Introduction

2-Aminothiophene-3-carboxylic acid esters and their 3-carbonitrile analogues are commonly used for the synthesis of 2-unsubstituted thieno[2,3-d]pyrimidines. These compounds are endowed with biological activity [1]. Several of the compounds showed antibacterial activity, in particular against Staphylococcus aureus. Also, some of the thieno[2,3-d]pyrimidines (esp. thienopyrimidin-4-one (thione) derivatives) were endowed with cytotoxic activity against hepatocellular carcinoma HepG2, cervix carcinoma HeLa, breast carcinoma MCF-7 and colorectal carcinoma MCT116 cells. Their 50 % inhibitory concentrations were in the lower micromolar/higher nanomolar range. A variety of 2-aminothiophene-3-carboxylates and carboxamides, in particular 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes with 3-carboxylate and 3-carboxamide substituents were found to behave as adenosine A1 receptor allosteric enhancers [2] and 2-amino-4,5,6,7-tetrahydro N-phenylbenzo[b]thiophene-3-carboxamides were endowed with anti-arrhythmic, serotonin antagonist and anti-anxiety activities [3]. Several 2-aminothiophene analogues were also shown to exhibit anti-inflammatory potential [4]. All above-mentioned compound classes were synthesized starting from 2-aminothiophene-3-carboxylic acids which resulted in the formation of thiophenes containing a fixed ring system. Interestingly, the fluorophenyl derivative of the thiophene 2-ureido-3-carboxylic acid amide TPCA-1 has recently been identified as a small molecule IKB kinase β (IKKβ) inhibitor [5, 6].

Recently, we have synthesized a series of 2-amino-3-aroyl-4- or 5-substituted thiophene derivatives as potential anti-proliferative agents [7, 8]. The rational was to replace the 2-aminobenzene moiety in the 2-aminobenzophenone part of the strong cytotoxic phenstatin derivative [9] by the bioisostere 2-aminothiophene system. The compounds were shown to inhibit tubulin polymerization, resulting in an accumulation of a proportion of the drug-exposed cells in the G2/M and sub-G1 phases of the cell cycle [8]. In order to design additional thiophene derivatives with more potent cytotoxic/static activity, we now further extended these studies by synthesizing 2-aminothiophene-3-carboxylic acid ester derivatives that were predominantly modified at the C-5 position of the thiophene ring upon introduction of a substituted phenyl ring either directly linked to the C-5 position of the thiophene or through an alkyl or alkynyl linker. Whereas it was found that most 2-aminothiophene-3-carboxylic acid ester derivatives were endowed with a rather moderate cytostatic activity (middle to higher micromolar range) against a broad panel of tumor cell lines, surprisingly, several compounds preferentially and potently inhibited the proliferation of tumor cells derived from T-lymphoma, prostate, kidney and liver cancer. They proved solely against these particular tumor cells selectively cytostatic in the lower micromolar to higher nanomolar range. Although their molecular mechanism of cytostatic action has not been fully clarified, the cytostatic specificity of this novel drug class for particular tumor cells is highly unusual and intriguing and justifies further in-depth studies on the molecular mechanism of their selective cytostatic action.

Materials and methods

Compounds

The synthesis and characterization of the compounds will be reported elsewhere.

Radiochemicals

The radiolabeled precursors [3H-methyl]dThd (49 Ci/mmol), [5-3H]Urd (27 Ci/mmol) and [4,5-3H]leu (140 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA).

Cytostatic assays

To each well of a 96-well microtiter plate were added 5 to 7.5 × 104 cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h to 96 h (depending on the nature of the tumor cell line) at 37 °C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter Counter (Coulter Electronics Ltd, Harpenden Herts, United Kingdom). The IC50 (50 % inhibitory concentration) was defined as the concentration of compound that reduced the number of viable cells by 50 %.

Flow cytometric analysis of the cell cycle

Prostate cancer (PC-3M) cells were seeded at 25,000 cells/cm2 in DMEM with 10 % FBS in the presence of different concentrations of compound 3 (100–10–1 μM). At different time points, the DNA of the cells was stained with propidium iodide (PI) using the CycleTEST PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA). Within 3 h after staining, the DNA content of the cells was measured by flow cytometry on a FACSCalibur flow cytometer and analyzed with the CellQuest software (BD Biosciences). Cell debris and clumps were excluded from the analysis by appropriate dot plot gating. Percentages of sub-G1, G1, S, and G2/M cells were quantified using appropriate region markers.

Fluorescence detection of caspase-3 activity in live cells

The sequence DEVD is cleaved by caspase-3 during cell death by apoptosis. NucViewTM 488-DEVD is a cell membrane-permeable fluorogenic caspase substrate designed for detecting caspase-3 activity within live cells in real time. This probe is a non-fluorescent substrate until it is cleaved by caspase-3 and allows the real-time imaging of caspase-3 activity in the nucleus of living cells. PC-3M and HeLa cells were seeded in micro-angiogenesis slides (IBIDI, München, Germany) at 50,000 cells/cm2 in DMEM with 10 % FBS. After 24 h, cells were incubated in DMEM containing 1 μg/ml of Hoechst 33342 (30 min at 37 °C) to stain the nucleic acids (blue). Next, the cells were washed and incubated in Hank’s Buffered Salt Solution (HBSS) supplemented with 10 % FBS and 10 mM HEPES (Invitrogen) containing different concentrations of compound 3 and 2 μM of the caspase-3 substrate NucViewTM 488-DEVD (Biotium, Hayward, CA). This substrate is non-fluorescent until it is cleaved by caspase-3 to release its dye, which stains the nucleus with bright green fluorescence. Real-time imaging of caspase-3 activity in the nucleus of living cells was performed every 30 min using a Carl Zeiss Axiovert 200 M inverted microscope (Zeiss, Göttingen, Germany) and a 20× objective.

Antimetabolic activity assays

Radiolabeled precursors of DNA synthesis (1 μCi [3H-methyl]dThd), RNA synthesis (1 μCi [5-3H]Urd), and protein synthesis (1 μCi [4,5-3H]leu) were added to 105 CEM cell cultures in the 200 μl-wells of a 96-well microtiter plate in the presence of varying concentrations of the test compounds (5-fold dilution series). The cells were allowed to proliferate for 20 h at 37 °C in a humidified, CO2-controled atmosphere. At the end of the incubation period, the contents of the wells were brought onto 25-mm glass fiber filters and further processed for measurement of acid-insoluble radioactivity.

Results

A series of 30 2-aminothiophene derivatives (Table 1) have been evaluated for their cytostatic activity against a broad variety of tumor cell lines in cell culture. Minor structural modifications resulted in a wide difference of antiproliferative activities, 3 being the most potent agent and 24 being the least active agent that shows a difference by up to 100- to 500-fold in cytostatic potential, compared with 3 (Tables 2, 3, 4 and 5).

Table 1 2-amino-3-carboxylic acid ester- and 2-amino-3-cyano-thiophene derivatives
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Table 2 Cytostatic activity of 2-aminothiophene derivatives against T-cell lymphoma, B-lymphoma and monocyte-derived cell lines
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Table 3 Cytostatic activity of 2-aminothiophene derivatives against a broad variety of tumor and non-tumorigenic cell lines
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Table 4 Cytostatic activity of 2-aminothiophene derivatives against kidney and liver cancer cell lines
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Table 5 Cytostatic activity of 2-aminothiophene derivatives against prostate cancer cell lines
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Cytostatic activity against immune cell-derived tumor cell lines

The compounds were first evaluated against a variety of different human T-lymphoma tumor cell lines (Table 2). Whereas the majority of the compounds showed rather narrow variations in their antiproliferative activity range for all these T-lymphoma cell lines, there were seven notable exceptions on this rule. These seven compounds (3, 1, 2, 7, 9, 14 and 13) (data indicated in grey background in Table 2) were endowed with moderate cytostatic activity against MT-4 and HUT-78 cells (IC50 range: 8.5 to 60 μM) but showed pronounced cytostatic activities against CEM, Molt4/C8, Sup T1, MT-2 and C8166 cells (IC50 range for these compounds against the T-lymphoma cell lines: 0.22 to 3.4 μM). Such a strikingly increased cytostatic activity against those five T-lymphoma tumor cell lines was not observed for any of the other 2-aminothiophene derivatives (Table 2; Fig. 1a). Also, the compounds were considerably less cytostatic against human B-lymphoma Raji and Daudi cells, as well as human monocytic U937 and THP-1 cells and promyelocytic HL-60 cells and the murine leukemia L1210 cells (Table 2).

Fig. 1
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Cytostatic activity of 2-aminothiophene derivatives against a variety of tumor and non-tumorigenic cell lines

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Cytostatic activity against solid tumor-derived cell lines and non-tumorigenic cell lines

A very modest cytostatic activity range (middle to higher micromolar concentrations) was observed against a variety of other tumor cell types including osteosarcoma HOS CD4 +, human breast carcinoma MCF-7 and murine mammary carcinoma FM3A, cervix carcinoma HeLa, colorectal carcinoma HT29, glioma U87 and U87 CD4+.CXCR4+.CCR5+ (U87 cells transduced by the CD4, CXCR4 and CCR5 genes) and non-tumorigenic kidney embryo fibroblast 293T, human embryonic lung fibroblast HEL and bovine aortic endothelial BAEC cells (Table 3; Fig. 1b).

However, when the compounds were evaluated for their inhibitory potential against the proliferation of three human hepatoma (Huh7, HepG2C3A, PLC/PRF/5), two human kidney (Caki-1, Caki-2) (Table 4) and seven different prostate cancer cell lines (Table 5), again, the same compounds that showed preferential inhibition of several T-cell lymphoma-derived tumor cell lines, also showed an increased cytostatic activity against the hepatoma Huh7 and kidney Caki-1 cells and an even more increased cytostatic potential against several of the prostate tumor cell lines [PC3 (rega), PC-3 (UZ), LNCaP (UZ)] (Table 4; Fig. 1c, d). In fact, a nanomolar cytostatic activity was observed against two different PC3 sources and against LNCaP cells, a low micromolar antiproliferative activity was shown for DU145 and 22Rv1 prostate cancer cells, and moderate cytostatic activity was shown against BPH-1 and PNT1A prostate cancer cells. In contrast with what had been observed for the lymphoma cell lines, the other non-selective group of compounds had a tendency to be somewhat more cytostatic to the PC3 and LNCaP cell lines than against the other prostate tumor cell lines.

Structure-activity relationship for the tumor-selective compounds

Based on a relatively limited number of compounds, some preliminary SAR parameters can be deduced. Common features for these unusually tumor-selective compounds are a carboxymethyl or carboxyethyl group at R1 (3-position) of the thiophene ring, no substituent or one single methoxy group on the aryl part of the molecule (a second methoxy or methyl group on the phenyl ring does not necessarily guarantee tumor selectivity) and a bridge between aryl and thiophene that consists of 2 carbons (either an ethylene or an acetylene). Following more detailed statements could be made: (i) The presence of a carbon bridge linking the thiophene and the phenyl group is necessary for the cytostatic selectivity. For example, when comparing compounds 3 and 15, 13 and 16 and 14 and 16, compounds 3, 13 and 14 containing an ethylene or acetylene bridge are selective, while their corresponding derivatives without a bridge (15 and 16) are not selective anymore against the lymphoma, hepatoma, kidney and prostate cancer cell lines. Also, a one-carbon bridge between the thiophene and the aryl moiety was not sufficient to display tumor selectivity (data not shown). (ii) It was surprising to notice that 2 containing a COOMe group at the 3 position of the thiophene is selective while the corresponding -COOEt derivative 11 is not, although the -COOEt group does not necessarily destroy the selectivity in compounds 13 and 14. However, replacing -COOalkyl by -C≡N annihilates the selectivity (compare 21 with 2, 22 with 3, and 1 with 20). (iii) The presence of a second -COOEt on the thiophene is detrimental for selectivity (compare 18 with 13). (iv) When the -COOMe at R1 (compound 3) was replaced by an amide (26), methylamide (24), or a free carboxylic acid (25) the selective cytostatic activity was markedly decreased. (v) When the NH2 at R0 on the thiophene of compound 3 was replaced by H (30), NHCH3 (28) or N(CH3)2 (29) the cytostatic potency/selectivity was also markedly compromised. Thus, a free amino group at the C-2 position of the thiophene is strictly required for tumor selectivity. (vi) The location of the methoxy group(s) also seems to play an instrumental role in tumor cell selectivity. For example, when the methoxy group is located at the R3 position of the phenyl (compound 2), tumor selectivity is present, but adding an additional MeO group at R6 (compound 5) annihilates selectivity. (vii) Finally, when the aryl was replaced by a thiazole (compound 9), selectivity was retained, but not when the aryl was replaced by a pyridine (compound 8). The fact that the free acid 25 lost its activity/selectivity may indicate that the active compound needs an intact carboxy ester moiety at the thiophene ring.

The replacement of the -COOalkyl group on the thiophene by a -C ≡ N group did not result in an improved antiproliferative activity since the cyano-containing compounds 21, 22 and 23 were inferior to their respective -COOMe-containing counterparts 1, 2 and 3. Also, the cytostatic activity of the tumor non-selective -COOalkyl containing compound 15 is superior to the tumor non-selective -C≡N containing compound 24. Although potent cytostatic activity was observed for the thiophene 3-ethyl ester compound 18 and the thiophene 3-methyl ester compound 5 containing two methoxy groups at R3 and R6 of the aryl moiety, no tumor selectivity could be observed.

Effect of tumor-selective compounds on cellular macromolecular synthesis

To reveal preliminary insights in the molecular mechanism of the cytostatic action of the compounds, the tumor-selective compounds, as well as a selection of tumor-nonselective compounds were investigated for their inhibitory activity against macromolecular DNA, RNA or protein synthesis. For this purpose, the inhibitory effect of the compounds was determined against incorporation of [3H]thymidine in DNA, [3H]uridine in RNA and [3H]leucine in proteins of T-lymphoma-derived CEM and B-lymphoma-derived Raji cells (Table 6). Virtually none of the compounds, including the tumor-selective compounds, showed pronounced inhibitory activity against DNA, RNA or protein synthesis in the B-lymphoma-derived Raji cells (Table 6). Instead, the inhibition of macromolecular (DNA, RNA and protein) synthesis in the T-lymphoma-derived CEM tumor cells was generally far more pronounced for the tumor-selective compounds (1, 2, 3, 7, 9, 13 and 14) than for the tumor-nonselective compounds (compare also ratio IC50 Raji/IC50 CEM for the tumor-selective compounds with those of the tumor-nonselective compounds 5, 12, 16, 19, 21) (Table 6). Protein synthesis (measured by [3H]leucine incorporation) seems to be somewhat more preferentially inhibited by the tumor-selective compounds than DNA or RNA synthesis (IC50 range for DNA synthesis, RNA and protein synthesis: 2.3–11 μM, 0.66–3.0 μM and 0.19–1.9 μM, respectively) (Table 6). Linear regression analysis determining the correlation between the cytostatic activity of the tumor-selective compounds 1, 2, 3, 7, 9, 13 and 14 (data from Table 2) and inhibition of DNA, RNA or protein synthesis (data from Table 6) revealed correlation coefficients of r = 0.70, r = 0.79 and r = 0.88, respectively.

Table 6 Inhibitory effect of test compounds against the incorporation of radiolabeled precursors in cellular macromolecules
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Effect of tumor-selective compound 3 on the cell cycle and apoptosis in PC-3M tumor cells

FACs analysis revealed that compound 3 exposure for 24 h to PC-3M prostate carcinoma cells (being a PC-3 cell clone that represents a highly metastatic variant of the parental PC-3) caused an accumulation of the tumor cells in the G1 phase of the cell cycle (Fig. 2). Whereas 55.7 % of the non-treated PC-3M cell cultures were in the G1 phase of their cell cycle, 1, 10 and 100 μM compound 3 exposure for 24 h dose-dependently increased the amount of cells in the G1 phase to 73.9 %. At the same time, the amount of cells in the S phase and G2/M phase dose-dependently decreased from 19.8 % (S) and 23.3 % (G2/M) to 8.2 % (S) and 15.7 % (G2/M), respectively (Fig. 2). A shorter exposure time of 6 h did not result in significant changes in cell phase distribution and an incubation period of 48 h resulted predominantly in dead tumor cells (data not shown). This is in contrast with the tubulin-targeting thiophene derivatives that are non-selective in terms of tumor cell specificity, but afford a selective accumulation of the tumor cells in their mitotic (G2/M) cell phase [8]. Also, treatment of PC-3M prostate carcinoma cells with 100 or 10 μM compound 3 resulted in the activation of caspase-3 after 6 h and 12 h, respectively (Fig. 3). No induction of apoptosis was observed in the presence of 1 μM compound 3. Under identical experimental conditions, activation of caspase-3 in HeLa cells (that are not selectively targeted by the tumor-selective compounds) was only observed after 12 h in the presence of 100 μM compound 3. Thus, compound 3 seemed to preferentially induce caspase-3-induced apoptosis in PC-3M cell cultures versus HeLa cell cultures.

Fig. 2
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PC-3M cell cycle analysis upon exposure of the tumor cells to different concentrations of compound 3

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Fig. 3
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Effect of compound 3 on apoptosis of PC-3M and HeLa cell cultures. PC-3M or HeLa cells were seeded in μ-angiogenesis slides at 50,000 cells/cm2 in DMEM with 10 % FBS. After 24 h, the cells were incubated in HBSS with 10 % FBS containing different concentrations of compound 3 and 2 μM of the caspase-3 substrate NucViewTM 488-DEVD. Real-time imaging of caspase-3 activity in the nucleus of living cells was performed every 30 min for 24 h

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Discussion

Among a series of closely-related 2-aminothiophene analogues seven derivatives have been identified that show a preferential cytostatic activity of at least 20- to 50-fold against several human T-cell lymphoma, kidney, hepatoma and prostate cancer-derived cell lines, but not against any other type of tumor cell included in this study. Also, the cytostatic selectivity was not observed for non-tumorigenic cell lines. 2-Aminothiophene-3-carboxylic acid esters have never been reported as potential cytostatic (anticancer) agents, and the observed selectivity for different tumor cell lines is rather unusual and surprising. Although several structural properties that are strictly required for tumor cell selectivity (i.e. a free amino group at C-2, an intact carboxyalkyl group at C-3 of the thiophene and a bridge containing at least two carbons between the thiophene and the aryl moiety) could be revealed, a complete structure-activity relationship (SAR) among the 2-aminothiophene-3-carboxylic ester derivatives is still not available. The linker between the thiophene and the aryl moiety, and, in particular, the aryl and its substituents should be subject of intensive modifications to optimize the tumor selectivity of these novel compounds. Whereas the free 3-carboxylic acid derivative 26 did not show cytostatic selectivity at all and proved rather poorly inhibitory to tumor cell proliferation, it cannot be excluded that the free carboxy acid derivative is the active intracellular species of the compounds, but does–as such–not efficiently penetrate into the tumor cells making esterification necessary for optimal tumor cell penetration. Further studies have to address this issue.

The molecular basis for the unusual selectivity of the compounds for the several T-cell lymphoma, kidney, hepatoma and prostate cancer cell lines is currently still not clear. It was interesting to observe that the prototype compound 3 dose-dependently afforded an accumulation of PC-3M tumor cells in the G1 phase (Fig. 2). In this respect, the tumor-selective compound(s) clearly differ in their molecular mechanism of action from the 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 G2/M phase of their cell cycle [7, 8]. Many different types of compounds have been described to induce G1 arrest and apoptosis in tumor cells. This can be afforded by a specific interaction of the drug with a variety of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events and/or that define a decision made in the G1 phase to initiate and complete a round of mitotic cell division [10]. For example, tunicamycin [11] and certain alkylphenols [12] both induce an endoplasmatic reticulum (ER) stress-mediated G1 arrest and induce apoptosis at higher concentrations. p27 seemed to be a critical mediator of ER stress-induced (G1) growth arrest [11]. Other compounds such as Gomisin A afford cell cycle arrest in the G1 phase by the down-regulation of cyclin DI expression and retinoblastoma (RB) phosphorylation [13]. Also Sasanquasaponin, a triterpenoid [14] and volatile oils [15] induce G1 phase arrest in the tumor cell cycle and trigger apoptosis by upregulation of p21 and down-modulation of cyclin DI. Yet, other drugs cause G1 arrest and apoptosis by still other molecular mechanisms than those described above. It will, therefore, be challenging to define the exact mechanism of cytostatic action of the novel class of 2-amino-3-carboxylic acid ester thiophene derivatives, and in particular their molecular basis of unusual tumor cell specificity.

In conclusion, an unusual cytostatic selectivity was observed for a number of 2-amino-3-carboxylic acid ester thiophene derivatives substituted at the 5-position of the thiophene ring. These compounds showed a preferential anti-proliferative activity against several human T-cell lymphoma, kidney carcinoma, hepatoma and prostate cancer cell lines. They tend to preferentially inhibit protein synthesis rather than DNA or RNA synthesis, and may specifically lead to an accumulation of the drug-exposed tumor cells in their G1 cell phase.