Design, Synthesis, and Evaluation of Novel Thienopyrrolizinones as Antitubulin Agents

Article abstract of DOI:10.1021/jm030961z

Herein, we describe the structure-activity relationship study of a new 3-aryl-8H-thieno[2,3-b]pyrrolizin-8-one series of antitubulin agents. The pharmacological results from the National Cancer Institute in vitro human disease oriented tumor cell line screening allowed us to identify compound 1d (NSC 676693) as a very efficient antitumoral drug in all cancer cell lines tested. This prompted us to define the structural requirements essential for this antiproliferative activity. Among all analogues synthesized in this study, compound lo was the most promising, being 10-fold more potent than compound Id. Its activity over a panel of nine tumoral cell lines was in the nanomolar range for all of the histological types tested, and surprisingly, the resistant KB-A1 cell line was also sensitive to this compound. Moreover, a flow cytometric study showed that L1210 cells treated by the most potent compounds were arrested in the G2/M phases of the cell cycle with a significant percentage of cells having reinitiated a cycle of DNA synthesis without cell division. This interesting pharmacological profile, resulting from inhibition of tubulin polymerization, encouraged us to perform preliminary in vivo studies that led to a new prodrug chemical approach.

Full text of DOI:10.1021/jm030961z

1448
J . Med. Chem. 2004, 47, 1448-1464
Design , Syn th esis, a n d Eva lu a tion of Novel Th ien op yr r olizin on es a s An titu bu lin
Agen ts
Vincent Lisowski,† Ste´phane Le´once,‡ Laurence Kraus-Berthier,‡ J ana Sopkova´-de Oliveira Santos,†
Alain Pierre´,‡ Ghanem Atassi,‡ Daniel-Henri Caignard,§ Pierre Renard,§ and Sylvain Rault*,†
Centre d’Etudes et de Recherche sur le Me´dicament de Normandie, UFR des Sciences Pharmaceutiques,
Universite´ de Caen, 1 Rue Vaube´nard, 14032 Caen Cedex, France, Institut de Recherche Servier, 11 Rue des Moulineaux,
92150 Suresnes, France, and Les Laboratoires Servier, 1 Rue Carle He´bert 92415 Courbevoie Cedex, France
Received J uly 21, 2003
Herein, we describe the structure-activity relationship study of a new 3-aryl-8H-thieno[2,3-
b]pyrrolizin-8-one series of antitubulin agents. The pharmacological results from the National
Cancer Institute in vitro human disease oriented tumor cell line screening allowed us to identify
compound 1d (NSC 676693) as a very efficient antitumoral drug in all cancer cell lines tested.
This prompted us to define the structural requirements essential for this antiproliferative
activity. Among all analogues synthesized in this study, compound 1o was the most promising,
being 10-fold more potent than compound 1d . Its activity over a panel of nine tumoral cell
lines was in the nanomolar range for all of the histological types tested, and surprisingly, the
resistant KB-A1 cell line was also sensitive to this compound. Moreover, a flow cytometric
study showed that L1210 cells treated by the most potent compounds were arrested in the
G2/M phases of the cell cycle with a significant percentage of cells having reinitiated a cycle of
DNA synthesis without cell division. This interesting pharmacological profile, resulting from
inhibition of tubulin polymerization, encouraged us to perform preliminary in vivo studies that
led to a new prodrug chemical approach.
In tr od u ction
Cancer today is still an important clinical problem
with its prognosis remaining relatively poor for the
majority of tumors. Surgery, radiotherapy, and chemo-
therapy all have an important role to play in the
treatment of cancer, either alone or combined with each
other to define more effective strategies. Moreover, we
have gained remarkable biological knowledge about the
exact steps necessary for cancer cells to grow, divide,
F igu r e 1.
and spread. This has opened the door for new prospects
in chemotherapy to stop or reverse this proliferative
process, especially using targeted approaches based on
regulation of the cancer cell cycle like tubulin dynamic
Cancer Institute in vitro human disease oriented tumor
cell line screening panel.7 One of these compounds, 1d
(NSC 676693), expressed a cytotoxic activity in all
inhibition. Indeed, the mitotic spindle of eukaryotic cells
cancer cell lines tested as shown in Table 1. These re-
is an attractive target for the development of compounds
sults obtained at submicromolar concentrations prompted
useful in anticancer therapy.1-3 Microtubules show
the National Cancer Institute to perform hollow fiber
highly dynamic instability and play an essential role
and xenograft evaluations for 1d and encouraged us to
further investigate the structural parameters associated
with its biological activities. Structure-activity rela-
tionship studies were started from compound 1d to
in mitosis.4 Chemicals that attack microtubules through
their major component, tubulin, disrupt or suppress
both microtubule structure and normal functions by
inhibition or promotion of microtubule assembly, result-
design and synthesize more potent analogues useful as
ing in cell arrest in mitosis.5
anticancer agents and to define the minimal required
In previous chemical studies aimed at researching
structure to maintain the cytotoxic activity. So we
new microtubule polymerization inhibitors, we per-
successively explored the importance of cycle D (route
formed the synthesis of 3-substituted 8H-thieno[2,3-b]-
1), thiophene A (route 2), and pyrrolizine BC (route 3)
pyrrolizin-8-ones 1a -d (Figure 1).6 To investigate the
(Figure 2).
potential antitumor interest of this original chemical
series, these products were submitted to the National
Ch em istr y
Rou te 1. First, we decided to study the influence of
* To whom correspondence should be addressed. E-mail: rault@
an aromatic substitution with the synthesis of three
compounds already described by our team: the unsub-
stituted thienopyrrolizinone 1e,8 the 3-methyl derivative
1f,9 and finally 1g,10 the isomer of 1d (Figure 1). The
pharmacie.unicaen.fr. Phone: 33 (0)2 31 93 64 58. Fax: 33 (0)2 31 93
11 88.
† Universite´ de Caen.
‡ Institut de Recherche Servier.
§ Les Laboratoires Servier.
10.1021/jm030961z CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/13/2004

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1449
Ta ble 1. Cytotoxicity of Compounds 1a -d from the NCI 60 Tumoral Cell Lines Panel (log GI50 Effects in Ma)
cell type:
cell line:
leukemia
CCRF-CEM
NSCLb
NCI-H522
colon
SW-620
CNSc
SF-295
melanoma
M-14
ovarian
OVCAR-3
renal
XF-393
prostate
PC-3
breast
MDA-N
1a (NSC 683507)
1b (NSC 683509)
1c (NSC 683508)
1d (NSC 676693)
-4.7
-4.0
-4.5
-7.5
-4.9
-5.5
-4.8
-7.8
-4.8
-4.0
-4.6
-7.6
-4.7
-4.0
-4.6
-7.4
-4.7
-4.3
-4.5
-7.5
-4.8
-4.0
-4.3
-7.4
-4.9
-4.0
-4.6
-7.2
-4.9
-4.0
-4.5
-7.4
-4.8
-5.1
-5.1
-7.9
a
b
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. Non-small-cell lung. c Central nervous
system.
Ta ble 2. In Vitro Antiproliferative Activity and L1210 Cell Cycle Effects of Thienopyrrolizinone Analogues (Route 1)
compd
A
B
GI50 (µM)
effect on the cell cyclea
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
7a
7b
7c
7d
7e
8a
8b
8c
8d
C6H4pOCH3
H
CH3
H
H
H
H
0.19
85
83% G2 + M + 8N at 0.5 µΜ
NTb
>100
NTb
C6H4pOCH3
9.4
NTb
C6H4oOCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
18.9
5.2
11.7
36.4
4.2
75% G2 + M + 8N at 50 µM
C6H4mOCH3
C6H3(m,pdiOCH3)
C6H2(o,m,ptriOCH3)
C6H3(m,pOCH2O)
C6H3(mOBn,pOCH3)
C6H3(mOCH3,pOBn)
C6H3(mOH,pOCH3)
C6H3(mOCH3,pOH)
C6H4oOH
94% G2 + M + 8N at 25 µM
86% G2 + M + 8N at 25 µM
NTb
88% G2 + M + 8N at 10 µM
5.4
80% G1 at 25µM
24.1
0.015
8
3.7
29
30.1
0.28
12
0.43
28.1
12.2
29.6
NTb
86% G2 + M + 8N at 0.5 µM
86% G2 + M + 8N at 25 µM
72% G1 at 50 µM
C6H4mOH
C6H4pOH
NTb
NTb
C6H3(m,pdiOH)
C6H2(o,m,ptriOH)
C6H4pOC2H5
C6H4pOnC3H7
C6H4pOiC3H7
C6H4pOnC4H9
86% G2 + M + 8N at 0.5 µM
NTb
88% G2 + M + 8N at 1 µM
NTb
NTb
NTb
a
b
Percent of untreated cell in the phases of the cycle: 41% (G1); 28% (S); 24% (G2 + M); 1% (8N). NT: not tested.
forded methyl 3-amino-4-arylthiophen-2-carboxylates
4h -n in good yields.13-15 Treatment with dimethox-
ytetrahydrofuran (dimethoxyTHF) and 4-chloropyridine
hydrochloride in dioxane gave the pyrrolylthiophene
carboxylates 5h -n , which led to the corresponding
carboxamides 6h -n in refluxing pyrrolidine. Cyclization
of the latter compounds was performed by action of
phosphorus oxychloride to give, via an intermediate
iminium salt subsequently hydrolyzed in alkaline condi-
tion, the tricyclic ketones 1h -n in 47-71% yields.
Phenolic compounds 1o-p were obtained from com-
pounds 1m -n in 53 and 58% yields, respectively, by
cleavage of the O-benzyl protecting group using a 33%
hydrobromic acid solution in glacial acetic acid (Scheme
1).
F igu r e 2.
in vitro antiproliferative activity of these three com-
pounds 1e-g on the L1210 cell line was 100-fold lower
than that of compound 1a (Table 2). This unsatisfactory
result together with that obtained by the National Can-
cer Institute for the phenyl derivative 1a and the
p-chloro and p-fluorophenyl derivatives 1b-c prompted
us to synthesize a series of compounds where the thieno-
pyrrolizinone moiety is substituted at position 3 by an
alkoxyphenyl or a hydroxyphenyl group (derivatives
1h -p , 7a -e, 8a -d , Schemes 1-3).11 In a preliminary
communication, we described a general route to prepare
3-substituted thienopyrrolizinones from suitable ary-
lacetonitriles (Scheme 1). These nitriles precursors are
commercially available excepted for those neces-
sary for the synthesis of compounds 1m -p , which were
prepared from O-benzyl-protected isovanillin and vanil-
lin.12 Then treatment of arylacetonitriles with ethyl
formate and sodium methoxide led to the corresponding
hydroxyacrylonitrile sodium salts 2h -n in 53-98%
yields. These were then protected by a benzenesulfonyl
group to give 3h -n . Reaction of the latter compounds
with methyl thioglycolate and sodium methoxide af-
The methoxyphenyl derivatives 1d ,h -k were dem-
ethylated, affording the corresponding phenols 7a -e
(Scheme 2). The best yields were obtained using 1 equiv
of boron tribromide for each methoxy group to cleave.16
Then, to explore relationship between cytotoxic activity
and steric parameters from alkoxy substituents, we
synthesized a series of p-alkoxyphenyl derivatives from
the 1d -derived phenolic compound 7c. This one readily
reacted with various commercial alkyl bromides in the
presence of sodium methoxide to yield the corresponding
ethers 8a -d in 63-77% yields (Scheme 3).
To continue our SAR study on compound 1d , we
decided to investigate the thieno[2,3-b]pyrrolizin-8-one
skeleton and more precisely the role played by the

1450 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
Sch em e 1a
a
Reaction conditions: (a) ethyl formate, MeONa, MeOH; (b) sulfonylbenzene chloride, DMF; (c) methyl thioglycolate, MeONa, MeOH;
(d) dimethoxyTHF, 4-chloropyridine hydrochloride, dioxane; (e) pyrrolidine; (f) (i) POCl3, (ii) 10% NaOH; (g) 33% HBr in AcOH.
Sch em e 2
sized the bicyclic pyrrolizinone analogue 14. Its prepar-
ation was performed starting from the isovanillin,
taking into account our previous work on the synthesis
of such pyrrolizinones17 and using the method of selec-
tive O-benzyl deprotection (Scheme 4).
Isovanillin was first protected by a benzyl group, pro-
viding the O-protected isovanillin 9 with excellent yield,
which gave in a one-pot reaction the corresponding â-
aminopropanoic acid 10 in 58% yield, using ammonium
acetate and malonic acid in ethanol.18,19 Compound 10
was then heated in acetic acid in the presence of 2,5-di-
methoxyTHF to provide pyrrole 11, which was converted
into the corresponding amide 12 by treatment with ethyl
chloroformate, triethylamine, and pyrrolidine. Intramo-
lecular ring closure was performed using phosphorus
oxychloride in toluene to yield a Vilsmeier salt, which
was then hydrolyzed in alkaline conditions into pyr-
rolizinone 13. Finally, deprotection of compounds 13
using a 33% hydrobromic acid solution in glacial acetic
acid gave 3-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-
1H-pyrrolizin-1-one 14 in 65% yield (Scheme 4).
Sch em e 3
thiophene (route 2) and the pyrrolizinone rings (route
3) in global cytotoxic activity (Figure 2).
Rou te 2. Before synthesizing isosteric analogues of
1d , we wanted to know what would be the activity of
one analogue where cycle D was directly linked to the
pyrrolizinone ring without fused thiophene.
So, considering the best cytotoxic compound 1o
(IC50(L1210) ) 0.015 µM) obtained in route 1 (see Biologi-
cal Results and Discussion part, Table 2), we synthe-
In the second part of route 2, a more exhaustive
chemical study was run with the synthesis of bioiso-
steric analogues in the furane, pyrrole, and benzene
series. The chemical strategy adopted for the heterocy-
clic ones (furane-pyrrole) is based on the general route
described for thiophene series (Scheme 1). First, we
needed to develop the original chemistry to synthesize
Sch em e 4a
a
Reaction conditions: (a) BnBr, K2CO3, MeOH; (b) malonic acid, ammonium acetate, EtOH; (c) 2,5-dimethoxyTHF, acetic acid; (d) (i)
ethyl chloroformate, NEt3, acetone, (ii) pyrrolidine; (e) (i) POCl3, toluene, (ii) 5% NaOH; (f) 33% HBr in AcOH.

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1451
Sch em e 5a
a
Reaction conditions: (a) diethyl chloromalonate, DMF; (b) DBN, MeOH; (c) AcOH, H2O; (d) AcONa, diethyl aminomalonate HCl,
MeOH/H2O; (e) MeONa, MeOH; (f) 2,5-dimethoxyTHF, 4-chloropyridine hydrochloride, dioxane; (g) pyrrolidine (h) (i) POCl3, (ii) 10%
NaOH.
Sch em e 6a
the key intermediates, i.e., methyl 3-amino-4-arylfuran-
2-carboxylate 15 and methyl 3-amino-4-aryl-1H-pyrrol-
2-carboxylate 16, respectively. We describe herein only
the synthesis of one compound for each series with 3,4-
dimethoxyphenyl as the substituent because these syn-
theses are very difficult and are currently under inves-
tigation. The route outlined in Scheme 5 started with
the hydroxyacrylonitrile sodium salt 2j. Condensation
of the latter with diethyl chloromalonate in N,N-di-
methylformamide (DMF) provided an unstable vinyl
ether intermediate that directly cyclized into 15 after
removal of the solvent and in the presence of 1,5-diaza-
bicyclo[4.3.0]non-5-ene (DBN) in methanol.20 The cor-
responding pyrrole 16 was obtained from 2j following
Elliot’s procedure.21 So compound 2j under acidic condi-
a
Reaction conditions: (a) 5% NaOH, 30% H2O2; (b) 2,5-
tions gave the corresponding enol 17, which was then
reacted with diethylaminomalonate hydrochloride to
provide the enamine 18. Ring closure was performed
by treatment with sodium methoxide in methanol in
85% yield. At the end, the expected aminoesters 15 and
16 were submitted successively to the Clauson-Kaas
reaction and condensation with pyrrolidine to yield the
corresponding amides 21 and 22, respectively. The latter
compound was treated with phosphorus oxychloride,
and a 10% aqueous sodium hydroxide solution gave the
tricyclic pyrrolizinones 23 and 24, respectively (Scheme
5).
dimethoxyTHF, 4-chloropyridine hydrochloride, dioxane; (c) SOCl2,
EtOH; (d) 1 M BBr3, CH2Cl2; (e) RB(OH)2, NaHCO3, Pd(PPh3)4,
DME/H2O.
Sch em e 7a
By analogy, the thiophene ring being considered as
the first bioisoster of the benzene ring, this prompted
us to prepare a series of 5-arylpyrroloindolones 29a -d
in which the phenyl ring was fused to pyrrolizinone
instead of thiophene. Indeed, molecular modeling stud-
ies showed that the three-dimensional structures of
these compounds were very close to those in the
thiophene series as assessed by preliminary X-ray
crystallographic data. So we synthesized the direct
analogues 29 in which the aryl substituent is in the
ortho position related to the pyrrole link and not in the
meta. By analysis of possible approaches to obtain 29,
a convergent pathway starting from the easily accessible
7-iodoisatin and based on our previous studies22 was
followed. As shown in Scheme 6, oxidative cleavage of
7-iodoisatin was readily performed in an alkaline me-
dium in the presence of hydrogen peroxide, giving the
expected anthranilic acid 25 in 81% yield.23 Introduction
of a pyrrole ring was accomplished by reaction with 2,5-
a
Reaction conditions: (a) NaBH4, MeOH; (b) NH2OH‚HCl,
pyridine; (c) (i) Cl(CH2)3N(CH3)2, K2CO3, HCl, acetone/H2O, (ii)
oxalic acid, isOH.
dimethoxyTHF. Initially it was thought that the inter-
mediate acid 26 could be cyclized directly using Vils-
meier-Haack conditions (DMF/POCl3), but all attempts
failed. So we considered a subsequent esterification by
treatment of 26 using thionyl chloride in ethanol.
Treatment of the corresponding carboxylate 27 by boron
tribromide in dichloromethane afforded 5-iodopyrroloin-

1452 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
Sch em e 8a
a
Reaction conditions: (a) NaOH, EtOH; (b) NEt3, NaN3, acetone; (c) 1,2-dichlorobenzene; (d) 1 M BBr3, CH2Cl2.
Ta ble 3. In Vitro Antiproliferative Activity on L1210 Cells
(Route 2)
dolones 28 in 62% yield.24 The last step was performed
via a Suzuki cross coupling with commercial arylboronic
acids providing 5-arylpyrroloindolones 29a -d in 58-
73% yields. So compounds 29a -d were synthesized in
five steps from 7-iodoisatin and with overall yields of
15-18% (Scheme 6).
compd
R
GI50 (µM)
Rou te 3. In the third route aimed at studying the
role played by the pyrrolizinone ring, the carbonyl group
of 1d was first reduced in alcohol 30 using sodium
borohydride, and then condensation with hydroxylamine
in pyridine gave the oxime 31 in 95% yield. The latter
was subsequently substituted by a chloroamine chain
under appropriate conditions to form the corresponding
oxime ether 32 (Scheme 7). To determine the importance
of the central ring size and the need for a planar tricyclic
system, pyrrolothienopyrazinones 35 and 36 were pre-
pared from ester 5j. Saponification of 5j leading to the
corresponding acid 33 allowed formation of azide 34 in
70% yield. Treatment of 34 in hot o-dichlorobenzene
provided the pyrazinone 35 by ring closure via a
modified Curtius rearrangement.25,26 Finally O-de-
methylation using boron tribromide gave the corre-
sponding diphenol 36 (Scheme 8).
14
C6H3(mOH,pOCH3)
25
compd
R
GI50 (µM)
29a
29b
29c
29d
C6H3(m,pdiOCH3)
C6H4pOCH3
2-furyl
72.2
3
27.6
71.9
2-thienyl
compd
X
R
GI50 (µM)
Biologica l Resu lts a n d Discu ssion
23
24
O
NH
C6H3(m,pdiOCH3)
C6H3(m,pdiOCH3)
24.2
11.8
In vitro evaluation of the compounds’ antitumor
activity and particularly their effects on the cell cycle
were studied on the L1210 cell line (Tables 2-4).
Analysis of the results obtained for the derivatives
resulting from our SAR study enables us to specify the
structural requirements to identify a new lead com-
pound and finally to begin understanding their mech-
anism of action.
Ta ble 4. In Vitro Antiproliferative Activity on L1210 Cells
(Route 3)
First, thienopyrrolizinone 1d (NSC 676693) confirmed
the promising results provided by the National Cancer
Institute because it expresses a GI50 of 0.19 µM on the
L1210 cell line and this activity seemed closely depend-
ent on the cycle in position 3. Indeed, absence of this
cycle or its replacement by a small alkyl substituent
totally abolished the activity (1e, GI50 ) 85 µM; 1f, GI50
> 100 µM). Similarly, 1g, the position isomer of 1d , was
found to be 100 times less active (GI50 ) 9.4 µM) (Table
2). Considering substitutions on the cycle in position 3,
it appeared, as indicated in Table 2, that weak struc-
tural modifications were responsible for the large GI50
variation and that the hydroxy (7a -d ) and methoxy
(1d ,h -l) series are not really comparable in term of
SAR. Let us note here the activity of the diphenol 7d ,
which expressed a GI50 of 0.28 µM, similar to those of
1d . However, the substituent size seemed to be a feature
closely related to the activity. Among the ethers 8a -d ,
only the ethyl one 8a (GI50 ) 0.43 µM) kept an activity
similar to that of 1d , and any additional steric hin-
drance harmed the activity (8b-d , GI50 > 10 µM).
compd
R
GI50 (µM)
30
31
32
sOH
dNOH
4.9
34.3
3.9
dNO(CH2)3N(CH3)2 (oxalate)
compd
R
GI50 (µM)
>100
8.8
35
36
OCH3
OH
Taking into account the fact that the two more active
compounds are 1d and 7d , we considered the synthesis
of “mixed” compounds bearing either a hydroxy or

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1453
Ta ble 5. In Vitro Antiproliferative Activity of 1o against a Panel of Nine Tumor Cell Lines
cell line:
L1210
15
B16
23
OVCAR3
31
A2780
40
DU145
43
A549
45
HT29
713
LS174T
887
KBA1
15
P388
21
1o GI50 (nM):
Ch a r t 1. X-ray Crystal Structure for 1o
Ch a r t 2. X-ray Crystal Structure for 1j
methoxy substituent in the 3 and 4 positions. These
vicinal methoxy and hydroxy groups on a phenyl ring
appear to be a recurrent theme among antitubulin
agents1,5 and more particularly with combretastatin A-4
and analogues.27 Our first attempts to prepare these
analogues starting from the dimethoxy derivative 1j
failed to produce selective O-demethylation and gave
only a mixture of monomethylated compounds. Simi-
larly, mono-O-methylation of the dihydroxy derivative
7d using various conditions also failed. This prompted
us to prepare the two isomers 1o and 1p by unequivocal
routes starting from vanillic and isovanillic derivatives,
respectively (Scheme 1). Pharmacological evaluation of
1o,p was beyond our expectations because one of the
isomers, the isovanillic 1o (GI50 ) 0.015 µM), is about
1000-fold more active than the vanillic 1p (GI50 ) 8 µM)
and is the best compound of the series, being 10-fold
more potent than the parent 4-methoxy derivative 1d .
This result was confirmed over a panel of nine tumoral
cell lines. Activities in the nanomolar range were
measured for all of the histological types tested, and a
surprising sensitivity for the multidrug resistant cell
line KB-A1 (Table 5) was measured.
In addition, the weak activity of 1p (GI50 ) 8 µM) is
comparable with that observed for 1j (GI50 ) 11.7 µM),
showing the harmful effect of the steric hindrance on
the phenol function in meta position. This assumption
is corroborated by the first X-ray crystallographic data
obtained for compounds 1o and 1j. We can thus note
very important differences in the relative positions of
the phenyl cycle and the tricyclic system plans. The
orientation observed in the best derivative 1o (44.3°)
(Chart 1) can be explained by the small steric hindrance
of the phenol function. It seems that such an orientation

1454 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
Ta ble 6. Anticancer Activity of 1d in the Hollow Fiber Assaya
toward the interior of the medium plan of the molecule
is essential for activity. This is confirmed by the fact
that in the case of the dimethoxyphenyl 1j analogue,
which is 1000-fold less active than 1o, we observed an
orientation of the phenyl cycle of (128° (Chart 2), which
positions in all the cases the meta methoxy substituent
toward the outside of the medium plan of the molecule.
We currently try to crystallize the vanillic isomer 1p
because its X-ray crystallographic analysis would enable
us to check this assumption.
compd
ip score
16
sc scoreb
0
total score
16
net cell kill
yes
1d
a
A total of 12 ip and 12 sc cell lines were tested in triplicate at
two dosage levels, and each cell line with a 50% or greater
b
reduction in viable cell line was given a score of 2. The ip and sc
scores are the sums of all of the ip and sc scores. A compound
with a combined ip + sc score g 20, a sc score g 8, or a net cell
kill of one or more cell lines is considered as active in this assay.
perform a tubulin polymerization inhibitory test on the
best compound 1o using deoxypodophyllotoxin as an
internal reference (IC50 ) 2.4 µM). The result was
beyond our expectations because 1o inhibited microtu-
bule polymerization with an IC50 of 2.9 µM. This
capacity to interact with the mitotic spindle confirms
the assumption of a powerful antimitotic activity, and
our best compounds 1d , 1o, and 7d were evaluated in
an in vivo study, even if additional tubulin assays for
compounds 7d (IC50 ) 21 µM) and 1j (IC50 ) 19 µM)
did not shown any correlation between their effect on
tubulin assembly and their cytotoxicity.
Moreover, the results from routes 2 and 3 allowed us
to refine the structural requirements of the family.
Study of the influence of thiophene revealed that this
cycle is essential for cytotoxicity, taking into account the
result for pyrrolizinone 14 (GI50 ) 25 µM). Its substitu-
tion by a benzene in the pyrroloindole series 29 did not
permit us to obtain activities similar to those in the
thiophene series. For instance, we noted that the best
compound 29b, which expressed a GI50 of 3 µM, was
more than 10 times less active than its thiophene
analogue 1d (GI50 ) 0.19 µM). On the other hand, if we
consider the furan and pyrrole series, it appears that
the nature of the heteroatom on the cycle has no
influence on the activity observed because compounds
23 and 24 expressed a GI50 of 24.2 and 11.8 µM,
respectively, while a GI50 of 11.7 µM was obtained for
the thiophene analogue 1j (Table 3). Last, the modifica-
tions made to the pyrrolizinone cycle, concerning the
extension of the central cycle (35 and 36 (GI50 > 5 µM))
or the carbon 8 hybridization (30, GI50 ) 4.9 µM), and
the nature of its substituents (31, 32; GI50 > 3 µM) led
to a reduction or a loss of the initial activity of 1d .
Compound 1d was first evaluated by the National
Cancer Institute in an in vivo model where polyvi-
nylidene fluoride hollow fibers containing various cancer
cell cultures were implanted intraperitoneally (ip) and
subcutaneously (sc) into mice. Compound 1d was ad-
ministered by the ip route,30 and its effects on the
reduction of tumoral cell mass were determined. The
compound was tested against a panel of 12 human
tumor cell lines as described previously.31 It was solu-
bilized in 10% DMSO in saline/Tween-80R and admin-
istered intraperitoneally once daily for a total of four
doses at each of two dose levels. The day after the last
dose, the fibers were collected and assessed for viable
cell mass. A score of 2 was assigned each time the
compound produced a 50% or greater reduction in viable
cell mass compared to the vehicle-treated controls. The
score for 1d is summed in Table 6 for the intraperitoneal
and subcutaneous fibers. Compound 1d expressed a
good ip score with, however, a total lack of subcutaneous
activity (Table 6). Taking into account the ip adminis-
tration, this profile indicates that the compound pre-
sented an insufficient bioavaibility due to a lack of
solubility in physiological conditions. Nevertheless,
evaluation of 1d is currently continued for further in
vivo testing in standard subcutaneous xenograft models
(LOX IMVI, MDA-MB-231, SF-295, NCI-H23) because
of its net cell kill effect found in the hollow fiber assay.
Finally, this SAR study indicated that our compounds
must possess a tripentacyclic skeleton with a phenyl in
position 3 bearing small substituents in the meta and
para positions. Moreover, discovery of the new lead
compound 1o prompted us to carry out pharmacological
investigations to elucidate the mechanism of action of
the best compounds.
Preliminary flow cytometric studies (Table 2) showed
that L1210 cells treated by the more potent compounds
are arrested in the G2/M phases of the cell cycle, with a
significant percentage of cells having reinitiated a cycle
of DNA synthesis without cell division (8N DNA con-
tent). Compound 1o led to an accumulation of 86% of
cells in G2/M at 0.5 µM with 66% of cells having a DNA
content greater than 4N chromosomes. This pharma-
cological profile is similar to that observed for the
majority of the antimitotic agents and kinase inhibitors
and led us to run the COMPARE program set up by
the National Cancer Institute, which permits us to
establish for any compound submitted to the human
cancer cell lines screen a correlation (expressed by a
Pearson correlation coefficient (PCC)) with the various
references recorded in NCI database.28,29 Any PCC value
greater than 0.55 can be regarded as significant to
assume a certain degree of similarity for the mechanism
of action, independent of the structural considerations.
Application of this program to 1d (NSC 676693) con-
firmed the pharmacological effect observed on the cycle
of L1210 leukemia cells because all PCC values greater
than 0.55 are observed for products having tubulin as
the biological target (e.g., paclitaxel, vinca alkaloids,
maytansin, rhizoxin). These results prompted us to
In parallel, the Institut de Recherche Servier under-
took on compounds 1o and 7d with strong in vitro
potentiality an evaluation of their in vivo activity in an
ip P388 murine leukaemia model. In this test, the
antitumor activity is expressed by the life span of mices
(i.e., the average survival time of the treated mice (T)
in comparison with the average survival time of mice
controls (C)) measured following a single injection of the
product by ip and iv routes. The result is significant
when ratio T/C (%) is above 125% (Table 7). The same
profile as that of 1d was observed for 1o and 7d with a
moderate ip activity at high concentrations but with no

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1455
Ta ble 7. Anticancer Activity of 1o and 7d against an ip P388
125.5. 125.3, 120.8, 120.6, 120.5, 114.7, 112.6, 110.5, 54.7.
Anal. (C16H11NO2S) C, H, N.
Murine Modela
3-(3-Me t h oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (1i). The thienopyrrolizinone 1i was isolated as an orange
ip (mg/kg)
iv (mg/kg)
compd
25 50 100 200 400 25 50 100 200 400
powder in 71% yield: mp 150-152°C; IR 1680 (CdO) cm-1
;
1H NMR (DMSO-d6) δ 8.13 (s, 1H, H2), 7.43 (m, 1H, H5′), 7.15
(m, 2H, H2′ and H6′), 7.03 (d, J 4′,5′ ) 8.0 Hz, 1H, H4′), 6.89 (d,
J 7,6 ) 3.8 Hz, 1H, H7), 6.75 (d, J 5,6 ) 2.6 Hz, 1H, H5), 6.11 (m,
1H, H6), 3.80 (s, 3H, OCH3); 13C NMR (CDCl3) δ 174.5, 156.3,
150.2, 135.2, 134.8, 130.4, 129.3, 125.9, 125.1, 121.7, 120.7,
120.4, 115.3, 112.3, 111.3, 55.4. Anal. (C16H11NO2S) C, H, N.
3-(3,4-Dim eth oxyp h en yl)-8H-th ien o[2,3-b]p yr r olizin -8-
on e (1j). The thienopyrrolizinone 1j was isolated as an orange
7d T/C (%) 111 118 134 133 149 95 101 105 103 21
1o T/C (%) 108 116 121 117 121 102 104 102 103 19
a
Mice were inoculated ip with 106 leukemic P388 cells, and the
two compounds were tested with increasing concentrations (25-
400 mg/kg) and administered the first day of the assay in a single
injection. A compound is considered as active when the ratio T/C
(%) is up to 125%.
powder in 53% yield: mp 190 °C; IR 1679 (CdO) cm-1 1H
;
iv activity. This confirmed the poor solubility of the
compounds, and we are currently investigating prodrug
approaches27d,32 (esters of malic acid or retinoic acid and
N-alkylcarbamate esters) in thiophene series (1o, 7d )
using the reactivity of the phenol function to obtain
products with sufficient aqueous solubility and stability
and high reconversion in vivo.
While additional kinetic in vivo studies are necessary
to have a better understanding of the behavior of our
compounds, these preliminary results are very encour-
aging. We have discovered a new antimitotic family
based on the arylthienopyrrolizinone molecular skeleton
with a growth inhibitory activity in the nanomolar range
for the most promising compounds. The first results of
this SAR study allowed us to specify structural require-
ments and prompted us to synthesize some new ana-
logues to improve the in vivo results. In particular, we
are currently developing an original prodrug chemistry
on the best compounds 1o and 7d .
NMR (DMSO-d6) δ 8.05 (s, 1H, H2), 7.16 (s, 1H, H2′), 7.09 (m,
2H, H5′ and H6′), 6.94 (m, 1H), 6.75 (m, 1H), 6.11 (m, 1H, H6),
3.80 (s, 6H, OCH3); 13C NMR (CDCl3) δ 179.8, 150.8, 149.4,
149.2, 135.9, 134.1, 129.5, 124.9, 123.2, 120.6, 120.4, 115.6,
113.3, 111.3, 111.1, 56.1, 55.9. Anal. (C17H13NO3S) C, H, N.
3-(3,4,5-Tr im eth oxyph en yl)-8H-th ien o[2,3-b]pyr r olizin -
8-on e (1k ). The thienopyrrolizinone 1k was isolated as a
yellow powder in 53% yield: mp 180-182 °C; IR 1680 (CdO)
1
cm-1; H NMR (DMSO-d6) δ 8.10 (s, 1H, H2), 7.02 (d, J 5,6
)
1.8 Hz, 1H, H5), 6.88 (s, 2H, H2′ and H6′), 6.75 (d, J 7,6 ) 3.3
Hz, 1H, H7), 6.11 (m, 1H, H6), 3.82 (s, 6H, OCH3), 3.71 (s, 3H,
OCH3); 13C NMR (CDCl3) δ 173.1, 150.3, 151.4, 146.5, 135.3,
135.2, 133.9, 130.1, 125.9, 121.8, 115.7, 113.6, 106.7. Anal.
(C18H15NO4S) C, H, N.
3-(3,4-Meth ylen edioxyph en yl)-8H-th ien o[2,3-b]pyr r oliz-
in -8-on e (1l). The thienopyrrolizinone 1l was isolated as an
orange powder in 47% yield: mp 168-169 °C; IR 1691 (CdO)
cm-1; 1H NMR (DMSO-d6) δ 8.11 (s, 1H, H2), 7.24 (s, 1H, H2′),
7.12 (m, 2H, H5′ and H6′), 6.96 (m, 1H, H5), 6.82 (m, 1H, H7),
6.18 (m, 3H, H6 and CH2); 13C NMR (CDCl3) δ 176.4, 152.5,
148.2, 148.0, 136.7, 134.9, 128.8, 126.5, 24.9, 121.8, 120.7,
115.8, 114.1, 112.5, 111.5, 87.9. Anal. (C16H9NO3S) C, H, N.
3-(3-Ben zyloxy-4-m eth oxyph en yl)-8H-th ien o[2,3-b]p yr -
r olizin -8-on e (1m ). The thienopyrrolizinone 1m was isolated
as a yellow powder in 71% yield: mp 138 °C; IR 1680 (CdO)
Exp er im en ta l Section
Ch em istr y. Commercial reagents were used as received
without additional purification. Melting points were deter-
mined on a Kofler melting point apparatus and are uncor-
rected. Elemental analyses for new compounds were within
(0.4% of theoretical values and were performed at the Institut
de Recherche en Chimie Organique Fine (Rouen, France). IR
spectra were recorded on a Genesis series FTIR infrared
cm-1; 1H NMR (CDCl3) δ 7.35 (m, 6H, Harom), 7.05 (dd, J 6′,5′
)
8.2 Hz, J 6′,2′ ) 1.9 Hz, 1H, H6′), 6.98 (d, J 2′,6′ ) 1.9 Hz, 1H,
H2′), 6.96 (d, J 5′,6′ ) 8.2 Hz, 1H, H5′), 6.62 (dd, J 7,6 ) 3.6 Hz,
J 7,5 ) 0.7 Hz, 1H, H7), 6.45 (dd, J 5,6 ) 2.6 Hz, J 5,7 ) 0.7 Hz,
1H, H5), 5.88 (dd, J 6,5 ) 2.6 Hz, J 6,7 ) 3.6 Hz, 1H, H6), 5.21 (s,
2H, CH2), 3.95 (s, 3H, OCH3); 13C NMR (CDCl3) δ 174.1, 150.7,
150.1, 148.1, 136.5, 135.8, 134.1, 129.4, 128.7, 128.0, 127.0,
126.9, 124.9, 121.1, 120.7, 115.5, 113.5, 113.3, 111.9, 70.8, 56.1.
Anal. (C23H17NO3S) C, H, N.
1
spectrometer using KBr pellets. The H and 13C NMR spectra
were obtained on a J EOL Lambda 400 spectrometer using
DMSO-d6 or CDCl3 as solvent and TMS as internal standard.
The chemical shifts (δ) are reported in ppm, and the coupling
constants are in hertz. Electron impact mass spectra (EIMS)
were obtained using a J EOL J MS GCMate spectrometer.
Reactions were monitored by thin-layer chromatography (TLC)
using 0.2 mm Polygram Sil silica gel G/UV254 precoated plates
with visualization by irradiation with a short-wavelength UV
light. Silica gel flash chromatography was performed using
63-200 µm Kieselgel Merck 60 silica gel.
Gen er al P r ocedu r e for th e Syn th esis of th e Th ien opyr -
r olizin on es 1h -n . A solution of the corresponding amide 6
(10 mmol) in phosphorus oxychloride (25 mL) was stirred at
70 °C for 2 h. After cooling, the reaction mixture was
concentrated to give a red solid that was filtered, washed with
Et2O, and dried. Then, this intermediary iminium salt was
added slowly to a 10% aqueous sodium hydroxide solution (70
mL) and the mixture was successively heated for 1 h to 50 °C
and filtered when returned to room temperature to provide
the crude product 1, which was purified by column chroma-
tography (chloroform).
3-(4-Ben zyloxy-3-m eth oxyph en yl)-8H-th ien o[2,3-b]p yr -
r olizin -8-on e (1n ). The thienopyrrolizinone 1n was isolated
as an orange powder in 52% yield: mp 188-190 °C; IR 1672
1
(CdO) cm-1; H NMR (CDCl3) δ 7.39 (m, 6H, Harom), 6.98 (m,
3H, H2′, H5′ and H6′), 6.75 (d, J 5,6 ) 2.2 Hz, 1H, H5), 6.67 (d,
J 7,6 ) 3.4 Hz, 1H, H7), 6.01 (dd, J 6,5 ) 2.2 Hz, J 6,7 ) 3.4 Hz,
1H, H6), 5.21 (s, 2H, CH2), 3.92 (s, 3H, OCH3); 13C NMR
(CDCl3) δ 174.2, 150.9, 150.3, 148.3, 136.7, 136.1, 134.7, 129.9,
129.3, 128.1, 127.1, 126.5, 125.7, 121.3, 120.9, 116.2, 113.7,
113.3, 111.9, 70.8, 55.9. Anal. (C23H17NO3S) C, H, N.
Gen er al P r ocedu r e for th e Syn th esis of th e Th ien opyr -
r olizin on es 1o,p . A solution of the corresponding O-benzylth-
ienopyrrolizinones 1m ,n (1.94 g, 5 mmol) in a 33% solution of
hydrobromic acid in glacial acetic acid (20 mL) was stirred at
room temperature for 1 h. After cooling, the reaction mixture
was diluted with water (50 mL) and the resulting precipitate
was extracted with ethyl acetate (3 × 30 mL). Then, the
organic layers were combined, washed with a 10% aqueous
NaHCO3 solution, dried (MgSO4), and evaporated to give an
orange solid. This residue was purified by column chromatog-
raphy, eluting by hexane/ethyl acetate (2:1) to provide 1o,p .
3-(3-Hyd r oxy-4-m eth oxyp h en yl)-8H-th ien o[2,3-b]p yr -
r olizin -8-on e (1o). The thienopyrrolizinone 1o was isolated
as an orange powder in 55% yield: mp 204 °C; IR 3427 (O-
H), 1678 (CdO) cm-1; 1H NMR (CDCl3) δ 7.36 (s, 1H, H2), 7.01
(d, J 2′,6′ ) 1.7 Hz, 1H, H2′), 6.92 (dd, J 6′,5′ ) 8.3 Hz, J 6′,2′ ) 1.7
3-(2-Me t h oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (1h ). The thienopyrrolizinone 1h was isolated as an
orange powder in 58% yield: mp 142 °C; IR 1685 (CdO) cm-1
;
1H NMR (DMSO-d6) δ 8.02 (s, 1H, H2), 7.48 (m, 1H, H4′), 7.41
(d, J 6′,5′ ) 7.5 Hz, 1H, H6′), 7.18 (d, J 3′,4′ ) 8.2 Hz, 1H, H3′),
7.07 (m, 1H, H5′), 6.72 (d, J 7,6 ) 3.8 Hz, 1H, H7), 6.49 (d, J 5,6
) 2.6 Hz, 1H, H5), 6.07 (m, 1H, H6), 3.77 (s, 3H, OCH3); 13C
NMR (CDCl3) δ 173.7, 155.8, 151.5, 135.4, 135.1, 130.1, 130.0,

1456 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
Hz, 1H, H6′), 6.86 (d, J 5′,6′ ) 8.3 Hz, 1H, H5′), 6.74 (d, J 5,6 ) 2.4
Hz, 1H, H5), 6.59 (d, J 7,6 ) 3.5 Hz, 1H, H7), 5.92 (m, 1H, H6),
5.78 (br s, 1H, D2O exchangeable, OH), 3.89 (s, 3H, OCH3);
13C NMR (CDCl3) δ 174.1, 150.8, 147.1, 146.1, 135.8, 134.1,
129.5, 126.9, 124.6, 121.0, 119.8, 115.7, 114.2, 113.2, 110.9,
56.1. Anal. (C16H11NO3S) C, H, N.
mp 102 °C; IR 2226 (CN), 1193 (SO2) cm-1; 1H NMR (DMSO-
d6) δ 7.99 (m, 6H, Harom), 6.75 (m, 2H, Harom), 3.70 (m, 9H,
OCH3).
2-Cya n o-2-(3,4-m et h ylen ed ioxyp h en yl)vin ylb en zen e
Su lfon a te (3l). 3l was obtained as a beige powder in 47%
yield: mp 110°C; IR 2221 (CN), 1195 (SO2) cm-1 1H NMR
;
(DMSO-d6) δ 7.96 (m, 4H, Harom), 7.01 (m, 5H, Harom), 6.06 (m,
3-(4-Hyd r oxy-3-m eth oxyp h en yl)-8H-th ien o[2,3-b]p yr -
r olizin -8-on e (1p ). The thienopyrrolizinone 1p was isolated
as a yellow powder in 58% yield: mp 190-191 °C; IR 3396
(O-H), 1679 (CdO) cm-1; 1H NMR (CDCl3) δ 7.36 (s, 1H, H2),
6.92 (m 3H, H2′, H5′, and H6′), 6.68 (d, J 5,6 ) 2.1 Hz, 1H, H5),
6.60 (d, J 7,6 ) 3.7 Hz, 1H, H7), 5.93 (m, 1H, H6), 5.77 (br s, 1H,
D2O exchangeable, OH), 3.86 (s, 3H, OCH3); 13C NMR (CDCl3)
δ 174.1, 150.9, 146.8, 146.2, 135.9, 134.1, 129.7, 126.8, 124.4,
2H, CH2).
2-Cya n o-2-(3-b en zyloxy-4-m et h oxyp h en yl)vin ylb en -
zen e Su lfon a te (3m ). 3m was obtained as a beige powder
(from methanol) in 50% yield: mp 136 °C; IR 2232 (CN), 1202
1
(SO2) cm-1; H NMR (CDCl3) δ 7.97 (m, 2H, Harom), 7.75 (m,
1H, Harom), 7.62 (m, 2H, Harom), 7.46 (s, 1H, Hvinyl), 7.35 (m,
5H, Harom), 6.95 (dd, J 6,5 ) 8.4 Hz, J 6,2 ) 1.6 Hz, 1H, H6), 6.90
(d, J 2,6 ) 1.6 Hz, 1H, H2), 6.85 (d, J 5,6 ) 8.4 Hz, 1H, H5), 5.12
(s, 2H, CH2), 3.87 (s, 3H, OCH3).
121.2, 120.7, 115.6, 114.9, 113.3, 110.5, 56.1. Anal. (C16H11
NO3S) C, H, N.
-
Gen er a l P r oced u r e for th e Syn th esis of 2-Ar yl-3-
h yd r oxya cr ylon itr iles Sod iu m Sa lts 2h -n . The corre-
sponding arylacetonitrile (100 mmol) was added dropwise to
a solution of ethyl formate (8.9 mL, 110 mmol) and sodium
methoxide (5.9 g, 110 mmol) in methanol (150 mL). The
mixture was refluxed for 2 h. The resulting precipitate was
collected, washed with diethyl ether (2 × 50 mL), and dried
under reduced pressure with P2O5 to give 2h -n , which were
used without further purification.
2-Cya n o-2-(4-b en zyloxy-3-m et h oxyp h en yl)vin ylb en -
zen e Su lfon a te (3n ). 3n was obtained as a white powder
(from methanol) in 52% yield: mp 104 °C; IR 2227 (CN), 1192
1
(SO2) cm-1; H NMR (CDCl3) δ 7.98 (m, 2H, Harom), 7.59 (m,
3H, Harom), 7.38 (m, 6H, Harom), 7.11 (m, 1H, Harom), 6.86 (m,
2H, Harom), 5.16 (s, 2H, CH2), 3.86 (s, 3H, OCH3).
Gen er al P r ocedu r e for th e Syn th esis of Meth yl 3-Am in o-
4-a r yl-2-th iop h en e Ca r boxyla tes 4h -n . Sodium methylate
was prepared from sodium (1.38 g, 60 mmol) in methanol (150
mL). After the mixture was cooled, methyl thioglycolate (2.16
mL, 24 mmol) and the sulfonate 3 (20 mmol) were slowly and
successively added, and the reaction mixture was heated at
60 °C for 2 h. It was then concentrated and diluted with water
(200 mL) and stirred overnight to afford a precipitate. The
latter was filtered, washed with water (3 × 100 mL), dried
over P2O5 under vacuum, and crystallized from ethanol to
afford 4.
Meth yl 3-Am in o-4-(2-m eth oxyph en yl)-2-th ioph en e Car -
boxyla te (4h ). 4h was obtained as a white powder in 58%
yield: mp 110-111 °C; IR 3486 (N-H), 3371 (N-H), 1678 (Cd
O) cm-1; 1H NMR (DMSO-d6) δ 7.49 (s, 1H, Hthiophene), 7.38 (m,
1H, H4), 7.19 (d, J 3,4 ) 7.1 Hz, 1H, H3), 7.10 (d, J 6,5 ) 8.2 Hz,
1H, H6 ), 7.02 (m, 1H, H5), 6.01 (br s, 2H, D2O exchangeable,
3-Hyd r oxy-2-(2-m eth oxyp h en yl)a cr ylon itr ile Sod iu m
Sa lt (2h ). 2h was obtained as a white powder in 85% yield:
mp > 260 °C; IR 2180 (CN) cm-1
.
3-Hyd r oxy-2-(3-m eth oxyp h en yl)a cr ylon itr ile Sod iu m
Sa lt (2i). 2i was obtained as a white powder in 73% yield:
mp > 260 °C; IR 2176 (CN) cm-1
.
3-Hyd r oxy-2-(3,4-d im eth oxyp h en yl)a cr ylon itr ile So-
d iu m Sa lt (2j). 2j was obtained as a white powder in 83%
yield: mp > 260 °C; IR 2166 (CN) cm-1
.
3-Hyd r oxy-2-(3,4,5-tr im eth oxyp h en yl)a cr ylon itr ile So-
d iu m Sa lt (2k ). 2k was obtained as a beige powder in 98%
yield: mp > 260 °C; IR 2179 (CN) cm-1
.
3-H yd r oxy-2-(3,4-m e t h yle n e d ioxyp h e n yl)a cr ylon i-
tr ile Sod iu m Sa lt (2l). 2l was obtained as a beige powder in
NH2), 3.75 (s, 3H, OCH3), 3.73 (s, 3H, OCH3). Anal. (C13H13
NO3S) C, H, N.
-
53% yield: mp > 260 °C; IR 2177 (CN) cm-1
.
3-Hyd r oxy-2-(3-ben zyloxy-4-m eth oxyp h en yl)a cr ylon i-
Meth yl 3-Am in o-4-(3-m eth oxyph en yl)-2-th ioph en e Car -
boxyla te (4i). 4i was obtained as a white powder in 52%
yield: mp 93 °C; IR 3453 (N-H), 3341 (N-H), 1685 (CdO)
tr ile Sod iu m Sa lt (2m ). 2m was obtained as a yellow powder
in 83% yield: mp ) 196 °C; IR 2172 (CN) cm-1
.
3-Hyd r oxy-2-(4-ben zyloxy-3-m eth oxyp h en yl)a cr ylon i-
cm-1 1H NMR (DMSO-d6) δ 7.67 (s, 1H, Hthiophene), 7.37 (m,
;
tr ile Sod iu m Sa lt (2n ). 2n was obtained as a yellow powder
1H, H4), 7.01 (m, 3H, Harom), 6.29 (br s, 2H, D2O exchangeable,
in 92% yield: mp ) 204 °C; IR 2175 (CN) cm-1
.
NH2), 3.79 (s, 3H, OCH3), 3.75 (s, 3H, OCH3). Anal. (C13H13
NO3S) C, H, N.
-
Gen er a l P r oced u r e for th e Syn th esis of 2-Cya n o-2-
a r ylvin ylben zen e Su lfon a tes 3h -n . A solution of the
corresponding sodium salt 2 (100 mmol) in DMF (150 mL) was
cooled with an ice bath to 0 °C before adding dropwise the
benzene sulfonyl chloride (15.3 mL, 120 mmol). The reaction
mixture was heated at 70 °C for 2 h and then diluted with
water (200 mL) and stirred overnight to afford a precipitate.
The latter was filtered, washed with water (3 × 100 mL), and
dried over P2O5 under vacuum to give 3h -n , which were used
without further purification.
2-Cya n o-2-(3-m eth oxyp h en yl)vin ylben zen e Su lfon a te
(3h ). 3h was obtained as a beige powder in 32% yield: mp 78
°C; IR 2221 (CN), 1193 (SO2) cm-1; 1H NMR (DMSO-d6) δ 8.29
(m, 1H, Harom), 8.10-7.75 (m, 5H, Harom), 7.41 (m, 2H, Harom),
7.07 (m, 2H, Harom), 3.71 (m, 3H, OCH3).
Meth yl 3-Am in o-4-(3,4-d im eth oxyp h en yl)-2-th iop h en e
Ca r boxyla te (4j). 4j was obtained as a white powder in 63%
yield: mp 154 °C; IR 3426 (N-H), 3331 (N-H), 1667 (CdO)
cm-1 1H NMR (DMSO-d6) δ 7.58 (s, 1H, Hthiophene), 6.99 (m,
;
3H, Harom), 6.25 (br s, 2H, D2O exchangeable, NH2), 3.79 (s,
3H, OCH3), 3.77 (s, 3H, OCH3), 3.74 (s, 3H, OCH3). Anal.
(C14H15NO4S) C, H, N.
Meth yl 3-Am in o-4-(3,4,5-tr im eth oxyph en yl)-2-th ioph en e
Ca r boxyla te (4k ). 4k was obtained as a white powder in 65%
yield: mp 146 °C; IR 3468 (N-H), 3361 (N-H), 1675 (CdO)
cm-1; 1H NMR (DMSO-d6) δ 7.65 (s, 1H, Hthiophene), 6.72 (s, 1H,
H
arom), 6.70 (s, 1H, Harom), 6.34 (br s, 2H, D2O exchangeable,
NH2), 3.75 (s, 6H, OCH3), 3.73 (m, 6H, OCH3). Anal. (C15H17
NO5S) C, H, N.
-
2-Cya n o-2-(2-m eth oxyp h en yl)Vin ylben zen e Su lfon a te
Meth yl 3-Am in o-4-(3,4-m eth ylen ed ioxyp h en yl)-2-th io-
p h en e Ca r boxyla te (4l). 4l was obtained as a beige powder
in 48% yield: mp 168 °C; IR 3460 (N-H), 3345 (N-H), 1687
(CdO) cm-1; 1H NMR (DMSO-d6) δ 7.57 (s, 1H, Hthiophene), 6.95
(m, 3H, Harom), 6.23 (br s, 2H, D2O exchangeable, NH2), 6.04
(s, 2H, CH2), 3.74 (s, 3H, OCH3). Anal. (C13H11NO4S) C, H, N.
Met h yl 3-Am in o-4-(3-ben zyloxy-4-m et h oxyp h en yl)-2-
th iop h en e Ca r boxyla te (4m ). 4m was obtained as a white
powder in 53% yield: mp 92 °C; IR 3464 (N-H), 3349 (N-H),
1677 (CdO) cm-1; 1H NMR (CDCl3) δ 7.35 (m, 5H, Harom), 7.10
(s, 1H, Hthiophene), 6.92 (m, 3H, H2, H5, and H6), 5.42 (br s, 2H,
D2O exchangeable, NH2), 5.18 (s, 2H, CH2), 3.91 (s, 3H, OCH3),
(3i). 3i was obtained as a beige powder in 50% yield: mp 67-
1
69 °C; IR 2222 (CN), 1215 (SO2) cm-1; H NMR (DMSO-d6) δ
8.03 (m, 1H, Harom), 7.94-7.71 (m, 5H, Harom), 7.32 (m, 3H,
H
arom), 7.03 (m, 1H, Harom), 3.67 (m, 3H, OCH3).
2-Cya n o-2-(3,4-d im et h oxyp h en yl)vin ylb en zen e Su l-
fon a te (3j). 3j was obtained as a white powder (from diethyl
ether) in 55% yield: mp 164 °C; IR 2233 (CN), 1196 (SO2) cm-1
;
1H NMR (DMSO-d6) δ 8.01 (m, 2H, Harom), 7.71 (m, 4H, Harom),
6.99 (m, 1H, Harom), 6.85 (m, 2H, Harom), 3.89 (m, 6H, OCH3).
2-Cya n o-2-(3,4,5-tr im eth oxyp h en yl)vin ylben zen e Su l-
fon a te (3k ). 3k was obtained as a beige powder in 58% yield:

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1457
3.82 (s, 3H, OCH3); 13C NMR (CDCl3) δ 164.9, 151.4, 149.4,
147.9, 136.6, 132.8, 128.5, 127.9, 127.8, 127.2, 126.7, 121.0,
113.9, 112.2, 100.8, 70.7, 55.9, 51.1. Anal. (C20H19NO4S) C, H,
N.
125.3, 122.4, 120.3, 112.0, 111.4, 109.3, 70.5, 55.8, 52.2. Anal.
(C24H21NO4S) C, H, N.
Meth yl 4-(4-Ben zyloxy-3-m eth oxyp h en yl)-3-(p yr r ol-1-
yl)-2-th iop h en e Ca r boxyla te (5n ). 5n was obtained as a
white powder in 76% yield: mp 124-126 °C; IR 1725 (CdO)
cm-1; 1H NMR (CDCl3) δ 7.34 (m, 6H, Harom), 6.80 (d, J 5,6 ) 8
Hz, 1H, H5), 6.71 (d, J 6,5 ) 8 Hz, 1H, H6), 6.63 (m, 2H, HRpyrrole),
6.25 (m, 2H, Hâpyrrole), 6.21 (s, 1H, H2), 5.12 (s, 2H, CH2), 3.78
(s, 3H, OCH3), 3.60 (s, 3H, OCH3); 13C NMR (CDCl3) δ 160.8,
149.2, 147.8, 141.3, 140.4, 136.8, 128.4, 127.8, 127.2, 126.6,
126.3, 125.9, 122.3, 119.7, 113.4, 109.9, 109.3, 70.7, 55.6, 52.1.
Anal. (C24H21NO4S) C, H, N.
Met h yl 3-Am in o-4-(4-b en zyloxy-3-m et h oxyp h en yl)-2-
th iop h en e Ca r boxyla te (4n ). 4n was obtained as a white
powder in 64% yield: mp 130-132 °C; IR 3420 (N-H), 3331
(N-H), 1676 (CdO) cm-1 1H NMR (CDCl3) δ 7.38 (m, 5H,
;
Harom), 7.18 (s, 1H, Hthiophene), 6.93 (m, 3H, H2, H5, and H6),
5.61 (br s, 2H, D2O exchangeable, NH2), 5.19 (s, 2H, CH2), 3.91
(s, 3H, OCH3), 3.85 (s, 3H, OCH3); 13C NMR (CDCl3) δ 174.5,
142.2, 141.5, 139.9, 138.5, 132.2, 128.9, 128.5, 127.3, 127.1,
Gen er a l P r oced u r e for th e Syn th esis of 4-Ar yl-3-
(p yr r ol-1-yl)th ien -2-ylp yr r olid in e Ca r boxa m id es 6h -n .
A solution of methyl 4-aryl-3-(pyrrol-1-yl)-2-thenoate 5 (10
mmol) in pyrrolidine (40 mL) was refluxed for 2 h and 30 min.
After the mixture was cooled and evaporated, the yellow oil
was dissolved in chloroform (150 mL) and the solution was
washed with an 1 N aqueous hydrochloric acid solution (2 ×
150 mL), dried (MgSO4), and evaporated to give, after cooling
in an ice bath, a beige solid washed with petroleum ether (2
× 100 mL), filtered, and dried over P2O5 under vacuum to give
6h -n .
120.3, 119.4, 114.1, 112.9, 111.7, 70.9, 56.1, 51.3. Anal. (C20H19
NO4S) C, H, N.
-
Gen er a l P r oced u r e for th e Syn th esis of Meth yl 4-Ar yl-
3-(p yr r ol-1-yl)-2-th iop h en e Ca r boxyla tes 5h -n . A solution
of 2,5-dimethoxytetrahydrofuran (1.42 mL, 11 mmol) in diox-
ane (100 mL) was stirred for 15 min with 4-chloropyridine
hydrochloride (1.65 g, 11 mmol). The methyl 3-amino-4-aryl-
2-thiophene carboxylate 4 (10 mmol) was added, and the
reaction mixture was refluxed for 3 h and filtered through a
small pad of Celite. The solvent was evaporated to give a brown
residue that was dissolved in methylene chloride (150 mL).
The solution was washed with an 1 N aqueous hydrochloric
acid solution (2 × 100 mL), dried (MgSO4), and evaporated to
give a beige solid. An analytical sample was crystallized from
diethyl ether.
Meth yl 4-(2-Meth oxyph en yl)-3-(pyr r ol-1-yl)-2-th ioph en e
Ca r boxyla te (5h ). 5h was obtained as a white powder in 72%
yield: mp 141-143 °C; IR 1723 (CdO) cm-1; 1H NMR (DMSO-
d6) δ 7.88 (s, 1H, Hthiophene), 7.26 (dd, J 4,3 ) 6.1 Hz, J 4,5 ) 7.1
Hz, 1H, H4), 6.95 (d, J 3,4 ) 6.1 Hz, 1H, H3), 6.90 (d, J 6,5 ) 8.3
Hz, 1H, H6 ), 6.84 (dd, J 5,6 ) 8.3 Hz, J 5,4 ) 7.1 Hz, 1H, H5),
6.53 (m, 2H, HRpyrrole), 5.95 (m, 2H, Hâpyrrole), 3.69 (s, 3H, OCH3),
3.48 (s, 3H, OCH3). Anal. (C17H15NO3S) C, H, N.
4-(2-Meth oxyp h en yl)-3-(p yr r ol-1-yl)th ien -2-ylp yr r oli-
d in e Ca r boxa m id e (6h ). 6h was obtained as a beige powder
in 89% yield: mp 108 °C; IR 1627 (CdO) cm-1 1H NMR
;
(DMSO-d6) δ 7.66 (s, 1H, Hthiophene), 7.29 (m, 1H, H4), 7.10 (d,
J 6,5 ) 6.8 Hz, 1H, H6), 6.92 (m, 2H, H5 and H3), 6.43 (m, 2H,
HRpyrrole), 6.02 (m, 2H, Hâpyrrole), 3.80 (s, 3H, OCH3), 3.30 (m,
2H, HRpyrrolidine), 2.57 (m, 2H, HRpyrrolidine), 1.64 (m, 2H, Hâpyrrolidine),
1.51 (m, 2H, Hâpyrrolidine). Anal. (C20H20N2O2S) C, H, N.
4-(3-Meth oxyp h en yl)-3-(p yr r ol-1-yl)th ien -2-ylp yr r oli-
d in e Ca r boxa m id e (6i). 6i was obtained as a beige powder
in 82% yield: mp 91-94 °C; IR 1615 (CdO) cm-1 1H NMR
;
(DMSO-d6) δ 7.86 (s, 1H, Hthiophene), 7.19 (t, J 5,4 ) 7.9 Hz, J 5,6
) 7.9 Hz, 1H, H5), 6.82 (d, J 4,5 ) 7.9 Hz, 1H, H4), 6.66 (dd, J 6,5
) 7.9 Hz, J 6,2 ) 2.2 Hz, 1H, H6), 6.57 (m, 2H, HRpyrrole), 6.44
(d, J 2,6 ) 2.2 Hz, 1H, H2), 6.13 (m, 2H, Hâpyrrole), 3.59 (s, 3H,
OCH3), 3.34 (m, 2H, HRpyrrolidine), 2.78 (m, 2H, HRpyrrolidine), 1.68
(m, 2H, Hâpyrrolidine), 1.59 (m, 2H, Hâpyrrolidine). Anal. (C20H20N2O2S)
C, H, N.
Meth yl 4-(3-Meth oxyph en yl)-3-(pyr r ol-1-yl)-2-th ioph en e
Ca r boxyla te (5i). 5i was obtained as a white powder in 84%
1
yield: mp 74 °C; IR 1715 (CdO) cm-1; H NMR (DMSO-d6) δ
8.11 (s, 1H, Hthiophene), 7.17 (m, 1H, H5), 6.82 (dd, J 6,5 ) 8.3
Hz, J 6,2 ) 2.2 Hz, 1H, H6), 6.68 (m, 2H, HRpyrrole), 6.64 (m, 1H,
H4), 6.36 (d, J 2,6 ) 2.2 Hz, 1H, H2), 6.12 (m, 2H, Hâpyrrole), 3.70
(s, 3H, OCH3), 3.58 (s, 3H, OCH3). Anal. (C17H15NO3S) C, H,
N.
4-(3,4-Dim eth oxyp h en yl)-3-(p yr r ol-1-yl)th ien -2-ylp yr -
r olid in e Ca r boxa m id e (6j). 6j was obtained as a beige
powder in 75% yield: mp 115 °C; IR 1633 (CdO) cm-1 1H
;
Meth yl 4-(3,4-Dim eth oxyp h en yl)-3-(p yr r ol-1-yl)-2-th io-
p h en e Ca r boxyla te (5j). 5j was obtained as a beige powder
in 92% yield: mp 122-124 °C; IR 1714 (CdO) cm-1; 1H NMR
(DMSO-d6) δ 8.04 (s, 1H, Hthiophene), 6.85 (m, 1H, Harom), 6.68
(m, 3H, Harom and 2HRpyrrole), 6.23 (m, 1H, Harom), 6.13 (m, 2H,
Hâpyrrole), 3.70 (s, 6H, OCH3), 3.49 (s, 3H, OCH3). Anal. (C18H17
NO4S) C, H, N.
Met h yl 4-(3,4,5-Tr im et h oxyp h en yl)-3-(p yr r ol-1-yl)-2-
NMR (DMSO-d6) δ 7.79 (s, 1H, Hthiophene), 6.87 (d, J 5,6 ) 8.5
Hz, 1H, H5), 6.69 (d, J 6,5 ) 8.5 Hz, 1H, H6), 6.59 (m, 2H,
HRpyrrole), 6.33 (s, 1H, H2), 6.14 (m, 2H, Hâpyrrole), 3.71 (s, 3H,
OCH3), 3.50 (s, 3H, OCH3), 3.34 (m, 2H, HRpyrrolidine), 2.80 (m,
2H, HRpyrrolidine), 1.68 (m, 2H, Hâpyrrolidine), 1.59 (m, 2H, Hâpyrrolidine).
Anal. (C21H22N2O3S) C, H, N.
-
4-(3,4,5-Tr im eth oxyph en yl)-3-(pyr r ol-1-yl)th ien -2-ylpyr -
r olid in e Ca r boxa m id e (6k ). 6k was obtained as a beige
1
powder in 83% yield: mp 68-72 °C; IR 1618 (CdO) cm-1; H
th iop h en e Ca r boxyla te (5k ). 5k was obtained as a white
powder in 77% yield: mp 110 °C; IR 1695 (CdO) cm-1 1H
;
NMR (DMSO-d6) δ 7.89 (s, 1H, Hthiophene), 6.62 (m, 2H, HRpyrrole),
6.25 (s, 2H, H2 and H6), 6.16 (m, 2H, Hâpyrrole), 3.60 (s, 9H,
OCH3), 3.32 (m, 2H, HRpyrrolidine), 2.83 (m, 2H, HRpyrrolidine), 1.68
(m, 2H, Hâpyrrolidine), 1.61 (m, 2H, Hâpyrrolidine). Anal. (C22H24N2O4S)
C, H, N.
NMR (DMSO-d6) δ 8.15 (s, 1H, Hthiophene), 6.70 (m, 2H, HRpyrrole),
6.21 (s, 2H, H2 and H6), 6.16 (m, 2H, Hâpyrrole), 3.69 (s, 3H,
OCH3), 3.60 (s, 9H, OCH3). Anal. (C19H19NO5S) C, H, N.
Meth yl 4-(3,4-Meth ylen ed ioxyp h en yl)-3-(p yr r ol-1-yl)-
2-th iop h en e Ca r boxyla te (5l). 5l was obtained as a white
4-(3,4-Met h ylen ed ioxyp h en yl)-3-(p yr r ol-1-yl)t h ien -2-
ylp yr r olid in e Ca r boxa m id e (6l). 6l was obtained as a beige
powder in 93% yield: mp 116 °C; IR 1728 (CdO) cm-1 1H
;
powder in 71% yield: mp 124 °C; IR 1625 (CdO) cm-1 1H
;
NMR (DMSO-d6) δ 8.01 (s, 1H, Hthiophene), 6.80 (d, J 5,6 ) 8.03
Hz, 1H, H5), 6.66 (m, 2H, HRpyrrole), 6.51 (dd, J 6,5 ) 8.03 Hz,
J 6,2 ) 1.4 Hz, 1H, H6), 6.35 (d, J 2,6 ) 1.4 Hz, 1H, H2), 6.12 (m,
2H, Hâpyrrole), 5.96 (s, 2H, CH2), 3.68 (s, 3H, OCH3). Anal.
(C17H13NO4S) C, H, N.
NMR (DMSO-d6) δ 7.76 (s, 1H, Hthiophene), 6.82 (d, J 5,6 ) 8.0
Hz, 1H, H5), 6.57 (m, 2H, HRpyrrole), 6.52 (d, J 6,5 ) 8.0 Hz, 1H,
H6), 6.44 (s, 1H, H2), 6.14 (m, 2H, Hâpyrrole), 5.98 (s, 2H, CH2O),
3.33 (m, 2H, HRpyrrolidine), 2.77 (m, 2H, HRpyrrolidine), 1.67 (m, 2H,
Hâpyrrolidine), 1.59 (m, 2H, Hâpyrrolidine). Anal. (C20H18N2O3S) C,
H, N.
Meth yl 4-(3-Ben zyloxy-4-m eth oxyp h en yl)-3-(p yr r ol-1-
yl)-2-th iop h en e Ca r boxyla te (5m ). 5m was obtained as a
white powder in 69% yield: mp 166 °C; IR 1725 (CdO) cm-1
;
4-(3-Ben zyloxy-4-m eth oxyp h en yl)-3-(p yr r ol-1-yl)th ien -
2-ylp yr r olid in e Ca r boxa m id e (6m ). 6m was obtained as a
1H NMR (CDCl3) δ 7.43 (s, 1H, Hthiohene), 7.33 (m, 5H, Harom),
6.81 (d, J 5,6 ) 8.4 Hz, 1H, H5), 6.75 (dd, J 6,5 ) 8.4 Hz, J 6,2
)
beige powder in 77% yield: mp 120 °C; IR 1628 (CdO) cm-1
;
2.1 Hz, 1H, H6), 6.62 (m, 2H, HRpyrrole), 6.28 (m, 2H, Hâpyrrole),
6.26 (d, J 2,6 ) 2.1 Hz, 1H, H2), 4.84 (s, 2H, CH2), 3.86 (s, 3H,
OCH3), 3.79 (s, 3H, OCH3); 13C NMR (CDCl3) δ 160.9, 149.3,
147.9, 141.5, 140.5, 136.9, 128.4, 127.7, 127.2, 126.5, 126.3,
1H NMR (CDCl3) δ 7.33 (m, 6H, Harom), 6.82 (d, J 5,6 ) 8.3 Hz,
1H, H5), 6.75 (dd, J 6,5 ) 8.3 Hz, J 6,2 ) 1.8 Hz, 1H, H6), 6.57
(m, 2H, HRpyrrole), 6.34 (d, J 2,6 ) 1.8 Hz, 1H, H2), 6.18 (m, 2H,
Hâpyrrole), 4.85 (s, 2H, CH2), 3.87 (s, 3H, OCH3), 3.49 (m, 2H,

1458 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
HRpyrrolidine), 2.75 (m, 2H, HRpyrrolidine), 1.76 (m, 2H, Hâpyrrolidine),
1.65 (m, 2H, Hâpyrrolidine); 13C NMR (CDCl3) δ 162.6, 149.1, 147.8,
137.6, 136.9, 134.8, 130.5, 128.4, 127.7, 127.2, 126.6, 123.1,
121.5, 120.5, 112.8, 111.4, 109.7, 70.5, 55.8, 47.4, 46.1, 25.6,
24.1. Anal. (C27H26N2O3S) C, H, N.
4-(4-Ben zyloxy-3-m eth oxyp h en yl)-3-(p yr r ol-1-yl)th ien -
2-ylp yr r olid in e Ca r boxa m id e (6n ). 6n was obtained as a
beige powder in 82% yield: mp 150-152 °C; IR 1626 (CdO)
cm-1; 1H NMR (CDCl3) δ 7.36 (m, 6H, Harom), 6.82 (d, J 5,6 ) 8
Hz, 1H, H5), 6.72 (dd, J 6,5 ) 8 Hz, J 6,2 ) 1.6 Hz, 1H, H6), 6.57
(m, 2H, HRpyrrole), 6.29 (m, 1H, H2), 6.16 (m, 2H, Hâpyrrole), 5.13
(s, 2H, CH2), 3.61 (s, 3H, OCH3), 3.50 (m, 2H, HRpyrrolidine), 2.76
(m, 2H, HRpyrrolidine), 1.78 (m, 2H, Hâpyrrolidine), 1.64 (m, 2H,
Hâpyrrolidine); 13C NMR (CDCl3) δ 161.5, 149.2, 147.7, 137.6,
136.8, 134.9, 130.4, 128.5, 127.8, 127.2, 127.1, 123.1, 121.5,
119.9, 113.5, 110.8, 109.7, 70.8, 55.9, 47.4, 46.1, 25.6, 24.1.
Anal. (C27H26N2O3S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(Hyd r oxy-
p h en yl)-8H-th ien o[2,3-b]p yr r olizin -8-on es 7a -e. A solu-
tion of 3-(methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 1
(3.5 mmol) in methylene chloride (30 mL) was stirred and
cooled to 0 °C with an ice bath. Then, a 1 M boron tribromide
solution in methylene chloride was added (3.5 mL, 3.5 mmol
for each methyl ether bond to cleave) and the reaction mixture
was stirred for 0.5 h to room temperature and diluted with
cold water (100 mL). After 3 h, the orange solid was filtered,
washed with methylene chloride (2 × 50 mL), dried over P2O5
under vacuum, and recrystallized from ethanol.
d6) δ 173.2, 151.4, 150.3, 146.5, 135.3, 135.2, 133.9, 130.1,
125.9, 121.8, 115.7, 113.6, 106.7. Anal. (C15H9NO4S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(Alk oxy-
p h en yl)-8H-th ien o[2,3-b]p yr r olizin -8-on es 8a -d . A solu-
tion of sodium (0.48 g, 2.1 mmol) in methanol (50 mL) was
stirred and cooled to 0 °C before adding successively 3-(4-
hydroxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 7c (0.5 g, 2.1
mmol) and the corresponding alkyl bromide (2.1 mmol). The
reaction mixture was refluxed for 3 h and concentrated under
reduced pressure, and the beige residue was stirred in a 0.5
N aqueous sodium hydroxide solution (100 mL) for 15 min.
The resulting precipitate was filtered, washed with water (2
× 50 mL), dried over P2O5 under vacuum, and recrystallized
from ethanol/methanol (1:2).
3-(4-E t h oxyp h en yl)-8H -t h ien o[2,3-b]p yr r olizin -8-on e
(8a ). 8a was obtained as an orange powder in 77% yield using
ethyl bromide (0.15 mL, 2.1 mmol) as reagent: mp ) 198-
1
201 °C; IR 1687 (CdO) cm-1; H NMR (DMSO-d6) δ 8.02 (s,
1H, H2), 7.50 (d, J ) 8.6 Hz, 2H, H2′ and H6′), 7.03 (d, J ) 8.6
Hz, 2H, H3′ and H5′), 6.86 (d, J 5,6 ) 2.5 Hz, 1H, H5), 6.75 (d,
J 7,6 ) 3.5 Hz, 1H, H7), 6.03 (m, 1H, H6), 4.08 (q, J ) 6.9 Hz,
2H, CH2), 1.35 (t, J ) 6.9 Hz, 3H, CH3); 13C NMR (CDCl3) δ
174.2, 159.3, 150.9, 135.9, 133.9, 129.4, 129.1, 126.8, 124.4,
120.8, 115.6, 114.8, 113.2, 63.6, 14.7. Anal. (C17H13NO2S) C,
H, N.
3-(4-n -P r op oxyp h en yl)-8H -t h ien o[2,3-b]p yr r olizin -8-
on e (8b). 8b was obtained as an orange powder in 74% yield
using n-propyl bromide (0.19 mL, 2.1 mmol) as reagent: mp
) 172 °C; IR 1680 (CdO) cm-1; 1H NMR (DMSO-d6) δ 8.03 (s,
1H, H2), 7.50 (d, J ) 7.6 Hz, 2H, H2′ and H6′), 7.06 (d, J ) 7.6
Hz, 2H, H3′ and H5′), 6.87 (d, J 5,6 ) 2.3 Hz, 1H, H5), 6.75 (d,
J 7,6 ) 3.4 Hz, 1H, H7), 6.11 (m, 1H, H6), 3.97 (m, 2H, OCH2),
1.75 (m, 2H, CH2), 0.98 (t, J ) 6.7 Hz, 3H, CH3); 13C NMR
(CDCl3) δ 174.1, 159.5, 150.3, 135.7, 133.3, 129.1, 129.2, 127.1,
3-(2-H yd r oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (7a ). 7a was obtained as an orange powder in 62% yield
starting from 3-(2-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-
1
8-one 1h : mp 192 °C; IR 3243 (O-H), 1660 (CdO) cm-1; H
NMR (DMSO-d6) δ 10.08 (br s, 1H, D2O exchangeable, OH),
7.99 (s, 1H, H2), 7.31 (m, 2H, Harom), 7.01 (m, 1H, Harom), 6.91
(m, 1H, Harom), 6.69 (m, 1H, Harom), 6.60 (m, 1H, Harom), 6.06
(m, 1H, H6); 13C NMR (CDCl3) δ 175.3, 153.1, 150.3, 136.2,
131.5, 131.2, 129.4, 126.1, 124.0, 121.5, 119.3, 119.1, 115.7,
113.8, 112.2. Anal. (C15H9NO2S) C, H, N.
124.8, 121.5, 115.9, 114.9, 112.7, 62.4, 31.6, 13.7. Anal. (C18H15
NO2S) C, H, N.
-
3-(4-i-P r op oxyp h en yl)-8H -t h ien o[2,3-b]p yr r olizin -8-
on e (8c). 8c was obtained as an orange powder in 63% yield
using isopropyl bromide (0.19 mL, 2.1 mmol) as reagent: mp
3-(3-H yd r oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (7b). 7b was obtained as an orange powder in 77% yield
starting from 3-(3-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-
8-one 1i: mp > 260 °C; IR 3293 (O-H), 1661 (CdO) cm-1; 1H
NMR (DMSO-d6) δ 9.78 (br s, 1H, D2O exchangeable, OH), 8.08
(s, 1H, H2), 7.31 (m, 1H, H5′), 6.97 (m, 2H, Harom), 6.90 (d, J 5,6
) 2.1 Hz, 1H, H5), 6.85 (m, 1H, Harom), 6.76 (d, J 7,6 ) 3.6 Hz,
1H, H7), 6.13 (m, 1H, H6); 13C NMR (CDCl3) δ 175.1, 152.6,
151.4, 137.3, 132.3, 131.6, 129.6, 126.3, 124.7, 122.2, 118.8,
119.5, 116.3, 113.9, 113.2. Anal. (C15H9NO2S) C, H, N.
3-(4-H yd r oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (7c). 7c was obtained as an orange powder in 88% yield
starting from 3-(4-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-
8-one 1d : mp > 260 °C; IR 3165 (O-H), 1662 (CdO) cm-1; 1H
NMR (DMSO-d6) δ 9.72 (br s, 1H, D2O exchangeable, OH), 7.89
(s, 1H, H2), 7.32 (d, J ) 8.5 Hz, 2H, H2′ and H6′), 6.82 (d, J )
8.5 Hz, 2H, H3′ and H5′), 6.80 (d, J 5,6 ) 2.4 Hz, 1H, H5), 6.66
(d, J 7,6 ) 3.4 Hz, 1H, H7), 6.03 (m, 1H, H6). Anal. (C15H9NO2S)
C, H, N.
3-(3,4-Dih yd r oxyp h en yl)-8H-th ien o[2,3-b]p yr r olizin -8-
on e (7d ). 7d was obtained as an orange powder in 68% yield
starting from 3-(3,4-dimethoxyphenyl)-8H-thieno[2,3-b]pyr-
rolizin-8-one 1j: mp > 260 °C; IR 3491 (O-H), 1655 (CdO)
cm-1; 1H NMR (DMSO-d6) δ 9.29 (br s, 2H, D2O exchangeable,
OH), 7.93 (s, 1H, H2), 6.84 (m, 5H, Harom), 6.11 (m, 1H, H6);
13C NMR (DMSO-d6) δ 173.1, 150.4, 146.1, 145.7, 135.3, 135.2,
129.7, 125.9, 122.7, 121.6, 119.1, 116.1, 115.6, 115.1, 113.6.
Anal. (C15H9NO3S) C, H, N.
1
) 132-133 °C; IR 1687 (CdO) cm-1; H NMR (DMSO-d6) δ
8.02 (s, 1H, H2), 7.48 (d, J ) 8.2 Hz, 2H, H2′ and H6′), 7.04 (d,
J ) 8.2 Hz, 2H, H3′ and H5′), 6.88 (d, J 5,6 ) 2.2 Hz, 1H, H5),
6.75 (d, J 7,6 ) 3.2 Hz, 1H, H7), 6.11 (m, 1H, H6), 3.67 (m, 1H,
OCH), 1.28 (d, J ) 5.9 Hz, 6H, CH3); 13C NMR (CDCl3) δ 174.2,
159.2, 150.8, 135.9, 133.6, 129.4, 129.2, 127.2, 125.0, 121.8,
116.3, 115.0, 112.8, 73.1, 19.6, 19.7. Anal. (C18H15NO2S) C, H,
N.
3-(4-n -Bu t oxyp h e n yl)-8H -t h ie n o[2,3-b]p yr r olizin -8-
on e (8d ). 8d was obtained as an orange powder in 75% yield
using n-butyl bromide (0.23 mL, 2.1 mmol) as reagent: mp )
1
129 °C; IR 1679 (CdO) cm-1; H NMR (DMSO-d6) δ 8.02 (s,
1H, H2), 7.50 (d, J ) 8.2 Hz, 2H, H2′ and H6′), 7.06 (d, J ) 8.2
Hz, 2H, H3′ and H5′), 6.87 (d, J 5,6 ) 2.0 Hz, 1H, H5), 6.75 (d,
J 7,6 ) 3.0 Hz, 1H, H7), 6.10 (m, 1H, H6), 4.01 (m, 2H, OCH2),
1.71 (m, 2H, CH2), 1.45 (m, 2H, CH2), 0.93 (m, 3H, CH3); 13C
NMR (CDCl3) δ 174.1, 159.5, 150.8, 135.8, 133.8, 129.5, 129.1,
126.8, 124.3, 120.7, 115.5, 114.9, 113.1, 67.7, 31.2, 19.2, 13.8.
Anal. (C19H17NO2S) C, H, N.
3-Ben zyloxy-4-m eth oxyben za ld eh yd e (9). Isovanillin
(62.5 g, 410 mmol) was dissolved in methanol (250 mL) in the
presence of potassium carbonate (68 g, 492 mmol) and benzyl
bromide (58.5 mL, 492 mmol). The mixture was refluxed for 2
h and filtered, and the filtrate was evaporated under reduced
pressure. The residue was taken up in chloroform (300 mL),
and the solution was washed with water (2 × 200 mL), dried
(MgSO4), and evaporated to afford a white solid in 98% yield:
mp ) 68-70 °C; IR 1678 (CdO) cm-1; 1H NMR (CDCl3) δ 9.80
(s, 1H, CHO), 7.36 (m, 7H, Harom), 6.97 (d, J 5,6 ) 8.5 Hz, 1H,
H5), 5.17 (s, 2H, CH2), 3.93 (s, 3H, OCH3); 13C NMR (CDCl3) δ
190.7, 154.8, 148.5, 136.1, 129.9, 128.5, 128.1, 127.3, 126.8,
111.1, 110.6, 70.6, 56.1. Anal. (C15H14O3) C, H, N.
3-(3,4,5-Tr ih yd r oxyp h en yl)-8H-th ien o[2,3-b]p yr r olizin -
8-on e (7e). 7e was obtained as an orange powder in 83% yield
starting from 3-(3,4,5-trimethoxyphenyl)-8H-thieno[2,3-b]pyr-
rolizin-8-one 1k : mp > 260 °C; IR 3385 (O-H), 1667 (CdO)
cm-1; 1H NMR (DMSO-d6) δ 9.23 (br s, 2H, D2O exchangeable,
OH3′ and OH5′), 8.49 (br s, 1H, D2O exchangeable, OH4′), 7.92
(s, 1H, H2), 7.01 (m, 1H, H5 or H7), 6.75 (m, 1H, H5 or H7),
6.47 (s, 2H, H2′ and H6′), 6.14 (m, 1H, H6); 13C NMR (DMSO-
3-Am in o-3-(3-ben zyloxy-4-m eth oxyp h en yl)p r op a n oic
Acid (10). The aldehyde 9 (33 g, 136 mmol) was dissolved in
ethanol (300 mL) with malonic acid (14.2 g, 136 mmol) and

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1459
ammonium acetate (21 g, 272 mmol). The reaction mixture
was refluxed for 24 h until the appearance of a yellow
precipitate. The latter was filtered, washed with hot ethanol
(150 mL), and dried in a laboratory oven (60 °C): mp ) 258
°C; IR 2103 (Zwitterion), 1676 (CdO) cm-1; 1H NMR (DMSO-
d6) δ 7.42 (m, 5H, Harom), 7.14 (m, 1H, Harom), 6.93 (m, 2H,
Harom), 5.07 (s, 2H, CH2), 4.18 (m, 1H, CH), 3.76 (s, 3H, OCH3),
2.68 (m, 2H, CH2). Anal. (C17H19NO4) C, H, N.
122.5, 117.7, 117.3, 112.2, 110.9, 107.5, 57.8, 55.9, 49.6; EIMS
m/z (relative intensity) 243 (M+, 72), 150 (100), 135 (36), 94
(12), 77,8 (12). Anal. (C14H13NO3) C, H, N.
Meth yl 3-Am in o-4-(3,4-d im eth oxyp h en yl)fu r a n -2-ca r -
boxyla te (15). To a solution of 3-hydroxy-2-(3,4-dimethoxy-
phenyl)acrylonitrile sodium salt 2j (2 g, 8.8 mmol) in DMF
(20 mL) was added diethyl chloromalonate (1.57 mL, 9.7
mmol). The reaction mixture was stirred for 5 h at room
temperature, and the organic solvent was then removed under
reduced pressure. The dark oil was extracted with methylene
chloride (50 mL), and the organic layer was washed with H2O
(2 × 100 mL), dried (MgSO4), and evaporated to give an orange
syrup. The orange syrup was dissolved in ethanol (50 mL) to
which 1,5-diazabicyclo[4.3.0]non-5-ene (1.2 mL, 9.7 mmol) was
added, and the mixture was stirred at room temperature
overnight. The solution was concentrated, and an analytical
sample was prepared by silica gel chromatography, eluting by
ethyl acetate/cyclohexane (1:2). The orange powder was re-
crystallized from methanol to give a yellow solid in 25% yield:
mp ) 121-123 °C; IR 3464 (N-H), 3371 (N-H), 1688 (CdO)
3-(3-Ben zyloxy-4-m et h oxyp h en yl)-3-(p yr r ol-1-yl)p r o-
p a n oic Acid (11). A solution of 2,5-dimethoxytetrahydrofuran
(2.37 mL, 18 mmol) in acetic acid (50 mL) was heated under
stirring at 80 °C for 1 h and 30 min with the propanoic acid
10 (5 g, 17 mmol). The solvent was evaporated to give a brown
residue. The latter was dissolved in methylene chloride (150
mL) and the solution was washed with a 1 N aqueous
hydrochloric acid solution (2 × 100 mL), dried (MgSO4), and
evaporated to give a beige solid. An analytical sample was
recrystallized from diethyl ether to afford a white solid in 72%
yield: mp ) 92 °C; IR 3103 (O-H), 1699 (CdO) cm-1; 1H NMR
(CDCl3) δ 7.22 (m, 5H, Harom), 6.71 (d, J 5′,6′ ) 8.3 Hz, 1H, H5′),
6.63 (d, J 6′,5′ ) 8.3 Hz, 1H, H6′), 6.55 (m, 3H, HRpyrrole and H2′),
6.04 (m, 2H, Hâpyrrole), 5.41 (m, 1H, CH), 4.95 (s, 2H, CH2), 3.73
(s, 3H, OCH3), 3.05 (dd, J 2a,2b ) 16.1 Hz, J 2a,3 ) 8.5 Hz, 1H,
1
cm-1; H NMR (CDCl3) δ 7.35 (s, 1H, Hfuran), 6.94 (m, 3H, H2,
H5, and H6), 4.69 (br s, 2H, D2O exchangeable, NH2), 3.91 (s,
9H, OCH3); 13C NMR (CDCl3) δ 160.1, 149.1, 148.5, 141.7,
126.0, 122.2, 119.9, 119.6, 111.4, 110.5, 55.9, 55.1; EIMS m/z
(relative intensity) 277 (M+, 100), 262 (40), 203 (50), 174 (31),
146 (32). Anal. (C14H15NO5) C, H, N.
H
2a), 2.96 (dd, J 2b,2a ) 16.1 Hz, J 2b,3 ) 6.6 Hz, 1H, H2b); 13C
NMR (CDCl3) δ 175.3, 149.3, 147.9, 136.6, 132.6, 128.4, 127.8,
127.4, 119.4, 118.9, 112.4, 111.6, 108.4, 70.9, 58.4, 55.8, 40.6.
Anal. (C21H21NO4) C, H, N.
Meth yl 3-Am in o-4-(3,4-d im eth oxyp h en yl)-1H-p yr r ole-
2-ca r boxyla te (16). To a solution of the enamine (3.85 g, 11
mmol) in methanol (80 mL) was added sodium methoxide (0.65
g, 12 mmol). The reaction mixture was refluxed for 45 min,
and the dark solution obtained was then stirred for 2 h at room
temperature until the precipitation of a beige solid. After
cooling, the mixture was filtered, washed with cold methanol
(2 × 50 mL), and dried over P2O5 under vacuum to afford a
white solid in 85% yield: mp ) 169 °C; IR 3463 (N-H), 3358
(N-H), 3275 (N-H), 1661 (CdO) cm-1; 1H NMR (CDCl3) δ 6.94
(m, 2H, H5, Harom), 6.86 (d, J ) 8.2 Hz, 1H, Harom), 6.77 (m,
1H, Harom), 4.53 (br s, 3H, D2O exchangeable, NH, NH2), 3.84
(s, 9H, OCH3); 13C NMR (CDCl3) δ 162.3, 149.2, 147.6, 128.6,
128.5, 127.7, 126.7, 119.4, 114.6, 111.7, 110.7, 55.9, 55.8, 50.9;
EIMS m/z (relative intensity) 276 (M+, 64), 244 (100). Anal.
(C14H16N2O4) C, H, N.
3-(3-Ben zyloxy-4-m et h oxyp h en yl)-3-(p yr r ol-1-yl)p yr -
r olidin e P r opion am ide (12). 3-(3-Benzyloxy-4-methoxyphen-
yl)-3-(pyrrol-1-yl)propionic acid 11 (2.1 g, 6 mmol) was dis-
solved in anhydrous acetone (50 mL). After the mixture was
cooled, TEA (0.836 mL, 6 mmol), ethyl chloroformate (0.576
mL, 6 mmol), and pyrrolidine (0.427 g, 6 mmol) were succes-
sively added slowly with a latent period of 15 min. The mixture
was refluxed for 1 h and 30 min. After the mixture was cooled,
the triethylammonium salt was filtered and the solvent was
evaporated to afford a colorless oil in 62% yield: IR 1668 (Cd
1
O) cm-1; H NMR (CDCl3) δ 7.18 (m, 5H, Harom), 6.65 (m, 3H,
H2′, H5′, and H6′), 6.53 (m, 2H, HRpyrrole), 5.97 (m, 2H, Hâpyrrole),
5.56 (m, 1H, CH), 4.90 (s, 2H, CH2), 3.66 (s, 3H, OCH3), 3.20
(m, 2H, HRpyrrolidine), 2.94 (m, 2H, CH2), 2.81 (m, 2H, HRpyrrolidine),
1.61 (m, 4H, Hâpyrrolidine). Anal. (C25H28N2O3) C, H, N.
2-(3,4-Dim eth oxyp h en yl)-3-h yd r oxya cr ylon itr ile (17).
A solution of 3-hydroxy-2-(3,4-dimethoxyphenyl)acrylonitrile
sodium salt 2j (10 g, 44 mmol) in water (100 mL) was acidified
by glacial acetic acid to pH 4. The resulting precipitate was
filtered, washed with water, and dried over P2O5 under
vacuum to afford a white solid in 96% yield which was used
without further purification: mp ) 120-122 °C; IR 3194 (O-
3-(3-Ben zyloxy-4-m eth oxyp h en yl)-2,3-d ih yd r o-1H-p yr -
r olizin -1-on e (13). A solution of 3-(3-benzyloxy-4-methoxy-
phenyl)-3-(pyrrol-1-yl)pyrrolidine propionamide 12 (1 g, 2.5
mmol) in toluene (50 mL) was cooled to 0 °C, and phosphorus
oxychloride (0.46 mL, 5 mmol) was added slowly. The mixture
was refluxed for 1 h, and the solvent was evaporated. The oily
residue was taken up in water (50 mL), stirred overnight, and
filtered. The aqueous layer was diluted with a 5% aqueous
sodium hydroxide solution to pH 10, and the precipitate was
dissolved in chloroform (70 mL). The organic layer was dried
(MgSO4) and evaporated to give a white solid in 53% yield:
1
H), 2208 (CN) cm-1; H NMR (CDCl3) δ 7.41 (s, 1H, H3), 7.20
(m, 1H, Harom), 6.88 (m, 2H, Harom), 3.90 (s, 6H, OCH3).
Dieth yl ((2-Cya n o-2-(3,4-d im eth oxyp h en yl)vin yl)a m i-
n o)m a lon a te (18). 2-(3,4-Dimethoxyphenyl)-3-hydroxyacryl-
onitrile 17 (8 g, 39 mmol) was dissolved in methanol/water
(5:1), and diethyl aminomalonate hydrochloride (12.4 g, 59
mmol) and sodium acetate (10.6 g, 78 mmol) were added. The
mixture was stirred for 48 h to room temperature, and the
solvent was concentrated under reduced pressure. The aqueous
solution was extracted with ethyl acetate (150 mL), and the
organic layer was washed with water (2 × 100 mL), dried
(MgSO4), and evaporated to give a yellow oil that crystallized
from diethyl ether. The white solid was obtained in 35%
yield: mp ) 120 °C; IR 3194 (O-H), 2208 (CN) cm-1; 1H NMR
(CDCl3) δ 7.06 (d, J H1,NH ) 12.9 Hz, 1H, H1), 6.82 (m, 3H,
1
IR 1681 (CdO) cm-1; H NMR (CDCl3) δ 7.22 (m, 5H, Harom),
6.73-6.38 (m, 6H, Harom), 5.27 (m, 1H, CH), 4.93 (s, 2H, CH2),
3.63 (s, 3H, OCH3), 3.32 (m, 1H, H2a), 2.73 (m, 1H, H2b). Anal.
(C21H19NO3) C, H, N.
3-(3-H yd r oxy-4-m et h oxyp h en yl)-2,3-d ih yd r o-1H -p yr -
r olizin -1-on e (14). A solution of the 3-(3-benzyloxy-4-meth-
oxyphenyl)-2,3-dihydro-1H-pyrrolizin-1-one 13 (0.4 g, 1.2 mmol)
in a 33% solution of hydrobromic acid in glacial acetic acid
(10 mL) was stirred at room temperature for 1 h. After cooling,
the reaction mixture was diluted with water (50 mL) and the
resulting precipitate was extracted with ethyl acetate (3 × 20
mL). Then, the organic layers were combined, washed with
an aqueous 10% NaHCO3 solution, dried (MgSO4), and evapo-
rated to give a white solid purified by column chromatography,
eluting by hexane/ethyl acetate (2:1) to provide 14 in 65%
H
arom), 5.72 (dd, J NH,H1 ) 12.9 Hz, J NH,CH ) 8.3 Hz, 1H, D2O
exchangeable, NH), 4.70 (d, J CH,NH ) 8.3 Hz, 1H, CH), 4.31
(m, 4H, CH2), 3.88 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 1.32 (t,
J ) 7.1 Hz, 6H, CH3); 13C NMR (CDCl3) δ 165.9, 149.2, 147.6,
144.5, 126.1, 117.8, 116.2, 111.6, 107.6, 84.5, 62.9, 62.3, 55.9,
55.8, 13.9. Anal. (C18H22N2O6) C, H, N.
yield: mp ) 186 °C; IR 3312 (O-H), 1683 (CdO) cm-1 1H
;
NMR (CDCl3) δ 6.81 (m, 3H, Harom), 6.63 (m, 2H, Harom), 6.52
(m, 1H, Harom), 5.84 (br s, 1H, D2O exchangeable, OH), 5.43
(m, 1H, CH), 3.89 (s, 3H, OCH3), 3.49 (dd, J 2a,2b ) 18 Hz, J 2a,3
) 7.7 Hz, 1H, H2a), 2.93 (dd, J 2b,2a ) 18 Hz, J 2b,3 ) 2.6 Hz, 1H,
H2b); 13C NMR (CDCl3) δ 188.4, 146.7, 146.2, 133.6, 133.1,
Meth yl 4-(3,4-Dim eth oxyp h en yl)-3-(p yr r ol-1-yl)fu r a n -
2-ca r boxyla te (19). A solution of 2,5-dimethoxytetrahydro-
furan (0.26 mL, 2 mmol) in dioxane (50 mL) was stirred for
15 min at room temperature with 4-chloropyridine hydrochlo-

1460 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
ride (0.3 g, 2 mmol). Methyl 3-amino-4-(3,4-dimethoxyphenyl)-
furan-2-carboxylate 15 (0.5 g, 1.8 mmol) was added to the
reaction mixture, which was heated under reflux for 2 h and
then filtered through a small pad of Celite. The filtrate was
evaporated to give a beige residue. The beige residue was
dissolved in methylene chloride (150 mL) and the solution was
washed with an aqueous 1 N hydrochloric acid solution (2 ×
100 mL), dried (MgSO4), and evaporated to give a beige solid
hydroxide solution (70 mL) and the mixture was heated for 1
h to 50 °C. The precipitate was extracted by diethyl ether (100
mL), and the organic layer was dried (MgSO4) and evaporated
under reduced pressure to give a brown residue purified by
column chromatography, eluting by ethyl acetate/cyclohexane
(1:2) to afford an orange powder in 38% yield: mp ) 132-134
1
°C; IR 1684 (CdO) cm-1; H NMR (CDCl3) δ 7.51 (s, 1H, H2),
6.98 (dd, J 6′,5′ ) 8.6 Hz, J 6′,2′ ) 1.7 Hz, 1H, H6′), 6.89 (m, 2H,
H2′ and H5′), 6.77 (d, J 5,6 ) 2.5 Hz, 1H, H5), 6.59 (d, J 7,6 ) 3.1
Hz, 1H, H7), 5.98 (m, 1H, H6), 3.82 (s, 6H, OCH3); 13C NMR
(CDCl3) δ 173.9, 150.8, 149.4, 149.2, 135.9, 134.1, 129.5, 126.8,
124.9, 120.6, 120.5, 115.6, 113.2, 111.4, 111.1, 56.0, 55.9; EIMS
m/z (relative intensity) 295 (M+, 51), 177 (81), 114 (100). Anal.
(C17H13NO4) C, H, N.
1
in 81% yield: IR 1710 (CdO) cm-1; H NMR (CDCl3) δ 7.71
(s, 1H, Hfuran), 6.79 (dd, J 6,5 ) 8.1 Hz, J 6,2 ) 1.8 Hz, 1H, H6),
6.71 (m, 3H, H5 and 2HRpyrrole), 6.27 (m, 2H, 2Hâpyrrole), 6.21 (d,
J 2,6 ) 1.8 Hz, 1H, H2), 3.84 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
3.62 (s, 3H, OCH3). Anal. (C18H17NO5) C, H, N.
Met h yl 4-(3,4-Dim et h oxyp h en yl)-3-(p yr r ol-1-yl)-1H -
p yr r ole-2-ca r boxyla te (20). A solution of 2,5-dimethoxytet-
rahydrofuran (1.48 mL, 11 mmol) and 4-chloropyridine hy-
drochloride (1.7 g, 11 mmol) in dioxane (50 mL) was stirred
for 15 min at room temperature. Methyl 3-amino-4-(3,4-
dimethoxyphenyl)-1H-pyrrole-2-carboxylate 16 (2.85 g, 10
mmol) was added to the reaction mixture, which was refluxed
for 3 h and 30 min and filtered through a small pad of Celite.
The filtrate was evaporated to give a beige residue. The latter
was dissolved in methylene chloride (200 mL), and the solution
was washed with a 1 N aqueous hydrochloric acid solution (2
× 100 mL), dried (MgSO4), and evaporated to give a beige solid
in 72% yield: mp ) 158-161 °C; IR 3385 (N-H), 1704 (CdO)
3-(3,4-Dim eth oxyph en yl)-1,8-dih ydr opyr r olo[2,3-b]pyr -
r olizin -8-on e (24). A solution of 22 (1.4 g, 3.8 mmol) in
phosphorus oxychloride (60 mL) was heated at 70 °C for 5 h.
After cooling, the reaction mixture was evaporated to give a
red solid, which was filtered, washed with Et2O, and dried.
Then, this intermediary iminium salt was added slowly to a
10% aqueous sodium hydroxide solution (100 mL) and the
mixture was heated for 3 h to 80 °C. The precipitate was
extracted by diethyl ether (2 × 100 mL), and the organic layers
were dried (MgSO4) and evaporated under reduced pressure
to give a brown residue purified by column chromatography,
eluting by ethyl acetate to afford a red powder in 62% yield:
cm-1 1H NMR (CDCl3) δ 9.68 (br s, 1H, D2O exchangeable,
;
mp ) 194 °C; IR 3142 (N-H), 1655 (CdO) cm-1 1H NMR
;
NH), 7.09 (d, J H5,NH ) 3.4 Hz, 1H, H5), 6.76 (s, 1H, H2′), 6.74
(d, J 6′,5′ ) 2 Hz, 1H, H6′), 6.70 (m, 2H, HRpyrrole), 6.26 (m, 2H,
Hâpyrrole), 6.21 (d, J 5′,6′ ) 2 Hz, 1H, H5′), 3.84 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 3.61 (s, 3H, OCH3); 13C NMR (CDCl3) δ
160.7, 148.7, 147.6, 127.7, 125.1, 123.1, 122.8, 118.9, 118.4,
117.0, 110.9, 109.3, 108.8, 55.6, 55.4, 51.6; EIMS m/z (relative
intensity) 326 (M+, 78), 294 (69), 136 (100), 83 (92). Anal.
(C18H18N2O4) C, H, N.
(CDCl3) δ 11.18 (br s, 1H, D2O exchangeable, NH), 6.93 (dd,
J 6′,5′ ) 8.2 Hz, J 6′,2′ ) 1.8 Hz, 1H, H6′), 6.91 (d, J H2,NH ) 3 Hz,
1H, H2), 6.88 (d, J 2′,6′ ) 1.8 Hz, 1H, H2′), 6.84 (d, J 5′,6′ ) 8.2
Hz, 1H, H5′), 6.81 (d, J 5,6 ) 2.2 Hz, 1H, H5), 6.47 (d, J 7,6 ) 3.6
Hz, 1H, H7), 5.88 (dd, J 6,5 ) 2.2 Hz, J 6,7 ) 3.6 Hz, 1H, H6),
3.84 (s, 6H, OCH3); 13C NMR (CDCl3) δ 171.4, 149.3, 148.4,
140.2, 136.2, 126.7, 125.1, 124.9, 121.7, 119.1, 114.6, 113.1,
112.2, 111.7, 110.1, 56.1. Anal. (C17H14N2O3) C, H, N.
4-(3,4-Dim eth oxyp h en yl)-3-(p yr r ol-1-yl)fu r a n -2-p yr r o-
lid in e Ca r boxa m id e (21). A solution of methyl 4-(3,4-
dimethoxyphenyl)-3-(pyrrol-1-yl)furan-2-carboxylate 19 (0.48
g, 1.5 mmol) in pyrrolidine (20 mL) was refluxed for 3 h. After
the mixture was cooled and evaporated, the yellow oil was
dissolved in methylene chloride (100 mL) and the solution was
washed with a 1 N aqueous hydrochloric acid solution (2 ×
100 mL), dried (MgSO4), and evaporated to give 21 as a yellow
oil in 63% yield: IR 1636 (CdO) cm-1; 1H NMR (CDCl3) δ 7.53
(s, 1H, Hfuran), 6.70 (dd, J 6,5 ) 8.3 Hz, J 6,2 ) 1.7 Hz, 1H, H6),
6.62 (d, J 5,6 ) 8.3 Hz, 1H, H5), 6.61 (m, 2H, HRpyrrole), 6.18 (d,
J 2,6 ) 1.7 Hz, 1H, H2), 6.14 (m, 2H, Hâpyrrole), 3.75 (s, 6H, OCH3),
3.47 (m, 2H, HRpyrrolidine), 3.12 (m, 2H, HRpyrrolidine), 1.72 (m, 4H,
Hâpyrrolidine).
4-(3,4-Dim eth oxyp h en yl)-3-(p yr r ol-1-yl)-1H-p yr r ole-2-
p yr r olid in e Ca r boxa m id e (22). A solution of methyl 4-(3,4-
dimethoxyphenyl)-3-(pyrrol-1-yl)-1H-pyrrole-2-carboxylate 20
(2.3 g, 7.1 mmol) in pyrrolidine (40 mL) was refluxed for 15 h.
After the mixture was cooled and evaporated, the yellow oil
was dissolved in methylene chloride (150 mL) and the solution
was washed with water (3 × 100 mL), dried (MgSO4), and
evaporated to give a residue purified by column chromatog-
raphy, eluting by ethyl acetate/cyclohexane (1:1) to afford a
white powder in 55% yield: mp ) 150-151 °C; IR 3223 (N-
H), 1593 (CdO) cm-1; 1H NMR (CDCl3) δ 11.01 (br s, 1H, D2O
exchangeable, NH), 6.96 (d, J H5,NH ) 2.7 Hz, 1H, H5), 6.69 (d,
J 5,6 ) 8.3 Hz, 1H, H5), 6.63 (d, J 6,5 ) 8.3 Hz, 1H, H6), 6.55 (m,
2H, HRpyrrole), 6.17 (s, 1H, H2), 6.12 (m, 2H, Hâpyrrole), 3.76 (s,
3H, OCH3), 3.52 (m, 5H, OCH3, HRpyrrolidine), 2.50 (m, 2H,
HRpyrrolidine), 1.69 (m, 2H, Hâpyrrolidine), 1.53 (m, 2H, Hâpyrrolidine);
13C NMR (CDCl3) δ 161.7, 148.6, 147.4, 126.1, 123.5, 122.2,
120.8, 120.5, 118.9, 117.9, 110.9, 109.9, 109.5, 55.7, 55.3, 46.6,
46.4, 26.0, 23.8. Anal. (C21H23N3O3) C, H, N.
2-Am in o-3-iod oben zoic Acid (25). To a stirred suspension
of 7-iodoisatin22 (1 g, 3.7 mmol) in a 5% aqueous sodium
hydroxide solution (15 mL) was added dropwise a 30% aqueous
hydrogen peroxide solution (15 mL). The reaction mixture was
stirred at 50 °C for 30 min and then allowed to reach room
temperature. The filtered solution was acidified to pH 4 with
an aqueous 1 N hydrochloric acid solution, and the white
precipitate 25 was collected by filtration and dried over P2O5
under vacuum with 81% yield: mp ) 182 °C; IR 3540 (O-H),
3230 (N-H), 3178 (N-H), 1665 (CdO) cm-1; 1H NMR (CDCl3)
δ 7.96 (d, J 4,5 ) 8 Hz, 1H, H4), 7.85 (d, J 6,5 ) 7.6 Hz, 1H, H6),
6.44 (dd, J 5,4 ) 8 Hz, J 5,6 ) 7.6 Hz, 1H, H5). Anal. (C7H6NO2I)
C, H, N.
3-Iod o-2-(p yr r ol-1-yl)ben zoic Acid (26). A solution of 2,5-
dimethoxytetrahydrofuran (1.25 mL, 9.6 mmol) and 4-chloro-
pyridine hydrochloride (1.44 g, 9.6 mmol) in dioxane (50 mL)
was stirred for 15 min at room temperature. 2-Amino-3-
iodobenzoic acid 25 (2.3 g, 8.7 mmol) was added to the reaction
mixture, which was refluxed for 2 h and filtered through a
small pad of Celite. The filtrate was evaporated to give a beige
residue. The beige residue was dissolved in diethyl ether (100
mL), and the solution was washed with a 1 N aqueous
hydrochloric acid solution (2 × 50 mL), dried (MgSO4), and
evaporated to give a white solid in 60% yield: mp ) 170-172
°C; IR 3100 (O-H), 1681 (CdO) cm-1; 1H NMR (CDCl3) δ 10.17
(br s, 1H, D2O exchangeable, OH), 8.01 (d, J 4,5 ) 7.6 Hz, 1H,
H4), 7.81 (d, J 6,5 ) 7.6 Hz, 1H, H6), 7.12 (t, J ) 7.6 Hz, 1H,
H5), 6.58 (m, 2H, HRpyrrole), 6.24 (m, 2H, Hâpyrrole). Anal. (C11H8-
NO2I) C, H, N.
Eth yl 3-Iod o-2-(p yr r ol-1-yl)ben zoa te (27). 3-Iodo-2-(pyr-
rol-1-yl)benzoic acid 26 (1.5 g, 4.8 mmol) was dissolved in
ethanol (50 mL) in the presence of thionyl chloride (0.35 mL,
4.8 mmol), and the mixture was refluxed for 5 h. The solvent
was evaporated under reduced pressure to afford an oil that
slowly crystallized from petroleum ether as a beige solid in
3-(3,4-Dim et h oxyp h en yl)-8H -fu r o[2,3-b]p yr r olizin -8-
on e (23). A solution of 21 (0.35 g, 0.96 mmol) in phosphorus
oxychloride (15 mL) was refluxed for 6 h. After cooling, the
reaction mixture was concentrated to give a red solid that was
filtered, washed with Et2O, and dried. Then, this intermediary
iminium salt was added slowly to a 10% aqueous sodium
83% yield: mp ) 82-84 °C; IR 1672 (CdO) cm-1 1H NMR
;
(CDCl3) δ 8.10 (d, J 4,5 ) 8 Hz, 1H, H4), 7.84 (d, J 6,5 ) 7.6 Hz,
1H, H6), 7.23 (dd, J 5,4 ) 8 Hz, J 5,6 ) 7.6 Hz, 1H, H5), 7.10 (m,

Thienopyrrolizinones as Antitubulin Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1461
2H, HRpyrrole), 6.36 (m, 2H, Hâpyrrole), 4.13 (q, J ) 6.8 Hz, 2H,
H7), 6.77 (d, J 1,2 ) 3.2 Hz, 1H, H1), 6.55 (d, J 3,2 ) 2.4 Hz, 1H,
H3), 6.12 (m, 1H, H2); 13C NMR (CDCl3) δ 179.1, 157.2, 142.1,
138.1, 132.4, 130.9, 127.8, 127.5, 127.1, 125.1, 124.2, 122.6,
119.8, 115.5, 114.1. Anal. (C15H9NOS) C, H, N.
CH2), 1.14 (t, J ) 6.8 Hz, 3H, CH3). Anal. (C13H12NO2I) C, H,
N.
5-Iod o-9H-p yr r olo[1,2-a ]in d ol-9-on e (28). A solution of
ethyl 3-iodo-2-(pyrrol-1-yl)benzoate 27 (1.3 g, 3.8 mmol) in
methylene chloride (50 mL) was cooled to 0 °C with an ice bath
before adding a 1 M solution of boron tribromide in methylene
chloride (4.2 mL, 4.2 mmol). The reaction mixture was stirred
for 45 min to room temperature and diluted with cold water
(100 mL). After 3 h, the organic layer was separated, washed
with water (2 × 50 mL), dried (MgSO4), and evaporated under
reduced pressure. The green residue was purified by column
chromatography, eluting by ethyl acetate/hexane (1:4) to afford
a yellow powder in 62% yield: mp ) 138-140 °C; IR 1700 (Cd
3-(4-Met h oxyp h en yl)-8H -t h ien o[2,3-b]p yr r olizin -8-ol
(30). 3-(4-Methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 1d
(0.5 g, 1.78 mmol) was dissolved in tetrahydrofuran (30 mL).
After the mixture was cooled to 0 °C with an ice bath, lithium
aluminum hydride (0.17 g, 4.44 mmol) was slowly added to
the mixture, which was stirred for 2 h at 0 °C before the
reaction was quenched with crushed ice. The solvent was
concentrated under reduced pressure, and the resulting sus-
pension was extracted with diethyl ether (2 × 50 mL). The
organic layers were collected, washed with water (2 × 50 mL),
dried (MgSO4), and evaporated to give a yellow residue purified
by column chromatography, eluting by ethyl acetate/cyclohex-
ane (1:2) to give 30 as a gray powder with 78% yield: mp )
1
O) cm-1; H NMR (CDCl3) δ 7.87 (dd, J 3,2 ) 2.8 Hz, J 3,1 ) 0.8
Hz, 1H, H3), 7.58 (dd, J 8,7 ) 8 Hz, J 8,6 ) 1 Hz, 1H, H8), 7.40
(dd, J 6,7 ) 7.3 Hz, J 6,8 ) 1 Hz, 1H, H6), 6.72 (m, 2H, H7 and
H1), 6.22 (dd, J 2,3 ) 2.8 Hz, J 2,1 ) 3.6 Hz, 1H, H2); 13C NMR
(CDCl3) δ 177, 145.2, 144.2, 132.3, 132.2, 126.5, 123.9, 121.6,
115.3, 114.7, 74.5. Anal. (C11H6NOI) C, H, N.
1
124 °C; IR 3386 (O-H) cm-1; H NMR (CDCl3) δ 7.35 (d, J )
7.9 Hz, 2H, H2′ and H6′), 7.19 (d, J ) 7.9 Hz, 2H, H3′ and H5′),
7.09 (s, 1H, Hthiophene), 6.70 (m, 1H, H5), 6.26 (m, 1H, H7), 6.04
(m, 2H, 1H D2O exchangeable, H6 and OH), 5.62 (s, 1H, H8),
3.84 (s, 3H, OCH3); 13C NMR (CDCl3) δ 142.8, 142.0, 137.9,
132.8, 130.8, 129.5, 128.1, 126.6, 113.5, 110.9, 106.6, 67.0, 55.5.
Anal. (C16H13NO2S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 5-Ar yl-9H-
p yr r olo[1,2-a ]in d ol-9-on es 29a -d . To a mixture of 5-iodo-
9H-pyrrolo[1,2-a]indol-9-one 28 (0.3 g, 1 mmol) and Pd(PPh3)4
(0.035 g, 0.03 mmol) in DME (30 mL) was added successively
the corresponding arylboronic acid (1.1 mmol) and sodium
hydrogen carbonate (0.17 g, 2 mmol) in water (30 mL). The
reaction mixture was refluxed with vigorous stirring under
argon, and the rate of the reaction was followed by TLC. After
the starting aryl halide 28 was consumed (8 h), the organic
solvent was removed under reduced pressure. The residue was
extracted with ethyl acetate (2 × 40 mL), and the organic
layers were dried (MgSO4) and evaporated. The orange solid
was purified by column chromatography, eluting by ethyl
acetate/hexane (1:4).
5-(3,4-Dim e t h oxyp h e n yl)-9H -p yr r olo[1,2-a ]in d ol-9-
on e (29a ). 29a was obtained as a yellow powder in 73% yield
using 3,4-dimethoxyphenylboronic acid (0.2 g, 1.1 mmol) as
reagent: mp ) 198 °C; IR 1688 (CdO) cm-1; 1H NMR (CDCl3)
δ 7.56 (d, J 8,7 ) 7.4 Hz, 1H, H8), 7.31 (d, J 6,7 ) 8.7 Hz, 1H,
H6), 7.16 (dd, J 7,6 ) 8.7 Hz, J 7,8 ) 7.4 Hz, 1H, H7), 7.01 (m,
3H, H2′, H5′, and H6′), 6.75 (d, J 1,2 ) 4 Hz, 1H, H1), 6.45 (d, J 3,2
) 2 Hz, 1H, H3), 6.08 (dd, J 2,1 ) 4 Hz, J 2,3 ) 2 Hz, 1H, H2),
3.97 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13C NMR (CDCl3) δ
179.0, 149.2, 148.9, 141.1, 136.1, 132.4, 130.7, 128.8, 127.1,
125.2, 123.1, 122.7, 121.2, 115.2, 113.8, 111.9, 111.2, 55.9, 55.8.
Anal. (C19H15NO3) C, H, N.
8-H yd r oxyim in o-3-(4-m et h oxyp h en yl)-8H -t h ien o[2,3-
b]p yr r olizin e (31). 3-(4-Methoxyphenyl)-8H-thieno[2,3-b]pyr-
rolizin-8-one 1d (1 g, 3.6 mmol) was dissolved in pyridine (10
mL), and hydroxylamine hydrochloride (0.55 g, 7.9 mmol) was
slowly added to the reaction mixture, which was refluxed for
3 h. The solvent was evaporated under reduced pressure, and
the residue was taken up in ethyl acetate (100 mL). The
solution was washed with a 1 N aqueous hydrochloric acid
solution (2 × 100 mL) and the organic layer was dried (MgSO4)
and evaporated to give a yellow residue purified by column
chromatography, eluting by ethyl acetate/cyclohexane (1:4). 31
was obtained as a yellow powder with 95% yield in a mixture
of Z and E isomers (30:70) not separated: mp ) 191-194 °C;
IR 3225 (O-H), 1607 (CdN) cm-1 1H NMR (DMSO-d6), Z
;
isomer, δ 12.07 (br s, 1H, D2O exchangeable, OH), 7.65 (s, 1H,
H2), 7.56 (d, J ) 8.3 Hz, 2H, H2′ and H6′), 7.12 (d, J ) 8.3 Hz,
2H, H3′ and H6′), 6.93 (m, 1H, H5), 6.72 (m, 1H, H7), 6.22 (m,
1H, H6), 3.85 (s, 3H, OCH3); 1H NMR (DMSO-d6), E isomer, δ
12.11 (br s, 1H, D2O exchangeable, OH), 7.79 (s, 1H, H2), 7.56
(d, J ) 8.3 Hz, 2H, H2′ and H6′), 7.12 (d, J ) 8.3 Hz, 2H, H3′
and H6′), 6.93 (m, 1H, H5), 6.55 (m, 1H, H7), 6.18 (m, 1H, H6),
3.85 (s, 3H, OCH3). Anal. (C16H12N2O2S) C, H, N.
5-(4-Meth oxyph en yl)-9H-pyr r olo[1,2-a ]in dol-9-on e (29b).
29b was obtained as a yellow powder in 65% yield using
4-methoxyphenylboronic acid (0.17 g, 1.1 mmol) as reagent:
8-(N,N-Dim eth yla m in op r op yloxyim in o)-3-(4-m eth oxy-
p h en yl)-8H -t h ien o[2,3-b]p yr r olizin e Mon ooxa la t e (32).
To a solution of the oxime 31 (0.5 g, 1.7 mmol) in anhydrous
acetone (50 mL) were added potassium carbonate (0.47 g, 3.4
mmol), 3-chloro-N,N-dimethylpropylamine hydrochloride (0.3
g, 1.9 mmol), and water (1 mL). The reaction mixture was
refluxed for 48 h and concentrated under reduced pressure.
The residue was taken up in diethyl ether (100 mL), and the
solution was washed with water (3 × 50 mL). The organic layer
was dried (MgSO4) and evaporated to give a yellow oil. The
yellow oil was dissolved in propan-2-ol, and oxalic acid (0.15
g, 1.7 mmol) was added to the suspension, which was refluxed
for 10 min. After the mixture was cooled, the precipitate was
filtered and dried with anhydrous diethyl ether (3 × 50 mL)
and then in a laboratory oven (40 °C) to afford yellow plates
with 45% yield in a mixture of Z and E isomers (30:70) not
separated: mp ) 107 °C; IR 3417 (O-H), 1719 (CdO), 1612
(CdN) cm-1; 1H NMR (DMSO-d6), Z isomer, δ 7.68 (s, 1H, H2),
7.51 (d, J ) 8.2 Hz, 2H, H2′ and H6′), 7.08 (d, J ) 8.2 Hz, 2H,
H3′ and H5′), 6.91 (m, 1H, H5), 6.74 (d, J 7,6 ) 3.4 Hz, 1H, H7),
6.16 (m, 1H, H6), 4.32 (m, 4H, D2O exchangeable, 2 × OH and
OCH2), 3.80 (s, 3H, OCH3), 3.12 (m, 2H, NCH2), 2.74 (s, 6H,
N(CH3)2), 2.10 (m, 2H, CH2); 1H NMR (DMSO-d6), E isomer, δ
7.83 (s, 1H, H2), 7.51 (d, J ) 8.2 Hz, 2H, H2′ and H6′), 7.08 (d,
1
mp ) 145-147 °C; IR 1696 (CdO) cm-1; H NMR (CDCl3) δ
7.56 (d, J 8,7 ) 7.3 Hz, 1H, H8), 7.39 (d, J ) 8.5 Hz, 2H, H2′ and
H6′), 7.27 (d, J 6,7 ) 7.8 Hz, 1H, H6), 7.14 (dd, J 7,6 ) 7.8 Hz, J 7,8
) 7.3 Hz, 1H, H7), 7.02 (d, J ) 8.5 Hz, 2H, H3′ and H5′), 6.75
(d, J 1,2 ) 3.5 Hz, 1H, H1), 6.45 (d, J 3,2 ) 2.2 Hz, 1H, H3), 6.07
(m, 1H, H2), 3.89 (s, 3H, OCH3); 13C NMR (CDCl3) δ 179.5,
159.8, 141.3, 136.2, 132.5, 130.8, 130.1, 128.7, 127.1, 125.2,
123.1, 122.7, 115.2, 114.2, 113.8, 55.3. Anal. (C18H13NO2) C,
H, N.
5-(F u r -2-yl)-9H-p yr r olo[1,2-a ]in d ol-9-on e (29c). 29c was
obtained as a yellow powder in 71% yield using fur-2-ylboronic
acid (0.12 g, 1.1 mmol) as reagent: mp ) 121 °C; IR 1692 (Cd
1
O) cm-1; H NMR (CDCl3) δ 7.52 (m, 1H, H5′), 7.47 (d, J 8,7
)
)
7.2 Hz, 1H, H8), 7.38 (d, J 6,7 ) 7.8 Hz, 1H, H6), 7.07 (dd, J 7,6
7.8 Hz, J 7,8 ) 7.2 Hz, 1H, H7), 7.02 (m, 1H, Harom), 6.74 (m,
1H, Harom), 6.58 (m, 1H, Harom), 6.51 (m, 1H, Harom), 6.13 (m,
1H, H2); 13C NMR (CDCl3) δ 179.1, 149.4, 142.6, 140.5, 134.5,
132.5, 131.6, 125.3, 124.1, 123.8, 116.4, 115.6, 114.3, 111.9,
109.6. Anal. (C15H9NO2) C, H, N.
5-(Th ien -2-yl)-9H-p yr r olo[1,2-a ]in d ol-9-on e (29d ). 29d
was obtained as a yellow powder in 58% yield using thien-2-
ylboronic acid (0.14 g, 1.1 mmol) as reagent: mp ) 112 °C; IR
1
1690 (CdO) cm-1; H NMR (CDCl3) δ 7.60 (m, 1H, H5′), 7.49
J ) 8.2 Hz, 2H, H3′ and H5′), 6.91 (m, 1H, H5), 6.56 (d, J 7,6
)
3.4 Hz, 1H, H7), 6.14 (m, 1H, H6), 4.32 (m, 4H, D2O exchange-
(d, J 8,7 ) 7.1 Hz, 1H, H8), 7.37 (d, J 6,7 ) 8.2 Hz, 1H, H6), 7.26
(m, 2H, H3′ and H4′), 7.17 (dd, J 7,6 ) 8.2 Hz, J 7,8 ) 7.1 Hz, 1H,
able, 2 × OH and OCH2), 3.80 (s, 3H, OCH3), 3.12 (m, 2H,

1462 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lisowski et al.
NCH2), 2.74 (s, 6H, N(CH3)2), 2.10 (m, 2H, CH2). Anal.
(C23H25N3O6S) C, H, N.
Sigma Chemical Co., St. Louis, MO) was added to each well
and the plates were incubated for 4 h at 37 °C. The medium
was aspirated, and the formazan was solubilized by 100 µL of
DMSO. The optical density (OD) was read at 540 nm with a
plate reader (Multiskan MCC, Labsystem) connected to a
computer. The percentage of growth was calculated for each
well: % growth ) [(OD treated cells)/(OD control cells)] × 100.
The percentages of growth of the tri- or hexaplicate were then
averaged and plotted as a function of the log of the concentra-
tion. The GI50 (concentration reducing by 50% of the OD) was
calculated by a linear regression performed on the linear zone
of the curve.
4-(3,4-Dim et h oxyp h en yl)-3-(p yr r ol-1-yl)-2-t h iop h en e
Ca r boxylic Acid (33). An aqueous 2.5% sodium hydroxide
solution (40 mL) was added dropwise to a stirred solution of
methyl 4-(3,4-dimethoxyphenyl)-3-(pyrrol-1-yl)-2-thenoate 5j
(5 g, 14.6 mmol) in ethanol (50 mL). After 10 h at room
temperature, water (50 mL) was added and the reaction
mixture was acidified with 12 N hydrochloric acid to pH 3.
The precipitate was extracted with ethyl acetate (50 mL); the
organic layer was washed with water (2 × 50 mL), dried
(MgSO4), and evaporated under reduced pressure to give a
white solid with 95% yield: mp ) 148-151 °C; IR 3100 (O-
Cell Cycle An a lysis. L1210 cells (2.5 × 105/mL) were
incubated for 21 h (approximately two doubling times) with
various concentrations of cytotoxic drugs. Cells were then fixed
by 70% ethanol, washed twice with PBS, and incubated for
30 min in PBS containing 100 µg/mL RNAse, 25 µg/mL PI.
For each sample, 10 000 cells were analyzed by flow cytometry.
Results are expressed as the percentage of cells accumulated
in the G2 + M + 8N phase of the cell cycle.
Tu bu lin Bin d in g Assa y. Calf brain tubulin was purified
according to the method of Shelanski,35 by three cycles of
assembly-disassembly and then dissolved in the assembly
buffer containing 0.1 M MES, 0.5 mM MgCl2, 2 mM EGTA,
and 1 mM GTP, pH 6.6 (the concentration of tubulin was about
2-3 mg/mL). Tubulin assembly was monitored and recorded
continuously by turbidimetry at 400 nM in a UV spectropho-
tometer, equipped with a thermostated cell at 37 °C.36 We
determined for drugs the IC50 values for their concentrations
that decreased by 50% the maximum assembly rate of the
tubulin without drug. The IC50 values were compared to the
IC50 of deoxypodophyllotoxin, measured the same day under
the same conditions.
In Vivo Eva lu a tion of An titu m or Activity. Female
B6D2F1 (C57B1/6 x DBA2) mice were used in murine tumor
models. All mice were purchased from Iffa Credo (Lyon,
France). They were aged 4-6 weeks and weighed 20-22 g at
the start of the experiment. These were conducted in ac-
cordance with the protocols published by the National Cancer
Institute (NCI) and European Organization for Research and
Treatment of Cancer (EORTC) members.37,38 All tumors used
for experiments were provided by the Division of Cancer
Treatment, Tumor Repository, NCI. Mice were inoculated ip
with 106 leukemic P388 cells, and drugs are administrated in
an ip or iv single injection with increasing concentrations (25-
400 mg/kg) the first day of the experiment. The evaluation of
antitumor activity was represented vs BCNU as an internal
reference by the life span of mices. The median survival time
(MST) of the treated group (T) was compared with that of the
control group (C), and the results were expressed as T/C (%).
T/C (%) ) [(MST of treated group)/(MST of control group)] ×
100.
1
H), 1659 (CdO) cm-1; H NMR (DMSO-d6) δ 13.07 (br s, 1H,
D2O exchangeable, OH), 7.97 (s, 1H, Hthiophene), 6.85 (m, 1H,
Harom), 6.68 (m, 3H, Harom and 2HRpyrrole), 6.22 (m, 1H, Harom),
6.11 (m, 2H, Hâpyrrole), 3.70 (s, 3H, OCH3), 3.49 (s, 3H, OCH3).
Anal. (C17H15NO4S) C, H, N.
4-(3,4-Dim et h oxyp h en yl)-3-(p yr r ol-1-yl)-2-t h iop h en e
Azide (34). 4-(3,4-Dimethoxyphenyl)-3-(pyrrol-1-yl)-2-thiophene
carboxylic acid 33 (1.5 g, 4.6 mmol) was dissolved in anhydrous
acetone (40 mL). The solution was cooled to 0 °C with an ice
bath, and TEA (0.7 mL, 5 mmol), ethyl chloroformate (0.5 mL,
5 mmol), and sodium azide (0.33 g, 5 mmol) were added
successively with a latent period of 15 min. After 2 h of stirring
at room temperature, the reaction mixture was filtered and
diluted with water (100 mL) and the resulting precipitate was
extracted with ethyl acetate (2 × 50 mL). The organic layers
were collected, washed with water (2 × 50 mL), dried (MgSO4),
and evaporated to give a pink solid with 70% yield, which was
used without further purification: mp < 100 °C; IR 2139 (N3),
1803 (CdO) cm-1
.
1-(3,4-Dim et h oxyp h en yl)-4,5-d ih yd r op yr r olo[1,2-a ]-
th ien o[2,3-e]p yr a zin -5-on e (35). The azide 34 (1.14 g, 3.2
mmol) was added to hot 1,2-dichlorobenzene (15 mL, 180 °C).
After 15 min, the mixture was cooled at room temperature
until the appearance of a precipitate. The precipitate was
filtered and recrystallized from methanol to give a white
powder with 76% yield: mp ) 242 °C; IR 3108 (N-H), 1642
(CdO) cm-1 1H NMR (DMSO-d6) δ 11.56 (br s, 1H, D2O
;
exchangeable, NH), 7.59 (s, 1H, H2), 7.36 (m, 1H, H8), 7.03
(m, 3H, Harom), 6.81 (m, 1H, H6), 6.39 (m, 1H, H7), 3.84 (s, 3H,
OCH3), 3.77 (s, 3H, OCH3); EIMS m/z (relative intensity) 326
(M+, 100), 311 (27), 283 (27), 251 (18), 163 (19), 91 (14). Anal.
(C17H14N2O3S) C, H, N.
1-(3,4-Dih yd r oxyp h en yl)-4,5-d ih yd r op yr r olo[1,2-a ]-
th ien o[2,3-e]pyr azin -5-on e (36).Asolution of1-(3,4-dimethoxy-
phenyl)-4,5-dihydropyrrolo[1,2-a]thieno[2,3-e]pyrazin-5-one 35
(0.25 g, 0.76 mmol) in methylene chloride (20 mL) was stirred
and cooled to 0 °C with an ice bath. Then, a 1 M solution of
boron tribromide in methylene chloride was added (1.5 mL,
1.5 mmol) to the reaction mixture, which was stirred for 0.5 h
to room temperature before being diluted with cold water (100
mL). After 3 h, the organic layer was extracted, washed with
water (2 × 50 mL), dried (MgSO4), and evaporated under
reduced pressure. The solid residue was crystallized from
X-r a y Cr ysta llogr a p h y. Suitable crystals of the title
compounds were obtained by slow evaporation from methylene
chloride/methanol (v/v) at room temperature, and they were
mounted on a glass fiber.
Diffraction data were collected on an Enraf-Nonius CAD4
diffractometer with Mo KR radiation (λ ) 0.710 73 Å) at room
temperature. Data were measured using θ-2θ scan. The data
treatment, polarization and decay corrections, was carried out
with J ANA98 program.39 The crystal structure was solved by
direct methods using the SHELX97 package.40 All non-
hydrogen atoms were refined anisotropically. For 1o, all H
atoms were determined via difference Fourier maps and
refined with isotropic atomic displacement parameters, with
the exception of H atoms of the methoxy group for which the
H atoms were fixed. For 1j, the majority of H atoms were fixed.
methanol: mp > 260 °C; IR 3231 (N-H), 1651 (CdO) cm-1
;
1H NMR (DMSO-d6) δ 11.43 (br s, 1H, D2O exchangeable, NH),
9.24 (br s, 2H, D2O exchangeable, OH), 6.95 (s, 1H, H2), 6.85
(m, 2H, Harom), 6.76 (m, 2H, Harom), 6.68 (m, 1H, Harom), 6.42
(m, 1H, H7); EIMS m/z (relative intensity) 298 (M+, 100), 251
(14), 140 (15), 112 (10). Anal. (C15H10N2O3S) C, H, N.
P h a r m a cology. Sta n d a r d P r olifer a tion Assa y. The
principle of this assay and its application to anticancer drug
screening have been the subject of publications.33 Adherent
cells were trypsinized and seeded in 96-well microplates at
the indicated densities, previously determined to maintain
control cells in the exponential phase of growth for the
duration of the experiment and to obtain a linear relationship
between the optical density and the number of viable cells.34
The plates were incubated with the tested compounds for four
doubling times, the maximum duration being 7 days. At the
end of this period, an amount of 15 µL of 5 mg/mL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Ack n ow led gm en t. The authors thank D. Gue´nard
from the Institut des Substances Naturelles, Gif sur
Yvette, for the inhibitory polymerization tubulin test
and the National Cancer Institute Bethesda, MD, for
the in vitro and in vivo preliminary studies. This work
was supported by FEDER (Fond Europe´en de De´vel-

Thienopyrrolizinones as Antitubulin Agents
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