Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes

Article abstract of DOI:10.1016/j.ejmech.2017.03.044

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4?h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives.

Full text of DOI:10.1016/j.ejmech.2017.03.044

Accepted Manuscript
Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-
methylcarboxylate thiophenes
Joice Thomas, Alenka Jecic, Els Vanstreels, Lizette van Berckelaer, Romeo
Romagnoli, Wim Dehaen, Sandra Liekens, Jan Balzarini
PII:
S0223-5234(17)30201-5
10.1016/j.ejmech.2017.03.044
EJMECH 9304
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 January 2017
Revised Date: 21 February 2017
Accepted Date: 22 March 2017
Please cite this article as: J. Thomas, A. Jecic, E. Vanstreels, L. van Berckelaer, R. Romagnoli, W.
Dehaen, S. Liekens, J. Balzarini, Pronounced anti-proliferative activity and tumor cell selectivity of 5-
alkyl-2-amino-3-methylcarboxylate thiophenes, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.03.044.
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ACCEPTED MANUSCRIPT
Table of Contents graphic

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Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-
methylcarboxylate thiophenes
a,
Ö
,1
b,
Ö
,2
b
b
Joice Thomas , Alenka Jecic , Els Vanstreels , Lizette van Berckelaer , Romeo
c
a,
b,
b,
Romagnoli , Wim Dehaen *, Sandra Liekens * & Jan Balzarini *
a
KU Leuven, Department of Chemistry, Celestijnenlaan 200f, B-3001 Leuven, Belgium
b
KU Leuven, Rega Institute for Medical Research, Herestraat 49, postbus 1043, B-3000
Leuven, Belgium
c
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, 44121 Ferrara,
Italy
_
_____________________
Contributed equally.
Ö
*
Corresponding authors.
E-mail addresses: wim.dehaen@kuleuven.be (W. Dehaen), Sandra.liekens@kuleuven.be (S.
Liekens), jan.balzarini@kuleuven.be (J. Balzarini)
1
Present address: Department of Chemistry, The Bridge@USC and Loker Hydrocarbon
Research Institute, University of Southern California, USA.
2
Present address: Department of Neurobiology, Care Sciences and Society, Center for
Alzheimer Research, Division for Neurogeriatrics, Karolinska Institutet, Huddinge, Sweden
Abbreviations used: : IC , 50% inhibitory concentration; THF, tetrahydrofuran; MeOH,
5
0
methanol; EtOH, ethanol; DMF, dimethylformamide; EtOAc, ethoxyacetate; CNS, central
nervous system; NBD, 7-nitrobenzofurazan or nitrobenzoxadiazole; RFP, red fluorescent
protein; ER, endoplasmic reticulum; DMEM, Dulbecco's minimal essential medium; NIH,
National Institutes of Health; NCI, National Cancer Institute.

2
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ABSTRACT
-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the
prototype drug of a recently discovered novel class of tumor-selective compounds that
preferentially inhibit the proliferation of specific tumor cell types (e.g.
5
leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the
molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-
3
-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at
least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the
mid-nanomolar range with 500- to 1,000-fold tumor cell selectivity. The compounds
preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3,
kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other
tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel
prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic
activity, already after 4h of exposure. The unusual tumor selectivity could not be explained
by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells.
Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the
cytoplasm, no visible appearance in the nucleus, and a predominant localization in the
endoplasmic reticulum. These observations may be helpful to narrow down the intracellular
localization and identification of the molecular target of the 5-substituted thiophene
derivatives.
Keywords: anti-proliferative activity; tumor selectivity; Gewald Reaction, 5-alkyl-2-
aminothiophenes; intracellular uptake

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1
. Introduction
Most clinically effective antitumor agents are inhibitors of DNA, RNA or protein
synthesis, or interfere with the metabolism of other cell components, frequently lacking or
displaying poor selective antitumor activity [1-7]. These traditional antitumor agents often
poorly discriminate between tumor and normal non-tumor cells, especially those with high
proliferative potential. A number of novel technologies, including antibody-drug conjugates
[
1], transporter-related tumor targeting of small anticancer drugs [2], prodrugs of anticancer
agents [3], targeting of agents to defined (cancer) tissues [4], or specific delivery systems for
anticancer drugs [5-7], have recently become subject of extensive investigations to improve
anticancer selectivity.
We recently identified 5-substituted methyl 2-amino-3-carboxylate thiophenes with
anti-proliferative activity against specific tumor cell lines [8,9]. The prototype compound
(TR560) contains a methyl 2-amino-3-carboxylate thiophene core linked at C-5 of the
thiophene to a 4-methoxyphenyl through a short alkyl (i.e. ethyl) linker [8] (Fig. 1). This
tumor-specific compound was originally synthesised with the aim to design and develop a
novel class of antitumor agents that target tubulin [10]. The most active derivatives indeed
markedly inhibited tubulin assembly and binding of radiolabeled colchicine to tubulin [10].
Most of the synthesised compounds showed pronounced anti-proliferative activity against
an extensive panel of tumor cell lines, and did not discriminate between T- and B-cell
lymphoma/leukemia, nor between several types of solid tumors including colon, mammary,
glioma, prostate and osteosarcoma [10]. Instead, TR560 inhibited the proliferation of
several human T-cell (but not B-cell lymphoma or monocyte-derived tumor cell lines), as well
as several solid tumor cell lines derived from prostate (i.e. PC-3), kidney (i.e. Caki-1) and
hepatoma (i.e. Huh-7) (but not colon, mammary, cervix or glioma). Moreover, the tumor-

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selective compound(s) showed poor, if any, anti-tubulin activity, and thus, may have
acquired a more specific molecular mechanism of action. TR560 inhibited the proliferation
of the sensitive tumor cell lines in the high nanomolar range while being poorly active
against several other tumor cell lines (IC values in the mid-micromolar range). As a result,
5
0
6
0-fold tumor selectivity for T-cell lymphoma tumor cells compared to HeLa cells was
obtained [8]. A slight increase in tumor selectivity could be achieved by further exploring and
optimizing the substituents on the 5-phenylethyl group of the methyl 2-amino-3-carboxylate
thiophene molecule [9]. In the current study, we investigated whether tumor selectivity
could be further increased by replacing the 5-phenyl moiety by a methyl 2-amino-3-
carboxylate thiophene thereby creating a symmetrical molecule, or by simply deleting the
aromatic phenyl moiety and introducing structural variability in the aliphatic alkyl linker
chain. We demonstrate that simplification of the prototype drug structure of TR560 to 5-
alkyl-substituted methyl 2-amino-3-carboxylate thiophenes resulted in a substantial increase
of anti-proliferative potency and markedly enhanced tumor selectivity (500- and 1,400-fold
for T-cell lymphoma versus B-cell lymphoma Raji and cervical carcinoma HeLa cells,
respectively).
2
. Results
2
.1. Chemistry
The Gewald three-component reaction, which results in the formation of a
compound containing a multisubstituted 2-aminothiophene moiety with an electron-
withdrawing entity in the 3-position has been widely applied for the synthesis and discovery
of large libraries of potentially bioactive molecules [11,12]. This reaction generally involves
an intermolecular Knoevenagel condensation reaction with an enolizable aldehyde and an
activated nitrile followed by a base-promoted reaction with elemental sulfur followed by

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ring closure. The synthetic methodology applied for the preparation of the target
compounds 3a-3ad is shown in Table 1 based upon this reaction. Thus, condensation of
various saturated and unsaturated aliphatic aldehydes with the methyl cyanoacetate in the
presence of elemental sulfur and triethylamine yielded alkyl-2-aminothiophene-3-
carboxylate derivatives 3a-3o, 3q and 3s-3ad in good to excellent yields. Similarly,
aminonitrile derivative 3p was prepared by replacing methyl cyanoacetate with a malonitrile
building block whereas 3-methyl aminothiophene derivative 3r was obtained by using the
corresponding ketone, decan-2-one, instead of the aldehyde derivative. However, the yields
of the last two reactions were very low as compared to the other reactions.
A
multi-step procedure was followed for the synthesis of 2-amino-5-(7-
aminoheptyl)thiophene derivative 6. This reaction at first involved the synthesis of
phthalimido derivative 5 in good yield via Gewald reaction followed by the removal of the
phthaloyl group by using hydrazine hydrate in refluxing ethanol over a period of 12 h
(Scheme 1).
Scheme 1. Synthesis of 2-amino-5-(7-aminoheptyl)thiophene derivative 6.
Next, we synthesized a series of compounds by replacing the ethylene spacer between the
alkyl chain and the 2-aminothiophene of the prototype compound 3 by a thiomethylene
bridge. This reaction involved the classical nucleophilic substitution reaction with various
alkyl thiols and phthalimido derivative 8 [9] in the presence of K CO as base in THF over a
2
3
period of 24 h which furnished phthaloyl-protected intermediates 9a-g in good yields. The
desired compounds 10a-g were obtained by the removal of the phthaloyl group by use of
hydrazine hydrate in refluxing ethanol over a period of 12 h (Table 2).

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An extension of the Gewald-multicomponent reaction where a double Gewald reaction was
performed on a bifunctional building block such as enolizable bis-aliphatic aldehydes
resulted in the synthesis of bis-2-aminothiophene derivatives 12a-12d in reasonable yields
(Table 3). As expected, a slightly longer reaction time is required for this reaction.
An analogue of compound 10 where the sulfur atom was directly connected at the 5-
position of 2-aminothiophene moiety 15 was synthesized by utilizing an efficient
Pd(OAc) /1,1'-bis(diisopropylphosphino)ferrocene-catalyzed cross-coupling reaction. In
2
order to investigate the significance of extending the ethylene spacer to an unsaturated
group, the Sonogashira cross-coupling reaction has been employed on to the 5-bromo
derivative of protected 2-aminothiophene 13 [10]. These reactions delivered the expected
compounds 14a and 14b after the removal of the protecting group.
COOMe
NH
1) SH-(CH
Pd (dba)
DPPF (2 mol%), iPr
2
)
7
CH
(1 mol%)
3
1)
R
COOMe
O
2
3
COOMe
NH
PdCl (PPh ) (10mol%),
CuI (10 mol%), NEt
2
3 2
2
NEt
S
2
3
R
S
N
O
R
Br
S
S
2
) NH
2
NH
2
2
2
) NH NH
2
2
2 4
14a, (29%), R = -(CH ) CH3
14b, (47%), R = -C H -(CH ) -CH
6 5 2 5 3
1
5, (59%), R = -(CH ) CH
3
2
7
1
3
Scheme 2. Miscellaneous derivatives of 2-amino-3-carboxymethylthiophene.
Finally, fluorescent derivatives of 6 such as its dansyl derivative 17 and 7-nitrobenzofurazan
derivative 19 were also synthesized by using the classical base-mediated reactions as shown
in Scheme 3.

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Scheme 3. Synthesis of fluorescent derivatives of 6.
2
2
.2. Biology
.2.1. Structure-activity relationship of the methyl 2-amino-3-carboxylate thiophene
derivatives
The prototype compound TR560 containing a methyl 2-amino-3-carboxylate
thiophene core linked at its C-5 position to a 4-methoxy-substituted phenyl ring through an
ethylene linker showed preferential anti-proliferative activity against T-cell
leukemia/lymphoma compared to B-cell lymphoma cells [8]. The compound also inhibited
the proliferation of several solid tumor cell lines, such as prostate PC-3, hepatoma Huh-7 and
kidney Caki-1 without affecting cervix carcinoma HeLa cells [8,9]. Earlier studies revealed the
crucial importance of an intact methyl 2-amino-3-carboxylate thiophene entity in the drug
molecule to preserve anti-proliferative activity [8,9]. We now sought to further explore the
structure-activity relationship of 5-phenylalkyl-methyl 2-amino-3-carboxylate thiophenes in
which the substituted phenyl group was replaced by a methyl 2-amino-3-carboxylate
thiophene entity, or methyl 2-amino-3-carboxylate thiophenes containing a simple
(un)substituted aliphatic alkyl/alkenyl/alkynyl/thioalkyl chain at the C-5 position .

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Given the importance of the methyl 2-amino-3-carboxylate thiophene core to
preserve the anti-proliferative activity [8,9], symmetrical molecules were designed
containing two methyl 2-amino-3-carboxylate thiophenes linked by an alkyl group with
variable length (Table 4). When replacing the 4-methoxyphenyl group by a methyl 2-amino-
3
-carboxylate thiophene keeping the original ethylene linker moiety (12a), a substantial
decrease in anti-proliferative activity was observed against the tumor cell lines tested.
However, expanding the ethyl to a butyl linker (12b) markedly increased the anti-
proliferative activity, in particular in T-lymphoma CEM and Molt/4 and in prostate cancer PC-
3
cells (Table 4). Further extension of the linker to a pentyl (12c) or an octyl (12d) chain fully
restored the initial tumor selectivity of the prototype compound for most tumor cell lines
tested, while maintaining the anti-proliferative activity observed for TR560 (Table 4).
To reveal the importance of the second methyl 2-amino-3-carboxylate thiophene in
the increased anti-proliferative activity of the symmetrical compounds, a large series of
methyl 2-amino-3-carboxylate thiophene derivatives containing unsubstituted alkyl chains at
the C-5 position of the thiophene ring were synthesized. Whereas the shortest methyl (3a)
and ethyl (3b) substituents at C-5 did not result in relevant anti-proliferative activity, the
propyl and butyl derivatives 3c and 3d were inhibitory in the low micromolar range (Table 5).
Higher alkyl derivatives (i.e. pentyl to nonyl) (viz. 3f, 3h, 3j, 3n and 3s) were invariably
inhibitory to all sensitive tumor cell lines at nanomolar concentrations. Longer alkyl chains
(i.e. decyl to hexadecyl) (viz. 3v, 3x, 3y, 3z, 3aa, 3ab, 3ac and 3ad) showed a progressively
lower anti-proliferative potency. Such a clear structure-anti-proliferative activity relationship
was evident for both T-lymphoma (i.e. CEM) and selected solid tumors (i.e. PC-3) (Fig. 2).
None of the 5-alkyl-substituted methyl 2-amino-3-carboxylate thiophenes were
inhibitory against B-lymphoma Raji or cervix carcinoma HeLa cell lines, indicating a tumor

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selectivity profile similar to the original prototype compound. In this respect, the tumor
selectivity of the most potent 5-alkyl-methyl 2-amino-3-carboxylate thiophene inhibitors
(containing 5 to 9 methylene units) ranged between 108 and 580 when comparing IC50
values for T-lymphocyte CEM versus B-lymphomas Raji cells, or between 132 and 248 when
comparing IC values for prostate PC-3 versus cervix carcinoma cells HeLa (Table 5).
5
0
Introduction of an unsaturated alkyl chain (i.e. 3g, 3l, 3m, 3r, 3u, 3w, 3ad) did not
improve the anti-proliferative activity. Functionalization of the alkyl chain by an end-
standing hydroxyl (3e, 3i, 3k, 3w) or amino (i.e. 6) group resulted in variable anti-
proliferative activities, i.e. decreased activity of 3e and 3i compared to the corresponding
unsubstituted alkyl derivatives, but equal or slightly superior anti-proliferative activity of 3k
and 3w, depending on the tumor cell line tested (Table 5).
Finally, given the improved potency and tumor selectivity of 5-alkyl derivatives and
our previous findings, which demonstrated that the ethylene linker between the methyl 2-
amino-3-carboxylate thiophene and the phenyl in the prototype compound could be
replaced by a methylthioether entity [9], a variety of 5-alkylthiomethyl-substituted methyl 2-
amino-3-carboxylate thiophene derivatives were synthesized (Table 6). The novel thioether-
alkyl derivatives showed a similar tumor selectivity profile as their corresponding alkyl
derivatives. However, they were endowed with comparable or inferior anti-proliferative
potency compared to the 5-alkyl derivatives, resulting in lower tumor selectivity indices.
2
.2.2. Anti-proliferative activity and selectivity of compound 3j
Based on its anti-proliferative potency and selectivity in 7 tumor cell lines (see higher),
compound 3j was chosen as the novel prototype drug for further biological studies. First, 3j
was evaluated in the NCI-60 anticancer screen of the US National Institutes of Health (NIH),

1
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containing 60 well-characterized tumor cell lines of different origins (Fig. 3). 3j inhibited the
growth of 15 cancer cell lines at nanomolar concentrations (IC50 < 1 µM). Besides
leukemia/lymphoma cell lines, nanomolar anti-proliferative activity (and even cytotoxicity at
1
6
00 µM) was also detected in several renal, liver, CNS and prostate cancer cell lines. The NCI-
0 screen also revealed a comparable tumor selectivity of 3j and the initial prototype
compound TR560 [8].Finally, the selectivity profile of 3j was not comparable to that of any
known antitumor drug present in the NIH database, pointing to a likely novel molecular
mechanism of action of 3j.
We also found a high correlation (r-value between 0.85 and 0.89) between the IC50
values of 30 analogues of this class of compounds against T-lymphoma CEM and Molt, CEM
and solid prostate PC-3, PC-3 and hepatoma Huh-7 and PC-3 and kidney Caki-1 tumor cell
lines (Suppl. Fig. 1, panels A-D). These findings again argue for a similar molecular
mechanism of action of the test compounds against the sensitive lymphoma and solid tumor
cell types included in the study.
2
.2.3. Cell growth kinetics
The IC values depicted in Tables 4 to 6 were calculated from the growth curves of
5
0
the tumor cells as depicted in Fig. 4. Compound 3j dose-dependently inhibited CEM (panel A)
and PC-3 (panel B) tumor cell proliferation, whereas neglectable inhibitory activity was
noted in HeLa cell cultures (panel C). Under these standard experimental conditions, the
compound was present at a fixed concentration for the whole incubation period. Similar
growth curves were found for the original prototype TR560, albeit at somewhat higher
concentrations compared with 3j (Suppl. Fig. 2, panels A-C).
Next, compound 3j was administered to PC3 prostate cancer cell cultures for limited
time periods. When exposed for 4, 2 or 1 h every day at 10 µM for up to five consecutive

1
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days, 3j completely suppressed PC-3 proliferation upon 4 h-drug exposure, and afforded >
8
0% and >70% suppression upon 2 h- or 1 h-drug exposure times, respectively. Also, at 2 µM
3
j (partially) suppressed tumor cell proliferation when measured at 5 days after the start of
the experiment (Fig. 4, panel D). Such drug exposure schedule may somewhat mimic daily
drug administration in vivo.
In conclusion, the tumor cell culture kinetic experiments revealed that the drug does
not have to be present continuously to afford a sustainable tumor cell suppression.
2
.2.4. Drug resistance selection
As already mentioned above, the identical tumor cell selectivity of the original
prototype TR560 derivative and the novel prototype compound 3j is strongly suggestive for a
similar mechanism of action. To further demonstrate this, CEM and PC-3 cell cultures were
exposed to escalating concentrations of TR560 or 3j over time, starting at a ∼ 2-fold IC drug
5
0
value, until 10 µM-concentrations were reached (Fig. 5A). After 40 to 46 subcultivations (3 to
days for each passage), PC-3 and CEM cell cultures that had been exposed to escalating
4
TR560 or 3j concentrations became highly resistant to their selecting drug, with high cross-
resistance to TR560, 3j and 3k (the closely related and potent 7-hydroxy derivative of 3j)
(
IC : 34 to > 50 µM) (Fig. 5B and Fig. 5C).
50
2
.2.5. Uptake and intracellular localization of tumor-sensitive thiophene derivatives
The molecular basis for the tumor selectivity of TR560 and 3j is currently still
unknown. To exclude differential uptake of the drugs by sensitive versus resistant tumor cell
lines as an underlying mechanism of tumor selectivity, fluorescent derivatives of 3j,
containing a dansyl (compound 17) or a 7-nitrobenzofurazan (NBD) moiety linked to the alkyl
end of 3j through an amine bond (compound 19) were synthesized. Both compounds
showed similar tumor selectivity properties as the parental prototype 3j. Compound 19 was

1
2
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virtually equally potent in inhibiting CEM cell proliferation as 3j (IC : 0.58 µM), 17 was
5
0
somewhat less inhibitory (IC : 1 µM). However, both fluorescent compounds were markedly
5
0
more inhibitory to CEM and PC-3 cells than to HeLa cells (IC : > 40 µM) and thus, still kept
5
0
pronounced tumor cell selectivity (Table 7). Moreover, both fluorescent compounds showed
cross-reactivity with the 3j- and 19-resistant cells. These observations indicate that uptake
and intracellular localization experiments carried out with the fluorescent drugs are reliable
and relevant for their non-fluorescent prototype congeners.
In a first set of uptake experiments drug-sensitive (wild-type) CEM and PC-3 and
naturally drug-resistant HeLa tumor cell cultures were exposed to 17 . In all cases, the
fluorescent drug was efficiently taken-up and quickly appeared in the cytoplasm of CEM
(panels A, B), PC-3 (panels C, D) and HeLa cells (panels E, F), excluding preferential uptake of
the drug by the sensitive tumor cells as an underlying mechanism to explain tumor
selectivity. No fluorescence was detected in the nucleus of the tumor cells (Fig. 6), even after
prolonged incubation times (not shown). .
In a second set of experiments, intracellular localization of 3j was investigated using
an NBD-carrying fluorescent derivative (19). This different fluorescent label was introduced
to rule out the possibility that localization of the drug was affected by the the fluorescent
dansyl moiety as such. Moreover, the NBD label can be excited with visible light (but not UV
light as required for dansyl excitation) avoiding cross-excitation of red fluorescent protein
(see below). We focused on PC-3 cells for these studies since the cytoplasmic area of
monolayer cells is much higher than that of suspension (CEM) cells. Alike the dansyl
derivative 17, the NBD fluorescent derivative 19 was abundantly taken-up into the PC-3
cytoplasm but not into the nuclear compartment.

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We then exposed PC-3 cell cultures transfected with constructs expressing red
fluorescent protein (RFP) conjugated to an organel-specific targeting signal, to 19, and
evaluated the localization of the red and the green signal (Fig. 7). Green fluorescence of 19
clearly did not colocalize with RFP targeted to the golgi (panels A-C), lysosomes (panels D-F),
early endosomes (panels G-I) or late endosomes (panels J-L). While a limited overlap with
RFP targeted to the mitochondria was observed (panels M-O), localization of 19 seemed to
overlap to a significant extent, but not entirely, with the endoplasmic reticulum (ER) (panels
P-R). Also, a rather sharp green lining around the nucleus was visible in most 19-treated
cells, pointing to its presence in, or immediately around, the nuclear membrane. Similar
localization of the compound was observed in 3j-resistant PC-3 cell cultures (Suppl. Fig. 3).
These observations implicate that tumor cells that acquired resistance to the drugs by long-
term exposure to escalating drug levels do not alter their drug uptake (influx) or efflux, as
also observed earlier for the naturally drug-resistant HeLa cells.
3
. Discussion
Structure-activity relationship (SAR) studies revealed that the tumor-selective
prototype drug 5-(2-(4-methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene
TR560 can be structurally simplified to 5-alkyl-substituted methyl 2-amino-3-carboxylate
thiophenes thereby markedly increasing both the cytostatic potency and tumor selectivity
(i.e. 3j). Such 5-alkyl-thiophene derivatives have never been reported as anticancer agents,
and the observed selective inhibition of certain well-defined tumor cell lines is quite unusual
and intriguing. Moreover, unlike most cytostatic/cytotoxic drugs, 3j did not show any toxicity
in normal cells, including normal human fibroblasts (HEL; human embryonic lung fibroblasts)
or blood cells (PBMC, human peripheral blood mononuclear cells) (IC > 50 µM, not shown).
5
0
Evaluation of 3j in the NCI-60 human tumor cell line panel revealed a unique tumor

1
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ACCEPTED MANUSCRIPT
selectivity pattern, which was similar to that of the original prototype TR560 [8], but did not
resemble any of the compounds included in the NCI database. This indicates that the
response pattern of 3j is unique and that the compound most likely interacts with a novel
drug target. Although the 5-alkyl-2-amino-3-methylcarboxylate thiophenes contain a PAINS
(Pan-Assay Interference substructure) that might affect the activity of a wide variety of
proteins, thereby generating false signals across a variety of assays, and might potentially
generate misleading results [15,16], it is unlikely that the data generated here are due to
aspecific effects of the molecules. In fact, the compounds from which these new analogues
originate target tubulin and are anti-proliferative agents that do not discriminate between
different types of tumor cells [10]. Instead, the new 5-substituted 2-amino-3-
methylcarboxylate thiophene derivatives do not target tubulin and showed a significant
selectivity among a wide variety of tumor cell lines (Fig. 3). Such selectivity amounted up to
5
00- to 1,500-fold for the best compounds of the series. Also, by varying the length of the 5-
alkyl side chain, a close SAR could be defined for both the highly sensitive T-lymphoma
CEM) and prostate (PC-3) tumor cell lines, resulting in optimal anti-proliferative activity for
(
an alkyl chain length between hexyl and nonyl, and markedly decreasing activities at shorter
and longer alkyl side chains, pointing to a high degree of selectivity and interaction with a
well-defined target. Also, although compounds with cytostatic/toxic properties were
frequently shown in the past to inhibit virus replication (due to the underlying cellular
toxicity, rather than a specific antiviral effect), the most active compounds did not show any
measurable antiviral activity when evaluated against a broad panel of DNA and RNA viruses
in at least five different cellular assay models (not shown). These observations and the above
mentioned properties of the compounds let us to believe that they do not behave as PAINS.
Altogether, 3j showed cytotoxicity in almost one third of all tested cancer cell lines with over

1
5
ACCEPTED MANUSCRIPT
6
00-fold selectivity, i.e. IC (50% inhibitory activity) of ∼ 100 nM in sensitive tumor cell lines
5
0
versus ∼ 60 µM in insensitive tumor cell lines.
A tight correlation (r-value ranging between 0.85 and 0.89) was found between the
anti-proliferative potencies of around 30 different 5-alkyl-methyl 2-amino-3-carboxylate
thiophenes in T-cell lymphoma CEM versus Molt/4, CEM versus PC-3 prostate, and PC-3
versus kidney Caki-1 and Huh-7 hepatoma tumor cells. This suggests that the molecular
target of the test compounds in leukemia/lymphoma and solid tumor cell lines is identical
(Suppl. Fig. 1).
Although the molecular mechanism of action is currently unknown and subject of
further studies, the fluorescently labeled thiophene derivatives were rapidly taken-up by
both sensitive (CEM, PC-3) and insensitive (HeLa) tumor cells. Thus, insensitivity cannot be
explained by a differential cellular drug uptake by the sensitive versus resistant tumor cells.
Interestingly, no measurable fluorescent signal could be detected in the nucleus of both
sensitive and resistant tumor cell lines, while the compound was abundantly present around
the nucleus and in the cytoplasm. It is therefore tempting to conclude that the eventual
molecular target of the novel thiophene drugs is located outside the nucleus, excluding a
wide variety of nuclear proteins and factors as potential targets. Whereas specific
association/accumulation of the drug could also be excluded for a number of organelles (i.e.
early and late endosomes, golgi, lysosomes, …), substantial colocalization within the ER could
be observed. These observations will help to exclude/confirm intracellular targets for these
thiophene derivatives.
4
. Conclusions
We identified a novel class of cytostatic/toxic agents with potent selectivity against
selected tumor cell types. However, investigations using the NCI-60 screen and our in-house

1
6
ACCEPTED MANUSCRIPT
tumor cell lines revealed that for the different organ-specific tumor cell classes,
leukemia/lymphoma tumor cells display the highest chance of sensitivity to the compounds.
The molecular mechanism of tumor cell inhibition is currently unknown, but a
fluorescently-labeled prototype drug revealed a fast and abundant intracellular uptake by
both sensitive and resistant tumor cell lines without measurable appearance in the nuclear
compartment , most likely excluding a nuclear target for these compounds.
Once the mechanism of action has been revealed and the intracellular target
identified, these thiophene derivatives may qualify for a so-called tailor-made cancer drug
application, to be used solely in patients who suffer from a cancer that expresses this
particular molecular target. Moreover, it may be expected that, due to their inherent tumor
selectivity and poor inhibitory activity against non-tumorigenic cells, these compounds will
be less associated with systemic toxic side-effects than traditional chemotherapeutics.
Indeed, in this respect, the new class of substituted 2-aminothiophenes differs from
traditional chemotherapeutic treatment modalities that are often not selective in their
antitumor activity and also affect normal cells in the tumor-bearing host.
Priority should now be given to reveal the molecular target of the compounds. This
can be performed by CRISPR/Cas technology or thermal shift assay [17,18]. This will allow
optimization of the lead compounds and will be helpful to better discriminate between
sensitive and non-sensitive tumors.
5
5
. Experimental Section
.1. Chemistry
5
.1.1. General Experimental Methods
NMR spectra were acquired on commercial instruments (Bruker Avance 300 MHz, Bruker
AMX 400 MHz or Bruker Avance II+ 600 MHz) and chemical shifts (δ) are reported in parts

1
7
ACCEPTED MANUSCRIPT
per million (ppm) referenced to tetramethylsilane (1H), or the internal (NMR) solvent signal
13C). Exact mass measurements were acquired on a Kratos MS50TC instrument (performed
in the EI mode at a resolution of 10000) or a Bruker Daltonics Apex2 FT-ICR instrument
performed in the ESI mode at a resolution of 60000). IR spectra were recorded on a Bruker-
(
(
Alpha T FTIR spectrometer with universal sampling module. Melting points (not corrected)
were determined using a Reichert Thermovar apparatus. For column chromatography 70-
2
30 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
received from commercial sources. The compounds purity have been estimated by HPLC.
The determination of purity was conducted on a Shimadzu LC-10AT vp system with Prevail
C18 column (4.6 mm × 250 mm,5 μm). Elution was performed with a gradient of water/
acetonitrile ata ratio of 50/50 for 20 min and staying at 100% acetonitrile for another 20
min. The flow rate was 1 ml/min. Peaks were detected at 254 nm. The peaks below
retention time of 2.0 min and at 6.30 min were invariably observed in each of the
chromotograms and is considered as an artifact but is not related to the synthesized
compounds.
5
5
.1.2. Synthesis
.1.2.1. General procedure for the synthesis of 2-amino-5-alkylthiophene-3-carboxylate
derivatives 3 as shown in Table 1.
A solution of aliphatic carbonyl compound 1 (2 mmol), activated nitrile (2 mol), elemental
sulfur (2 mmol) and triethylamine (2 mmol) in MeOH (15 ml) was heated at reflux
temperature for 12 h under argon atmosphere. The MeOH was evaporated under reduced
pressure and the residue was extracted with dichloromethane (3 × 50ml). The combined
organic phases were washed with a saturated solution of NaHCO , dried over magnesium
3

1
8
ACCEPTED MANUSCRIPT
sulfate and the solvent was evaporated under reduced pressure. Purification by column
chromatography (silica, eluent CH Cl ) afforded 3 as an off-white solid or a semi-solid.
2
2
[
13]
Methyl 2-amino-5-ethylthiophene-3-carboxylate (3b) . Off-white solid (41%, 154 mg); m.p.
1
4
0
1
6 - 47˚C; H NMR (300 MHz, CDCl ) δ 6.62 (s, 1H), 5.82 (s, 2H), 3.79 (s, 3H), 2.60 (dd, J = 7.5,
3
13
.9 Hz, 2H), 1.22 (t, J = 7.5 Hz, 3H); C NMR (75 MHz, CDCl ) δ 165.87, 161.47, 128.65,
3
+
20.59, 106.01, 50.99, 23.11, 15.42; Peak: 96.13%; HRMS (ESI ): m/z calcd for C H NO S
8
11
2
+
[
M+H] : 186.0583, found 186.0587.
Methyl 2-amino-5-propylthiophene-3-carboxylate (3c). Off-white solid (53%, 212 mg); m.p.
1
4
0 - 41˚C; H NMR (300 MHz, CDCl ) δ 6.61 (s, 1H), 5.71 (s, 2H), 3.79 (s, 3H), 2.55 (t, J = 7.4
3
1
3
Hz, 2H), 1.68 – 1.51 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H); C NMR (75 MHz, CDCl ) δ 165.87,
3
+
1
61.52, 126.93, 121.52, 106.09, 51.01, 31.86, 24.37, 13.64; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 200.0739, found 200.0733.
9
13
2
[
14]
Methyl 2-amino-5-butylthiophene-3-carboxylate (3d) . Off-white solid (72%, 308 mg); m.p.
1
4
7 - 48˚C; H NMR (300 MHz, CDCl ) δ 6.61 (s, 1H), 5.80 (s, 2H), 3.79 (s, 3H), 2.57 (t, J = 7.1
3
1
3
Hz, 2H), 1.65 – 1.47 (m, 2H), 1.47 – 1.27 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); C NMR (75 MHz,
CDCl ) δ 165.88, 161.49, 127.17, 121.38, 106.08, 51.00, 33.25, 29.48, 22.13, 13.92; Peak:
3
+
+
9
9.89%; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 214.0896, found 214.0899.
10 15 2
Methyl 2-amino-5-(4-hydroxybutyl)thiophene-3-carboxylate (3e).Off-white solid (56%, 256
1
mg); m.p. 50 - 51˚C; H NMR (300 MHz, CDCl ) δ 6.62 (s, 1H), 5.70 (s, 1H), 3.78 (s, 2H), 3.65
3
13
(
t, J = 5.9 Hz, 2H), 2.61 (t, J = 6.6 Hz, 1H), 1.75 – 1.50 (m, 3H); C NMR (75 MHz, CDCl ) δ
3
1
65.84, 161.64, 126.45, 121.69, 105.97, 62.65, 51.01, 32.00, 29.52, 27.27; Peak: 99.67%;
+
+
HRMS (ESI ): m/z calcd for C H NO S [M+H] : 230.0845, found 230.0841.
1
0
15
3
[14]
Methyl 2-amino-5-pentylthiophene-3-carboxylate (3f)
. Off-white solid (77%, 350 mg);
1
m.p. 49 - 50˚C; H NMR (300 MHz, CDCl ) δ 6.61 (s, 1H), 5.80 (s, 2H), 3.79 (s, 3H), 2.56 (t, J =
3

1
9
ACCEPTED MANUSCRIPT
7
.1 Hz, 2H), 1.70 – 1.48 (m, 2H), 1.43 – 1.22 (m, 4H), 0.89 (t, J = 6.7 Hz, 3H); Purity: 96.90%;
+
+
HRMS (ESI ): m/z calcd for C H NO S [M+H] : 228.1052, found 228.1053.
1
1
17
2
Methyl (Z)-2-amino-5-(pent-2-en-1-yl)thiophene-3-carboxylate (3g). Off-white solid (86%,
1
3
88 mg); m.p. 47 - 48˚C;
H NMR (300 MHz, CDCl ) δ 6.62 (s, 1H), 5.88 (s, 2H), 5.58 – 5.39
3
1
3
(
m, 2H), 3.77 (s, 3H), 3.31 (d, J = 6.0 Hz, 2H), 2.30 – 1.94 (m, 2H), 0.99 (t, J = 7.5 Hz, 3H); C
NMR (75 MHz, CDCl ) δ 165.82, 161.93, 133.56, 126.06, 125.40, 121.49, 105.97, 50.95,
3
+
+
2
2
7.43, 20.56, 14.28; Purity: 95.21%; HRMS (ESI ): m/z calcd for C H NO S [M+H] :
11 15 2
26.0896, found 226.0891.
Methyl 2-amino-5-hexylthiophene-3-carboxylate (3h). Off-white solid (85%, 410 mg); m.p. 49
1
-
50˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.79 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.2 Hz,
3
1
3
2
H), 1.70 – 1.47 (m, 2H), 1.47 – 1.16 (m, 6H), 0.88 (t, J = 5.9 Hz, 3H); C NMR (75 MHz, CDCl )
3
δ 165.88, 161.49, 127.23, 121.36, 106.08, 50.99, 31.68, 31.12, 29.82, 28.74, 22.69, 14.20;
+
+
Purity: 96.23%; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 242.1209, found 242.1201.
1
2
19
2
Methyl 2-amino-5-(6-hydroxyhexyl)thiophene-3-carboxylate (3i). Off-white solid (73%, 376
1
mg); m.p. 46 - 47˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.64 (s, 2H), 3.78 (s, 3H), 3.63
3
1
3
(
t, J = 6.4 Hz, 2H), 2.57 (t, J = 7.3 Hz, 2H), 1.54 – 1.57 (s, 4H), 1.35 – 1.39 (s, 4H); C NMR (75
MHz, CDCl ) δ 165.86, 161.56, 126.91, 121.44, 105.97, 63.00, 51.01, 32.71, 31.02, 29.69,
3
+
+
2
8.75, 25.56; Purity: 96.23%; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 258.1158,
12 19 3
found 258.1156.
Methyl 2-amino-5-heptylthiophene-3-carboxylate (3j). Off-white solid (79%, 404 mg); m.p.
4
7
2
4 - 45˚C; IR (ATR): 3425, 3309, 3167, 2925, 2850, 1655, 1500, 1445, 1257, 1191, 1129, 993,
1
76, 752; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.79 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4 Hz,
3
13
H), 1.74 – 1.39 (m, 2H), 1.44 – 1.16 (m, 8H), 0.88 (t, J = 6.6 Hz, 3H); C NMR (75 MHz, CDCl )
3
δ 165.87, 161.49, 127.22, 121.34, 106.05, 50.99, 31.87, 31.15, 29.81, 29.14, 29.02, 22.76,

2
0
ACCEPTED MANUSCRIPT
+
+
1
2
4.21; Purity: 95.67%; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 256.1365, found
1
3
21
2
56.1669.
Methyl 2-amino-5-(7-hydroxyheptyl)thiophene-3-carboxylate (3k). Off-white solid (78%, 424
mg); m.p. 80 - 81˚C; IR (ATR): 3447, 3412, 3286, 3183, 2925, 2849, 1659, 1603, 1502, 1444,
1
1
5
6
3
391, 1356, 1253, 1186, 1095, 1068, 1037, 854, 779; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H),
3
.76 (s, 2H), 3.78 (s, 3H), 3.63 (t, J = 6.4 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 1.56 (s, 4H), 1.34 (s,
1
3
H); C NMR (75 MHz, CDCl ) δ 165.84, 161.59, 126.93, 121.35, 105.87, 62.99, 50.97, 32.76,
3
+
0.97, 29.71, 29.18, 28.91, 25.70; Purity: 97.74%; HRMS (ESI ): m/z calcd for C H NO S
1
3
21
3
+
1
13
[
M+H] : 272.1314, found 272.1317. For H NMR and C NMR spectra, see also Suppl. Fig. 4.
Methyl (Z)-2-amino-5-(hept-4-en-1-yl)thiophene-3-carboxylate (3l). Off-white solid (82%,
1
4
16 mg); m.p. 46 - 47˚C;
H NMR (300 MHz, CDCl ) δ 6.61 (s, 1H), 5.85 (s, 1H), 5.88 – 5.00
3
(m, 1H), 3.78 (s, 1H), 2.57 (t, J = 7.5 Hz, 1H), 2.23 – 1.90 (m, 1H), 1.76 – 1.45 (m, 1H), 0.95 (t, J
1
3
=
1
7.5 Hz, 1H); C NMR (75 MHz, CDCl ) δ 165.82, 161.60, 132.48, 128.26, 126.64, 121.51,
3
+
05.90, 50.93, 31.04, 29.22, 26.37, 20.63, 14.41; Purity: 97.13 %; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 254.1209, found 254.1211.
1
3
19
2
Methyl 2-amino-5-(6-phenylhexyl)thiophene-3-carboxylate (3m). Off-white solid (56%, 128
1
mg); m.p. 72 - 73˚C;
H NMR (300 MHz, CDCl ) δ 7.40 – 7.08 (m, 5H), 6.60 (s, 1H), 5.78 (s,
3
1
3
2
H), 3.78 (s, 3H), 2.69 – 2.50 (m, 4H), 1.765 – 1.55 (m, 4H), 1.39 – 1.30 (m, 4H); C NMR (75
MHz, CDCl ) δ 165.85, 161.48, 142.86, 128.51, 128.35, 127.06, 125.71, 121.41, 106.06, 51.00,
3
+
3
6.02, 31.47, 31.03, 29.78, 29.09, 28.87; Purity: 99.18%; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 318.1522, found 318.1529.
1
8
23
2
Methyl 2-amino-5-octylthiophene-3-carboxylate (3n). Off-white solid (89%, 480 mg); m.p. 45
1
-
46˚C; H NMR (300 MHz, CDCl ) δ 6.56 (s, 1H), 5.74 (s, 2H), 3.75 (s, 3H), 2.51 (s, 2H), 1.51 (s,
3
1
3
2
H), 1.25 – 1.20 (m, 10H), 0.83 (s, 3H); C NMR (75 MHz, CDCl ) δ 165.88, 161.48, 127.24,
3

2
1
ACCEPTED MANUSCRIPT
1
21.36, 106.09, 50.99, 31.98, 31.15, 29.82, 29.44, 29.33, 29.07, 22.79, 14.22; Purity: 100%;
+
+
HRMS (ESI ): m/z calcd for C H NO S [M+H] : 270.1522, found 270.1523.
1
4
23
2
.
Methyl 2-amino-5-(6-methylhept-5-en-2-yl)thiophene-3-carboxylate (3o) Off-white solid
1
(
73%, 392 mg); m.p. 49 - 50˚C; H NMR (300 MHz, CDCl ) δ 6.62 (s, 1H), 5.80 (s, 2H), 5.08 (t, J
3
=
1
1
1
2
2
2
-
7.1 Hz, 1H), 3.79 (s, 3H), 2.94 – 2.81 (m, 2H), 2.82 – 2.72 (m, 1H), 1.96 (q, J = 7.4 Hz, 2H),
1
3
.68 (s, 3H), 1.60 – 1.52 (m, 4H), 1.22 (d, J = 6.9 Hz, 3H); C NMR (75 MHz, CDCl ) δ 169.55,
3
65.93, 164.89, 161.27, 136.84, 135.59, 133.22, 131.94, 127.80, 124.18, 123.84, 120.22,
09.55, 105.90, 81.21, 80.79, 80.37, 54.58, 50.97, 42.34, 38.73, 38.50, 29.52, 26.26, 25.90,
+
+
5.83, 22.63, 21.47, 17.84; Purity: 96.70%; HRMS (ESI ): m/z calcd for C H NO S [M+H] :
1
4
21
2
68.1365, found 268.1359.
-Amino-5-(non-8-en-1-yl)thiophene-3-carbonitrile (3p). Off-white solid (3%, 15 mg); m.p. 42
1
43˚C; H NMR (300 MHz, CDCl ) δ 6.36 (s, 1H), 5.88– 5.73 (m, 1H), 5.03 – 4.90 (m, 2H), 4.62
3
(s, 2H), 2.58 (t, J = 7.4 Hz, 2H), 2.08– 1.98 (m, 2H), 1.65 – 1.50 (m, 2H), 1.42 – 1.22 (m, 8H);
1
3
C NMR (75 MHz, CDCl ) δ 160.72, 139.24, 130.97, 121.19, 115.83, 114.36, 87.56, 33.88,
3
+
+
3
2
1.09, 29.78, 29.24, 29.09, 28.97, 28.92; ; HRMS (ESI ): m/z calcd for C H N S [M+H] :
14 20 2
49.1419, found 249.1422.
Methyl 2-amino-5-(non-8-en-1-yl)thiophene-3-carboxylate (3q). Off-white solid (85%, 479
1
mg); m.p. 38 - 39˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.88 – 5.74 (m, 3H), 505 – 4.90
3
(m, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4 Hz, 2H), 2.04 (q, J = 6.8 Hz, 2H), 1.66 – 1.48 (m, 2H), 1.40
1
3
–
1
1.25 (s, 8H); C NMR (75 MHz, CDCl ) δ 95.87, 91.48, 69.30, 57.19, 51.42, 44.31, 36.14, -
3
+
9.00, -36.09, -38.87, -40.19, -40.69, -40.86, -40.99; Purity: 97.69%; HRMS (ESI ): m/z calcd
+
for C H NO S [M+H] : 282.1522, found 282.1527.
1
5
23
2
Methyl 2-amino-4-methyl-5-octylthiophene-3-carboxylate (3r). Off-white solid (6%, 34 mg);
1
m.p. 40 - 41˚C H NMR (300 MHz, CDCl ) δ 5.91 (s, 1H), 3.78 (s, 3H), 2.53 (t, J = 7.5 Hz, 2H),
3

2
2
ACCEPTED MANUSCRIPT
1
3
2
.16 (s, 3H), 1.52 – 1.45 (m, 2H), 1.35 – 1.23 (m, 10H), 0.89 (s, 3H); C NMR (75 MHz, CDCl )
3
δ 166.68, 161.41, 141.80, 129.86, 120.50, 50.75, 31.99, 31.42, 29.51, 29.38, 29.18, 27.16,
+
+
2
2
.
2.78, 14.89, 14.22. HRMS (ESI ): m/z calcd for C H NO S [M+H] : 284.1678, found
15 25 2
84.1671.
1
(3s). Off-white solid (92%, 522 mg); m.p.56 - 57˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s,
3
1
1
1
H), 5.80 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4 Hz, 2H), 1.60 – 1.41 (m, 2H), 1.36 – 1.20 (m,
1
3
2H), 0.88 (t, J = 6.5 Hz, 3H); C NMR (75 MHz, CDCl ) δ 165.88, 161.48, 127.24, 121.36,
3
06.08, 50.99, 32.01, 31.15, 29.82, 29.63, 29.48, 29.43, 29.06, 22.80, 14.24; Purity: 96.95%;
+
+
HRMS (ESI ): m/z calcd for C H NO S [M+H] : 284.1678, found 284.1678.
1
5
25
2
Methyl 2-amino-5-(non-8-yn-1-yl)thiophene-3-carboxylate (3t). Off-white solid (27%, 150
1
mg); m.p. 46 - 47˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.81 (s, 2H), 3.78 (s, 3H), 2.56
3
(t, J = 7.1 Hz, 2H), 2.18 (td, J = 7.0, 2.6 Hz, 2H), 1.94 (t, J = 2.6 Hz, 1H), 1.62 – 1.45 (m, 4H),
1
3
1
8
.45 – 1.23 (m, 6H); C NMR (75 MHz, CDCl ) δ 165.84, 161.49, 127.02, 121.40, 106.02,
3
4.82, 68.26, 50.99, 31.04, 29.75, 28.90, 28.85, 28.71, 28.51, 18.47; Purity: 96.11%; HRMS
+
+
(
ESI ): m/z calcd for C H NO S [M+H] : 280.1365, found 280.1367.
15 21 2
Methyl 2-amino-5-(non-7-yn-1-yl)thiophene-3-carboxylate (3u). Off-white solid (85%, 476
1
mg); m.p. 42 - 43˚C; H NMR (300 MHz, CDCl ) δ 6.59 (s, 1H), 5.79 (s, 2H), 3.77 (s, 3H), 2.56
3
(t, J = 7.5 Hz, 3H), 2.10 (t, J = 6.9 Hz, 2H), 1.77 (t, J = 2.5 Hz, 3H), 1.68 – 1.49 (m, 2H), 1.49 –
1
3
1
7
.26 (m, 6H); C NMR (75 MHz, CDCl ) δ 165.85, 161.48, 127.04, 121.43, 106.08, 79.39,
3
+
5.60, 51.01, 31.02, 29.76, 29.04, 28.67, 28.57, 18.82, 3.61; Purity: 100%; HRMS (ESI ): m/z
+
calcd for C H NO S [M+H] : 280.1365, found 280.1368.
1
5
21
2
Methyl 2-amino-5-decylthiophene-3-carboxylate (3v). Off-white solid (93%, 550 mg); m.p. 42
1
-
43˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.78 (s, 2H), 3.79 (s, 3H), 2.56 (t, J = 7.5 Hz,
3
1
3
2
H), 1.63 – 1.50 (m, 2H), 1.26 (s, 14H), 0.88 (t, J = 6.6 Hz, 3H); C NMR (75 MHz, CDCl ) δ
3

2
3
ACCEPTED MANUSCRIPT
1
2
2
65.89, 161.46, 127.29, 121.38, 106.15, 51.02, 32.04, 31.17, 29.84, 29.73, 29.68, 29.49,
+
+
9.46, 29.08, 22.83, 14.26; Purity: 95.26%; HRMS (ESI ): m/z calcd for C H NO S [M+H] :
1
6
27
2
98.1835, found 298.1833.
Methyl 2-amino-5-(10-hydroxydecyl)thiophene-3-carboxylate (3w). Semi-solid (76%, 477
1
mg); H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.85 (s, 2H), 3.78 (s, 3H), 3.63 (t, J = 6.6 Hz, 2H),
3
1
3
2
1
2
3
.56 (t, J = 7.4 Hz, 2H), 1.63 – 1.50 (m, 4H), 1.28 (s, 12H); C NMR (75 MHz, CDCl ) δ 165.85,
3
61.55, 127.10, 121.33, 105.93, 63.11, 50.98, 32.86, 31.07, 29.77, 29.61, 29.50, 29.37, 28.95,
+
+
5.82; Purity: 99.17; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 314.1784, found
1
6
27
3
14.1789.
Methyl 2-amino-5-undecylthiophene-3-carboxylate (3x). Off-white solid (85%, 530 mg); m.p.
1
3
6 - 37˚C; H NMR (300 MHz, CDCl ) δ 6.77 (s, 1H), 5.72 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4
3
1
3
Hz, 2H), 1.66 – 1.50 (m, 2H), 1.40 – 1.18 (s, 16H), 0.88 (t, J = 6.7 Hz, 3H); C NMR (75 MHz,
CDCl ) δ 165.89, 161.48, 127.26, 121.36, 106.10, 51.01, 32.05, 31.16, 29.83, 29.7 29.76,
3
+
2
9.68, 29.48, 29.08, 22.83, 14.26; Purity: 99. 77%; HRMS (ESI ): m/z calcd for C H NO S
1
7
29
2
+
[
M+H] : 312.1991, found 312.1987.
Methyl 2-amino-5-dodecylthiophene-3-carboxylate (3y). Off-white solid (91%, 591 mg); m.p.
1
5
0 - 51˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.78 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.1
3
1
3
Hz, 2H), 1.61 – 1.53 (m, 2H), 1.40 – 1.22 (m, 18H), 0.89 (t, J = 8.8, 4.7 Hz, 3H); C NMR (75
MHz, CDCl ) δ 165.89, 161.47, 127.27, 121.37, 106.12, 51.01, 32.06, 31.17, 29.84, 29.80,
3
+
2
9.78, 29.68, 29.49, 29.08, 22.83, 14.26; Purity: 96.81%; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 326.2148, found 326.2141.
1
8
31
2
Methyl 2-amino-5-tridecylthiophene-3-carboxylate (3z) . Off-white solid (89%, 605 mg); m.p.
1
6
0 - 61˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.74 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.2
3
1
3
Hz, 2H), 1.70 – 1.45 (m, 2H), 1.38 – 1.20 (s, 20H), 0.88 (t, J = 6.6 Hz, 3H); C NMR (75 MHz,

2
4
ACCEPTED MANUSCRIPT
CDCl ) δ 165.89, 161.48, 127.27, 121.37, 106.11, 51.00, 32.06, 31.17, 29.82, 29.79, 29.68,
3
+
+
2
3
9.49, 29.08, 22.83, 14.26; Purity: 96.07%; HRMS (ESI ): m/z calcd for C H NO S [M+H] :
19 33 2
40.2304, found 340.2309.
Methyl 2-amino-5-tetradecylthiophene-3-carboxylate (3aa) . Off-solid (94%, 665 mg); m.p.
1
4
5 - 46˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.79 (s, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4
3
1
3
Hz, 2H), 1.62 – 1.50 (m, 2H), 1.35 – 1.21 (s, 22H), 0.89 (t, J = 6.6 Hz, 3H); C NMR (75 MHz,
CDCl ) δ 165.88, 161.48, 127.24, 121.35, 106.08, 51.00, 32.06, 31.16, 29.82, 29.68, 29.50,
3
+
+
2
3
9.08, 22.83, 14.26; Purity: 97.23%; HRMS (ESI ): m/z calcd for C H NO S [M+H] :
20 35 2
54.2461, found 354.2461.
Methyl (Z)-2-amino-5-(tetradec-9-en-1-yl)thiophene-3-carboxylate (3ab). Off-white solid
1
(
90%, 633 mg); m.p. 32 - 33˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.83 (s, 2H), 5.65 –
3
5
1
1
2
.10 (m, 2H), 3.78 (s, 3H), 2.56 (t, J = 7.4 Hz, 2H), 2.10 – 1.94 (m, 4H), 1.64 – 1.52 (m, 2H),
1
3
.44 – 1.20 (m, 14H), 0.90 (t, J = 4.9 Hz, 3H); C NMR (151 MHz, CDCl ) δ 165.86, 161.50,
3
29.99, 129.89, 127.14, 121.33, 106.00, 50.98, 32.07, 31.13, 29.85, 29.80, 29.53, 29.43,
+
9.37, 29.04, 27.29, 27.03, 22.46, 14.13; Purity: 96.48%; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 352.2304, found 270.1523.
2
0
33
2
Methyl 2-amino-5-pentadecylthiophene-3-carboxylate (3ac). Off-white solid (91%, 667 mg);
1
m.p. 55 - 56˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.79 (s, 2H), 3.78 (s, 3H), 2.56 (t, J =
3
1
3
7
.3 Hz, 3H), 1.60 – 1.48 (m, 2H), 1.37 – 1.19 (s, 24H), 0.88 (t, J = 6.6 Hz, 3H); C NMR (75
MHz, CDCl ) δ 165.89, 161.49, 127.25, 121.36, 106.09, 51.00, 32.06, 31.17, 29.83, 29.80,
3
+
2
9.68, 29.50, 29.09, 22.83, 14.26; Purity: 96.91 %; HRMS (ESI ): m/z calcd for C H NO S
2
1
37
2
+
[
M+H] : 368.2617, found 368.2614.
Methyl 2-amino-5-hexadecylthiophene-3-carboxylate (3ad). Off-white solid (94%, 718 mg);
1
m.p. 60 - 61˚C; H NMR (300 MHz, CDCl ) δ 6.60 (s, 1H), 5.80 (s, 2H), 3.78 (s, 3H), 2.56 (t, J =
3

2
5
ACCEPTED MANUSCRIPT
1
3
7
.3 Hz, 2H), 1.61 – 1.52 (m, 2H), 1.37 – 1.18 (s, 26H), 0.88 (t, J = 6.6 Hz, 3H); C NMR (75
MHz, CDCl ) δ 165.88, 161.50, 127.23, 121.36, 106.08, 50.99, 32.06, 31.16, 29.83, 29.80,
3
+
+
2
9.68, 29.50, 29.08, 22.83, 14.25; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 382.2774,
22 39 2
found 352.2776.
5
.1.2.2. Synthesis of methyl 2-amino-5-(7-(1,3-dioxoisoindolin-2-yl)heptyl)thiophene-3-
carboxylate (5). A solution of 4 (1g, 3.5 mmol) , methyl cyanoacetate (350mg, 3.5 mol) and
triethylamine (351mg, 3.5 mmol) in MeOH (40 ml) was heated at reflux temperature for 12 h
under argon atmosphere. The MeOH was evaporated under reduced pressure and the
residue was extracted with dichloromethane (3 × 50ml). The combined organic phases were
washed with a saturated solution of NaHCO , dried over magnesium sulfate and the solvent
3
was evaporated under reduced pressure. Purification by column chromatography (silica,
1
eluent CH Cl ) afforded 5 as a pale yellow solid. White solid (84%, 1.19 g); m.p. 60 - 61˚C; H
2
2
NMR (400 MHz, CDCl ) δ 7.94 – 7.77 (m, 2H), 7.76 – 7.64 (m, 2H), 6.59 (s, 1H), 5.81 (s, 2H),
3
3
.78 (s, 3H), 3.67 (t, J = 7.3 Hz, 2H), 2.62 – 2.50 (m, 2H), 1.67 (dd, J = 13.7, 7.5 Hz, 2H), 1.56
1
3
(
dd, J = 14.1, 7.0 Hz, 2H), 1.34 (s, 6H); C NMR (101 MHz, CDCl ) δ 168.60, 165.85, 161.52,
3
1
2
5
33.97, 132.29, 127.00, 123.28, 121.44, 106.06, 50.97, 38.13, 30.97, 29.72, 28.97, 28.80,
+
+
8.66, 26.82. HRMS (ESI ): m/z calcd for C H N O S [M+H] : 401.1529, found 401.1533.
2
1 24 2 4
.1.2.3. Synthesis of methyl 2-amino-5-(7-aminoheptyl)thiophene-3-carboxylate (6). To a
stirred suspension of the compound 5 (1g, 2.5 mmol), in 30 ml EtOH , hydrazine hydrate (1.5
equiv) was added. The reaction mixture was then refluxed for 3h. After this, the reaction
mixture was cooled to room temperature and keep aside for another 3 h. The resulting
precipitate was filtered off and the filtrate was evaporated to dryness afforded the
1
corresponding product 6 as a semi-solid. Semi- solid (63%, 1.19 g); H NMR (400 MHz, CDCl ) δ
3
7
.94 – 7.77 (m, 2H), 7.76 – 7.64 (m, 2H), 6.59 (s, 1H), 5.81 (s, 2H), 3.78 (s, 3H), 3.67 (t, J = 7.3 Hz, 2H),

2
6
ACCEPTED MANUSCRIPT
1
3
2
.62 – 2.50 (m, 2H), 1.67 (dd, J = 13.7, 7.5 Hz, 2H), 1.56 (dd, J = 14.1, 7.0 Hz, 2H), 1.34 (s, 6H); C
NMR (101 MHz, CDCl ) δ 168.60, 165.85, 161.52, 133.97, 132.29, 127.00, 123.28, 121.44, 106.06,
3
+
5
0.97, 38.13, 30.97, 29.72, 28.97, 28.80, 28.66, 26.82; purity: 96.60%; HRMS (ESI ): m/z calcd for
+
C H N O S [M+H] : 271.1474, found 271.1479.
13
22
2
2
5
.1.2.4. General procedure for the synthesis of 10. To a mixture of 8 (300 mg, 0.79 mmol)
and K CO (0.79 mmol) in THF (10 mL) was added corresponding alky thiol 7 (0.79 mmol).
2
3
After stirring the resulting mixture for another 24 h at 45 °C, the reaction mixture was added
to water (30 ml). CH Cl (30 ml) was added and the organic solution was washed with
2
2
distilled water (3 ×30 ml) and then evaporated to dryness to afford the crude product
mixture of 9. This crude reaction mixture was then again dissolved in 10 ml of EtOH and
hydrazine (1.2 mmol) was then added into it. The reaction mixture was refluxed for 3h.
CH Cl (30 ml) was added and the organic solution was washed with distilled water (3 ×30
2
2
ml), dried over MgSO , filtered, and then evaporated to dryness to afford the crude product
4
mixture. Purification by column chromatography (silica, eluent CH Cl ) afforded
2
2
corresponding product 10 as a semi-solid.
Methyl 2-amino-5-((butylthio)methyl)thiophene-3-carboxylate (10a). Semi-solid (33%, 68
1
mg); H NMR (300 MHz, CDCl ) δ 6.75 (s, 1H), 5.93 (s, 2H), 3.79 (s, 3H), 3.68 (s, 2H), 2.46 (t, J
3
1
3
=
7.3 Hz, 2H), 1.68 - 1.48 (m, 2H), 1.44 - 1.28 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H); C NMR (101
MHz, CDCl ) δ 165.70, 162.77, 123.90, 123.84, 105.55, 51.06, 31.25, 31.09, 30.93, 22.08,
3
+
+
1
2
3.79; Purity: 95.44 %; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 260.07734, found
11 17 2 2
60.07739.
Methyl 2-amino-5-((pentylthio)methyl)thiophene-3-carboxylate (10b). Semi-solid (39%, 83
1
mg); H NMR (300 MHz, CDCl ) δ 6.75 (s, 1H), 5.93 (s, 2H), 3.79 (s, 3H), 3.69 (s, 2H), 2.45 (t, J
3
1
3
=
7.4 Hz, 2H), 1.61 – 1.50 (m, 2H), 1.39 – 1.23 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); C NMR (75

2
7
ACCEPTED MANUSCRIPT
MHz, CDCl ) δ 165.73, 162.77, 123.92, 123.87, 105.58, 51.08, 31.24, 31.15, 31.11, 28.86,
3
+
+
2
2.41, 14.08; Purity: 95.365; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 274.0929,
12 19 2 2
found 274.0921.
Methyl 2-amino-5-((isopentylthio)methyl)thiophene-3-carboxylate (10c). Semi-solid (36%, 78
1
mg); H NMR (300 MHz, CDCl ) δ 6.76 (s, 1H), 5.91 (s, 2H), 3.79 (s, 3H), 3.69 (s, 2H), 2.45 (t, J
3
1
3
=
7.8 Hz, 2H), 1.71 – 1.58 (m, 1H), 1.48 – 1.39 (m, 2H), 0.88 (t, J = 6.6 Hz, 6H); C NMR (75
MHz, CDCl ) δ 165.74, 162.75, 123.96, 123.84, 105.61, 51.10, 38.17, 31.08, 29.27, 27.55,
3
+
+
2
2
2.42; Purity: 99.18%; HRMS (ESI ): m/z calcd for C H NO S [M+H] : 274.0929, found
12 19 2 2
74.0935.
Methyl 2-amino-5-((hexylthio)methyl)thiophene-3-carboxylate (10d). Off-white solid (43%,
1
9
3
7 mg); m.p. 71 - 72˚C; H NMR (300 MHz, CDCl ) δ 6.75 (s, 1H), 5.91 (s, 2H), 3.77 (s, 3H),
3
.69 (s, 2H), 2.46 (t, J = 7.3 Hz, 2H), 1.74 – 1.48 (m, 2H), 1.41 – 1.20 (m, 6H), 0.88 (t, J = 6.6
1
3
Hz, 3H); C NMR (101 MHz, CDCl ) δ 165.75, 162.75, 123.97, 105.72, 51.08, 31.56, 31.34,
3
+
3
1.16, 29.20, 28.67, 22.68, 14.15; Purity: 95.33%; HRMS (ESI ): m/z calcd for C H NO S
1
3
21
2 2
+
[
M+H] : 288.1086, found 288.1088.
Methyl 2-amino-5-((heptylthio)methyl)thiophene-3-carboxylate (10e). Off-white solid (39%,
1
9
3
3 mg); m.p. 110 - 111˚C; H NMR (300 MHz, CDCl ) δ 6.75 (s, 1H), 5.93 (s, 2H), 3.79 (s, 3H),
3
.68 (s, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.62 – 1.54 (m, 2H), 1.39 – 1.23 (m, 8H), 0.87 (t, J = 6.5
1
3
Hz, 3H); C NMR (75 MHz, CDCl ) δ 165.72, 162.78, 123.92, 123.87, 105.56, 51.07, 31.83,
3
+
3
1.24, 31.10, 29.17, 28.99, 28.93, 22.72, 14.19; Purity: 97.12%; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 302.1243, found 302.1249.
1
4
23
2 2
Methyl 2-amino-5-((octylthio)methyl)thiophene-3-carboxylate (10f). Off-white solid (47%,
1
1
3
17 mg); m.p. 116 - 117˚C;
H NMR (600 MHz, CDCl ) δ 6.75 (s, 1H), 5.92 (s, 2H), 3.79 (s,
3
H), 3.69 (s, 2H), 2.45 (t, J = 7.4 Hz, 2H), 1.59 – 1.52 (m, 2H), 1.31 – 1.21 (m, 10H), 0.88 (t, J =

2
8
ACCEPTED MANUSCRIPT
1
3
6
3
.9 Hz, 3H); C NMR (101 MHz, CDCl ) δ 165.73, 162.75, 123.95, 105.69, 51.07, 31.95, 31.34,
3
+
1.16, 29.31, 29.23, 29.00, 22.79, 14.23; Purity: 96.11%; HRMS (ESI ): m/z calcd for
+
C H NO S [M+H] : 316.1399, found 316.1391.
1
5
25
2 2
Methyl 2-amino-5-((decylthio)methyl)thiophene-3-carboxylate (10g). Off-white solid (49%,
1
1
3
33 mg); m.p. 106 - 107˚C; H NMR (300 MHz, CDCl ) δ 6.75 (s, 1H), 5.91 (s, 2H), 3.79 (s, 3H),
3
.68 (s, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.62 – 1.48 (m, 2H), 1.38 – 1.22 (m, 14H), 0.87 (t, J = 6.4
1
3
Hz, 3H); C NMR (75 MHz, CDCl ) δ 165.73, 162.77, 123.92, 123.89, 105.58, 51.08, 32.02,
3
3
1.25, 31.10, 29.68, 29.65, 29.44, 29.35, 29.18, 28.99, 22.81, 14.25; Purity: 97.39%; HRMS
+
+
(
ESI ): m/z calcd for C H NO S [M+H] : 344.1712, found 344.1718.
17 29 2 2
5
.1.2.5. General procedure for the synthesis of bis-methyl 2-amino-5-alkylthiophene-3-
carboxylate derivatives 12 as shown in Table 3. A solution of bis-aliphatic aldehyde 11 (1
mmol), activated nitrile (2 mmol), elemental sulfur (2 mmol) and triethylamine (2 mmol) in
MeOH (15 ml) was heated at reflux temperature for 16 h under argon atmosphere. The
MeOH was evaporated under reduced pressure and the residue was extracted with
dichloromethane (3 × 30 ml). The combined organic phases were washed with a saturated
solution of NaHCO , dried over magnesium sulfate and the solvent was evaporated under
3
reduced pressure. Purification by column chromatography (silica, eluent CH Cl ) afforded 12
2
2
as an off-white solid.
Dimethyl 5,5'-(ethane-1,2-diyl)bis(2-aminothiophene-3-carboxylate) (12a). Off-white solid
1
(
33%, 107 mg); m.p. 53 - 51˚C; H NMR (300 MHz, CDCl ) δ 6.58 (s, 2H), 5.75 (s, 4H), 3.71 (s,
3
1
3
6
3
3
H), 2.78 (s, 4H); C NMR (75 MHz, CDCl ) δ 165.84, 161.76, 124.78, 122.51, 106.16, 51.07,
3
+
+
1.48;Purity: 96.81%; HRMS (ESI ): m/z calcd for C H N O S [M+H] : 341.0624, found
1
4 16 2 4 2
41.0629.