Synthesis and biological evaluation of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)-6-substituted-4 ,5,6,7-tetrahydrothieno[2,3-c]pyridine derivatives as antimitotic agents and inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bmcl.2008.08.006

Microtubules are among the most successful targets of compounds potentially useful for cancer therapy. A new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-4,5,6,7-tetrahydrothieno[b]pyridine molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-amino-3-(3,4,5-trimethoxybenzoyl)-6-methoxycarbonyl-4,5,6,7-tetrahydro thieno[b]pyridine, which inhibits cancer cell growth with IC₅₀-values ranging from 25 to 90 nM against a panel of four cancer cell lines, and interacts strongly with tubulin by binding to the colchicine site. In this series of N⁶-carbamate derivatives, any further increase in the length and in the size of the alkyl chain resulted in reduced activity.

Full text of DOI:10.1016/j.bmcl.2008.08.006

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5041–5045
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 2-amino-3-(30,40,50-trimethoxybenzoyl)-
6-substituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivatives
as antimitotic agents and inhibitors of tubulin polymerization
a,
a
a
a
Romeo Romagnoli a, , Pier Giovanni Baraldi , Maria Dora Carrion , Olga Cruz-Lopez , Carlota Lopez Cara ,
Manlio Tolomeo b, Stefania Grimaudo c, Antonietta Di Cristina c, Maria Rosa Pipitone c, Jan Balzarini d,
Sahar Kandil e, Andrea Brancale e, Taradas Sarkar f, Ernest Hamel f
*
*
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
b Centro Interdipartimentale di Ricerca in Oncologia (C. I. R. O. C.) e Servizio AIDS, Dipartimento di Oncologia, Policlinico ‘‘P. Giaccone”, Università di Palermo, Palermo, Italy
c Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
d Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
e The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF, UK
f Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Microtubules are among the most successful targets of compounds potentially useful for cancer therapy.
A new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-
4,5,6,7-tetrahydrothieno[b]pyridine molecular skeleton was synthesized and evaluated for antiprolifera-
tive activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in
this series was 2-amino-3-(3,4,5-trimethoxybenzoyl)-6-methoxycarbonyl-4,5,6,7-tetrahydrothie-
no[b]pyridine, which inhibits cancer cell growth with IC50-values ranging from 25 to 90 nM against a
panel of four cancer cell lines, and interacts strongly with tubulin by binding to the colchicine site. In this
series of N6-carbamate derivatives, any further increase in the length and in the size of the alkyl chain
resulted in reduced activity.
Received 18 July 2008
Revised 31 July 2008
Accepted 1 August 2008
Available online 6 August 2008
Keywords:
Microtubules
Combretastatin-A4
Tubulin polymerization
4,5,6,7-Tetrahydrothieno[b]pyridine
Colchicine
Ó 2008 Elsevier Ltd. All rights reserved.
The mitotic spindle is formed by microtubules that are gener-
ated by the polymerization of tubulin, and the spindle is an attrac-
tive target for the development of compounds useful in anticancer
chemotherapy.1 Besides being critical for cell division, the microtu-
bule system of eukaryotic cells is involved in many fundamental
cellular functions, including cell signaling, secretion, cell architec-
ture in interphase, and intracellular transport.2 A large number of
antimitotic drugs displaying wide structural diversity, derived
from natural sources or by screening compound libraries in combi-
nation with traditional medicinal chemistry, have been identified
and shown to interfere with the tubulin system.3
During our studies directed at the synthesis of new antitubulin
agents, we previously reported the potent in vitro antitumor activity
of a series of molecules with general structure 2, characterized by
the presence of a 2-amino-3-(3,4,5-trimethoxybenzoyl)-benzo[b]thi-
ophene skeleton.7 These compounds strongly inhibited tumor cell
growth, as well as tubulin polymerization by binding to the colchicine
site of tubulin, and caused arrest in the G2/M phase of the cell cycle.
The trimethoxybenzoyl moiety is crucial for retaining potency in this
and other series of molecules which occupy the colchicine site.8
Previously, investigators at Altana Pharma reported a series of
2-amido-3-cyano-4,5,6,7-tetrahydrothieno[2,3-b]pyridine
ana-
One of the most important naturally occurring tubulin-binding
agents is combretastatin A-4 (CA-4, 1; Chart 1). CA-4, isolated from
the bark of the South African tree Combretum caffrum,4 strongly
inhibits the polymerization of tubulin by binding to the colchicine
site.5 Because of its simple structure, a wide number of CA-4 ana-
logues have been developed and evaluated in SAR studies.6
logues with general structure 3, active at micromolar concentra-
tions (IC50 = 0.2–5 lM) as antiproliferative agents against human
colon adenocarcinoma (RKOp27) cells.9
As a part of our search for novel antimitotic agents, these findings
prompted us to synthesize a new series of 2-amino-3-(3,4,5-trim-
ethoxybenzoyl)-4,5,6,7-tetrahydrothieno[b]pyridine derivatives
with general structure 4, obtained by combining the 2-amino-3-
(30,40,50-trimethoxybenzoyl)thiophene portion of compound with
general structure 2 with the N6-substituted-4,5,6,7-tetrahydropyr-
idine nucleus of general structure 3.
* Corresponding authors. Tel.: +39 0532 455303; fax: +39 0532 455953 (R.R.),
Tel.: +39 0532 291293; fax: +39 0532 455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.006

5042
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5041–5045
OMe
O
H3CO
H3CO
CN
O
A
OMe
R1
B
O
N
N
H
X
CH3O
R2
R
S
OMe
NH2
OH
OCH3
S
O
3
2
Combretastatin A-4 (CA-4), 1
X=-CH=CH- or nothing
R1=substituted or unsubstituted phenyl,
R=H, Me, OMe
2a, R=H
5 or 6-membered heterocyle
R2=straight or branched alkyl chain
H3CO
H3CO
OCH3
H3CO
H3CO
OCH3
O
O
NH2
N
R1
S
NH2
N
S
4
5
R1=CONHCH(CH3)2, 4m
R1=CONH(CH2)3CH3, 4n
R1=CONHC6H11, 4o
R1=CONHC6H5, 4p
R1=CONHCH2C6H5, 4q
R1=CONHCH2C6H4F-p, 4r
R1=CSNHCH3, 4s
R1=CSNHCH2CH3, 4t
R1=CSNHCH(CH3)2, 4u
R1=CSNHC(CH3)3, 4v
R1=CSNHC6H11, 4w
R1=CH3, 4a
R1=C2H5, 4b
R1=n-C3H7, 4c
R1=CH2C6H5, 4d
R1=COCH3, 4e
R1=COOCH3, 4f
R1=COOCH2CH3, 4g
R1=COO(CH2)2CH3, 4h
R1=COOCH2CH(CH3)2, 4i
R1=COOC(CH3)3, 4j
R1=COOC6H5, 4k
R1=COOCH2C6H5, 4l
Chart 1. Inhibitors of tubulin polymerization.
To the best of our knowledge, there have been no reports that
molecules characterized by the presence of the 4,5,6,7-tetrahydro-
thieno[b]pyridine skeleton can inhibit tubulin polymerization. In
order to explore the structure–activity relationships (SARs) at the
N6-position of the 2-amino-3-(3,4,5-trimethoxybenzoyl)-4,5,6,7-
tetrahydrothieno[b]pyridine nucleus, we embarked upon the
synthesis of different series of N6-substituted derivatives, repre-
sented by alkyl compounds 4a–d, amide 4e, carbamates 4f–l, ureas
4m–r, and thioureas 4s–w, characterized by the presence of alkyl
chains of varying size. In addition, we explored the effect of
bioisosteric replacement of the C-6 carbon atom of derivative 2a
with a nitrogen atom, to furnish the 2-amino-3-(30,40,50-trim-
ethoxybenzoyl)thieno[2,3-c]pyridine derivative 5.
Synthesis of derivatives 4a–w and 5 was carried out by the gen-
eral methodology shown in Scheme 1. The Gewald reaction10, ap-
plied to 3-(3,4,5-trimethoxyphenyl)-3-oxopropanenitrile,11 sulfur,
and N-substituted 4-piperidone 6a–l12 in the presence of triethyl-
amine and ethanol at reflux, furnished the 2-amino-3-(30,40,50-
trimethoxybenzoyl)-6-substituted-4,5,6,7-tetrahydrobenzo[b]-
thiophenes 4a–l. 13 The N6-tert-butoxycarbonyl (Boc) derivative 4j
was used as starting material for the synthesis of urea and thiourea
derivatives 4m–r and 4s–w, respectively. Acetylation of the amino
group of 4j, using acetyl chloride and pyridine, yielded 7j, which,
followed by removal of the N6-Boc protecting group with trifluoro-
acetic acid (TFA), afforded the derivative 8j. The subsequent aro-
matization, by treatment with manganese dioxide (MnO2) in
refluxing toluene, furnished the corresponding N2-acetyl-thie-
no[2,3-c]pyridine derivative, transformed by saponification into
the final product 5.
corresponding ureas 9m– and thioureas 9s–w, which were trans-
formed by hydrolysis with NaOH into the final products 4m–r
and 4s–w, respectively, in good yields.
Table 1 summarizes the antiproliferative activity of N6-substi-
tuted-4,5,6,7-tetrahydrothieno[b]pyridine derivatives 4a–w and
the thieno[2,3-c]pyridine 5 against the growth of murine leukemia
(L1210), murine mammary carcinoma (FM3A), and human T-lym-
phoblastoid (Molt/4 and CEM) cells, using CA-4 (1) and the
benzo[b]thiophene derivative 2a as reference compounds. The re-
sults indicated that N6-methyl and ethyl carbamates 4f and 4g,14
respectively, showed the most potent antiproliferative activities,
while N6-branched alkyl or aryl carbamates as well as alkyl, acetyl,
urea, and thiourea functionalities decreased activity drastically.
Of all the tested compounds, the N6-methyl carbamate deriva-
tive 4f possessed the highest potency, inhibiting the growth of
L1210, FM3A, Molt/4, and CEM cancer cell lines with IC50s of 25,
46, 45, and 90 nM, respectively.
The results indicated that a non-basic nitrogen atom at the N6-
position of 4,5,6,7-tetrahydrothieno[b]pyridine nucleus was
important for inhibition of cell growth. In fact, in the series of
N6-alkyl derivatives 4a–d, only the ethyl derivative 4b showed
moderate potency (IC50 = 1.7–2.1
propyl, and benzyl analogues (compounds 4a, 4c, and 4d, respec-
tively), the IC50 was greater than 10 M in all four cell lines. The
N6-acetyl derivative 4e also showed moderate antiproliferative
activity, with IC50-values of 1.1–3.9 M:
lM), whereas for the methyl,
l
l
In the series of carbamates 4f–l, a small substituent size was
important for good activity. Only the methyl and ethyl derivatives
4f and 4g, respectively, showed potent antiproliferative activity,
with 4f more active than 4g. Specifically, 4f and 4g had similar
activity against FM3A cells, but 4f was 4-, 5-, and 6-fold more po-
Compound 8j was further condensed with different isocyanates
or isothiocyanates, in the presence of triethylamine, to afford the

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5041–5045
5043
H3CO
H3CO
OCH3
H3CO
H3CO
OCH3
H3CO
H3CO
OCH3
O
O
c
b
a
O
O
N
NHCOCH3
NH2
NHCOCH3
R1
N
N
HN
R1
R1
S
S
S
8j
7j
4a-l
6a-l
f, e
R1=CH3, 4a, 6a
d
R1=C2H5, 4b, 6b
H3CO
OCH3
R1=n-C3H7, 4c, 6c
H3CO
H3CO
OCH3
R1=CH2C6H5, 4c, 6d
R1=COCH3, 4e, 6e
R1=COOCH3, 4f, 6f
H3CO
O
O
R1=COOCH2CH3, 4g, 6g
R1=COO(CH2)2CH3, 4h, 6h
NH2
H
N
N
S
NHCOCH3
N
R2
S
R1=COOCH2CH(CH3)2, 4i, 6i
R1=COOC(CH3)3, 4j, 6j
R1=COOC6H5, 4k, 6k
5
X
9m-w
R1=COOCH2C6H5, 4l, 6l
e
X=O, R2=CH(CH3)2, 4m, 9m
X=O, R2=(CH2)3CH3, 4n, 9n
X=O, R2=C6H11, 4o, 9o
X=O, R2=C6H5, 4p, 9p
H3CO
H3CO
OCH3
X=O, R2=CH2C6H5, 4q, 9q
X=O, R2=p-F-C6H4CH2, 4r, 9r
X=S, R2=CH3, 4s, 9s
X=S, R2=CH2CH3, 4t, 9t
X=S, R2=CH(CH3)2, 4u, 9u
X=S, R2=C(CH3)3, 4v, 9v
X=S, R2=C6H11, 4w, 9w
O
H
N
NH2
N
R2
S
X
4m-w
Scheme 1.
tent then 4g against L1210, CEM and Molt4 cells, respectively. A
further increase in the length of the straight alkyl chain, to furnish
the propyl derivative 4h, caused 10-, 25-, 1.5-, and 3-fold reduc-
tions in activity with the L1210, FM3A, Molt-4, and CEM cells,
respectively. With still bulkier carbamate moieties, there was
essentially total loss of activity. All urea derivatives 4m–r were
Table 1
In vitro inhibitory effects of compounds 2a, 4a-w, 5, and CA-4 (1) against the
proliferation of murine leukemia (L1210), murine mammary carcinoma (FM3A), and
human T-lymphocyte (Molt/4 and CEM) cells
a
Compound
IC50 (nM)
L1210
FM3A
Molt4/C8
CEM
also inactive (IC50 > 10 lM).
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
>10,000
2100 100
>10,000
>10,000
3900 290
>10,000
1900 0.0
>10,000
>10,000
1200 100
46 1.3
>10,000
1800 0.0
>10,000
>10,000
1100 60
45 3.1
290 25
470 0.00
>10,000
>10,000
>10,000
7500 140
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1600 60
4800 370
>10,000
>10,000
750 58
340 40
>10,000
1700 0.0
>10,000
>10,000
1400 20
90 1.7
It is noteworthy that for the active compounds 4f and 4g, the
replacement of the carbamate group with a thiourea, to furnish
the corresponding derivatives 4s and 4t, produced a dramatic drop
in potency (IC50 = 1.6–4.9 lM and 4.8–6.9 lM for 4s and 4t, respec-
tively, versus IC50 = 25–90 nM and 57–440 nM for 4f and 4g,
25
1
95 3.3
1100 80
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
4900 470
6800 430
>10,000
>10,000
1100 60
370 160
57 3.8
440 30
1200 30
>10,000
>10,000
>10,000
8300 500
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1800 70
6700 550
>10,000
>10,000
730 71
1000 900
74 15
respectively).
1400 100
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2800 110
6900 290
>10,000
>10,000
1400 50
400 170
In the series of thiourea derivatives 4s–w, the cyclohexyl thioc-
arbamoyl derivative 4w resulted in the most active compound,
Table 2
Inhibition of tubulin polymerization and colchicine binding by compounds 2a, 4f-g, 5,
and CA-4
Compound
Tubulin assemblya
Colchicine bindingb SD (%)
4s
4t
IC50 SD (lM)
1
l
M inhibitor
2
5
l
M inhibitor
4u
4v
4w
5
2a
CA-4 (1)
2a
4f
4g
5
1.9 0.1
0.6 0.02
0.6 0.03
5.4 0.4
1.2 0.1
25
71
84
82
27 0.1
99 0.7
1
3
2
71 0.6
67
n.d.
90
2
1
90
3
100
42
0
6
73
9
CA-4 (1)
2.8 1.1
1.6 1.4
1.9 1.6
n.d., not done.
a
a
IC50 = compound concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as means SE from the dose–response curves of at least
three independent experiments.
Inhibition of tubulin polymerization. Tubulin was at 10
l
M.
Inhibition of [3H]colchicine binding. Tubulin and colchicine were at 1 and 5
respectively, and the tested compound was at the indicated concentration.
b
lM,

5044
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5041–5045
To investigate whether the antiproliferative activities of these
compounds were related to an interaction with the microtubule
system, compounds 4f–g and 5 and reference derivatives 2a and
CA-4 were evaluated for inhibitory effects on tubulin polymeriza-
tion, and on the binding, of [3H]colchicine to tubulin (Table
2).15,16 For compounds 2a and 5, there was a positive correlation
between inhibition of both tubulin polymerization and colchicine
binding, and antiproliferative activity. However, relative to 2a,
both 4f and 4g were disproportionately more active as assembly
inhibitors, and these compounds also had greater activity as inhib-
itors of colchicine binding. The IC50s of 0.6
and 4g are among the lowest ever observed in this assembly assay,
and half that obtained in simultaneous experiments for CA-4 (IC50
1.2 M). Nonetheless, CA-4 had greater antiproliferative activity
lM obtained with 4f
,
l
and a greater inhibitory effect on the four cell lines than both 4f
and 4g. In addition, we should note that the similar effects of 4f
and 4g in the tubulin-based assays differed from the greater activ-
ity observed with 4f in the antiproliferative studies. This could de-
rive from preferential cellular uptake of 4f relative to 4g.
Alternatively, it is possible that cellular tubulin differs from the
neural tubulin used in the biochemical assays in its affinities for
the two compounds.
Because molecules exhibiting effects on tubulin assembly
should cause alteration of cell cycle parameters, with preferential
G2-M blockade, flow cytometry analysis was performed to deter-
mine the effect of the most active compounds on K562 (human
chronic myelogenous leukemia) cells.17 Cells were cultured for
24 h in the presence of each compound at the IC50 determined
for 24 h of growth (4f = 70 nM, 4g = 80 nM, 5 = 500 nM). Figure 1
shows that these molecules caused a marked increase in the per-
centage of cells blocked in the G2-M phase of the cell cycle, with
a simultaneous decrease of cells in S and G0-G1. These data con-
firm that this class of derivatives acts selectively on the G2-M
phase of the cell cycle, as expected for inhibitors of tubulin
assembly.
The proposed mechanism of action is also supported by docking
studies of compound 4g in the colchicine site of tubulin18 (method-
ology reported previously).7 Figure 2 shows how the trimethoxy-
phenyl moiety of 4g is situated in the same pocket on b-tubulin
as the structurally analogous ring A of the co-crystallized DAMA-
colchicine. In this model, the carbonyl group of the carbamate of
4g overlaps the carbonyl group of ring C of DAMA-colchicine. Fur-
thermore, the alkyl substituent of the carbamate lies in a small
Figure 1. Effects of compounds 4f (panel b), 4g (panel c), and 5 (panel d) on DNA
content/cell following treatment of K562 cells for 24 h. The cells were cultured
without compound (panel a) or with compound used at the concentration leading
to 50% cell growth inhibition after 24 h of treatment. Cell cycle distribution was
analyzed by the standard propidium iodide procedure. Sub-G0-G1 (apoptotic peak,
A), G0-G1, S, and G2-M cells are indicated in the panel (a).
with IC50s of 0.73–1.1 lM. Comparing urea and thiourea deriva-
tives with the same substitution at the N6-position (4m vs 4u
and 4o vs 4w), while the isopropyl derivatives 4m and 4u were
both inactive, the cyclohexyl thiourea derivative 4w was more ac-
tive than the urea derivative 4o.
Finally, comparing the benzo[b]thiophene 2a with the thie-
no[2,3-c]pyridine 5, replacement of the benzene with the bioisos-
teric pyridine ring led to a significant loss of activity. Compound
5 was 4- to 6-fold less active than 2a against L1210, FM3A, and
Molt-4 cells. Reduction in potency was even more pronounced
with the CEM cells, with 5 being 13-fold less potent than 2a. More-
over, the corresponding tetrahydrothieno[b]pyridine analogue of 5
resulted inactive (IC50 > 10
lM), indicating that the aromaticity of
the pyridine ring fused with the thiophene was critical for activity.
Figure 2. Docking pose of compound 4g in the colchicine site. DAMA-colchicine in green.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5041–5045
5045
Opin. Ther. Pat. 2006, 16, 647; (c) Nam, N. H. Curr. Med. Chem. 2003, 10, 1697;
(d) Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Curr. Pharm. Des. 2005, 11, 1655.
7. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Lopez Cara, C.; Preti, D.; Fruttarolo,
F.; Pavani, M. G.; Tabrizi, M. A.; Tolomeo, M.; Grimaudo, S.; Di Antonietta, C.;
Balzarini, J.; Hadfield, J. A.; Brancale, A.; Hamel, E. J. Med. Chem. 2007, 50, 2273.
8. Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.-M.; Lin, C. M.; Hamel, E. J.
Med. Chem. 1992, 35, 2293.
9. (a) Bartels, B.; Gimmnich, P.; Pekari, K.; Baer, T.; Schmidt, M.; Beckers, T. WO
2005118592, 2005; Chem. Abstr. 2005, 2568718.; (b) Pekari, K.; Baer, T.; Bertels,
B.; Schmidt, M.; Beckers, T. WO 2005118071, 2005; Chem. Abstr. 2005, 2567648.
10. Tinney, F. J.; Sanchez, J. P.; Nogas, J. A. J. Med. Chem. 1974, 17, 624.
11. Seneci, P.; Nicola, M.; Inglesi, M.; Vanotti, E.; Resnati, G. Synth. Commun. 1999,
2, 311.
12. Phenyl isocyanate, benzyl isocyanate, 4-fluorobenzyl isocyanate, isopropyl
isocynate, butyl isocyanate, cyclohexyl isocyanate, methyl isothiocyanate,
ethyl isothiocyanate, isopropyl isothiocyanate, cyclohexyl isothiocyanate, tert-
butyl isothiocyanate, 1-methyl-4-piperidone (6a), 1-ethyl-4-piperidone (6b), 1-
n-propyl-4-piperidone (6c), 1-benzyl-4-piperidone (6d), 1-acetyl-4-piperidone
(6e), 1-ethoxycarbonyl-4-piperidone (6g), n-propyl 4-oxopiperidine-1-
carboxylate (6h), isobutyl 4-oxopiperidine-1-carboxylate (6i), 1-tert-
butyloxycarbonyl-4-piperidone (6j), and 1-benzyloxycarbonyl-4-piperidone
(6l) are commercially available and were used as received. For the synthesis of
4-oxo-piperidine-1-carboxylic acid methyl ester (6f) see: (a) Xu, G.; Kannan, A.;
Hartman, T. L.; Wargo, H.; Watson, K.; Turpin, J. A.; Buckheit, R. W.; Johnson, A. A.;
Pommier, Y.; Cushman, M. Bioorg. Med. Chem. 2002, 10, 2807; For the synthesis of
4-oxo-piperidine-1-carboxylic acid phenyl ester (6k) see: (b) Mauleon, D.;
Antunez, S.; Rosell, G. Farmaco 1989, 44, 1109.
hydrophobic pocket deep in the binding cavity, which can only
accommodate a small group like methyl or ethyl. This model thus
is consistent with the SARs observed in the antiproliferative stud-
ies, and is in accord with previously reported results.19
In conclusion, the synthesis and the SAR of a series of 2-amino-
3-(3,4,5-trimethoxybenzoyl)-6-substituted-4,5,6,7-tetrahydrothie-
no[b]pyridines, which incorporated partial structures of both
2-amino-3-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene and 2-acet-
amido-3-cyano-6-alkoxycarbonyl-4,5,6,7-tetrahydrothieno [2,3-
b]pyridine with general structures 2 and 3, respectively, are
described. In particular, compounds 4f and 4g are the best amal-
gamation of structures 2 and 3. Derivatives 4f and 4g were highly
active as inhibitors of tubulin assembly, with IC50s half that of
CA-4. They also were strong inhibitors of the binding of colchicine
to tubulin, although somewhat less active than CA-4. Consistent
with their antitubulin activity, both 4f and 4g caused cells to arrest
in the G2/M phase of the cell cycle.
Molecular docking studies with 4g into the colchicine site18
provided a rationale for our observations. The trimethoxybenzene
ring and the carbamate carbonyl of 4g could bind in the same man-
ner as the trimethoxybenzene ring A and the ring C carbonyl,
respectively, of DAMA-colchicine in the crystal structure.18 An
adjacent pocket in b-tubulin could readily accommodate only a
methyl or ethyl group, consistent with the SAR observations.
Finally, we should note that the synthesis of 4f was efficient and
produced the compound in high yield. Thus, 4f represents the lead
compound of an interesting new class of antitubulin agents with
potential to be developed clinically for anticancer chemotherapy.
13. General procedure for the synthesis of compounds (4a–l). To a suspension of 3-
oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile
(2.55 g.,
10 mmol),
TEA
(1.54 mL, 11 mmol), and sulfur (352 mg, 11 mmol) in EtOH (50 mL) was
added the appropriate 1-substituted-4-piperidone 6a–l (10 mmol). After
stirring for 2 h at 70 °C, the solvent was evaporated and the residue diluted
with DCM (15 mL). After washing with water (2 ꢀ 5 mL) and brine (5 mL), the
organic layer was dried and evaporated. The crude product was purified by
column chromatography and crystallized from petroleum ether.
14. Characterization of compound 4f. Yellow solid, mp 85–87 °C. 1H NMR (CDCl3)
d: 2.07 (t, J = 5.0 Hz, 2H), 3.44 (t, J = 5.0 Hz, 2H), 3.74 (s, 3H), 3.86 (s, 6H), 3.90 (s,
3H), 3.95 (bs, 2H), 4.43 (s, 2H), 6.72 (s, 2H). Characterization of compound 4g.
Yellow solid, mp 65–67 °C. 1H NMR (CDCl3) d: 1.26 (t, J = 7.0 Hz, 3H), 2.14 (t,
J = 5.4 Hz, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.86 (s, 6H), 3.89 (s, 3H), 3.90 (bs, 2H),
4.18 (q, J = 7.0 Hz, 2H), 4.42 (s, 2H), 6.72 (s, 2H).
Supplementary data
15. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
Detailed synthesis and spectroscopic data for compounds
4a–w, 5, 6h–i, 7–8j, and 9m–w can be found in the online version.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.006.
16. Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu,
M.; White, J. D.; Falck, J. R.; Gerwick, W. H.; Day, B. W.; Hamel, E. Mol.
Pharmacol. 1998, 53, 62.
17. Flow cytometric analysis of cell cycle distribution. The effects of the most
active compounds of the series on cell cycle distribution were studied on K562
cells by flow cytometric analysis after staining with propidium iodide. Cells
were exposed 24 h to each compound used at a concentration corresponding to
the IC50 evaluated after a 24 h incubation. After treatment, the cells were
washed once in ice-cold PBS and resuspended at 1 ꢀ 106 per mL in a hypotonic
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