC–MS (ESI): tR = 12.03 min; m/z
[M+H]+: 509.17.
4.3.3. 3-(3-Benzyloxy-4-methoxyphenyl)-6-(4-
methoxyphenyl)-thieno[2,3-b]pyrrolizin-8-one (14a)
m
= 2958, 2924, 2869, 2836, 1682(CO), 1520, 1498, 1436, 1413,
1376, 1254, 1240, 1141, 1010, 912, 802, 760, 699, 586, 581 cmꢀ1
1H NMR (400 MHz, CDCl3): d = 7.36 (m, 6H, Hphenyl + H2), 7.01 (m,
A solution of tripentone 10b (50 mg, 0.11 mmol) in DMF (2 mL)
under argon was added to a mixture of Pd(PPh3)4 (5.3 ꢁ 10ꢀ3 mmol,
5%), 4-methoxyphenylboronic acid (0.21 mmol, 2 equiv) and K3PO4
(0.27 mmol, 2.5 equiv). The mixture was then irradiated at 120 °C
for 100 min using a Biotage microwave oven. The mixture was then
diluted with 50 mL water and extracted with diethyl ether
(2 ꢁ 75 mL). The combined organic layers were washed with brine
(2 ꢁ 100 mL), dried (MgSO4) and evaporated to give a yellow oil
which was purified by silica gel chromatography, eluted by cyclo-
hexane/ethyl acetate (3:1) to furnish tripentone 14a as a red solid
.
0
0
0
3H, H6 + H5 + H2 ), 6.74 (s, 1H, H5), 6.72 (s, 1H, H7), 5.18 (s, 2H,
OCH2Ph), 3.97 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3):
d = 172.9, 150.8, 150.4, 148.6, 136.7, 136.5, 134.7, 129.6, 128.7,
128.6, 128.2, 127.3, 124.4, 124.1, 121.2, 121.0, 113.9, 112.0,
71.35. MS (EI+) m/z: 512.8 (M, 38), 386.0 (27).
4.3. Typical metallo-catalyzed reaction
in 75% yield. Mp 134 °C. IR (KBr):
m = 3429, 2924, 2853, 1673(CO),
4.3.1. 3-[3-(3-Benzyloxy-4-methoxyphenyl)-8-oxo-8H-
1526, 1505, 1455, 1367, 1259, 1136, 1080, 1023, 801, 743,
thieno[2,3-b]pyrrolizin-6-yl]-acrylic acid methyl ester (12a)
A solution of palladium acetate (9.7 ꢁ 10ꢀ6 mol, 0.05 equiv), tri-
phenylphosphine (1.9 ꢁ 10ꢀ5 mol, 1 equiv) and triethylamine
(1.9 ꢁ 10ꢀ4 mol, 1 equiv) in dioxane was stirred under argon for
5 min. 100 mg (1.9 ꢁ 10ꢀ4 mol, 1 equiv) of tripentone 11a was then
added and the reaction mixture was stirred for 10 min. Methyl
acrylate (9.7 ꢁ 10ꢀ4 mol, 5 equiv) was then added and the reaction
mixture was heated under reflux for 12 h. 150 mL water was then
added and the reaction mixture was extracted with ethyl acetate
(2 ꢁ 100 mL). The combined organic layers were washed with brine
(2 ꢁ 100 mL), dried (MgSO4) and evaporated to give a yellow oil
which was purified by silica gel chromatography, eluting by cyclo-
hexane/ethyl acetate (4:1) to furnish tripentone 12a as a yellow so-
696 cmꢀ1 1H NMR (400 MHz, CDCl3): d = 7.32 (m, 5H, Harom), 6.98
.
(m, 2H, H5 + H6 ), 6.75 (d, 2H, 4J = 3.8 Hz, H2 + H6 ), 6.42 (d, 1H,
0
0
00
00
4
0
J = 1.9 Hz, H2 ), 6.30 (s, 1H, H5 or H7), 6.09 (s, 1H, H5 or H7), 5.90
(d, 2H, 4J = 3.8 Hz, H3 + H5 ), 4.82 (s, 2H, OCH2Ph), 3.93 (s, 3H,
OCH3). 3.57 (s, 3H, OCH3). MS (EI+) m/z 368.4 (15), 236.2 (19),
152.2 (18), 111.1 (47), 97.1 (91), 83.4 (100). LC–MS (ESI):
tR = 12.75 min; m/z [M+H]+: 494.75.
00
00
4.3.4. 3-(3-Benzyloxy-4-methoxyphenyl)-6-(3,4-
dimethoxyphenyl)-thieno[2,3-b]pyrrolizin-8-one (15a)
This compound was obtained from 10a as described for 14a as a
red solid and directly engaged in the debenzylation step.
lid in 54% yield. Mp 130 °C. IR (KBr):
m
= 2922, 2851, 1693(CO),
4.3.5. 3-(3-Benzyloxy-4-methoxyphenyl)-6-(3,4,5-
trimethoxyphenyl)-thieno[2,3-b]pyrrolizin-8-one (16a)
This compound was obtained from 10a as described for 14a as a
1635(CO), 1475, 1260, 11,65, 1116, 1024 cmꢀ1
.
1H NMR
(400 MHz, CDCl3): d = 7.38 (m, 7H, Harom + H2 + Hethyl), 7.04 (dd,
3
3
1H, 4J = 1.7 Hz, J = 8.0 Hz, H6 ), 7.00 (d, 1H, J = 8.0 Hz, H5 ), 6.97
red solid in 58% yield. Mp 148 °C. IR (KBr):
(CO), 1585, 1470, 1416, 1379, 1327, 1250, 1120, 1004, 844, 802,
740, 697 cmꢀ1. 1H NMR (400 MHz, CDCl3): d = 7.31 (s, 2H,
m
= 3078, 2928, 1673
0
0
4
0
(d, 1H, J = 1.7 Hz, H2 ), 6.85 (s, 1H, H5); 6.73 (s, 1H, H7), 6.05 (d,
1H, J = 15.8 Hz, Hethyl), 5.21 (s, 2H, OCH2), 3.98 (s, 3H, OCH3), 3.77
(s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): d = 170.01, 167.63,
150.45, 148.49, 145.08, 137.11, 136.94, 136.53, 135.18, 129.74,
128.77, 128.31, 127.13, 124.98, 124.36, 121.25, 121.07, 114.93,
113.69, 112.53, 112.02, 71.12, 56.13, 51.56. MS (EI+) m/z: 471.2
(M, 5), 91.1 (100). LC–MS (ESI): tR = 11.72 min; m/z [M+H]+: 472.01.
.
00
00
0
0
0
H2 + H6 ), 7.22 (m, 6H, Harom, + H2), 6.95 (m, 3H, H2 , H5 , H6 ),
6.64 (s, 2H, H5 + H7), 5.19 (s, 2H, OCH2), 3.97 (s, 3H, OCH3), 3.84
(s, 3H, OCH3), 3.83 (s, 6H, OCH3).13C NMR (100 MHz, CDCl3):
d = 174.0, 153.6, 150.8, 150.4, 148.6, 137.4, 136.5, 136.4, 134.5,
128.6, 127.2, 126.8, 124.9, 121.1, 116.6, 113.9, 113.3, 112.1,
104.7, 102.8, 71.4, 65.8, 60.9, 56.3. LC–MS (ESI): tR = 13.67 min;
m/z [M+H]+: 534.57.
4.3.2. 3-(3-Benzyloxy-4-methoxyphenyl)-6-(4-
methoxyphenylamino)-thieno[2,3-b]pyrrolizin-8-one (13a)
To a mixture of p-anisidine (53 mg, 0.43 mmol), Pd(OAc)2
(1.93 mg, 8.86 ꢁ 10ꢀ6 mol), Cs2CO3 (279 mg, 0.86 mmol) and 4,5-
bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos, 7.4 mg,
0.013 mmol) was added tripentone 10a (200 mg, 0.43 mmol) in
4.3.6. 3,6-Bis-(3-benzyloxy-4-methoxy-phenyl)-thieno[2,3-
b]pyrrolizin-8-one (17a)
This compound was obtained from 10a as described for 14a as an
orange solid in 46% yield. Mp 155 °C. IR (KBr): m = 3094, 3030, 2090,

V. Perri et al. / Bioorg. Med. Chem. 17 (2009) 7783–7788
7787
2833, 1678 (CO), 1605, 1562, 1454, 1372, 1259,1215, 1022, 967,
156.72, 147.56, 146.81, 146.50, 135.46, 133.85, 131.17, 130.55,
125.70, 125.18, 120.29, 119.40, 113.05, 112.70, 109.68, 108.00,
55.94, 50.14. LC–MS (ESI): tR = 9.72 min; m/z [M+H]+: 382.03.
HRMS (EI): calcd (M+Å) for C20H15NO5S: 381.06707, found:
381.06861.
747 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d = 7.32 (m, 1H, Harom + H2),
3
7.09 (dd, 1H, 4J = 1.96 Hz, J = 7.79 Hz, H6 ), 7.04 (d, 1H, 4J =
0
1.96 Hz, H2 ), 7.01 (d, 1H, J = 7.79 Hz, H5 ), 6.90 (dd, 1H, 4J =
3
0
0
1.96 Hz, 3J = 7.79 Hz, H6 ), 6.87 (d, 1H, 4J = 1.96 Hz, H2 ), 6.65 (d,
00
00
1H, 3J = 7.79 Hz, H5 ), 6.47 (d, 2H, J = 2.91 Hz, H5 + H7), 5.19 (s, 2H,
00
OCH2), 5.14 (s, 2H, OCH2), 3.98 (s, 3H, OCH3), 3.89 (s, 3H, OCH3).13
C
4.4.3. 3-(3-Hydroxy-4-methoxyphenyl)-6-(4-
NMR (100 MHz, CDCl3): d = 174.1, 150.8, 150.2, 150.1, 148.7,
148.1, 143.4, 136.8, 136.3, 134.9, 129.6, 129.3, 128.7, 128.5, 128.4,
127.8, 127.7, 127.3, 127.1, 126.9, 126.4, 124.7, 120.9, 118.0, 116.3,
113.6, 113.1, 112.0, 111.8, 111.6, 106.5, 102.8, 71.2, 70.7, 56.7,
56.0. MS (EI+) m/z 599.3 (100), 509.2 (53), 480.2 (39).
methoxyphenylamino)-thieno[2,3-b]pyrrolizin-8-one (13b)
This compound was obtained from 13a as described for 10b as a
yellow solid in 43% yield. Mp 145 °C. IR (KBr):
(NH), 2929, 2836, 2360, 2343, 1646 (CO), 1600, 1538, 1512, 1482,
1439, 1387, 1299, 1271, 1243, 1174, 821, 799, 731 cmꢀ1 1H NMR
(400 MHz, CDCl3): d = 7.27 (s, 1H, H2), 7.13 (d, 2H, J = 8.79 Hz,
m = 3390 (OH), 3108
.
00
00
4.3.7. 3-(3-Benzyloxy-4-methoxyphenyl)-6-pyridin-3-yl-
thieno[2,3-b]pyrrolizin-8-one (18a)
H2 + H6 ), 6.95 (s, 1H, NH), 6.87 (d, 1H, J = 1,96 Hz, H5), 6.77 (d,
3
00
00
0
2H, J = 8.79 Hz, H3 + H5 ), 6.74 (d, 1H, J = 8.8 Hz, H5 ), 6.67 (d,
4
0
This compound was obtained from 10a as described for 14a as a
1H, J = 1.96 Hz, H2 ), 6.58(d, 1H, J = 1,96 Hz, H7), 6.45 (dd, 1H,
3
4J = 1.96 Hz, J = 8.8 Hz, H6 ), 3.82 (s, 3H, OCH3), 3.75 (s, 3H,
0
yellow solid in 40% yield. Mp 140 °C. IR (KBr):
m
= 2956, 2922, 2853,
1682 (CO), 1620, 1579, 1562, 1504, 1462, 1312, 1225, 931, 606 cmꢀ1
.
OCH3). 13C NMR (100 MHz, CDCl3): d = 157.44, 157.17, 154.86,
147.80, 145.68, 137.78, 135.88, 133.41, 129.48, 128.76, 128.44,
127.70, 126.87, 126.75, 126.63, 122.36, 120.96, 120.66, 114.88,
112.15, 111.53, 56.74, 56.22. LC–MS (ESI): tR = 9.52 min; m/z
[M+H]+: 419.23. HRMS (EI): calcd (M+Å) for C23H18N2O4S:
418.0987, found: 418.09924.
1H NMR (400 MHz, CDCl3): d = 8.49 (m, 1H, Hpyridine), 7.83 (m, 2H,
0
Hpyridine), 7.55 (m, 2H, Hpyridine + H5 ), 7.32 (m, 6H, Haromatic + H2),
4
7.14 (d, 1H, J = 1.96 Hz, H2 ), 7.05 (dd, 1H, 3J = 1.96 Hz, 4J = 8,8 Hz,
0
0
H6 ), 6.92 (s, 1H, H5 or H7), 6.89 (s, 1H, H7 or H5), 5.20 (s, 2H,
OCH2), 3.99 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): d = 147.13,
146.97, 136.10, 133.10, 132.28, 131.10, 130.72, 128.23, 127.63,
127.03, 125,26, 125,51, 125,13, 120.23, 118.81, 117.32, 116.28,
113.90, 71.02, 55.50. LC–MS (ESI): tR = 10.05 min; m/z [M+H]+:
465.04.
4.4.4. 3-(3-Hydroxy-4-methoxyphenyl)-6-(4-methoxyphenyl)-
thieno[2,3-b]pyrrolizin-8-one (14b)
This compound was obtained from 14a as described for 10b as a
red solid in 60% yield. Mp 122 °C. IR (KBr):
m = 3422, 2961, 2926,
4.4. O-Debenzylation procedure
2855, 1668 (CO), 1563, 1502, 1438, 1381, 1279, 1248, 1131,
1086, 1041, 883, 803, 785, 758, 603 cmꢀ1 1H NMR (400 MHz,
.
CDCl3): d = 7.46 (s, 1H, H2), 7.31 (d, 2H, 4J = 8.7 Hz, H2 + H6 ),
00
00
4.4.1. 3-(3-Hydroxy-4-methoxyphenyl)-6-bromo-thieno[2,3-
b]pyrrolizin-8-one (10b)
4
0
7.18 (s, 1H, H5 or H7), 7.11 (d, 1H, J = 2.2 Hz, H2 ), 7.04 (s, 1H, H5
3
0
0
A solution of 10a (0.04 g, 0.086 mmol) in a 33% solution of
hydrobromic acid in glacial acetic acid (15 mL) was stirred at room
temperature for 1 h. After cooling, the reaction mixture was di-
luted with water (50 mL) and the resulting precipitate was ex-
tracted with ethyl acetate (3 ꢁ 20 mL). Then, the organic layers
were combined, washed with water (2 ꢁ 100 mL), dried (MgSO4)
and evaporated to give a dark red solid. The residue was diluted
in methanol (10 mL) and a molar aqueous solution of NaOH is
added (5 mL) before to be stirred for 1 h. The reaction mixture
was concentrated under vacuum, diluted with water, acidified with
aqueous 1 M HCl and extracted with ethyl acetate (2 ꢁ 100 mL).
The organic layers were combined, washed with water
(2 ꢁ 100 mL), dried (MgSO4) and evaporated to give a dark red so-
lid which was purified by silica gel chromatography, eluting by
cyclohexane/ethyl acetate (3:2) to furnish thienopyrrolizinone
or H7), 6.98 (d, 1H, J = 7.0 Hz, H5 ), 6.94 (m, 1H, H6 ), 6.88 (d, 2H,
4J = 8.7 Hz, H3 + H5 ), 5.7 (bl, 1H, OH), 3.98 (s, 3H, OCH3), 3.96 (s,
3H, OCH3).13C NMR (100 MHz, CDCl3): d = 158.5, 150.7, 149.6,
146.1, 136.5, 134.4, 132.6, 132.5, 129.5; 129.4, 126.2, 125.6,
122.2, 120.0, 119.8, 116.5, 114.2, 113.1, 110.9. LC–MS (ESI):
tR = 10.83 min; m/z [M+H]+: 404.19. HRMS (EI): calcd (M+Å) for
C23H17NO4S: 403.08781, found: 403.08626.
00
00
4.4.5. 6-(3,4-Dimethoxyphenyl)-3-(3-hydroxy-4-
methoxyphenyl)-thieno[2,3-b]pyrrolizin-8-one (15b)
This compound was obtained from 15a as described for 10b as a
red solid in 40% yield. Mp 190 °C. IR (KBr):
1688 (CO), 1567, 1508, 1477, 1436, 1256, 1224, 1135, 1032, 958,
803, 771, 762, 587 cmꢀ1 1H NMR (400 MHz, CDCl3): d = 7.46 (s,
1H, H2), 7.12 (d, 1H, 4J = 1.9 Hz, H2 or H2 ), 7.03 (dd, 1H,
m = 2955, 2932, 2854,
.
0
00
3J = 8.2 Hz, 4J = 1.9 Hz, H6 or H6 ), 6.99 (d, 1H, 4J = 1.9 Hz, H5 or
0
00
10b as a red solid (12 mg, 24%). Mp 154 °C. IR (KBr):
2924, 2853, 1678(CO), 1526, 1436, 1282, 1220, 1022, 922, 776,
581 cmꢀ1 1H NMR (400 MHz, CDCl3): d = 7.52 (s, 1H, H2), 7.25
m
= 3411,
H7), 6.96 (d, 1H, 3J = 8.2 Hz, H5 or H5 ), 6.94 (dd, 1H, 3J = 8.2 Hz,
0
00
4J = 1.9 Hz, H6 or H6 ), 6.90 (d, 1H, 4J = 1.9 Hz, H5 or H7), 6.88 (d,
0
00
.
3
(m, 3H, H2 , H5 , H6 ), 6.81 (s, 1H, H5), 6.65 (s, 1H, H7), 5.76 (bl,
1H, OH), 3.97 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3):
d = 173.8, 151.3, 154.2, 147.6, 146.5, 144.9, 135.2, 132.7, 129.9,
126.5, 125.6, 120.2, 117.1, 114.5, 111.4. 56.5. HRMS (EI): calcd
(M+Å) for C16H10BrNO3S: 374.95642, found: 374.956402.
1H, 4J = 1.9 Hz, H2 or H2 ). 6.84 (d, 1H, J = 8.2 Hz, H5 or H5 ), 5.8
(bl, 1H, OH), 3.98 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3). 13C NMR (100 MHz, CDCl3): d = 119.8, 117.6, 116.6, 114.2,
113.1, 111.7, 111.0, 109.1, 56.1, 56.0, 41.4. MS (EI+) m/z: 433.1
(M, 60), 418.1 (32), 368.3 (46), 341.2 (25), 326.2 (100). LC–MS
(ESI): tR = 10.30 min; m/z [M+H]+: 433.80. HRMS (EI): calcd (M+Å)
for C24H19NO5S: 433.09837, found: 433.09656.
0
0
0
0
00
0
00
4.4.2. 3-[3-(3-Hydroxy-4-methoxyphenyl)-8-oxo-8H-
thieno[2,3-b]pyrrolizin-6-yl]-acrylic acid methyl ester (12b)
This compound was obtained from 12a as described for 10b as
4.4.6. 3-(3-Hydroxy-4-methoxyphenyl)-6-(3,4,5-
an orange solid in 61% yield. Mp 153 °C. IR (KBr):
m
= 3134, 2934,
trimethoxyphenyl)-thieno[2,3-b]pyrrolizin-8-one (16b)
This compound was obtained from 16a as described for 10b as a
2895, 1694(CO), 1635(CO), 1476, 1243, 1100, 1067 cmꢀ1
.
1H
NMR (400 MHz, CDCl3): d = 7,55 (d, 1H, J = 15.8 Hz), 7.48 (dd, 1H,
red solid in 60% yield. Mp 156 °C. IR (KBr): m = 3328, 2927, 2853,
3
3
4J = 1.7 Hz, J = 8.0 Hz, H6 ), 6.99 (d, 1H, J = 8.0 Hz, H5 ), 6.98 (d,
2360, 2341, 1614 (CO), 1509, 1463, 1348, 1264, 1112, 1025, 803,
0
0
4
1H, J = 1.7 Hz, H2 ), 6.87 (s, 1H, H5), 6.31 (s, 1H, H7), 6.06 (d, 1H,
670 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d = 7.39 (s, 2H, H2 + H6 ),
0
00
00
4
0
0
J = 15.8 Hz, Hethyl), 5.39 (br s, 1H, OH), 3.99 (s, 3H, OCH3), 3.76 (s,
7.30 (s, 1H, H2), 7.11 (d, J = 1,96 Hz, 1H, H2 ), 7.03 (m, 2H, H5 ,
3H, OCH3). 13C NMR (100 MHz, CDCl3): d = 173.31, 167.85,
H6 ), 6.72 (s, 2H, H5 + H7), 5.4 (br s, 1H, OH), 3.98 (s, 3H, OCH3),
0

7788
V. Perri et al. / Bioorg. Med. Chem. 17 (2009) 7783–7788
3.84 (s, 3H, OCH3), 3.82 (s, 6H, OCH3). 13C NMR (100 MHz, CDCl3):
d = 170.2, 154.2, 152.4, 150.6, 149.9, 138.7, 137.5, 136.1, 134.2,
128.9, 128.6, 127.4, 126.6, 125.4, 124.2, 122.8, 121.3, 117.1,
113.7, 112.1, 104.6, 60.87, 55.97, 55.96. LC–MS (ESI):
tR = 10.52 min; m/z [M+H]+: 464.72. HRMS (EI): calcd (M+Å) for
C25H21NO6 S: 463.10892, found: 463.10785.
ney) and MRC5 (human fetal lung) were purchased from ECACC
(Salisbury, UK) and HL60 (promyeocytic leukaemia) cells from
ATCC. MCF7 (breast adenocarcinoma) were given by Dr Matthias
Kassack (Bonn University, Germany). KB, Vero and MRC5 cells were
grown in D-MEM medium supplemented with 10% fetal calf serum,
in the presence of penicilline, streptomycine and fungizone in
75 cm2 flask under 5% CO2, whereas HCT116, HCT15, MCF7, HL60
were grown in RPMI medium. Resistant MCF7 and HL60 cells were
obtained by prolonged treatment with doxorubicine.
4.4.7. 3,6-Bis-(3-hydroxy-4-methoxyphenyl)-thieno[2,3-
b]pyrrolizin-8-one (17b)
This compound was obtained from 17a as described for 10b as a
yellow solid in 48% yield. Mp 142 °C. IR (KBr):
m
= 3429, 3379, 2957,
4.5.2. Cell proliferation assay
2843, 2360, 1657(CO); 1532, 1511, 1485, 1466,1378, 1288,1267,
Cells were plated in 96-well tissue culture plates in 200
ium and treated 24 h later with compounds dissolved in DMSO
with compound concentrations ranged 0.05 nM to 1 M using a
ll med-
1176,974, 865 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d = 7.42 (s, 1H,
3
H2), 7.22 (dd, 1H, 4J = 1.96 Hz, J = 7.79 Hz, H6 ), 7.17 (d, 1H,
l
0
4
3
0
0
J = 1.96 Hz, H2 ), 7.04 (d, 1H, J = 7.79 Hz, H5 ), 6.97 (dd, 1H,
Biomek 3000 (Beckman). Controls received the same volume of
DMSO (1% final volume). After 72 h exposure to the drug, MTS re-
agent (Promega) was added and incubated for 3 h at 37 °C: the
absorbance was monitored at 490 nm and results expressed as
the inhibition of cell proliferation calculated as the ratio
[(1 ꢀ (OD490 treated/OD490 control)) ꢁ 100]. For IC50 determina-
tions (50% inhibition of cell proliferation) experiments were per-
formed in separate duplicate.
4J = 1.96 Hz, 3J = 7.79 Hz, H6 ), 6.87 (d, 1H, 4J = 1.96 Hz, H2 ), 6.65
00
00
(d, 1H, 3J = 7.79 Hz, H5 ), 6.45 (d, 2H, J = 2.91 Hz, H5 + H7), 3.93 (s,
00
3H, OCH3), 3.82 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): 158.1,
157.6, 147.3, 146.8, 145.7, 145.1, 137.4, 132.3, 130.7, 128.2,
125.9, 125.0, 120.4, 120.2, 120.0, 119.1, 117.2, 116.9, 115.6,
109.4, 55.7, 55.5. LC–MS (ESI): tR = 9.73 min; m/z [MꢀH]ꢀ:
418.97. HRMS (EI): calcd (M+Å) for C23H17NO5S: 419.08274, found:
419.08299.
References and notes
4.4.8. 3-(3-Hydroxy-4-methoxyphenyl)-6-pyridin-3-yl-
thieno[2,3-b]pyrrolizin-8-one (18b)
1. Lisowski, V.; Enguehard, C.; Lancelot, J. C.; Caignard, D.-H.; Lambel, S.; Leonce,
S.; Pierré, A.; Atassi, G.; Renard, P.; Rault, S. Bioorg. Med. Chem. Lett. 2001, 11,
2205.
This compound was obtained from 18a as described for 10b as a
yellow solid in 33% yield. Mp 152 °C. IR (KBr): 3114, 2965, 2932,
2. Lisowski, V.; Leonce, S.; Kraus-Berthier, L.; Sopkova-de Oliveira Santos, J.;
Pierré, A.; Atassi, G.; Caignard, D.-H.; Renard, P.; Rault, S. J. Med. Chem. 2004, 47,
1448.
3. (a) Rochais, C.; Lisowski, V.; Dallemagne, P.; Rault, S. Tetrahedron Lett. 2004, 45,
6353; (b) Rochais, C.; Sopkova-de Oliveira Santos, J.; Dallemagne, P.; Rault, S.
Heterocycles 2006, 68, 2063.
4. Rochais, C.; Dallemagne, P.; Rault, S. Anti-Cancer Agents Med. Chem. 2009, 9, 369.
5. Clauson-Kaas, N.; Zdenek, T. Acta Chem. Scand. 1952, 6, 667.
6. (a) Belanger, P. Tetrahedron Lett. 1979, 27, 2505; (b) Sonnet, P. J. Org. Chem.
1971, 36, 1005.
7. Gupton, J. T.; Miller, R. B.; Krumpe, K. E.; Clough, S. C.; Banner, E. J.; Kanters, R. P.
F.; Du, K. X.; Keertikar, K. M.; Lauerman, N. E.; Solano, J. M.; Adams, B. R.;
Callahan, D. W.; Little, B. A.; Scharf, A. B.; Sikorski, J. A. Tetrahedron 2005, 61,
1845.
2883, 1673 (CO), 1641, 1539, 1421, 1151, 1014, 792, 707 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d = 8.58 (m, 1H, Hpyridine), 7.86 (m,
0
2H, Hpyridine), 7.58 (m, 2H, Hpyridine + H5 ), 7.53 (s, 1H, H2), 7.17 (d,
4
4
1H, J = 1.96 Hz, H2 ), 7.09 (dd, 1H, 3J = 1.96 Hz, J = 8,8 Hz, H6 ),
6.95 (s, 1H, H5 or H7), 6.89 (s, 1H, H7 or H5), 5.4 (br s, 1H, OH),
3,98 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): d = 147.59;
147.24; 146.28; 134.78; 132.78; 132.21; 128.62;128.52; 127.48;
127.32; 123.62; 119.80; 116.96; 114.26; 111.77; 111.00; 56.34.
LC–MS (ESI): tR = 7.75 min; m/z [M+H]+: 375.97. HRMS (EI): calcd
(M+Å) for C21H14N2O3S: 374.07249, found: 374.07362.
0
0
8. Collot, V.; Varlet, D.; Rault, S. Tetrahedron 2000, 41, 4363.
9. Henry, Q.; Zhang, H. Q.; Xia, Z.; Vasudevan, A.; Djuric, S. W. Tetrahedron Lett.
2006, 47, 4881.
4.5. Pharmacology
10. For reviews see: (a) Bellina, F.; Carpita, A.; Rossi, R. Synthesis 2004, 2419; (b)
Hassan, J.; Sevignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M. Chem. Rev. 2002, 102,
1359.
4.5.1. Cell culture
The human cell lines KB (month epidermoid carcinoma),
HCT116 and HCT15 (colon adenocarcinoma), Vero (monkey kid-