Structure-activity relationship of tumor-selective 5-substituted 2-amino-3-carboxymethylthiophene derivatives

Article abstract of DOI:10.1002/cmdc.201402274

Methyl-2-amino-5-[2-(4-methoxyphenethyl)]thiophene-3-carboxylate (8c) is the prototype of a well-defined class of tumorselective agents. Compound 8c preferentially inhibited the proliferation of a number of tumor cell lines including many human T-lymphoma/leukemia cells, but also several prostate, renal, central nervous system and liver tumor cell types. Instead, a broad variety of other tumor cell lines including B-lymphomas and HeLa cells were not affected. The tumor selectivity (TS; selectivity index or preferential suppression of CEM lymphoma (IC₅₀=0.90 mm) versus HeLa tumor cell carcinoma (IC₅₀=39 mm)) amounted up to ～43 for 8c. At higher concentrations, the compound proved cytotoxic rather than cytostatic. The antiproliferative potency and selectivity of 8c could be preserved by replacing the ethyl linker between the 2-amino- 3-carboxymethylthiophene and the substituted aryl by a thioalkyl but not by an oxyalkyl nor an aminoalkyl. Among >50 novel 8c derivatives, the 5-(4-ethyl- and 4-isopropylarylmethylthio) thiophene analogues, methyl-2-amino-5-((4-ethylphenylthio) methyl)thiophene-3-carboxylate (13m) and methyl-2- amino-5-((4-isopropylphenylthio)methyl)thiophene-3-carboxylate (13n), were more potent (IC₅₀ : 0.3-0.4 mm) and selective (TS: 100-144) anti-T-lymphoma/leukemia agents than the prototype compound.

Full text of DOI:10.1002/cmdc.201402274

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DOI: 10.1002/cmdc.201402274
Structure–Activity Relationship of Tumor-Selective
5
-Substituted 2-Amino-3-carboxymethylthiophene
Derivatives
[a]
[b]
[b]
[c]
[b]
Joice Thomas, Alenka Jejcic, Peter Vervaeke, Romeo Romagnoli, Sandra Liekens,
Jan Balzarini,* and Wim Dehaen
[b]
[a]
Methyl-2-amino-5-[2-(4-methoxyphenethyl)]thiophene-3-car-
boxylate (8c) is the prototype of a well-defined class of tumor-
selective agents. Compound 8c preferentially inhibited the
proliferation of a number of tumor cell lines including many
human T-lymphoma/leukemia cells, but also several prostate,
renal, central nervous system and liver tumor cell types. In-
stead, a broad variety of other tumor cell lines including B-lym-
phomas and HeLa cells were not affected. The tumor selectivi-
ty (TS; selectivity index or preferential suppression of CEM lym-
phoma (IC =0.90 mm) versus HeLa tumor cell carcinoma
ic. The antiproliferative potency and selectivity of 8c could be
preserved by replacing the ethyl linker between the 2-amino-
3-carboxymethylthiophene and the substituted aryl by a thio-
alkyl but not by an oxyalkyl nor an aminoalkyl. Among >50
novel 8c derivatives, the 5-(4-ethyl- and 4-isopropylarylme-
thylthio)thiophene analogues, methyl-2-amino-5-((4-ethylphe-
nylthio)methyl)thiophene-3-carboxylate (13m) and methyl-2-
amino-5-((4-isopropylphenylthio)methyl)thiophene-3-carboxyl-
ate (13n), were more potent (IC : 0.3–0.4 mm) and selective
50
(TS: 100–144) anti-T-lymphoma/leukemia agents than the pro-
totype compound.
50
(
IC =39 mm)) amounted up to ~43 for 8c. At higher concen-
50
trations, the compound proved cytotoxic rather than cytostat-
Introduction
Multisubstituted 2-aminothiophene heterocycles have recently
been accepted as a privileged scaffold that plays an important
role in the discovery of several pharmaceutically active mole-
cules endowed with anti-inflammatory, antimicrobial, antiviral
and analgesic activity. Figure 1 illustrates a number of promis-
ing drugs (1–6) that contain the 2-aminothiophene fragment
[
1–8]
in their molecular skeleton.
The classical bioisosteric equivalence between benzene and
thiophene motivated us to replace the methyl salicylate group
[
9]
in the potent anticancer agent SDZ LAP 977 (7) by a methyl
-aminothiophene-3-carboxylate moiety. This replacement re-
2
sulted in the discovery of a series of new tumor-selective com-
pounds (8) that consisted of a thiophene ring substituted at
C-2 by a free amino group, at C-3 by a carboxymethyl- or car-
boxyethyl moiety and at C-5 by a substituted aryl through
Figure 1. Structure of pharmaceutical agents containing 2-aminothiophene
fragments.
[
10–12]
a short alkyl bridge.
shown in Figure 2.
Some representative examples are
Our previous studies revealed that these molecules were en-
dowed with a marked antiproliferative effect (IC <1 mm)
5
0
against particular well-defined tumor cell lines. They were not
substantially inhibitory (IC >10 mm) against a wide variety of
50
[
a] Dr. J. Thomas, Prof. W. Dehaen
Department of Chemistry, KU Leuven
001Heverlee (Belgium)
other tumor cell types (including human cervix carcinoma
HeLa). Our preliminary structure–activity relationship (SAR)
studies revealed that the presence of the 2-amino-3-carboxy-
methylthiophene scaffold is strictly necessary to preserve the
cytostatic selectivity between the drug-sensitive and -insensi-
tive tumor cell lines. Replacing the ÀCOOMe group by
ÀCOOEt, nitrile, amide, methylamide or a free carboxylic acid
group at the 3-position of the thiophene heterocycle usually
decreased the tumor selectivity. From a first set of 30 com-
pounds, methyl-2-amino-5-[2-(4-methoxyphenethyl)]thiophene-
3
[b] Dr. A. Jejcic, P. Vervaeke, Prof. S. Liekens, Prof. J. Balzarini
Rega Institute for Medical Research, KU Leuven
3
000 Leuven (Belgium)
E-mail: jan.balzarini@rega.kuleuven.be
[
c] Prof. R. Romagnoli
Dipartimento di ScienzeChimiche e Farmaceutiche, Universitꢀ di Ferrara
4
4121 Ferrara (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402274.
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type compound is unusual compared with known antitumor
drugs, likely pointing to a novel mechanism of antiproliferative
action.
Based on our previous studies, we wanted to further explore
the SAR of substituted thiophenes for tumor cell selectivity,
and focused on the nature of the bridge between the thio-
phene and the aryl group, and also on the modifications that
could be made at the aryl moiety. For all the new compounds,
the 2-amino-3-carboxymethylthiophene part was kept unal-
tered as this was earlier shown to be an essential part for both
Figure 2. Structures of SDZ LAP 977 and 2-aminothiophene-3-carboxylic acid
ester derivatives.
[10,12]
the cytostatic activity and selectivity.
We studied the effect caused by replacing the ethylene
spacer between the substituted benzene ring and the 2-ami-
nothiophene of the prototype compound 8c by an amino-
methyl (13a), an oxymethyl (13b) or a thiomethyl (13i) bridge.
Additionally, the significance of extending the ethylene spacer
to a methylthiomethyl group was investigated. In order to
evaluate the role of diverse substituents on the cytostatic se-
lectivity of the synthesized compounds, different electron-with-
drawing and -donating groups were introduced at various po-
sitions of the aryl ring. The effect on the antiproliferative activi-
ty caused by the replacement of the aryl group by other het-
erocycles, such as pyridine, furan or thiophene, was also exam-
ined. We could define several novel compounds with more
pronounced cytostatic activities and improved tumor cell se-
lectivities compared with the parent lead drug against human
lymphoma CEM tumor cells.
3
-carboxylate (8c) was earlier found to have the most potent
cytostatic activity against human CEM and PC-3 tumor cells,
and its antiproliferative activity was highly selective (poor cyto-
[10]
static activity against other tumor cell lines such as HeLa).
The cytostatic activity and tumor selectivity were completely
lost when the amino group of 8c was replaced by a hydrogen,
N-methylamino or N,N-dimethylamino group. Moreover, intro-
duction of a functional group at the C-4 position of the thio-
phene resulted in a significant reduction of the cytostatic activ-
ity. The importance of the ethyl spacer between the thiophene
and the aryl moiety was also confirmed in the earlier SAR stud-
[
10,11]
ies.
Finally, 8c was found to be stable when exposed to
human T-lymphocyte CEM cell extracts (6 h), and to tumor cell
culture medium containing 10% fetal calf serum (24 h) (J. Bal-
zarini, unpublished data). The prototype compound 8c was
evaluated in the broad NCI-60 anticancer screen of the US Na-
tional Institutes of Health (NIH, National Cancer Institute). A
wide variety of tumor cell lines including leukemias/lympho-
mas, non-small cell lung cancer, colon cancer, CNS cancer,
prostate cancer, ovarian cancer, renal cancer, melanoma and
breast cancer were exposed to a five-dose drug regimen of 8c.
The dose–response curves for all tumor cell lines are plotted in
Figure 3A, whereas the activity of the compound against indi-
vidual tumor cell lines ranked according to their origin is
shown in Figure 3B. Compound 8c proved inhibitory against
Results and Discussion
Chemistry
The synthetic protocol employed for the preparation of target
compounds 13a–x is shown in Scheme 1. The multisubstituted
2-aminothiophene 9 with an electron-withdrawing ester func-
tionality in the 3-position and a methyl group in the 5-position
was prepared by utilizing the Gewald three-component reac-
tion starting from methyl cyanoacetate, propionaldehyde and
1
4 (sensitive: IC ꢀ1 mm; insensitive: IC >10 mm) out of 54
50
50
[1]
cancer cell lines with IC50 values ranging from 30 nm to 1 mm.
elemental sulfur under basic conditions. This compound was
[10]
When also taking into account our own antitumor cell data,
c showed particular selectivity
then converted to phthalimido derivative 10 in good yield by
8
towards different leukemia/lym-
phoma tumor cell types, al-
though also several renal, liver,
CNS and prostate cancer cell
types proved sensitive to the
compound. At the highest com-
pound concentration tested (i.e.,
100 mm) cytotoxicity (lower cell
number at the end of the tumor
cell-drug incubation period than
the initial cell count) was ob-
served in 28 out of 54 tumor cell
lines (Figure 3B). Consultation of
the NIH drug data base revealed
that the tumor cell spectrum
that is inhibited by the proto-
[
1]
Scheme 1. Reagents and conditions: a) NEt
7%; c) NBS (1.0equiv), CCl , reflux, 48 h, 60%; d) K
nol or 4-methoxyaniline (1.0 equiv); e) NH NH (1.5 equiv), MeOH.
3
, MeOH, reflux; b) phthalic anhydride (1.5 equiv), AcOH, reflux, 30 h,
8
4
2
CO , THF, corresponding aryl thiols, alky thiols, 4-methoxyphe-
3
2
2
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Figure 3. Growth curves of tumor cell lines in the presence of five concentrations of prototype compound 8c. A) Growth curves of 54 different tumor cell
lines in the presence of 8c. Leukemia/lymphoma: red; non-small cell lung cancer: blue; colon cancer: green; CNS cancer: gray; melanoma: fuchsia; ovarian
cancer: purple; renal cancer: ochre; prostate cancer: cyan; breast cancer: light red. B) Growth curves presented in panel A were ranked according to nine dif-
ferent tumor cell type categories.
using phthalic anhydride in refluxing acetic acid. The subse-
a period of 24 h, which furnished phthaloyl-protected inter-
mediate derivatives 12c–x in good yields. It is noteworthy that
the reaction with an electron-donating group on the aryl thiol
or phenylthiol itself proceeded effectively at room temperature
whereas aryl thiol with electron-withdrawing groups needed
a slightly elevated temperature. Many functional groups, such
as nitro, esters, halides, amines and methoxy groups are well-
tolerated under these reaction conditions. Final compounds
13c–x were obtained by removal of the phthaloyl group by
quent regioselective monobromination at the C-5 methyl of 10
with N-bromosuccinimide in refluxing CCl afforded the crucial
4
intermediate product 11 in reasonably good yield. Compound
11 then served as the starting material for the synthesis of all
the designed compounds. After some optimization, intermedi-
ate 11 was subjected to nucleophilic substitution reactions
with different substituted thiophenols or heteroaryl thiols in
the presence of K CO as base in tetrahydrofuran (THF) over
2
3
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use of hydrazine in refluxing ethanol over a period of 12 h. An
aminomethyl and an oxymethyl spacer between the aryl group
and 2-aminothiophene was introduced by the nucleophilic re-
action of 4-methoxyaniline or 4-methoxyphenol with the
cancer model exists in mice). The prototype compound 8c in
which the 2-amino-3-carboxymethylthiophene and the 4-me-
thoxyphenyl are separated by an ethylene bridge, is cytostatic
+
against human CD4 T-lymphocyte CEM tumor cells at an IC50
5-bromo derivative 11 followed by the reaction with hydrazine
value of 0.9 mm (Table 2). Tumor selectivity (TS) was calculated
to afford final compounds 13a and 13b, respectively. However,
the final products so obtained were not very stable and de-
composed over a period of 24 h at room temperature.
Table 2. Antiproliferative activity of test compounds against murine leu-
kemia L1210, human T-lymphocyte CEM and human cervix carcinoma
HeLa cells.
It is very important to note that the crucial step in the syn-
thesis of prototype compound 8c involved a Sonogashira cou-
[
a]
[b]
Compound
IC50 [mm]
CEM
TS
[11]
pling reaction with the appropriate terminal arylacetylene.
L1210
HeLa
However, the lack of structural diversity in aryl alkynyl building
blocks limited the application of this method in SAR studies.
Interestingly, the present methodology provides an economi-
cally viable procedure to synthesize derivatives diversely func-
tionalized at the C-5 position of the 2-amino-3-carboxyme-
thylthiophene heterocycle from inexpensive commercially
available building blocks (Table 1). This facilitates the SAR stud-
ies of this novel class of compounds.
8c (prototype)
9
11 Æ1
0.90Æ0.43
39Æ11
43
<1
<1
<1
0.8
1.6
35
5.5
7.0
1.2
201Æ49
>250
>250
ꢁ250
>250
>250
>250
>250
>250
1
0
1
1
1
3a
83Æ2
96Æ16
78Æ15
13b
13c
48Æ14
19Æ0
43Æ13
2.0Æ1.1
17Æ9
68Æ13
71Æ24
94Æ7
1
1
1
1
1
1
1
1
1
1
1
3d
3e
3 f
3g
3h
3i
3j
3k
3l
110 Æ10
163Æ68
21Æ5
27Æ10
190Æ84
46Æ15
108Æ7
72Æ30
58Æ1
39Æ24
60Æ4
1.3Æ0.6
1.5Æ0.3
0.62Æ0.33
1.7Æ1.2
3.1Æ1.8
12Æ6
83
23Æ4
48
93
25
33
7.5Æ1.1
19Æ0
Biological results
42Æ5
54Æ14
17Æ3
102Æ1
16Æ1
Given the unusual selectivity of the prototype compound 8c,
it would be of interest to obtain more insight in the SAR of
this class of compounds to further optimize the potency and/
or tumor cell selectivity. Therefore, all compounds were initially
evaluated against human T-lymphocyte CEM cells (one of the
most sensitive tumor cell lines), human cervix carcinoma HeLa
1.3
3m
3n
7.5Æ2.7
65Æ1.2
82Æ6
0.34Æ0.17
0.46Æ0.05
4.1Æ1.7
124Æ16
3.5Æ1.1
3.9Æ1.4
1.2Æ0.7
4.9Æ0.1
3.3Æ0.9
111Æ31
0.90Æ0.04
1.2Æ0.1
49Æ16 144
46Æ4
108Æ0
75Æ15
61Æ38
88Æ10
74Æ15
102Æ6
99Æ5
100
27
0.6
17
23
62
21
30
13o
1
1
1
1
3p
3q
3r
117 Æ15
64Æ13
44Æ13
43Æ4
(one of the least sensitive tumor cell lines), and murine leuke-
3s
mia L1210 cells (a murine cell line for which a suitable in vivo
13t
96Æ14
95Æ5
13u
13v
13w
13x
124Æ4
22Æ2
93Æ6
0.84
83
45
74Æ6
Table 1. 2-Amino-3-carboxymethylthiophene derivatives.
3.8Æ0.5
55Æ20
1
2
3
4
5
Compound
R
R
R
R
R
X
n
[a] 50% inhibitory concentration or compound concentration required to
inhibit cell proliferation by 50%. Data are the mean ÆSD of two to three
independent experiments. [b] Tumor cell selectivity (TS) calculated as
IC50(HeLa)/IC50(CEM). The bold values represent test compounds with
a TSꢁ20.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2a, 13a
2b, 13b
2c, 13c
2d, 13d
2e, 13e
2 f, 13 f
2g, 13g
2h, 13h
2i, 13i
2j, 13j
2k, 13k
2l, 13l
2m, 13m
2n, 13n
2o, 13o
2p, 13p
2q, 13q
2r, 13r
H
H
CH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
NH
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
2
1
1
0
0
0
3
COOMe
NH
H
H
H
H
H
H
H
H
H
H
H
H
H
–
2
H
H
as the ratio of IC50 for the prototype drug-insensitive HeLa
tumor cells versus the IC50 value for the prototype drug-sensi-
tive CEM cells. For 8c, the TS value was 43. To explore modifi-
cations in the bridge moiety, the alkyl (ethylene) bridge be-
tween 2-amino-3-carboxymethylthiophene and 4-methoxy-
phenyl was first replaced by an aminomethyl (13a), an oxy-
methyl (13b) or a thiomethyl (13i) bridge. Whereas the oxy-
methyl and aminomethyl derivatives showed a ~50- to 100-
fold decreased cytostatic activity against CEM cells (IC : 43–
CH
3
OMe
Cl
F
NO
2
ethyl
isopropyl
H
H
H
OMe
H
–
–
3
COOH
H
H
H
–
–
–
5
0
9
6 mm) and completely lost tumor selectivity (TS: 0.8–1.6), the
thiomethyl-bridged derivative 13i was slightly more cytostatic
than the parent lead drug (IC =0.62 mm) and slightly (~2-fold)
gained tumor selectivity (TS=93 versus 43 for 8c) (Table 2).
Lengthening the thiomethyl bridge to a methylthiomethyl
2s, 13s
2t, 13t
2u, 13u
2v, 13v
[
a]
[
b]
–
–
–
H
–
–
–
H
–
–
–
H
50
[
c]
–
–
Phe
[
d]
2w, 13w
–
H
[
e]
2x, 13x
(
13r) decreased the cytostatic activity against CEM cells by
4–5-fold (IC =3.9 mm) and decreased the tumor selectivity
[
[
a] Phenyl is replaced by 2-furyl. [b] Phenyl is replaced by 2-thienyl.
c] Phenyl is replaced by 2-naphthyl. [d] Phenyl is replaced by 4-pyridinyl.
~
5
0
(
TS=23). When the aryl moiety was unsubstituted (13g),
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lengthening of the bridge to a methylthiomethyl (13q) nega-
tively affected the cytostatic potential (IC =3.5 mm for 13q
may indicate that the phthalimide derivatives act as partially
hydrolysable prodrugs of the free 2-amino-3-carboxymethylthi-
ophenes or, alternatively, are less efficient cytostatic agents
due to decreased cellular uptake and/or less efficient recogni-
tion by their cellular target.
50
versus IC =1.3 mm for 13g and TS=17 for 13q versus TS=
50
83 for 13g). But further lengthening the bridge to a methyl-
thioethyl (13s) increased the cytostatic activity (IC =1.2 mm)
50
and tumor selectivity (TS=62) again, when compared with the
methylthiomethyl-bridged derivative.
The most active derivatives in this new series of compounds
(endowed with TSꢁ20) were also evaluated for their antiproli-
ferative potential against human prostate PC-3, liver Huh7 and
kidney Caki-1 cancer cells. These particular tumor cells were
chosen because our earlier studies revealed that the tumor-se-
lective thiophenes were, besides against T-lymphocyte tumor
cells, also highly cytostatic against PC-3, Huh7 and Caki-
Next the effect of the nature of the aryl groups was investi-
gated. When the 4-methoxyphenyl in 13r was replaced by an
unsubstituted phenyl (13q), 2-thienyl (13u) or 2-furyl (13t), cy-
tostatic activity remained virtually unaltered (IC : 3.9, 3.5, 3.3
50
and 4.9 mm, respectively), resulting in TS values between 21
and 30. Replacement of the aryl in 13g by a naphthyl (13w) or
biphenyl (13x) still resulted in pronounced cytostatic activity
and tumor selectivity (IC : 0.90–1.2 mm; TS: 45–83), whereas
[10]
1 tumor cells.
The tumor-selective compounds showed
a comparable cytostatic activity in CEM, PC-3 and Huh7 tumor
cell cultures, but were somewhat less cytostatic against Caki-
1 tumor cell cultures (~2–6-fold decreased activity, see
Table 3). Also, non-tumorigenic human embryonic lung fibro-
blast (HEL), human embryonic kidney 293T and human micro-
vascular endothelial (HMVEC-d) cells were included in the
study to further estimate the tumor-selectivity of the test com-
pounds. HEL cells represent human embryonic lung fibroblasts,
293T cells are human embryonic kidney fibroblasts and
HMVEC-d cells are primary human dermal microvascular endo-
thelial cells. In general, the compounds proved markedly less
inhibitory against non-cancerogenic HEL (~3–50-fold) and
50
a pyridyl group (13v) annihilated tumor selectivity (Table 2).
We then decided to keep the aryl group linked through a thi-
omethyl bridge to the 2-amino-3-carboxymethylthiophene un-
altered, and to explore further the influence of a wide variety
of substituents on the phenyl group. No substitution (13g) or
a p-methoxy (13i) or o-methyl (13c) substitution afforded at
most threefold differences in the antiproliferative activity (IC :
50
0
9
.62–2.0) and showed pronounced tumor selectivity (TS: 35–
3). m-Methyl-substituted 13o was slightly less cytostatic
(
IC =4.1 mm; TS=27). However, the o-methoxyphenyl deriva-
50
tive 13d markedly lost inhibitory
activity (IC =17 mm; TS=5.5).
50
Instead, a p-ethyl (13m) or p-iso-
propyl (13n) group substantially
improved the cytostatic activity
Table 3. Antiproliferative activity of test compounds that showed a tumor selectivity factor of >20 for CEM
versus HeLa cell cultures.
[a]
IC50 [mm]
(
IC : 0.34 and 0.46 mm, respec-
CEM
PC-3
Huh7
Caki-1
293T
39Æ0
HEL
HMVEC-d
50
tively) resulting in the most
potent and tumor-selective com-
pounds found in the current
subseries of new 8c derivatives
with a TS value of 144 and 100,
1
13g
13h
3c
2.0Æ1.1
1.3Æ0.6
1.5Æ0.3
0.62Æ0.33
1.7Æ1.2
0.34Æ0.17
0.46Æ0.05
4.1Æ1.7
3.8Æ0.9
2.6Æ0.9
1.0Æ0.4
1.1Æ0.2
3.9Æ1.0
1.3Æ0.2
1.4Æ0.3
4.8Æ0.4
4.2Æ1.1
2.3Æ0.4
3.8Æ0.6
4.3Æ0.07
2.0Æ0.3
3.9Æ1.1
0.19Æ0.04
1.9Æ0.14
1.5Æ0.28
0.46Æ0.042
0.35Æ0.028
1.5Æ0.14
0.47Æ0.064
0.61Æ0.078
2.6Æ0.85
4.9Æ0.21
1.1Æ0.33
5.5Æ0.35
4.9Æ0.64
1.1Æ0.16
1.9Æ0.28
0.95Æ0.21
7.2Æ0.71
8.4Æ1.4
3.5Æ0.92
2.8Æ0.71
6.5Æ0.99
2.9Æ0.85
2.7Æ0.071
8.4Æ2.3
13Æ7.7
23Æ2.1
37Æ5.7
20Æ4.9
7.0Æ1.4
26Æ2.1
8.1Æ0.21
22Æ0
12Æ2.8
11Æ0.1
2.8Æ0.2
2.5Æ0.3
7.5Æ1.1
2.5Æ0.1
2.8Æ0.1
24Æ8.5
11Æ1.4
6.5Æ4.2
9.4Æ0.4
11Æ1.8
7.0Æ1.8
9.6Æ0.6
2.0Æ0.2
72Æ40
38Æ1.4
26Æ9.2
28Æ3.5
23Æ13
33Æ9.2
1
1
1
3i
3j
3m
respectively.
Compounds
in
13n
13o
which the p-phenyl substituent
was replaced by fluorine (13k)
or chlorine (13j) reasonably kept
>100
74Æ34
19Æ7.8
25Æ0.71
15Æ2.1
24Æ2.8
23Æ1.4
23Æ7.8
52Æ25
13r
13s
13t
3.9Æ1.4
1.2Æ0.7
4.9Æ0.1
3.3Æ0.9
0.90Æ0.04
1.2Æ0.1
0.90Æ0.43
43Æ7.1
38Æ0.71
52Æ0
4.1Æ0
11Æ4.6
cytostatic potential, but
a
13u
13w
9.6Æ6.3
4.3Æ0.28
5.0Æ2.0
1.7Æ1.1
50Æ6.4
35Æ1.4
35Æ5.7
33Æ25
p-NO2 group (13l) virtually de-
stroyed the tumor selectivity of
the compound. Also, an o-car-
boxymethyl (13e) or a m-carbox-
ylic acid (13p) and an o-amino
1
8
3x
c
[
a] 50% inhibitory concentration or compound concentration required to inhibit cell proliferation by 50%. Data
are the mean ÆSD of two to three independent experiments.
(
13 f) seriously compromised the
cytostatic activity and tumor selectivity properties of the com-
pounds.
293T (~11–70-fold) cells. However, they only showed 2–8-fold
less cytostatic activity against HMVEC-d cells than against CEM
cells. Since endothelial cells are usually non-proliferative in
adults, except during wound healing and the female reproduc-
tive cycle, and highly activated in tumors where blood vessel
formation is stimulated, the moderate antiproliferative activity
of the compounds against endothelial cells might offer an
extra advantage to target both tumor cells and the intratumor-
al blood vessels. It should be noticed that the higher or lesser
sensitivity of tumor cell lines for the test compounds is not re-
As a general rule, all corresponding phthalimide-protected
derivatives of the above-mentioned 2-amino-3-carboxyme-
thylthiophenes were endowed with at least a 5- to 20-fold
lower cytostatic activity and markedly lower TS values (see
table 1 in the Supporting Information). The phthaloyl-protect-
ed derivatives of the most active thiophenes (i.e., 12n versus
13n; 12m versus 13m; and 12g versus 13g) were also the
most active and selective “diketo”-substituted derivatives. This
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lated to differences in growth rates and generation times of
the particular tumor cell lines. In fact, the fastest proliferating
cell line (L1210) is clearly less sensitive to the antiproliferative
effect of the compounds than the CEM tumor cell line. Also,
the HeLa cells, being among the least sensitive tumor lines,
have a generation time close to the highly sensitive CEM cell
line.
Finally, the compounds have also been evaluated for their
antiproliferative activity against murine leukemia L1210 cells
(Table 2). Although the tumor-selective compounds were in
most cases somewhat more inhibitory to L1210 than to HeLa
cell proliferation, they were clearly inferior against the murine
L1210 leukemia cells when compared to their antiproliferative
activity against the human CEM lymphoma cell cultures. There-
fore, the murine L1210 tumor cell model in mice does not
seem to be an appropriate cancer model to evaluate the anti-
tumor efficacy of the 2-amino-3-carboxymethylthiophene de-
rivatives.
The IC50 values for the tumor-selective compounds (TSꢁ20)
against T-lymphocyte CEM tumor cells were plotted against
the corresponding IC50 values for the drug-sensitive PC-3,
Huh7 and Caki-1 tumor cells, and the insensitive HeLa and
non-tumorigenic HEL and 293T cells. It became evident that
the IC50 correlation plots in Figure 4 for the drug-sensitive
tumor cells have a rather steep regression line showing a tight
correlation between drug modification and potency of the
tumor-selective compounds. Indeed, an increase of antiprolifer-
ative potency against CEM tumor cells by one of the 8c deriva-
tives resulted in a similarly increased activity against the other
drug-sensitive tumor cells.
Figure 4. Correlation between the IC50 values of the compounds against
CEM tumor cells and their IC50 values against the drug-sensitive PC-3 (A),
Huh7 (B) and Caki-1 (C) tumor cells, the insensitive HeLa (D) tumor cells and
the insensitive non-tumorigenic HEL (E) and 293T (F) cells.
These correlation curves likely indicate a specific and similar
mechanism of antiproliferative activity of the thiophene deriva-
tives in the drug-sensitive tumor cell lines. Instead, the regres-
sion lines for the IC50 values of sensitive (CEM) versus insensi-
tive (HeLa, HEL, 293T) (tumor) cells are more flat lines, imple-
menting a poor, or virtual lack of, correlation between drug
modification and cytotoxic response of the insensitive (tumor)
cell lines. Although the exact mechanism of antiproliferative
activity is currently unknown, it is clear that these compounds,
alike the prototype compound 8c, differ in their molecular
mechanism of action from 2-amino-3-aroyl 4- or 5-substituted
thiophene derivatives that were previously shown to interact
with tubulin and arrest the tumor cells in the G2M phase of
terms of cytostatic potential and tumor selectivity by concomi-
tantly adding alkyl (i.e., ethyl/isopropyl) substitutions in the
para position of the aryl ring and replacing the ethylene
bridge by a thiomethyl entity. These compounds kept a similar
tumor selectivity against human T-lymphoma/leukemia cells,
such as CEM, renal cancer (i.e., Caki-1), prostate cancer (i.e.,
PC-3) and liver cancer (i.e., Huh7) cells, but lacked appreciable
cytostatic activity against a wide variety of other tumor types
including human cervical carcinoma HeLa cells and the non-
cancerous human fibroblast HEL and 293T cells. The NIH drug
database revealed that the molecular basis of the antiprolifera-
tive activity of this class of compounds is different from exist-
ing anticancer drugs. In-depth studies are required to reveal
the molecular mechanism of action of this novel class of com-
pounds.
[
13,14]
their cell cycle.
In fact, tubulin polymerization targeting
drugs have never shown to be very selective in their antitumor
action, and thus, differ from the current novel class of 5-substi-
tuted 2-amino-3-carboxymethylthiophenes.
Conclusions
Experimental Section
The recently described tumor-selective 2-amino-3-carboxyme-
thylthiophene-based lead compound 8c showed a pronounced
selectivity for a number of tumor cell types. Most T-lymphoma/
leukemia tumor cell types tested were preferentially sensitive
to the antiproliferative activity of the prototype drug. The lead
compound proved cytotoxic at the highest concentrations
tested. Compound 8c could be further improved both in
Biological assays
Beside the tumor cell lines used in the NCI-60 anticancer screen to
evaluate the cytostatic activity of the prototype (8c) compound,
following cell lines were included in the evaluation of the newly
synthesized compounds: murine leukemia (L1210) and human
+
CD4 T-lymphocyte CEM, cervix carcinoma HeLa, prostate cancer
PC3, hepatoma Huh7, renal (kidney) carcinoma Caki-1 and the
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non-tumorigenic kidney fibroblast 293T, embryonic lung HEL and
vascular endothelial HMVEC-d cells.
the case of 12c,d, 12 f–i and 12m–o the reaction mixture was
stirred at RT. Two equivalents of K CO was used in the case of
2
3
1
2p. After stirring the resulting mixture for another 24 h the reac-
The antiproliferative activity determination of the test compounds
was performed as follows. To each well of a 96-well microtiter
plate were added an appropriate amount of cells and a 5-fold dilu-
tion series of the test compounds (highest concentration tested:
tion mixture was added to H O (10 mL). CH Cl (15 mL) was added,
and the organic solution was washed with distilled H O (3ꢁ15 mL),
dried over MgSO , filtered, and evaporated to dryness to afford the
crude product mixture. Purification by column chromatography
2
2
2
2
4
2
50 mm). The cells were allowed to proliferate 48–96 h (depending
(silica, CH Cl ) afforded the corresponding product.
2 2
on the nature of the (tumor) cell lines) at 378C in a humidified
CO -controlled atmosphere. At the end of the incubation period,
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((4-methoxyphenylamino)-
2
the cells were counted in a Coulter counter (Coulter Electronics
Ltd., Harmenden, Herts, UK). The IC50 value (50% cytostatic/toxic
concentration) was defined as the compound concentration that
reduced the number of viable cells by 50%.
methyl)thiophene-3-carboxylate (12a). Off-white solid (131 mg,
1
59%): H NMR (300 MHz, CDCl ): d=3.70 (s, 3H), 3.75 (s, 3H), 4.47
3
(s, 2H), 6.65 (d, 2H, J=8.8 Hz), 6.79 (d, 2H, J=8.9 Hz), 7.40 (s, 1H),
+
7.81–7.79 ppm (m, 2H), 7.98–7.95 (m, 2H); MS (EI): m/z: 422 [M] ;
+
HRMS (EI): m/z [M] calcd for C H N O S: 422.0936, found:
22
18
2
5
4
22.0934.
Chemistry
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((4-methoxyphenoxy)me-
thyl)thiophene-3-carboxylate (12b). Off-white solid (159 mg,
NMR spectra were acquired on commercial instruments (Bruker
Avance 300 MHz or Bruker AMX 400 MHz) and chemical shifts (d)
are reported in parts per million (ppm) referenced to tetramethylsi-
lane ( H), or the internal (NMR) solvent signal ( C). Mass spectra
were run using a HP5989A apparatus (EI, 70 eV ionization energy)
with Apollo 300 data system, a Micromass Quattro II apparatus
1
7
1%): H NMR (300 MHz, CDCl ): d=3.71 (s, 3H), 3.78 (s, 3H), 5.12
3
(
s, 2H), 6.84 (d, 2H, J=9.2 Hz), 6.93 (d, 2H, J=9.0 Hz), 7.47 (s, 1H),
+
1
13
7.82–7.79 (m, 2H), 8.01–7.97 ppm (m, 2H); MS (EI): m/z: 423 [M] ;
HRMS (EI): m/z [M] calcd for C H NO S: 423.0777, found:
+
22
17
6
4
23.0771.
(
ESI) with MASSLYNX data system or a Thermo Finnigan LCQ Ad-
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-(o-tolylthiomethyl)thio-
phene-3-carboxylate (12c). Off-white solid (127 mg, 57%): H NMR
1
vantage apparatus (ESI). Exact mass measurements were acquired
on a Kratos MS50TC instrument (performed in the EI mode at a res-
olution of 10000). Melting points (not corrected) were determined
using a Reichert Thermovar apparatus. For column chromatogra-
phy, 70–230 mesh silica 60 (E. M. Merck) was used as the stationary
phase. Chemicals received from commercial sources were used
without further purification. Reaction solvents were used as re-
ceived from commercial sources.
(
300 MHz, CDCl ): d=2.39 (s, 3H), 3.68 (s, 3H), 4.23 (s, 2H), 7.20–
3
7
7
.14 (m, 3H), 7.27 (s, 1H), 7.36–7.33 (m, 1H), 7.81–7.79 (m, 2H),
.98–7.95 ppm (m, 2H); MS (EI): m/z: 423 [M] ; HRMS (EI): m/z [M]
+
+
calcd for C H NO S : 423.0599, found: 423.0561.
22
17
4 2
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((2-methoxyphenylthio)me-
thyl)thiophene-3-carboxylate (12d). Off-white solid (188 mg,
1
8
1%): H NMR (300 MHz, CDCl ): d=3.68 (s, 3H), 3.91 (s, 3H), 4.24
3
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-methylthiophene-3-carbox-
(
7
s, 2H), 6.94–6.87 (m, 2H), 7.27 (s, 1H), 7.36 (d, 1H, J=7.7 Hz),
[1]
+
ylate (10). To a suspension of 9 (5 g, 29.23 mmol) in acetic acid
80 mL) was added phthalic anhydride (6.4 g, 43.24 mmol). After
.83–7.78 (m, 3H), 7.97–7.94 ppm (m, 2H); MS (EI): m/z: 439 [M] ;
+
(
HRMS (EI): m/z [M] calcd for C H NO S : 439.0548, found:
22
17
5 2
stirring for 30 h at reflux, the solvent was evaporated and the resi-
due dissolved in CH Cl (30 mL). The organic solution was washed
4
39.0467.
2
2
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((2-(methoxycarbonyl)phe-
nylthio)methyl)thiophene-3-carboxylate (12e). Off-white solid
(96 mg, 39%): H NMR (300 MHz, CDCl ): d=3.69 (s, 3H), 3.94 (s,
3
with saturated aq NaHCO (50 mL), H O (50 mL), dried and concen-
3
2
trated. Purification by column chromatography (silica, CH Cl ) af-
2
2
1
1
3
forded 10 as an off-white solid (7.7 g, 87%): H NMR (300 MHz,
H), 4.33 (s, 2H), 7.22 (t, 1H, J=7.1 Hz), 7.34 (d, 1H, J=7.7 Hz),
CDCl ): d=2.51 (s, 3H), 3.69 (s, 3H), 7.19 (s, 1H), 7.81–7.87 (m, 2H);
3
+
+
7.49–7.44 (m, 2H), 7.81–7.78 (m, 2H), 8.00–7.95 ppm (m, 3H); MS
7
.98–7.95 ppm (m, 2H); MS (EI): m/z: 301 [M] ; HRMS (EI): m/z [M]
+
+
(EI): m/z: 467 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
23 17 6 2
calcd for C H NO S: 301.0409, found: 301.0428.
1
5
11
4
4
67.0497, found: 467.0499.
Methyl-5-(bromomethyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-
Methyl-5-((2-aminophenylthio)methyl)-2-(1,3-dioxoisoindolin-2-
3
1
-carboxylate (11). To a solution of protected 2-aminothiophene
0 (2.5 g, 8.33 mmol), N-bromosuccinimide (1.55 g, 8.74 mmol) and
yl)thiophene-3-carboxylate (12 f). Off-white solid (105 mg, 47%):
1
H NMR (300 MHz, CDCl ): d=3.67 (s, 3H), 4.05 (s, 2H), 4.32 (s ,
3
b
benzoyl peroxide (20 mg) in CCl (50 mL) was heated at reflux for
4
2
7
H), 6.73–6.68 (m, 2H), 7.16–7.10 (m, 2H), 7.34 (d, 1H, J=7.6 Hz),
4
8 h under argon atmosphere. CCl was evaporated under reduced
4
+
.81–7.78 (m, 2H), 7.97–7.94 ppm (m, 2H); MS (EI): m/z: 424 [M] ;
pressure, and the residue was extracted with CH Cl2 (3ꢁ50 mL).
2
+
HRMS (EI): m/z [M] calcd for C H N O S : 424.0551, found:
21
16
2
4 2
The combined organic phases were washed with saturated aq
4
24.0580.
NaHCO , dried over magnesium sulfate and the solvent was evapo-
3
rated under reduced pressure. Purification by column chromatog-
Methyl-2-phthalimido-5-(2-(phenyl)sulfanylmethyl)thiophene-3-
carboxylate (12g). Off-white solid (156 mg, 72%): H NMR
raphy (silica, eluent CH Cl ) afforded 11 as an off-white solid (1.9 g,
1
2
2
1
6
0%): H NMR (300 MHz, CDCl ): d=3.72 (s, 3H), 4.66 (s, 2H), 7.51
3
(300 MHz, CDCl ) 3.68 (s, 3H), 4.25 (s, 2H), 7.26 (m, 4H), 7.39 (d,
3
(
3
s, 1H), 7.83–7.81 (m, 2H), 8.01–7.97 ppm (m, 2H); MS (EI): m/z:
2
H, J=7.1 Hz), 7.81–7.78 (m, 2H), 7.97–7.94 ppm (m, 2H); MS (EI):
+
+
80 [M] ; HRMS (EI): m/z [M] calcd for C H NO S: 378.9514,
+
+
15
11
4
m/z: 409 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
21 15 4 2
found: 380.9391.
4
09.0442, found: 409.0441.
General procedure for the synthesis of 12a–x. To a mixture of 11
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-(p-tolylthiomethyl)thio-
(
200 mg, 0.529 mmol) and K CO3 (73 mg, 0.529 mmol) in THF
2
phene-3-carboxylate (12h). Off-white solid (159 mg, 71%):
(
4 mL) was added corresponding aryl thiols, alky thiols, 4-methoxy-
1
H NMR (300 MHz, CDCl ): d=2.31 (s, 3H), 3.67 (s, 3H), 4.20 (s, 2H),
3
phenol or 4-methoxyaniline (1.0 equiv). In the case of 12a,b,e,j,k
and 12p–x, the reaction mixture was stirred at 458C whereas in
7
.12 (d, 2H, J=7.9 Hz), 7.26 (s, 1H), 7.30 (d, 2H, J=8.1 Hz), 7.82–
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+
7
(
.79 (m, 2H), 7.98–7.94 ppm (m, 2H); MS (EI): m/z: 423 [M] ; HRMS
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((4-methoxybenzylthio)me-
+
EI): m/z [M] calcd for C H NO S : 423.0599, found: 423.0589.
thyl)thiophene-3-carboxylate (12r). Off-white solid (163 mg,
22
17
4 2
1
6
8%): H NMR (300 MHz, CDCl ): d=3.71 (s , 5H), 3.72 (s, 2H), 3.80
3
b
Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thi-
ophene-3-carboxylate (12i). Off-white solid (146 mg, 63%):
H NMR (300 MHz, CDCl ): d=3.68 (s, 3H), 3.79 (s, 3H), 4.13 (s, 2H),
(
2
s, 3H), 6.86 (d, 2H, J=8.7 Hz), 7.28–7.21 (m, 3H), 7.82–7.79 (m,
+
H), 8.00–7.96 ppm (m, 2H); MS (EI): m/z: 453 [M] ; HRMS (EI): m/z
1
3
+
[M] calcd for C H NO S : 453.0705, found: 453.0714.
23 19 5 2
6
7
(
.85 (d, 2H, J=8.6 Hz), 7.18 (s, 1H); 7.37 (d, 2H, J=8.6 Hz), 7.81–
+
.78 (m, 2H), 7.97–7.94 ppm (m, 2H); MS (EI): m/z: 439 [M] ; HRMS
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-(phenethylthiomethyl)thio-
+
1
EI): m/z [M] calcd for C H NO S : 439.0548, found: 439.0541.
phene-3-carboxylate (12 s). Off-white solid (92 mg, 41%): H NMR
22
17
5 2
(
7
300 MHz, CDCl ): d=2.91–2.82 (m, 4H), 3.71 (s, 2H), 3.82 (s, 3H),
3
Methyl-5-((4-chlorophenylthio)methyl)-2-(1,3-dioxoisoindolin-2-
.30–7.20 (m, 6H), 7.82–7.79 (m, 2H) 7.97–7.92 ppm (m, 2H); MS
yl)thiophene-3-carboxylate (12i). Off-white solid (138 mg, 59%):
+
+
(EI): m/z: 437 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
23 19 4 2
1
H NMR (300 MHz, CDCl ): d=3.69 (s, 3H), 4.22 (s, 3H), 7.26 (s, 2H),
3
4
37.0755, found: 437.0752.
7
.30–7.29 (m, 3H), 7.82–7.79 (m, 2H), 7.97–7.95 ppm (m, 2H); MS
+
+
(
EI): m/z: 443 [M] ; HRMS (EI): m/z [M] calcd for C H ClNO S :
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((furan-2-ylmethylthio)me-
21
14
4 2
4
43.0053, found: 443.0121.
thyl)thiophene-3-carboxylate (12t). Off-white solid (107 mg,
1
4
9%): H NMR (300 MHz, CDCl ): d=3.71 (s, 3H), 3.74 (s, 2H), 3.85
3
Methyl-5-((4-fluorophenylthio)methyl)-2-(1,3-dioxoisoindolin-2-
(s, 2H), 6.20 (d, 1H, J=3.0 Hz), 6.33 (t, 1H, J=2.4 Hz), 7.35 (s, 1H),
yl)thiophene-3-carboxylate (12k). Off-white solid (137 mg, 61%):
7
.39 (d, 1H, J=1.1 Hz), 7.82–7.79 (m, 2H), 7.99–7.94 ppm (m, 2H);
1
H NMR (300 MHz, CDCl ): d=3.68 (s, 3H), 4.18 (s, 3H), 7.04–6.98
+
+
3
MS (EI): m/z: 413 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
20
15
5 2
(
m, 2H), 7.20 (s, 1H), 7.41–7.36 (m, 2H), 7.82–7.79 (m, 2H), 7.98–
4
13.0392, found: 413.0381.
+
+
7
.95 ppm (m, 2H); MS (EI): m/z: 427 [M] ; HRMS (EI): m/z [M]
calcd for C H FNO S : 427.0348, found: 427.0344.
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((thiophen-2-ylmethylthio)-
2
1
14
4 2
methyl)thiophene-3-carboxylate (12u). Off-white solid (93 mg,
Methyl-5-((4-nitrophenylthio)methyl)-2-(1,3-dioxoisoindolin-2-
1
4
1%): H NMR (300 MHz, CDCl ): d=3.71 (s, 3H), 3.82 (s, 2H), 3.95
3
yl)thiophene-3-carboxylate (12l). Yellow solid (103 mg, 43%):
(
7
s, 2H), 6.95 (d, 2H, J=3.8 Hz), 7.26–7.23 (m, 1H), 7.32 (s, 1H),
1
H NMR (300 MHz, CDCl ): d=3.70 (s, 3H), 4.40 (s, 3H), 7.42–7.39
+
3
.82–7.79 (m, 2H), 7.99–7.96 ppm (m, 2H); MS (EI): m/z: 429 [M] ;
(
8
m, 3H), 7.82–7.79 (m, 2H), 7.98–7.95 (m, 2H), 8.16 ppm (d, 2H, J=
.9 Hz); MS (EI): m/z: 454 [M] ; HRMS (EI): m/z [M] calcd for
+
HRMS (EI): m/z [M] calcd for C H NO S : 429.0163, found:
+
+
20 15
4 3
4
29.0173.
C H N O S : 454.0293, found: 454.0277.
2
1
14
2
6 2
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((naphthalen-2-ylthio)me-
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((4-ethylphenylthio)me-
thyl)thiophene-3-carboxylate (12m). Yellow solid (146 mg, 63%):
d=1.21 (t, 3H, J=7.5 Hz), 2.62 (q, 2H, J=7.4 Hz), 3.68 (s, 3H), 4.21
thyl)thiophene-3-carboxylate (12v). Off-white solid (187 mg,
1
7
7%): H NMR (300 MHz, CDCl ): d=3.64 (s, 3H), 4.33 (s, 2H), 7.28
3
(
2
s, 1H); 7.48–7.44 (m, 3H); 7.83–7.76 (m, 6H), 7.96–7.94 ppm (m,
H); MS (EI): m/z: 459 [M] ; HRMS (EI): m/z [M] calcd for
(
7
s, 2H), 7.14 (d, 2H, J=7.5 Hz), 7.24 (s, 1H), 7.32 (d, 2H, J=8.3 Hz),
+
+
+
.81–7.79 (m, 2H), 7.97–7.95 ppm (m, 2H); MS (EI): m/z: 437 [M] ;
C H NO S : 459.0599; found: 459.0581.
+
25 17
4 2
HRMS (EI): m/z [M] calcd for C H NO S : 437.0755, found:
23
19
4 2
4
37.0754.
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((pyridin-4-ylthio)me-
thyl)thiophene-3-carboxylate (12w). Off-white solid (137 mg,
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-((4-isopropylphenylthio)-
1
6
3%): H NMR (300 MHz, CDCl ): d=3.72 (s, 3H), 4.37 (s, 2H), 7.12
3
methyl)thiophene-3-carboxylate (12n). Off-white solid (157 mg,
(
(
d, 2H, J=6.1 Hz), 7.37 (s, 1H), 8.50 (d, 1H, J=6.2 Hz), 7.82–7.76
1
6
6%): H NMR (300 MHz, CDCl ): d=1.22 (d, 6H, J=6.8 Hz), 2.92–
+
3
m, 6H), 7.99–7.97 ppm (m, 2H); MS (EI): m/z: 410 [M] ; HRMS (EI):
2
.83 (m, 1H), 3.68 (s, 3H), 4.21 (s, 2H), 7.17 (d, 2H, J=7.9 Hz), 7.24
+
m/z [M] calcd for C H N O S : 410.0395, found: 410.0379.
20
14
2
4 2
(
(
s, 1H), 7.33 (d, 2H, J=8.1 Hz), 7.80–7.77 (m, 2H), 7.97–7.94 ppm
m, 2H); MS (EI): m/z: 451 [M] ; HRMS (EI): m/z [M] calcd for
+
+
Methyl-5-((biphenyl-4-ylthio)methyl)-2-(1,3-dioxoisoindolin-2-
C H NO S : 451.0912, found: 451.0931.
yl)thiophene-3-carboxylate (12x). Off-white solid (146 mg, 57%):
2
4
21
4 2
1
H NMR (300 MHz, CDCl ) 3.68 (s, 3H), 4.28 (s, 2H), 7.36–7.18 (m,
3
Methyl-2-(1,3-dioxoisoindolin-2-yl)-5-(m-tolylthiomethyl)thio-
2
7
H), 7.467–7.40 (m, 4H), 7.58–7.53 (m, 5H), 7.82–7.77 (m, 2H),
.99–7.96 ppm (m, 2H); MS (EI): m/z: 458 [M] ; HRMS (EI): m/z [M]
phene-3-carboxylate (12o). Off-white solid (150 mg, 67%):
+
+
1
H NMR (300 MHz, CDCl ): d=2.33 (s, 3H), 3.68 (s, 3H), 4.24 (s, 2H),
3
calcd for C H NO S : 458.0755, found: 485.0767.
27
19
4 2
7
.06–7.12 (m, 1H), 7.19 (d, 2H, J=8.1 Hz), 7.27 (m, 2H), 7.80–7.76
+
(
m, 2H), 7.99–7.96 ppm (m, 2H); MS (EI): m/z: 423 [M] ; HRMS (EI):
General procedure for the synthesis of 13a–x. To a stirred sus-
pension of the compound having the general formula 12
+
m/z [M] calcd for C H NO S : 423.0599, found: 423.0559.
2
2
17
4 2
(
0.25 mmol, 1 equiv) in EtOH (5 mL), hydrazine (1.5 equiv) was
3
-((5-(1,3-Dioxoisoindolin-2-yl)-4-methoxycarbonyl)thiophen-2-
added. In the case of thiophenol derivative, the reaction mixture
was held at reflux for 12 h whereas in the case of p-methoxyphe-
nol or p-anisidine compound, the reaction mixture was stirred at
RT for 2 h. The reaction mixture was added to H O (10 mL). CH Cl
2
yl)methylthio)benzoic acid (12p). Off-white solid (53 mg, 22%):
1
H NMR (300 MHz, CDCl ): d=3.68 (s, 3H), 4.32 (s, 3H), 7.32 (d, 2H,
3
J=6.2 Hz), 7.41 (t, 1H, J=7.7 Hz), 7.61–7.56 (m, 2H), 7.80–7.77 (m,
2
HRMS (EI): m/z [M] calcd for C H NO S : 453.0341, found:
+
2
2
H), 7.96–7.93 (m, 2H), 8.13 ppm (s, 1H); MS (EI): m/z: 453 [M] ;
(10 mL) was added, and the organic solution was washed with dis-
+
22
15
6 2
tilled H O (3ꢁ10 mL), dried over MgSO , filtered, and evaporated
to dryness to afford the crude product mixture. Purification by
2
4
4
53.0240.
Methyl-5-(benzylthiomethyl)-2-(1,3-dioxoisoindolin-2-yl)thio-
column chromatography (silica, CH
products as solids or semi-solids.
Cl ) afforded the corresponding
2 2
phene-3-carboxylate (12q). Off-white solid (127 mg, 57%):
1
H NMR (300 MHz, CDCl ): d=3.70 (s, 3H), 3.72 (s, 2H), 3.74 (s, 3H),
3
Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thio-
phene-3-carboxylate (13a). Semi-solid (1.5 mg, 2%): H NMR
7
.36–7.26 (m, 5H), 7.81–7.78 (m, 2H), 7.98–7.95 ppm (m, 2H); MS
1
+
+
(
EI): m/z: 423 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
22 17 4 2
(
300 MHz, CDCl ): d=3.74 (s, 3H), 3.79 (s, 3H), 4.22 (s, 2H), 5.90 (s ,
3
b
4
23.0599, found: 423.0562.
ꢀ
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&
8
&
ÞÞ
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1
3
2
1
H), 6.63 (d, 2H, J=8.3 Hz), 6.78 (d, 2H, J=8.2 Hz), 6.84 ppm (s,
H). This compound is not stable at RT.
(d, 1H, J=8.6 Hz); C NMR (75 MHz, CDCl ): d=34.6, 51.0, 51.1,
3
105.8, 115.0, 118.6, 124.5, 130.4, 136.5, 148.6, 162.8, 165.7 ppm; MS
EI): m/z: 309 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
09.0493, found: 309.0482.
+
+
(
14 15 3 2
Methyl-2-amino-5-((4-methoxyphenoxy)methyl)thiophene-3-car-
boxylate (13b). Semi-solid (3 mg, 4%): H NMR (300 MHz, CDCl ):
3
1
3
d=3.78 (s, 3H), 3.83 (s, 3H), 4.94 (s, 2H), 5.93 (s , 2H), 6.71 (d, 2H,
J=8.3 Hz), 6.87 (s, 1H), 6.92 ppm (d, 2H, J=8.2 Hz). This com-
Methyl 2-amino-5-((4-chlorophenylthio)methyl)thiophene-3-car-
boxylate (13j). White solid (48 mg, 62%): mp: 98–998C; H NMR
b
1
pound is not stable at RT.
(300 MHz, CDCl ): d=3.77 (s, 3H), 4.04 (s, 2H), 5.91 (s , 2H), 6.69 (s,
3
b
13
1
1
H), 7.25 ppm (s , 4H); C NMR (75 MHz, CDCl ): d=34.6, 51.0,
b 3
Methyl-2-amino-5-(o-tolylthiomethyl)thiophene-3-carboxylate
05.7, 121.8, 124.5, 129.1, 132.0, 133.0, 133.7, 162.6, 165.5 ppm; MS
1
(13c). White solid (41 mg, 56%): mp: 68–698C; H NMR (300 MHz,
+
+
(EI): m/z: 312 [M] ; HRMS (EI): m/z [M] calcd for C H ClNO S :
13 12 2 2
CDCl ): d=2.37 (s, 3H), 3.76 (s, 3H), 4.05 (s, 2H), 5.89 (s , 2H), 6.73
3
b
3
12.9998, found: 312.9979.
13
(
s, 1H), 7.16–7.11 (m, 3H), 7.40–7.26 ppm (m, 1H); C NMR
(
1
2
75 MHz, CDCl ): d=20.6, 33.5, 51.1, 105.6, 121.9, 124.3, 126.5,
Methyl-2-amino-5-((4-fluorophenylthio)methyl)thiophene-3-car-
boxylate (13k). White solid (38 mg, 51%): mp: 77–788C; H NMR
3
1
26.6, 129.6, 130.2, 134.9, 138.4, 162.5, 165.6 ppm; MS (EI): m/z:
+
+
93 [M] ; HRMS (EI): m/z [M] calcd for C H NO S : 293.0544,
(300 MHz, CDCl ): d=3.76 (s, 3H), 4.00 (s, 2H), 5.92 (s , 2H), 6.62 (s,
14
15
2
2
3
b
1
3
found: 293.0542.
1H), 6.97 (t, 2H, J=8.6 Hz), 7.35–7.30 ppm (m, 2H); C NMR
75 MHz, CDCl ): d=35.7, 51.1, 105.7, 116.0, 116.3, 122.3, 124.5,
(
3
Methyl-2-amino-5-((2-methoxyphenylthio)methyl)thiophene-3-
1
30.3, 130.1, 133.9, 134.0, 160.8, 162.7, 164.1, 165.7 ppm; MS (EI):
carboxylate (13d). White solid (56 mg, 72%): mp: 89–908C;
+
+
m/z: 297 [M] ; HRMS (EI): m/z [M] calcd for C H FNO S :
1
13 12
2 2
H NMR (300 MHz, CDCl ): d=3.78 (s, 3H), 3.91 (s, 3H), 4.15 (s, 2H),
3
2
97.0293, found: 297.0286.
5
7
.88 (s , 2H), 6.89 (s, 1H), 7.18 (t, 1H, J=7.9 Hz), 7.34 (d, 1H, J=
b
.5 Hz), 7.42 (t, 1H, J=8.1 Hz), 7.96 ppm (d, 1H, J=8.5 Hz);
Methyl-2-amino-5-((4-nitrophenylthio)methyl)thiophene-3-car-
1
3
1
C NMR (75 MHz, CDCl ): d=32.3, 52.1, 52.3, 106.2, 121.0, 124.7,
boxylate (13l). Yellow solid (55 mg, 68%): mp: 88–898C; H NMR
(300 MHz, CDCl ): d=3.78 (s, 3H), 4.22 (s, 2H), 5.95 (s , 2H), 6.86 (s,
3
1
24.8, 126.5, 128.2, 131.4, 132.5, 140.7, 162.6, 165.7, 167.0 ppm; MS
3
b
+
+
13
(
EI): m/z: 309 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
1H), 7.37 (d, 2H, J=8.7 Hz), 8.12 ppm (d, 2H, J=8.6 Hz); C NMR
14
15
3 2
3
09.0493, found: 309.0482.
(75 MHz, CDCl ): d=32.5, 51.2, 106.0, 120.1, 124.1, 124.6, 125.3,
3
+
1
27.5, 146.1, 162.8, 165.5 ppm; MS (EI): m/z: 324 [M] ; HRMS (EI):
Methyl-2-amino-5-((2-(methoxycarbonyl)phenylthio)methyl)thio-
phene-3-carboxylate (13e). White solid (69 mg, 82%): mp: 67–
6
+
m/z [M] calcd for C H N O S : 324.0238, found: 324.0235.
13
12
2
4 2
1
88C; H NMR (300 MHz, CDCl ): d=3.69 (s, 3H), 3.94 (s, 3H), 4.34
Methyl-2-amino-5-((4-ethyllphenylthio)methyl)thiophene-3-car-
3
1
(
(
s, 2H), 5.88 (s , 2H), 6.89 (s, 1H), 7.18 (t, 1H, J=7.9 Hz), 7.44–7.36
m, 2H), 7.92 ppm (d, 1H, J=8.3 Hz); C NMR (75 MHz, CDCl ): d=
boxylate (13m). White solid (55 mg, 71%): mp: 66–678C; H NMR
b
13
(300 MHz, CDCl ): d=1.20 (t, 3H, J=7.5 Hz), 2.60 (q, 2H, J=7.5 Hz),
3
3
2
1
9.7, 32.2, 51.0, 52.2, 106.0, 120.9, 124.5, 126.4, 128.1, 131.2, 132.4,
3.75 (s, 3H), 4.03 (s, 2H), 5.89 (s , 2H), 6.67 (s, 1H), 7.12 (d, 2H, J=
b
+
13
40.6, 162.5, 165.6, 166.9 ppm; MS (EI): m/z: 337 [M] ; HRMS (EI):
7.7 Hz), 7.25 ppm (d, 2H, J=8.0 Hz); C NMR (75 MHz, CDCl ): d=
3
+
m/z [M] calcd for C H NO S : 337.0442, found: 337.0459.
15.5, 28.6, 35.0, 51.0, 105.9, 122.7, 124.3, 128.6, 131.3, 132.0, 143.5,
1
5
15
3
2
+
+
1
62.7, 165.7 ppm; MS (EI): m/z: 307 [M] ; HRMS (EI): m/z [M]
Methyl-2-amino-5-((2-aminophenylthio)methyl)thiophene-3-car-
boxylate (13 f). White solid (64 mg, 87%): mp: 65–668C; H NMR
calcd for C H NO S : 307.0701, found: 307.0724.
1
15 17
2 2
(
300 MHz, CDCl ): d=3.74 (s, 3H), 3.89 (s, 2H), 4.33 (s , 2H), 5.89
Methyl-2-amino-5-((4-isopropylphenylthio)methyl)thiophene-3-
3
b
(
s , 2H), 6.59 (s, 1H), 6.72–6.62 (m, 2H), 7.13 (t, 1H, J=7.9 Hz),
carboxylate (13n). White solid (53 mg, 66%): mp: 84–858C;
b
13
1
7
5
1
.27 ppm (d, 1H, J=7.6 Hz); C NMR (75 MHz, CDCl ): d=34.6,
H NMR (300 MHz, CDCl ): d=1.22 (d, 6H, J=6.4 Hz), 2.88–2.82 (m,
3
3
1.0, 105.8, 115.0, 117.0, 118.6, 122.4, 124.5, 130.4, 136.5, 148.6,
1H), 3.75 (s, 3H), 4.04 (s, 2H), 5.89 (s , 2H), 6.67 (s, 1H), 7.13(d, 6H,
b
+
+
13
62.8, 165.7 ppm; MS (EI): m/z: 294 [M] ; HRMS (EI): m/z [M]
J=6.7 Hz), 7.27 ppm (d, 6H, J=7.5 Hz); C NMR (75 MHz, CDCl3):
calcd for C H N O S : 294.0497, found: 294.0479.
d=24.0, 33.9, 35.0, 51.1, 105.8, 122.7, 124.3, 127.2, 131.2, 132.2,
1
3
14
2
2
2
+
1
48.1, 162.7, 165.7 ppm; MS (EI): m/z: 321 [M] ; HRMS (EI): m/z
Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-car-
boxylate (13g). White solid (58 mg, 83%): mp: 63–648C; H NMR
+
[M] calcd for C H NO S : 321.0857, found: 321.0853.
1
16 19 2 2
(
300 MHz, CDCl ): d=3.76 (s, 3H), 4.07 (s, 2H), 5.89 (s , 2H), 6.71 (s,
Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-
3
b
13
1
5
1
H), 7.35–7.25 ppm (m, 5H); C NMR (75 MHz, CDCl ): d=34.4,
3-carboxylate(13o). White solid (65 mg, 89%): mp: 68–698C;
3
1
1.1, 105.8, 122.4, 124.3, 126.9, 129.0, 130.5, 135.5, 162.7,
H NMR (300 MHz, CDCl ): d=2.30 (s, 3H), 3.76 (s, 3H), 4.07 (s, 2H),
3
+
13
65.7 ppm; MS (EI): m/z: 279 [M] ; C NMR (75 MHz, CDCl ) HRMS
5.89 (s , 2H), 6.72 (s, 1H), 7.02 (t, 1H, J=6.8 Hz), 7.16–7.12 ppm (m,
3
b
+
13
(
EI): m/z [M] calcd for C H N O S : 279.0388, found: 279.0393.
3H); C NMR (75 MHz, CDCl ): d=21.4, 34.3, 51.1, 105.9, 122.4,
13
13
2
2
2
3
1
24.4, 127.3, 127.8, 128.9, 131.1, 135.3, 138.8, 162.7, 165.7 ppm; MS
Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-
-carboxylate (13h). White solid (56 mg, 76%): mp: 75–768C;
+
+
(EI): m/z: 293 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
14 15 2 2
3
2
93.0544, found: 293.0543.
1
H NMR (300 MHz, CDCl ): d=2.31 (s, 3H), 3.76 (s, 3H), 4.03 (s, 2H),
3
5
.88 (s , 2H), 6.68 (s, 1H), 7.08 (d, 2H, J=7.9 Hz), 7.24 ppm (d, 2H,
3-((5-Amino-4-(methoxycarbonyl)thiophen-2-yl)methylthio)ben-
zoic acid (13p). White solid (6.5 mg, 8%): mp: 128–1298C; H NMR
b
1
3
1
J=7.9 Hz); C NMR (75 MHz, CDCl ): d=21.2, 35.1, 51.1, 105.8,
3
1
22.7, 124.3, 130.0, 131.3, 131.7, 137.2, 162.7, 165.7 ppm; MS (EI):
(300 MHz, CDCl ): d=3.68 (s, 3H), 412 (s, 3H), 6.07 (s , 2H), 6.71 (s,
3
b
+
+
m/z: 293 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
2
1H); 7.40 (t, 1H, J=8.3 Hz), 7.82–7.77 (m, 2H), 7.92 ppm (d, 1H, J=
14
15
2 2
13
93.0544, found: 293.0551.
8.2 Hz); C NMR (75 MHz, CDCl ): d=30.1, 32.5, 51.2, 106.0, 121.1,
3
1
24.8, 126.7, 128.5, 131.6, 132.7, 140.9, 162.7, 165.9, 167.3 ppm; MS
Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thio-
+
+
(EI): m/z: 323 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
14
13
4
2
phene-3-carboxylate (13i). White solid (66 mg, 85%): mp: 67–
3
23.0286, found: 323.0259.
1
6
88C; H NMR (300 MHz, CDCl ): d=3.65 (s, 3H), 3.79 (s, 3H), 3.96
3
(
s, 2H), 5.85 (s , 2H), 6.62 (s, 1H), 6.82 (d, 2H, J=8.6 Hz), 7.32 ppm
b
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
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9
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
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Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-
Acknowledgements
carboxylate (13q). White solid (31 mg, 42%): mp: 60–618C;
1
H NMR (300 MHz, CDCl ): d=3.57 (s, 2H), 3.66 (s, 2H), 3.80 (s, 3H),
.93 (s , 2H), 6.74 (s, 1H), 7.32–7.26 ppm (m, 5H); C NMR
b
3
We are grateful to Mrs. C. Callebaut for dedicated editorial assis-
tance and to Mrs. L. van Berckelaer, K. Minner and R. Van Berwa-
er for excellent technical assistance. The research was supported
by KU Leuven (GOA 10/14). The cytostatic/toxic evaluation of the
prototype compound in the NCI-60 screen by the US National In-
stitutes of Health (National Cancer Institute) is highly appreciat-
ed.
13
5
(
1
75 MHz, CDCl ): d=30.4, 35.4, 51.1, 105.7, 123.4, 124.4, 127.2,
3
+
28.6, 129.2, 137.9, 162.8, 165.7 ppm; MS (EI): m/z: 293 [M] ; HRMS
+
(
EI): m/z [M] calcd for C H NO S : 293.0544, found: 293.0541.
14 15 2 2
Methyl-2-amino-5-((4-methoxyphenylthio)methyl)thiophene-3-
carboxylate (13r). White solid (27 mg, 33%): mp: 65–668C;
1
H NMR (300 MHz, CDCl ): d=3.58 (s, 2H), 3.61 (s, 2H), 3.79 (s, 3H),
3
5
.94 (s , 2H), 6.73 (s, 1H), 6.84 (d, 2H, J=8.4 Hz), 7.20 ppm (d, 2H,
b
1
3
J=8.5 Hz); C NMR (75 MHz, CDCl ): d=30.3, 34.8, 51.1, 55.4,
3
Keywords: 2-amino-3-carboxymethylthiophene analogues
antitumor agents · lymphoma/leukemia · structure–activity
relationships · tumor selectivity
·
1
05.6, 114.0, 123.5, 124.3, 129.9, 130.2, 158.8, 162.8, 165.7 ppm; MS
+
+
(
EI): m/z: 323 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
15 17 3 2
3
23.0650, found: 323.0644.
Methyl-2-amino-5-(phenethylthiomethyl)thiophene-3-carboxyl-
1
ate (13s). Semi-solid in (24 mg, 31%): H NMR (300 MHz, CDCl3):
d=2.71 (t, 2H, J=6.4 Hz), 2.83 (t, 2H, J=6.4 Hz), 3.67 (s, 2H), 3.79
1
3
(
(
1
s, 2H), 5.93 (s , 2H), 6.75 (s, 1H), 7.21–7.09 ppm (m, 5H); C NMR
b
75 MHz, CDCl ): d=31.2, 32.4, 35.9, 51.2, 105.5, 123.4, 124.2, 126.5,
[1] Y. Huang, A. Dçmling, Mol. Diversity 2011, 15, 3–33.
[2] G. Kazuhiro, H. Masao, I. Hiroshi, Biochem. Pharmacol. 1977, 26, 11–18.
[3] X. Li, D. Conklin, H. L. Pan, J. C. Eisenach, J. Pharmacol. Exp. Ther. 2003,
3
+
28.5, 128.7, 140.5, 162.8, 165.7 ppm; MS (EI): m/z: 307 [M] ; HRMS
+
(
EI): m/z [M] calcd for C H NO S : 307.0701, found: 307.0720.
15 17 2 2
3
05, 950–955.
Methyl-2-amino-5-((furan-2-ylmethylthio)methyl)thiophene-3-
[4] a) R. F. Bruns, J. H. Fergus, Mol. Pharmacol. 1990, 38, 939–949; b) B.
Musser, R. V. Mudumbi, J. Liu, R. D. Olson, R. E. Vestal, J. Pharmacol. Exp.
Ther. 1999, 288, 446–454.
carboxylate (13t). White solid (54 mg, 77%): mp: 62–638C;
1
H NMR (300 MHz, CDCl ): d=3.65 (s, 2H), 3.67 (s, 2H), 3.79 (s, 3H),
3
[
[
[
5] P. L. Podolin, J. F. Callahan, B. J. Bolognese, Y. H. Li, K. Carlson, T. G.
Davis, G. W. Mellor, C. Evans, A. K. Roshak, J. Pharmacol. Exp. Ther. 2005,
5
1
3
1
.98 (s , 2H), 6.16 (d, 1H, J=3.2 Hz), 6.31 (t, 1H, J=2.8 Hz), 6.79 (s,
b
13
H), 7.31 ppm (d, 1H, J=1 Hz); C NMR (75 MHz, CDCl ): d=27.2,
3
3
12, 373–381.
0.6, 51.1, 105.5, 107.9, 110.5, 122.7, 124.6, 142.3, 151.3, 162.9,
6] A. Walburger, A. Koul, G. Ferrari, L. Nguyen, C. Prescianotto-Baschong, K.
Huygen, B. Klebl, C. Thompson, G. Bacher, J. Pieters, Science 2004, 304,
1800–1804.
+
+
65.7 ppm; MS (EI): m/z: 283 [M] ; HRMS (EI): m/z [M] calcd for
C H NO S : 283.0337, found: 283.0331.
1
2
13
3 2
7] a) J. M. Quintela, C. Peinador, M. J. Moreira, A. Alfonso, L. M. Botana, R.
Riguera, Eur. J. Med. Chem. 2001, 36, 321–332; b) H. R. Heyman, P. F.
Bousquet, G. A. Cunha, M. D. Moskey, A. A. Ahmed, N. B. Soni, P. A. Mar-
cotte, L. J. Pease, K. B. Glaser, M. Yates, J. J. Bouska, D. H. Albert, C. L.
Black-Schaefer, P. J. Dandliker, K. D. Stewart, P. Rafferty, S. K. Davidsen,
M. R. Michaelides, M. L. Curtin, Bioorg. Med. Chem. Lett. 2007, 17, 1246–
1249; c) B. V. Ashalatha, B. Narayana, K. K. V. Raj, N. S. Kumari, Eur. J.
Med. Chem. 2007, 42, 719–728; d) J. Katada, K. Lijima, M. Muramatsu,
M. Takami, E. Yasuda, M. Hayashi, M. Hattori, Y. Hayashi, Bioorg. Med.
Chem. Lett. 1999, 9, 797–802.
Methyl-2-amino-5-((thiophen-2-ylmethylthio)methyl)thiophene-
-carboxylate (13u). White solid (35 mg, 47%): mp: 66–678C;
3
1
H NMR (300 MHz, CDCl ): d=3.65 (s, 2H), 3.80 (s, 3H), 3.86 (s, 3H),
.96 (s, 2H), 6.76 (s, 1H), 6.91 (s , 2H), 7.21 ppm (s , 1H); C NMR
b b
3
13
5
(
1
75 MHz, CDCl ): d=29.5, 30.4, 51.1, 105.6, 122.7, 124.7, 125.1,
3
+
26.5, 126.8, 141.4, 162.9, 165.7 ppm; MS (EI): m/z: 299 [M] ; HRMS
+
(
EI): m/z [M] calcd for C H NO S : 299.0108, found: 299.0121.
12 13 2 3
Methyl-2-amino-5-((naphthalen-2-ylthio)methyl)thiophene-3-car-
1
boxylate (13v). White solid (65 mg, 79%): mp: 95–968C; H NMR
[8] H. Y. Meltzer, H. C. Fibiger, Neuropsychopharmacology 1996, 14, 83–85.
[9] a) P. Nussbaumer, A. P. Winiski, S. Cammisuli, P. Hiestand, G. Weckbecker,
A. Stꢂtz, J. Med. Chem. 1994, 37, 4079–4084; b) S. Cammisuli, A. Winiski,
P. Nussbaumer, P. Hiestand, P. Stꢂtz, G. Weckbecker, Int. J. Cancer 1996,
(
1
300 MHz, CDCl ): d=3.71 (s, 3H), 4.16 (s, 2H), 5.88 (s , 2H), 6.73 (s,
H), 7.45–7.39 (m, 3H), 7.77–7.70 ppm (m, 4H); C NMR (75 MHz,
3
b
13
CDCl ): d=34.2, 51.0, 105.8, 122.1, 124.5, 126.0, 126.6, 127.4, 127.8,
3
6
5, 351–359.
1
28.0, 128.6, 128.7, 132.2, 133.0, 133.7, 162.7, 165.6 ppm; MS (EI):
+
+
[10] J. Balzarini, J. Thomas, S. Liekens, S. Noppen, W. Dehaen, R. Romagnoli,
m/z: 329 [M] ; HRMS (EI): m/z [M] calcd for C H NO S :
17
15
2 2
Invest. New Drugs 2014, 32, 200–210.
3
29.0544, found: 329.0549.
[
11] R. Romagnoli, G. P. Baraldi, C. Lopez-Cara, K. M. Salvador, D. Preti, A. M.
Tabrizi, J. Balzarini, P.Nussbaumer, M. Bassetto, A. Brancale, H.-X. Fu, Y.
Gao, J. Li, Z.-S. Zhang, E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Bioorg.
Med. Chem. 2014, DOI: 10.1016/j.bmc.2013.12.030.
12] Novel anti-cancer compounds, J. Balzarini, W. Dehaen, J. Thomas, S. Liek-
ens, R. Romagnoli, P. G. Baraldi (Katholieke Universiteit Leuven, Belgium,
Universita Degli Studi Di Ferrara, Italy), PCT Int. Appl. WO
2013190137A2, December, 2013.
Methyl-2-amino-5-((pyridin-4-ylthio)methyl)thiophene-3-carbox-
1
ylate (13w). White solid (41 mg, 59%): mp: 123–1248C; H NMR
(
1
400 MHz, CDCl ) d 3.78 (s, 3H), 4.19 (s, 2H), 6.07 (s, 2H), 6.89 (s,
H), 7.14 (d, 2H, J=6.0 Hz), 8.51 ppm (d, 1H, J=6.1 Hz); C NMR
3 H
[
13
(
1
100 MHz, CDCl ): d=31.0, 51.1, 105.9, 119.9, 121.3, 125.1, 148.4,
3
+
49.2, 162.9, 165.5 ppm; MS (EI): m/z: 280 [M] ; HRMS (EI): m/z
+
[M] calcd for C H N O S : 280.0340, found: 280.0350.
[13] R. Romagnoli, P. G. Baraldi, M. G. Pavani, M. A. Tabrizi, D. Preti, F. Fruttar-
olo, L. Piccagli, M. K. Jung, E. Hamel, M. Borgatti, R. Gambari, J. Med.
Chem. 2006, 49, 3906–3915.
1
2
12
2
2 2
Methyl-2-amino-5-((biphenyl-4-ylthio)methyl)thiophene-3-car-
1
boxylate(13x). White solid (76 mg, 86%): mp 101–1028C; H NMR
[
14] R. Romagnoli, P. G. Baraldi, M. G. Pavani, O. Cruz-Lopez, E. Hamel, J. Bal-
(
7
300 MHz, CDCl ) 3.75 (s, 3H), 4.10 (s, 2H), 5.91 (s, 2H), 6.73 (s, 1H),
.44–7.33 (m, 5H), 7.57–7.49 ppm (m, 4H); C NMR (75 MHz,
zarini, E. Brognara, C. Zuccato, R. Gambari, Med. Chem. 2010, 6, 329–
343.
3
13
CDCl ): d=34.3, 51.2, 105.7, 122.8, 124.3, 126.9, 127.4, 127.6, 128.8,
3
+
1
(
30.6, 137.9, 141.4, 162.6, 165.7 ppm; MS (EI): m/z: 355 [M] ; HRMS
EI): m/z [M] calcd for C H N O S : 355.0701, found: 355.0694.
Received: July 4, 2014
Published online on && &&, 0000
+
19
17
2
2 2
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
&10
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
Cytostatic potential & tumor selectivi-
ty (TS) of a prototype 2-aminothio-
phene is retained by replacing the ethyl
linker between the thiophene and the
substituted aryl by a thioalkyl moiety. A
SAR of this class of compounds revealed
that the 4-alkylphenyl derivatives are
more potent and selective anti-T-lym-
phoma/leukemia agents than the proto-
type.
J. Thomas, A. Jejcic, P. Vervaeke,
R. Romagnoli, S. Liekens, J. Balzarini,*
W. Dehaen
&& – &&
Structure–Activity Relationship of
Tumor-Selective
5-Substituted 2-Amino-3-
carboxymethylthiophene Derivatives
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
&11
&
ÞÞ
These are not the final page numbers!