# Patent

## Title
Anti-cancer compounds

## Abstract

The present invention relates to compounds having cytostatic activity against tumor cells. The compounds of the invention are of formula (I), or derivatives hereof, wherein R0, R1, R2, A, and X have defined meanings as described in claim 1.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national stage entry under 35 U.S.C. §371 of PCT International Patent Application No. PCT/EP2013/063082, filed Jun. 21, 2013, which claims priority to Great Britain Patent Application No. 1222113.1, filed Dec. 10, 2012 and Great Britain Patent Application No. 1211086.2, filed Jun. 22, 2012, the contents of all of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The invention relates to compounds and compositions containing said compounds having cytostatic (antiproliferative) activity against tumor cells, more specifically against T-cell lymphoma, hepatoma and prostate cancer cells. The invention also provides processes for the preparation of the disclosed compounds and compositions containing said compounds. The invention further relates to the use of said compounds as biologically active ingredients, more specifically as a medicine for the treatment of cancer.
BACKGROUND OF THE INVENTION
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 were endowed with biological activity). 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 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. Several 2-aminothiophene analogues were also shown to exhibit anti-inflammatory potential. 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.
Recently, we have synthesized a series of 2-amino-3-aroyl-4-substituted thiophene derivatives as anti-proliferative agents. They seem 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 (Romagnoli et al., 2010) (1). However the compounds discovered in Romagnoli et al., 2010 showed no selectivity. Thus there is still a big need for very active and selective anti-proliferative compounds in this field and such selective and active compounds are useful for treating hyperproliferative disorders.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected finding that certain combinations of substituents at different positions of 2-amino,3-carboxylic ester thiophenes and their derivatives, said combinations not being suggested by the prior art, are able to meet one or more of the medical needs, and to show unexpected biological properties, in particular have significant and selective anticancer activity.
The present invention concerns a compound of formula I, Ia, or Ib:




wherein,

    
    
        A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-;
    
    


R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;

    
    
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
        X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        
            and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof.
        
        
    
    


In some embodiments, said compound is not Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitrile; 2-Amino-5-benzylthiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.
The present invention also concerns a compound having formula I, Ia or Ib, for use as a medicine.
The present invention also concerns a compound having formula I, Ia or Ib, for use as a medicine for the prevention or treatment of a proliferative disorder, including cancer in an animal, preferably in a mammal. In an embodiment, said mammal is a human being.
The present invention also concerns the use of a compound having formula I, Ia or Ib for use as a medicine for the prevention or treatment of proliferative disorders, including cancer, in an animal, preferably a mammal, and more preferably a human. The present invention also concerns the use of a compound having formula I, Ia or Ib for the manufacture of a medicament for the prevention or treatment of a proliferative disorder such as cancer in an animal, preferably a mammal, and more preferably a human.
In more specific embodiments of the invention, said proliferative disorder is cancer. In a more particular embodiment of the invention, said cancer is a hematological malignancy, such as leukemia (e.g. Lymphoblastic T cell leukemia, Chronic myelogenous leukemia (CML), Chronic lymphocytic/lymphoid leukemia (CLL), Hairy-cell leukemia, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), myelodysplastic syndrome, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, Plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, Acute megakaryocytic leukemia, promyelocytic leukemia and Erythroleukemia) and lymphoma, more specifically malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma and follicular lymphoma, MALT1 lymphomas, Hodgkin lymphomas, B-cell non-Hodgkin lymphoma- and marginal zone lymphoma. In a more particular embodiment of the invention, said cancer is selected from the group of hematological malignancies comprising acute leukemia, chronic leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome. In a more particular embodiment of the invention, said chronic leukemia is myeloid or lymphoid. In another more particular embodiment of the invention, said lymphoma is Hodgkin's or non-Hodgkin's lymphoma.
In another particular embodiment of the present invention, said cancer is a non-hematological cancer or solid tumor cancer such as cancer of the prostate, lung, breast, rectal, colon, lymph node, bladder, kidney, pancreatic, liver, ovarian, uterine, brain, skin, sarcoma, meningioma, glioblastoma, multiforme, skin, stomach, including all kinds of neuroblastoma, gastric carcinoma, renal cell carcinoma, neuroblastoma, gastric carcinoma, renal cell carcinoma, uterine cancer and muscle cancer. In another more particular embodiment of the present invention, said cancer is prostate cancer. In yet another more particular embodiment of the present invention, said cancer is liver cancer.
The present invention also concerns a pharmaceutical composition comprising a therapeutically effective amount of a compound having formula I, Ia or Ib, and one or more pharmaceutically acceptable excipients for use as a medicine for the prevention or treatment of a proliferative disorder such as cancer in an animal, mammal or human. Said composition may further comprise one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
The present invention also concerns a method of prevention or treatment of proliferative disorder, including cancer such as hematological malignancies, including acute leukemia, chronic leukemia (myeloid or lymphoid), lymphoma (Hodgkin's or non-Hodgkin's), multiple myeloma, myelodysplastic syndrome, or non-hematological cancers such as prostate carcinoma or liver carcinoma, in an animal, comprising the administration of a therapeutically effective amount of a compound having formula I, Ia or Ib, optionally in combination with one or more pharmaceutically acceptable excipients.
The present invention also concerns a process for preparation of the compounds of formula I, Ia or Ib comprising the steps of:

    
    
        (a) a Gewald reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group;
        (b) protection of the 5-amino group as phthalimide and further transformations at the 2-position of the thiophene starting from bromo or 2-bromomethyl derivatives; thus introducing the aromatic, heterocyclic or lipophilic side chain; and
        (c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.
    
    


Numbered statements of this invention are:

    1. A compound having the general formula I, Ia, or Ib:






wherein

    
    
        A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        
            R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-;
            R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;
        
        
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
        X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof;
        with the proviso that said compound is not Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitrile; 2-Amino-5-benzylthiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.
    
    
    2. The compound according to statement 1, wherein A is C1-7 alkyl or C2-7 alkenyl.
    3. The compound according to statement 1, wherein A is phenyl or thiophene.
    4. The compound according to any of statements 1 to 3, wherein R1 is carboxyC1-7alkyl, preferably a carboxymethyl or carboxyethyl group.
    5. The compound according to any of statements 1, 3 and 4, wherein A is an aryl group substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group.
    6. The compound according to any of statements 1 to 5, wherein X is selected from the group consisting of a —(CH2)2, an ethylene and an acetylene group.
    7. The compound according to any of statements 1 to 6, wherein n is 2.
    8. The compound according to any of statements 1 to 7, wherein R0 is amino.
    9. The compound according to any of statements 1 to 8, wherein R2 is hydrogen.
    10. The compound according to statement 1, wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-4-ethoxycarbonyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Methyl-2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene; Ethyl-2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene; Ethyl-2-amino-4-ethoxycarbonyl-5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; N-Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; Methyl-2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; and Methyl-2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate.
    11. The compound according to statements 1 or 10 wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methyl phenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; and Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate.
    12. A compound according to any of statements 1 to 11 or a compound selected from
    
        Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitrile; 2-Amino-5-benzylthiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; for use as a medicine.
    
    
    13. A compound according to any of statements 1 to 12, or a compound selected from
    
        Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; for use as a medicine for the prevention or treatment of cancer in a subject.
    
    
    14. A compound according to statement 13, wherein said cancer is a lymphoma, liver cancer or prostate cancer.
    15. A compound according to statement 14, wherein said lymphoma is a T-cell lymphoma.
    16. A compound according to any of statements 13 to 15, wherein said subject is an animal or a human.
    17. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of statements 1 to 11 or a compound selected from
    
        Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; and one or more pharmaceutically acceptable excipients.
    
    
    18. A pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier and as active ingredients a compound according to any of statements 1 to 11 or a compound selected from
    
        Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.
    
    
    19. The pharmaceutical composition according to statement 17 or 18, further comprising one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
    20. A method of prevention or treatment of cancer in an animal, comprising the administration of a therapeutically effective amount of a compound according to any of statements 1 to 11, or a compound selected from
    
        Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; optionally in combination with one or more pharmaceutically acceptable excipients.
    
    
    21. A process for preparing a compound of formula I, Ia and Ib, comprising the steps of:
    (a) a Gewald reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group;
    (b) protection of the 5-amino group as phthalimide and further transformations at the 2-position of the thiophene starting from bromo or 2-bromomethyl derivatives; thus introducing the aromatic, heterocyclic or lipophilic side chain; and
    (c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.
    22. A compound selected from the group consisting of:
    
        methyl 2-amino-5-propyl-thiophene-3-carboxylate; methyl 2-amino-5-(3-chloropropyl)thiophene-3-carboxylate; methyl 2-amino-5-butyl-thiophene-3-carboxylate; methyl 2-amino-5-pentyl-thiophene-3-carboxylate; methyl 2-amino-5-hexyl-thiophene-3-carboxylate; methyl 2-amino-5-heptyl-thiophene-3-carboxylate; methyl 2-amino-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-(1,5-dimethylhex-4-enyl)thiophene-3-carboxylate; 2-amino-5-non-8-enyl-thiophene-3-carbonitrile; methyl 2-amino-4-methyl-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-nonyl-thiophene-3-carboxylate; methyl 2-amino-5-decyl-thiophene-3-carboxylate; methyl 2-amino-5-dodecyl-thiophene-3-carboxylate; methyl 2-amino-5-tridecyl-thiophene-3-carboxylate; methyl 2-amino-5-tetradecyl-thiophene-3-carboxylate; methyl 2-amino-5-pentadecyl-thiophene-3-carboxylate; methyl 2-amino-5-hexadecyl-thiophene-3-carboxylate; methyl 2-amino-5-(butylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(pentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(isopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(hexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(heptylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(octylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclohexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(phenylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(p-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(o-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(m-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(2-aminophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxycarbonylphenyl)thiomethyl]thiophene-3-carboxylate; 3-((5-amino-4-(methoxycarbonyl)thiophen-2-yl)methylthio)benzoic acid; methyl 2-amino-5-[(4-fluorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-chlorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-nitrophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-ethylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-isopropylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(phenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(phenethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(biphenyl-4-yl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(2-naphthylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(4-pyridylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-thienylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-furylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(3-cyclohexylthiopropyl)thiophene-3-carboxylate; methyl 2-amino-5-[3-(4-methoxyphenyl)thiopropyl]thiophene-3-carboxylate.
    
    
    23. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to statement 22 and one or more pharmaceutically acceptable excipients.
    24. The pharmaceutical composition according to statement 23, further comprising one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
    25. A compound according to statement 22, or a pharmaceutical composition according to statement 23 or 24 for use as a medicine.
    26. A compound according to statement 22, or a pharmaceutical composition according to statement 23 or 24, for use as a medicine for the prevention or treatment of cancer in a subject, preferably wherein said subject is an animal or human.
    27. A compound according to statement 22, or a pharmaceutical composition according to statement 23 or 24, for use as a medicine for the prevention or treatment of cancer in a subject, wherein said cancer is a lymphoma, liver cancer or prostate cancer; preferably a T-cell lymphoma, preferably wherein said subject is an animal or human.
    28. A compound having the general formula I, Ia, or Ib: wherein
    
        A is selected from the group consisting of heteroaryl and aryl groups; C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        R0 is independently selected from the group consisting of hydrogen, amino, (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-;
        R1 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
        X is independently selected from the group consisting of —(CH2)n, —(CH2)n-acetylene, —(CH2)n-ethylene, —S(CH2)n—, —O(CH2)n—, —NH(CH2)n—; —(CH2)nS(CH2)n—, wherein n is 0, 1, 2, 3, 4, 5, 6 or 7;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
    
    
    and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof.
    29. The compound according to statement 28, wherein A is phenyl or thiophene.
    30. The compound according to statement 28 or 29, wherein R1 is a carboxymethyl or carboxyethyl group.
    31. The compound according to any of statements 28 to 30, wherein A is an aryl group substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group.
    32. The compound according to any of statements 28 to 31, wherein X is selected from the group consisting of a —(CH2)2—, an ethylene and an acetylene group.
    33. The compound according to any of statements 28 to 32, wherein n is 2.
    34. The compound according to any of statements 28 to 33, wherein R0 is amino.
    35. The compound according to any of statements 28 to 34, wherein R2 is hydrogen.
    36. The compound according to statement 28, wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methyl phenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl 2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-4-ethoxycarbonyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Methyl-2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene; Ethyl 2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene; Ethyl 2-amino-4-ethoxycarbonyl 5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; N-Methyl 2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; Methyl-2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; and Methyl-2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate.
    37. The compound according to statement 28 or 36 wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; and Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate.
    38. A compound according to any of statements 28 to 37 for use as a medicine.
    39. A compound according to any of statements 28 to 38 for use as a medicine for the prevention or treatment of cancer in a subject.
    40. A compound according to statement 39, wherein said cancer is a lymphoma, liver cancer or prostate cancer.
    41. A compound according to statement 40, wherein said lymphoma is a T-cell lymphoma.
    42. A compound according to any of statements 39 to 41, wherein said subject is an animal or a human.
    43. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of statements 28 to 37 and one or more pharmaceutically acceptable excipients.
    44. A pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier and as active ingredients a compound according to any of statements 28 to 37.
    45. The pharmaceutical composition according to statement 43 or 44, further comprising one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
    46. A method of prevention or treatment of cancer in an animal, comprising the administration of a therapeutically effective amount of a compound according to any of statements 28 to 37, optionally in combination with one or more pharmaceutically acceptable excipients.
    47. A process for preparing a compound of formula I, Ia and Ib according to any of statements 28 to 37, comprising the steps of:
    
        (a) a Gewald reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group;
        (b) protection of the 5-amino group as phthalimide and further transformations at the 2-position of the thiophene starting from bromo or 2-bromomethyl derivatives; thus introducing the aromatic, heterocyclic or lipophilic side chain; and
        (c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.
    
    
    48. A compound having the general formula I, Ia, or Ib:






wherein

    
    
        A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo-C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7-alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-;
        R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;
        wherein when X is C2 alkynyl (acetylene bridge) in formula I, then R1 is carboxyC1-7 alkyl;
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
        X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
    
    
    and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof.
    49. The compound according to statement 48, wherein A is C1-7 alkyl or C2-7 alkenyl.
    50. The compound according to statement 48, wherein A is phenyl or thiophene.
    51. The compound according to any of statements 48 to 50, wherein R1 is a carboxymethyl or carboxyethyl group.
    52. The compound according to any of statements 48, 50 and 51, wherein A is an aryl group substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group.
    53. The compound according to any of statements 48 to 52, wherein X is selected from the group consisting of a —(CH2)2—, an ethylene and an acetylene group.
    54. The compound according to any of statements 48 to 53, wherein n is 2.
    55. The compound according to any of statements 48 to 54, wherein R0 is amino.
    56. The compound according to any of statements 48 to 55, wherein R2 is hydrogen.
    57. The compound according to statement 48, wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methyl phenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-4-ethoxycarbonyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Methyl-2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene; Ethyl-2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene; Ethyl-2-amino-4-ethoxycarbonyl-5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; N-Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; Methyl-2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; and Methyl-2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate.
    58. The compound according to statement 48 or 57 wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methyl phenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; and Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate.
    59. A compound according to any of statements 48 to 58 for use as a medicine.
    60. A compound according to any of statements 48 to 58 for use as a medicine for the prevention or treatment of cancer in a subject.
    61. A compound according to statement 60, wherein said cancer is a lymphoma, liver cancer or prostate cancer.
    62. A compound according to statement 61, wherein said lymphoma is a T-cell lymphoma.
    63. A compound according to any of statements 60 to 62, wherein said subject is an animal or a human.
    64. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of statements 48 to 58 and one or more pharmaceutically acceptable excipients.
    65. A pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier and as active ingredients a compound according to any of statements 48 to 58.
    66. The pharmaceutical composition according to statement 64 or 65, further comprising one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
    67. A method of prevention or treatment of cancer in an animal, comprising the administration of a therapeutically effective amount of a compound according to any of statements 48 to 58, optionally in combination with one or more pharmaceutically acceptable excipients.
    68. A process for preparing a compound of formula I, Ia and Ib, comprising the steps of:
    (a) a Gewald reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group;
    (b) protection of the 5-amino group as phthalimide and further transformations at the 2-position of the thiophene starting from bromo or 2-bromomethyl derivatives; thus introducing the aromatic, heterocyclic or lipophilic side chain; and
    (c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.


The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Cytostatic activity of 2-aminothiophene derivatives against a variety of tumor and non-tumorigenic cell lines.
FIG. 2. 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.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of this invention concerns the compounds of formula I, Ia and Ib:




wherein,

    
    
        A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido; for example A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents, for example 1, 2, or 3 substituents each independently selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino; for example A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; thio C1-7 alkyl; thioC3-10cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halogen, C1-7 alkyl, halo C1-7 alkyl, nitro, hydroxyl, amino, C1-7 alkoxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, amino;
        R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-; preferably R0 is independently selected from the group consisting of amino; (mono- or di) C1-4 alkylamino; (mono- or di) arylamino; preferably R0 is independently selected from the group consisting of amino; (mono- or di) C1-4 alkylamino; more preferably R0 is amino;
        R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl; preferably R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl; preferably R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl; preferably R1 is independently selected from carboxyC1-6alkyl; or cyano; preferably R1 is independently selected from carboxyC1-4alkyl; or cyano; preferably R1 is independently selected from carboxyC1-2alkyl; or cyano;
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2; preferably R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; COOH; preferably R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; preferably R2 is independently selected from the group consisting of hydrogen; carboxyC1-6 alkyl, C1-6alkyl; preferably R2 is independently selected from the group consisting of hydrogen; carboxyC1-4 alkyl, C1-4alkyl; preferably R2 is independently selected from the group consisting of hydrogen; carboxyC1-2 alkyl, C1-2alkyl;
        X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7; preferably X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —(CH2)mS(CH2)p—, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7; preferably X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —(CH2)mS(CH2)p—, wherein m is 0, 1, 2, 3, 4, 5, or 6, and wherein p is 0, 1, 2, 3, 4, 5, or 6, and wherein n is 2, 3, 4, 5, or 6; preferably wherein m is 0, 1, 2, 3, 4, or 5, and wherein p is 0, 1, 2, 3, 4, or 5, and wherein n is 2, 3, 4, or 5;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido; preferably R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino; preferably R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-6alkyl; C1-6alkoxy; halogen; amino; hydroxyl; and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, or hydroxyl; preferably R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-6alkyl; C1-6alkoxy; halogen; preferably R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-4alkyl; C1-4alkoxy; and halogen;
    
    
    and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof.


In an embodiment, said compounds of formula (I), (Ia) or (Ib) is not Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.
One embodiment of the present invention concerns the compounds of formula I, wherein said group A can be any lipophilic moiety as these moieties are well known to the skilled person, including optionally substituted aryl heteroaryl cycloalkyl groups and the like.
One embodiment of the present invention concerns the compounds of formula I, wherein said group A is C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, C3-10 cycloalkyl or heterocyclic-substituted alkyl. In another embodiment said group A is C1-7 alkyl. In another embodiment said group A is C2-7 alkenyl. In another embodiment said group A is C2-7 alkynyl. In another embodiment said group A is halo C1-7 alkyl. In another embodiment said group A is C3-10 cycloalkyl. In another embodiment said group A is heterocyclic-substituted alkyl.
One embodiment of the present invention concerns the compounds of formula I, wherein said group A is thio C1-7 alkyl, thio C3-10 cycloalkyl, arylthio or arylalkylthio. In another embodiment said group A is thioC1-7 alkyl. In another embodiment said group A is thio C3-10 cycloalkyl. In another embodiment said group A is arylthio. In another embodiment said group A is arylalkylthio.
One embodiment of the present invention concerns the compounds of formula I, wherein said group A is phenyl, thiophene, cyclohexyl or cyclohexenyl. One embodiment of the present invention concerns the compounds of formula I, wherein said group A is phenyl, naphthlyl, biphenylyl, thiophene, cyclohexyl or cyclohexenyl. In another embodiment said group A is phenyl. In another embodiment said group A is cyclohexenyl. In another embodiment said group A is cyclohexyl. In another embodiment said group A is a thiophene. In another embodiment, said group A is an aryl group optionally substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group. In another embodiment, said group A is an aryl group substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group; and in a further more specified embodiment said substituent is a methoxy group; and in an even more specified embodiment said methoxy group is on the R5 or R3 position of said aryl or phenyl ring. In another embodiment said 1 or 2 substituents are on the R5 and/or R3 position of said aryl or phenyl ring.
Another embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein R1 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2, heteroaryl and aryl groups; and C3-10 cycloalkyl groups, wherein R, Ra and Rb are each independently selected from the group consisting of H, C1-7alkyl, C3-10 cycloalkyl and aryl.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R1 is a carboxyC1-7alkyl. One more specific embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R1 is a carboxymethyl or a carboxy-ethyl group. In another embodiment said R1 is a carboxymethyl group. In another embodiment said R1 is a carboxyethyl group.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R0 is selected from the group consisting of hydrogen; amino; (mono- or di) C1-7alkylamino, including —NHCH3, and —N(CH3)2; (mono- or di) arylamino, and 1H-Isoindole-1,3(2H)-dione,2-methyl-. In a more specific embodiment said R0 is amino.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R2 is selected from the group consisting of hydrogen, carboxyethyl and methyl. In a more specific embodiment said R2 is hydrogen.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R1 is a carboxymethyl or a carboxy-ethyl group, and wherein said R0 is amino. A more specific embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ia, wherein said R1 is a carboxymethyl or a carboxy-ethyl group, and wherein said R0 is amino, and wherein said R2 is hydrogen.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I, Ia and Ib, wherein said R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula Ia, wherein n is 2, 3, 4, 5, 6 or 7. In a more specific embodiment said n is 2 or 3. In an even more specific embodiment said n is 2.
One embodiment of the present invention concerns the compounds of this invention, including the compounds of formula I and Ib, wherein X is a spacer or a linker consisting of 1, 2, or 3, 4, 5, 6 or 7 carbon atoms. In a more specific embodiment said X is a spacer or a linker consisting of 1, 2, or 3 carbon atoms. In an even more specific embodiment said X is a spacer or a linker consisting of 2 carbon atoms. In another specific embodiment, said X is selected from the group consisting of —(CH2)2—, acetylene, ethylene, —SCH2—, —OCH2—, —NHCH2— and —CH2SCH2—.
The present invention also encompasses a compound having the general formula I, Ia, or Ib: wherein

    
    
        A is selected from the group consisting of C1-10 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, and wherein said C1-10alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, carboxyl, C1-7alkyloxycarbonyl; halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said C1-7alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-methyl-;
        R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; CONRaRb; SO2R; SO2NHR; SO2NRaRb, SO2OR; PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;
        R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
        X is independently selected from the group consisting of —(CReRf)n, —(CReRf)m-acetylene, -acetylene-(CReRf)m—, —(CReRf)m— ethylene, -ethylene-(CReRf)m—, —S(CReRf)m—, —(CReRf)mS—, —O(CReRf)m—, —(CReRf)mO—, —NH(CReRf)m—; —(CReRf)mNH—, —(CReRf)mS(CReRf)p—, —(CReRf)mO(CReRf)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7; wherein each Re and Rf is independently selected from hydrogen or C1-7alkyl; preferably X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene-, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7;
        R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7alkyl; C1-7 alkoxy; halogen; amino; (mono- or di) C1-7 alkylamino, (mono- or di) arylamino; azido; hydroxyl; C3-10 cycloalkyl, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
        and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof;
        with the proviso that said compound is not Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.
    
    


In some embodiments, A is C1-7 alkyl or C2-7 alkenyl. In some embodiments, A is phenyl or thiophene. In some embodiments, R1 is carboxyC1-7alkyl, preferably a carboxymethyl or carboxyethyl group. In some embodiments, A is an aryl group optionally substituted with 1 or 2 substituents selected from the group consisting of C1-6alkoxy and C1-6alkyl. In some embodiments, A is an aryl group optionally substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group. In some embodiments, X is selected from the group consisting of a —(CH2)2—, an ethylene and an acetylene group. In some embodiments, n is 2. In some embodiments, R0 is amino. In some embodiments, R2 is hydrogen.
The present invention also encompasses a compound selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-4-ethoxycarbonyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Methyl-2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene; Ethyl-2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene; Ethyl-2-amino-4-ethoxycarbonyl-5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; N-Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; Methyl-2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; and Methyl-2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; methyl 2-amino-5-propyl-thiophene-3-carboxylate; methyl 2-amino-5-(3-chloropropyl)thiophene-3-carboxylate; methyl 2-amino-5-butyl-thiophene-3-carboxylate; methyl 2-amino-5-pentyl-thiophene-3-carboxylate; methyl 2-amino-5-hexyl-thiophene-3-carboxylate; methyl 2-amino-5-heptyl-thiophene-3-carboxylate; methyl 2-amino-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-(1,5-dimethylhex-4-enyl)thiophene-3-carboxylate; 2-amino-5-non-8-enyl-thiophene-3-carbonitrile; methyl 2-amino-4-methyl-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-nonyl-thiophene-3-carboxylate; methyl 2-amino-5-decyl-thiophene-3-carboxylate; methyl 2-amino-5-dodecyl-thiophene-3-carboxylate; methyl 2-amino-5-tridecyl-thiophene-3-carboxylate; methyl 2-amino-5-tetradecyl-thiophene-3-carboxylate; methyl 2-amino-5-pentadecyl-thiophene-3-carboxylate; methyl 2-amino-5-hexadecyl-thiophene-3-carboxylate; methyl 2-amino-5-(butylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(pentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(isopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(hexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(heptylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(octylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclohexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(phenylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(p-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(o-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(m-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(2-aminophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxycarbonylphenyl)thiomethyl]thiophene-3-carboxylate; 3-((5-amino-4-(methoxycarbonyl)thiophen-2-yl)methylthio)benzoic acid; methyl 2-amino-5-[(4-fluorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-chlorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-nitrophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-ethylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-isopropylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(phenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(phenethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(biphenyl-4-yl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(2-naphthylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(4-pyridylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-thienylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-furylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(3-cyclohexylthiopropyl)thiophene-3-carboxylate; methyl 2-amino-5-[3-(4-methoxyphenyl)thiopropyl]thiophene-3-carboxylate.
The present invention also concerns the compounds of the present invention, including the compounds of formula I, Ia, and Ib, for use as a medicine.
One aspect of this invention relates to the compounds of this invention and their use as a medicament for the treatment of cancer, including lymphoma, hepatoma and prostate cancer.
In certain embodiments of this invention, the compounds of this invention are used as a medicament for the treatment or prevention of lymphomas. In certain more specific embodiments of the present invention, said lymphoma is a T-cell lymphoma.
In certain embodiments of this invention, the compounds of this invention are used as a medicament for the treatment or prevention of hepatomas or liver cancer.
One aspect of the disclosure relates to treatment or prevention of prostate disease. In some embodiments, the compounds of this invention are used as a medicament for treatment or prevention of abnormal prostatic growths in a subject. The growths may be benign, such as benign prostatic hyperplasia, or may be associated with prostate cancer, for example, as an early or precursor stage of prostate cancer. One exemplary condition associated with development of prostate cancer is prostatic intraepithelial neoplasia.
In certain embodiments, the compounds of formula (I) are used as a medicament for treatment or prevention of prostate cancer. In certain embodiments, the prostate cancer is a metastatic and/or aggressive form of prostate cancer. In some embodiments, the prostate cancer is adenocarcinoma. Adenocarcinoma is the most common form of prostate cancer, and is typically characterized by its origin in the peripheral zone (main glandular zone) of the prostate, and its development from epithelial cells. In further embodiments, the prostate cancer is a basal cell carcinoma, small cell carcinoma, squamous cell carcinoma, sarcoma, transitional cell carcinoma, or any combination of these. In addition, the prostate cancer may have developed from primary tumors that arose in other locations such as the bladder or urethra, and spread to the prostate.
In some embodiments, the prostate cancer is in stage T1 or T2, according to the four-stage Tumor/Nodes/Metastasis (TNM) system. In certain embodiments, the prostate cancer may have spread outside of the prostate, and may be a stage T3 or T4 cancer. For example, a prostate cancer may have spread to the lymph nodes, bladder, urethra, rectum, bones, or other organs. In some embodiments, the medicaments described herein may prevent and/or slow a prostate cancer's progression to the next stage.
In some embodiments, the prostate cancer has taken a form that does not respond to treatment and/or has become refractory. Often, prostate cancer is treated by administering compounds that disrupt the androgen signaling pathways essential for growth and survival of prostate cancer cells. For example, androgen receptor (AR) antagonists, also called anti-androgens, are administered in order to block binding of testosterone (T) and dihydrotestosterone (DHT) to the AR on prostate cancer cells. Initially, treatment with AR antagonists can prove successful in reducing the growth of prostate cancer cells. However, the prostate cancer can become refractive, and resume growth in spite of the treatment. When this occurs, the prostate cancer is known as a castration-resistant prostate cancer. This form of prostate cancer has also been previously termed “hormone refractory prostate cancer” and “androgen-independent prostate cancer.” Castration-resistant prostate cancer may emerge at any time after initiating treatment of prostate cancer. In some embodiments, the castration-resistant prostate cancer emerges within at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 months of treatment, or may emerge within 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 months, or within 2, 3, 4, or 5 years of treatment.
Thus, in certain embodiments, the compounds of formula I and/or formula Ia and/or Ib are used as a medicament for treatment or prevention of castration-resistant prostate cancer. In some embodiments, the castration-resistant prostate cancer was previously treated with at least one AR antagonist, for example, at least one steroidal and/or at least one nonsteroidal AR antagonist. Exemplary nonsteroidal AR antagonists include but are not limited to Bicalutamide (Bic), Hydroxyflutamide (HOFl), Nilutamide, MDV3100, or its variant RD-162, and ARN-509. Accordingly, the prostate cancer may no longer respond to the dosages of an AR antagonist such as Bic, HOFl, Nilutamide, MDV3100, RD-162 and/or ARN-509 that had been therapeutically effective in the past.
The present invention also concerns a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention, including a compound of formula I, Ia or Ib, and one or more pharmaceutically acceptable excipients for use as a medicine for the prevention or treatment of a proliferative disorder such as cancer in an animal, mammal or human. Said composition may further comprise one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.
The present invention also concerns a method of prevention or treatment of proliferative disorder, including cancer such as hematological malignancies, including acute leukemia, chronic leukemia (myeloid or lymphoid), lymphoma (Hodgkin's or non-Hodgkin's), multiple myeloma, myelodysplastic syndrome, or non-hematological cancers such as prostate carcinoma or liver carcinoma, in an animal, comprising the administration of a therapeutically effective amount of a compound of the present invention, including a compound of formula I, Ia or Ib, optionally in combination with one or more pharmaceutically acceptable excipients.
The present invention also encompasses processes for the preparation of compounds of Formula I, Ia, and Ib. In a certain embodiment of the present invention said process for preparation of the compounds of formula I, Ia or Ib comprises the steps of:

    (a) a Gewald reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group
    (b) protection of the 5-amino group as phthalimide and further transformations at the 2-position of the thiophene starting from bromo or 2-bromomethyl derivatives; thus introducing the aromatic, heterocyclic or lipophilic side chain and
    (c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.


DEFINITIONS
When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C1-7 alkyl” means straight and branched chain saturated acyclic hydrocarbon monovalent radicals having from 1 to 7 carbon atoms such as, for example, methyl, ethyl, propyl, n-butyl, 1-methylethyl (isopropyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (ter-butyl), 2-methylbutyl, n-pentyl, dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, n-heptyl and the like. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, C1-6alkyl groups include all linear, or branched alkyl groups having 1 to 6 carbon atoms, and thus includes for example methyl, ethyl, n-propyl, i-propyl, 2-methyl-ethyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C1-7 alkylene” means the divalent hydrocarbon radical corresponding to the above defined C1-7 alkyl, such as methylene, bis(methylene), tris(methylene), tetramethylene, hexamethylene and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C3-10 cycloalkyl” means a mono- or polycyclic saturated hydrocarbon monovalent radical having from 3 to 10 carbon atoms, such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like, or a C7-10 polycyclic saturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “aryl” designate any mono- or polycyclic aromatic monovalent hydrocarbon radical having from 6 up to 30 carbon atoms such as but not limited to phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, biphenyl, indacenyl, benzocyclobutenyl, benzocyclooctenyl and the like, including fused benzo-C4-β cycloalkyl radicals (the latter being as defined above) such as, for instance, indanyl, tetrahydronaphthyl, fluorenyl and the like, all of the said radicals being optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, trifluoromethyl, hydroxyl, sulfhydryl and nitro, such as for instance 4-fluorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 4-cyanophenyl, 2,6-dichlorophenyl, 2-fluorophenyl, 3-chlorophenyl, 3,5-dichlorophenyl and the like.
As used herein with respect to a substituting radical (including the combination of substituents in certain positions of the compounds of formula I, Ia, Ib, or group A), and unless otherwise stated, the term “heterocyclic” means a mono- or polycyclic, saturated or mono-unsaturated or polyunsaturated monovalent hydrocarbon radical having from 2 up to 15 carbon atoms and including one or more heteroatoms in one or more heterocyclic rings, each of said rings having from 3 to 10 atoms (and optionally further including one or more heteroatoms attached to one or more carbon atoms of said ring, for instance in the form of a carbonyl or thiocarbonyl or selenocarbonyl group, and/or to one or more heteroatoms of said ring, for instance in the form of a sulfone, sulfoxide, N-oxide, phosphate, phosphonate or selenium oxide group), each of said heteroatoms being independently selected from the group consisting of nitrogen, oxygen, sulfur, selenium and phosphorus, also including radicals wherein a heterocyclic ring is fused to one or more aromatic hydrocarbon rings for instance in the form of benzo-fused, dibenzo-fused and naphtho-fused heterocyclic radicals; within this definition are included heterocyclic radicals such as, but not limited to, diazepinyl, oxadiazinyl, thiadiazinyl, dithiazinyl, triazolonyl, diazepinonyl, triazepinyl, triazepinonyl, tetrazepinonyl, benzoquinolinyl, benzothiazinyl, benzothiazinonyl, benzoxa-thiinyl, benzodioxinyl, benzodithiinyl, benzoxazepinyl, benzothiazepinyl, benzodiazepine, benzodioxepinyl, benzodithiepinyl, berrzoxazocinyl, benzo-thiazocinyl, benzodiazocinyl, benzoxathiocinyl, benzodioxocinyl, benzotrioxepinyl, benzoxathiazepinyl, benzoxadiazepinyl, benzothia-diazepinyl, benzotriazepinyl, benzoxathiepinyl, benzotriazinonyl, benzoxazolinonyl, azetidinonyl, azaspiroundecyl, dithiaspirodecyl, selenazinyl, selenazolyl, selenophenyl, hypoxanthinyl, azahypo-xanthinyl, bipyrazinyl, bipyridinyl, oxazolidinyl, diselenopyrimidinyl, benzodioxocinyl, benzopyrenyl, benzopyranonyl, benzophenazinyl, benzoquinolizinyl, dibenzo-carbazolyl, dibenzoacridinyl, dibenzophenazinyl, dibenzothiepinyl, dibenzoxepinyl, dibenzopyranonyl, dibenzoquinoxalinyl, dibenzothiazepinyl, dibenzisoquinolinyl, tetraazaadamantyl, thiatetraazaadamantyl, oxauracil, oxazinyl, dibenzothiophenyl, dibenzofuranyl, oxazolinyl, oxazolonyl, azaindolyl, azolonyl, thiazolinyl, thiazolonyl, thiazolidinyl, thiazanyl, pyrimidonyl, thiopyrimidonyl, thiamorpholinyl, azlactonyl, naphtindazolyl, naphtindolyl, naphtothiazolyl, naphtothioxolyl, naphtoxindolyl, naphto-triazolyl, naphtopyranyl, oxabicycloheptyl, azabenzimidazolyl, azacycloheptyl, azacyclooctyl, azacyclononyl, azabicyclononyl, tetrahydrofuryl, tetrahydropyrarryl, tetrahydro-pyronyl, tetrahydroquinoleinyl, tetrahydrothienyl and dioxide thereof, dihydrothienyl dioxide, dioxindolyl, dioxinyl, dioxenyl, dioxazinyl, thioxanyl, thioxolyl, thiourazolyl, thiotriazolyl, thiopyranyl, thiopyronyl, coumarinyl, quinoleinyl, oxyquinoleinyl, quinuclidinyl, xanthinyl, dihydropyranyl, benzodihydrofuryl, benzothiopyronyl, benzothiopyranyl, benzoxazinyl, benzoxazolyl, benzodioxolyl, benzodioxanyl, benzothiadiazolyl, benzotriazinyl, benzothiazolyl, benzoxazolyl, phenothioxinyl, phenothiazolyl, phenothienyl (benzothiofuranyl), phenopyronyl, phenoxazolyl, pyridinyl, dihydropyridinyl, tetrahydropyridinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, tetrazinyl, triazolyl, benzotriazolyl, tetrazolyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, pyrrolyl, furyl, dihydrofutyl, furoyl, hydantoinyl, dioxolanyl, dioxolyl, dithianyl, dithienyl, dithiinyl, thienyl, indolyl, indazolyl, benzofutyl, quinolyl, quinazolinyl, quinoxalinyl, carbazolyl, phenoxazinyl, phenothiazinyl, xanthenyl, purinyl, benzothienyl, naphtothienyl, thianthrenyl, pyranyl, pyronyl, benzopyronyl, isobenzofuranyl, chromenyl, phenoxathiinyl, indolizinyl, quinolizinyl, isoquinolyl, phthalazinyl, naphthiridinyl, cinnolinyl, pteridinyl, carbolinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, imidazolinyl, imidazolidinyl, benzimidazolyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, piperazinyl, uridinyl, thymidinyl, cytidinyl, azirinyl, aziridinyl, diazirinyl, diaziridinyl, oxiranyl, oxaziridinyl, dioxiranyl, thiiranyl, azetyl, dihydroazetyl, azetidinyl, oxetyl, oxetanyl, oxetanonyl, homopiperazinyl, homopiperidinyl, thietyl, thietanyl, diazabicyclooctyl, diazetyl, diaziridinonyl, diaziridinethionyl, chromanyl, chromanonyl, thiochromanyl, thiochromanonyl, thiochromenyl, benzofuranyl, benzisothiazolyl, benzocarbazolyl, benzochromonyl, benzisoalloxazinyl, benzocoumarinyl, thiocoumarinyl, pheno-metoxazinyl, phenoparoxazinyl, phentriazinyl, thiodiazinyl, thiodiazolyl, indoxyl, thioindoxyl, benzodiazinyl (e.g. phthalazinyl), phthalidyl, phthalimidinyl, phthalazonyl, alloxazinyl, dibenzopyronyl (i.e. xanthonyl), xanthionyl, isatyl, isopyrazolyl, isopyrazolonyl, urazolyl, urazinyl, uretinyl, uretidinyl, succinyl, succinimido, benzylsulfinyl, benzylsulfanyl and the like, including all possible isomeric forms thereof, wherein each carbon atom of said heterocyclic ring may furthermore be independently substituted with a substituent selected from the group consisting of halogen, nitro, C1-7 alkyl (optionally containing one or more functions or radicals selected from the group consisting of carbonyl (oxo), alcohol (hydroxyl), ether (alkoxy), acetal, amino, imino, oximino, alkyloximino, amino-acid, cyano, carboxylic acid ester or amide, nitro, thio C1-7 alkyl, thio C3-10 cycloalkyl, C1-7 alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino, arylamino, arylalkyl-amino, hydroxylalkylamino, mercaptoalkylamino, heterocyclic-substituted alkylamino, heterocyclic amino, heterocyclic-substituted arylamino, hydrazino, alkylhydrazino, phenylhydrazino, sulfonyl, sulfonamido and halogen), C3-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, C3-10 cycloalkyl, aryl, arylalkyl, alkylaryl, alkylacyl, arylacyl, hydroxyl, amino, C1-7 alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino, arylamino, arylalkylamino, hydroxyalkylamino, mercaptoalkylamino, heterocyclic-substituted alkylamino, heterocyclic amino, heterocyclic-substituted arylamino, hydrazino, alkylhydrazino, phenylhydrazino, sulfhydryl, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C1-7 alkyl, thio C3-10 cycloalkyl, thioaryl, thioheterocyclic, arylalkylthio, heterocyclic-substituted alkylthio, formyl, hydroxylamino, cyano, carboxylic acid or esters or thioesters or amides thereof, tricarboxylic acid or esters or thioesters or amides thereof; depending upon the number of unsaturations in the 3 to 10 atoms ring, heterocyclic radicals may be sub-divided into heteroaromatic (or “heteroaryl”) radicals and non-aromatic heterocyclic radicals; when a heteroatom of said non-aromatic heterocyclic radical is nitrogen, the latter may be substituted with a substituent selected from the group consisting of C1-7 alkyl, C3-10 cycloalkyl, aryl, arylalkyl and alkylaryl.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “C1-7 alkoxy”, “C3-10 cycloalkoxy”, “aryloxy”, “arylalkyloxy”, “oxyheterocyclic”, “thio C1-7 alkyl”, “thio C3-10 cycloalkyl”, “arylthio”, “arylalkylthio” and “thioheterocyclic” refer to substituents wherein a carbon atom of a C1-7 alkyl, respectively a C3-10 cycloalkyl, aryl, arylalkyl or heterocyclic radical (each of them such as defined herein), is attached to an oxygen atom or a divalent sulfur atom through a single bond such as, but not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiocyclopropyl, thiocyclobutyl, thiocyclopentyl, thiophenyl, phenyloxy, benzyloxy, mercaptobenzyl, cresoxy, and the like.
As used herein with respect to a substituting atom, and unless otherwise stated, the term “halogen” or “halo” means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “halo C1-7 alkyl” means a C1-7 alkyl radical (such as above defined) in which one or more hydrogen atoms are independently replaced by one or more halogens (preferably fluorine, chlorine or bromine), such as but not limited to difluoromethyl, trifluoromethyl, trifluoroethyl, octafluoropentyl, dodecafluoroheptyl, dichloromethyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “C2-7 alkenyl” designate a straight and branched acyclic hydrocarbon monovalent radical having one or more ethylenic unsaturations and having from 2 to 7 carbon atoms such as, for example, vinyl, 1-propenyl, 2-propenyl (allyl), 1-butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, 3-hexenyl, 2-hexenyl, 2-heptenyl, 1,3-butadienyl, pentadienyl, hexadienyl, heptadienyl, heptatrienyl and the like, including all possible isomers thereof.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C2-7 alkynyl” defines straight and branched chain hydrocarbon radicals containing one or more triple bonds and optionally at least one double bond and having from 2 to 7 carbon atoms such as, for example, acetylenyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 2-pentynyl, 1-pentynyl, 3-methyl-2-butynyl, 3-hexynyl, 2-hexynyl, 1-penten-4-ynyl, 3-penten-1-ynyl, 1,3-hexadien-1-ynyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “arylalkyl”, “arylalkenyl” and “heterocyclic-substituted alkyl” refer to an aliphatic saturated or ethylenically unsaturated hydrocarbon monovalent radical (preferably a C1-7 alkyl or C2-7 alkenyl radical such as defined above) onto which an aryl or heterocyclic radical (such as defined above) is already bonded via a carbon atom, and wherein the said aliphatic radical and/or the said aryl or heterocyclic radical may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, hydroxyl, sulfhydryl, C1-7 alkyl, C1-7 alkoxy, trifluoromethyl and nitro, such as but not limited to benzyl, 4-chlorobenzyl, 4-fluorobenzyl, 2-fluorobenzyl, 3,4-dichlorobenzyl, 2,6-dichlorobenzyl, 3-methylbenzyl, 4-methylbenzyl, 4-ter-butylbenzyl, phenylpropyl, 1-naphthylmethyl, phenylethyl, 1-amino-2-phenylethyl, 1-amino-2-[4-hydroxy-phenyl]ethyl, 1-amino-2-[indol-2-yl]ethyl, styryl, pyridylmethyl (including all isomers thereof), pyridylethyl, 2-(2-pyridyl)isopropyl, oxazolylbutyl, 2-thienylmethyl, pyrrolylethyl, morpholinylethyl, imidazol-1-yl-ethyl, benzodioxolylmethyl and 2-furylmethyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “carboxyC1-7 alkyl” refers to a group of formula —C(═O)—O—Rd, wherein Rd is C1-7 alkyl such as defined above. As used herein with respect to a substituting radical, and unless otherwise stated, the term “carboxymethyl” refers to —C(═O)—O—CH3. As used herein with respect to a substituting radical, and unless otherwise stated, the term “carboxyethyl” refers to —C(═O)—O—CH2—CH3.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “ethylene” refers to the group —CH═CH—.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “—(CH2)m-ethylene” refers to the group of formula —(CH2)m—CH═CH—, wherein m has the same meaning as that defined herein.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “-ethylene-(CH2)m—” refers to the group of formula —CH═CH—(CH2)m—, wherein m has the same meaning as that defined herein.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “acetylene” refers to the group —C≡C—.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “—(CH2)m-acetylene” refers to the group of formula —(CH2)m—C≡C—, wherein m has the same meaning as that defined herein.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “-acetylene-(CH2)m—” refers to the group of formula —C≡C—(CH2)m—, wherein m has the same meaning as that defined herein.
“Lymphoma” is a cancer of the lymphocytes. Typically, lymphoma is present as a solid tumor of lymphoid cells. The malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs, in which case it is referred to as extranodal lymphoma. Extranodal sites include the skin, brain, bowels and bone. Lymphomas are closely related to lymphoid leukemias, which are also contemplated by the cancers to be prevented or treated by pharmaceutical compositions of this invention and these lymphoid leukemias also originate in lymphocytes but typically involve only circulating blood and the bone marrow and do not usually form static tumors. There are many types of lymphomas, and in turn, lymphomas are a part of the broad group of diseases called hematological neoplasms. Lymphomas can be divided in four broad groups: mature B-cell neoplasms, mature T-cell and natural killer cell neoplasms, Hodgkin lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Lymphomas include but are not limited to T-cell lymphomas and B-Cell lymphomas.
“T-cell lymphoma” is a disease in which cells in the lymphoid system called T cells (or T lymphocytes) become malignant. They can be associated with Epstein Barr virus and human T-cell leukemia virus-1. The T-cell proliferative disease can be peripheral T-cell lymphoma, (precursor T-cell) lymphoblastic lymphoma, cutaneous T-cell lymphoma, extranodal natural killer T-cell lymphoma, adult T-cell leukemia/lymphoma, T-cell acute lymphoblastic leukemia (T-ALL), T-cell chronic lymphoblastic leukemia (T-CLL), anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, mycosis fungoides or cutaneous T-cell lymphoma (CTCL), Sezary syndrome (the leukemic phase of CTCL), or subcutaneous panniculitis-like T-cell lymphoma.
“Hepatoma” or “liver cancer” can occur in many forms, although many cancers found in the liver are metastases from other tumors, frequently of the GI tract (like colon cancer, carcinoid tumors mainly of the appendix, etc.), but also from breast cancer, ovarian cancer, lung cancer, renal cancer, prostate cancer, etc. Liver cancers include but are not limited to the following examples. Hepatocellular carcinoma (HCC, also called malignant hepatoma) is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis. This tumor also has a variant type that consists of both HCC and cholangiocarcinoma components. Cancers which arise from the blood vessel cells in the liver are known as hemangioendotheliomas. As well as mixed tumors, rarer forms of liver cancer include: mesenchymal tissue; sarcoma; hepatoblastoma (a rare malignant tumor, primarily developing in children); cholangiocarcinoma (bile duct cancers); angiosarcoma and hemangiosarcoma; and lymphoma of liver.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and/or materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical composition is contemplated.
As used herein, “treating” or “treat” includes (i) preventing a pathologic condition from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; (iii) relieving the pathologic condition; and/or diminishing symptoms associated with the pathologic condition.
EXAMPLES
Example 1
Experimental Procedures
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, Calif.).
Cytostatic Assays.
To each well of a 96-well microtiter plate were added 5-7.5×104 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 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.
CEM cells were seeded at 25,000 cells/cm2 in DMEM with 10% FBS in the presence of different concentrations of TR560 (100-10-1 μM) were added. 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, Calif.). 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, HeLa, U87 or CEM cells were seeded in μ-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, Calif.). 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 ([3H-methyl]dThd), RNA synthesis ([5-3H]Urd), and protein synthesis ([4,5-3H]leu) were added to 105 CEM cell cultures in the 200 μl-wells of a microtiter plate at 1 μCi for each precursor in the presence of varying concentrations of the test compounds (5-fold dilution series). The cells were allowed to proliferate for 20 hrs at 37° C. in a humidified, CO2-controlled 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.
Example 2
Chemical Synthesis of Compounds of this Invention
Synthesis of Compounds of Formula (Ia) Wherein R2 is Hydrogen, R0 is Amino- and n is 2 is Shown in Scheme 1.




2-Amino-3-substituted-thiophene derivatives with general structure 7 were synthesized as shown in the reaction sequence reported in Scheme 1. 2-Amino-3-substituted thiophenes with general formula 2 were obtained by a one-step procedure (Gewald reaction, Huang Y and Dömling A., 2011 (2)) applied to malonitrile, methyl 2-cyanoacetate or ethyl 2-cyanoacetate 1 with the dimer of 2-mercaptoacetaldehyde (1,4-dithiane-2,5-diol) and triethylamine (TEA) as base in refluxing ethanol. The 5-unsubstituted thiophene derivative 2 was transformed almost quantitatively into the corresponding N-phthalimido derivative 3 using phthalic anhydride in refluxing acetic acid. The subsequent regioselective bromination of 3 in a mixture of acetic acid and sodium acetate, using a stoichiometric amount of bromine, furnished the intermediate 5-bromothiophene derivative with general structure 4 in good yield. This latter compound was coupled by a standard Sonogashira cross-coupling reaction with the appropriate alkyne in the presence of cuprous iodide (CuI), bis(triphenylphosphine)-palladium chloride [PdCl2(PPh3)2] and a mixture of TEA and DMF, to afford the arylacetylenic derivative 5. Compounds with general formula 6, characterized by the presence of a flexible ethyl linker, were prepared starting from derivatives 5, by catalytic hydrogenation of the triple bond over 10% palladium on charcoal (Pd/C). Removal of the N-protected phthaloyl group was accomplished by the use of hydrazine in refluxing ethanol, to afford the final compounds with general structure 7.
Synthesis of Further Compounds of the Present Invention, Including Compounds of Formula (Ib) (Scheme 2, 3, and 4)




We modified the free amino group and the ester group of the derivative 7 in Scheme 2 (compound 3 in Table 1a) to study the effect of these functional groups on the antiproliferative activity (Scheme 2). The methylation of the 2-aminothiophene derivative 7 was carried out by using MeI in presence of NaH as base. The use of one equivalent of MeI resulted in N-methyl-2-aminothiophene derivative 8 (compound 28 in Table 1a) whereas as an excess of MeI resulted in N,N-dimethylated derivative 9 (compound 29 in Table 1a). The free carboxylic acid derivative 10 (compound 26 in Table 1a) was synthesized by the hydrolysis of derivative 7 using 1N solution of NaOH as base in refluxing methanol followed by acidification. The 2-unsubstituted thiophene derivative 11 (compound 30 in Table 1a) was obtained by the reduction of the diazonium salt of derivative 7 at the expense of H3PO2 as the reducing agent.




Gewald multicomponent reaction between the aldehyde 12 and the elemental sulfur with cyanoacetamide 13 or N-methylcyanoacetamide 14 using NEt3 as base resulted in the corresponding 2-amino thiophene-3-amide derivative 15 (compound 27 in Table 1a) or derivative 16 (compound 25 in Table 1a) in reasonable yield (Scheme 3).




2-Aminothiophene-3-carboxylic acid ester derivatives having a thiomethyl (—SCH2), oxymethyl (—OCH2—), aminomethyl (—NHCH2—) or methylthiomethyl (—CH2SCH2—) group directly linked at the C-5 position of the thiophene ring instead of an ethylene bridge were synthesized as shown in the reaction sequence reported in Scheme 4. The classical Gewald reaction between propionaldehyde 17 and methyl cyanoacetate 18 in the presence of sulfur and NEt3 gives 2-amino-5-methylthiophene-3-carboxylate 19 in good yield. Subsequently, the 2-amino group of 19 was N-protected by transforming almost quantitatively into the corresponding N-phthalimido derivative 20 using phthalic anhydride in refluxing acetic acid. The bromination of the later compound with NBS yielded the corresponding bromide 21 in good yield. Further substitution reaction on to the latter compound with different nucleophiles such as benzyl mercaptan, p-methoxyphenol or p-anisidine gives the compounds having general structure 22 in reasonable to good yields.
Finally, the N-protected phthaloyl group was removed by the use of methylhydrazine in refluxing ethanol, furnishing final compounds with general structure 23.




Example 3
Biological Activities
A series of 31 2-aminothiophene derivatives have been evaluated for their cytostatic activity against a broad variety of tumor cell lines in cell culture (Table 1a, Tables 2-4).







TABLE 1a







2-Amino-3-carboxylic acid ester- and 2-amino-3-cyano-thiophene derivatives


(Ia)








 

















Compound nr









IC50 μM


and code
R0
R1
R2
R3
R4
R5
R6
R7
n
(CEM)





3 TR560
NH2
—COOMe
H
H
H
MeO
H
H
2 
   0.90


1 TR575
NH2
—COOMe
H
H
H
H
H
H
2 
   1.5


TJ109
NH2
—COOMe
H
H
H
H
H
H
1 
   8.4


2 TR567
NH2
—COOMe
H
MeO
H
H
H
H
2 
   1.8


7 TR583
NH2
—COOMe
H
Me
H
MeO
H
H
2 
   1.5


9 TR595a
NH2
—COOMe
H
—
—
—
—
—
2 
   1.7


14 TR530
NH2
—COOEt
H
H
H
MeO
H
H
2d
   0.7


13 TR532
NH2
—COOEt
H
H
H
MeO
H
H
2 
   1.9


16a TR525
NH2
—COOEt
H
H
H
MeO
H
H
0 
 40


17 TR531
NH2
—COOEt
H
H
H
H
H
MeO
0 
 27


12 TR542
NH2
—COOEt
H
MeO
H
H
H
H
2 
 20


18 TR547
NH2
—COOEt
—COOEt
H
H
MeO
H
H
2 
 72


15 TR572
NH2
—COOMe
H
H
H
MeO
H
H
0 
 74


4 TR581
NH2
—COOMe
H
H
H
H
H
MeO
0 
 55


23 TR582
NH2
—CN
H
H
H
MeO
H
H
0 
233


5 TR584
NH2
—COOMe
H
MeO
H
H
MeO
H
2 
   1.6


21 TR585
NH2
—CN
H
MeO
H
H
H
H
2 
   8.3


22 TR586
NH2
—CN
H
H
H
MeO
H
H
2 
 16


16b TR587
NH2
—COOEt
H
MeO
H
H
MeO
H
2 
   0.76


20 TR588
NH2
—CN
H
H
H
H
H
H
2 
 47


24 TR589
NH2
—CN
H
MeO
H
H
MeO
H
2 
    0.015


19 TR592
NH2
—COOEt
—COOEt
MeO
H
H
MeO
H
2 
 20


10 TR596b
NH2
—COOMe
H
—
—
—
—
—
2 
 63


11 TR597c
NH2
—COOMe
H
—
—
—
—
—
2 
 50


8 TR599
NH2
—COOMe
Me
MeO
H
H
MeO
H
2 
 44


6 TR601
NH2
—COOMe
H
MeO
H
H
MeO
H
0 
 42


25 TJ19
NH2
—CONHMe
H
H
H
MeO
H
H
2 
 31


26 TJ8
NH2
—COOH
H
H
H
MeO
H
H
2 
 31


27 TJ9
NH2
—CONH2
H
H
H
MeO
H
H
2 
 39


28 TJ6A
NHCH3
—COOMe
H
H
H
MeO
H
H
2 
 23


29 TJ6B
N(CH3)2
—COOMe
H
H
H
MeO
H
H
2 
 18


30 TJ7
H
—COOMe
H
H
H
MeO
H
H
2 
 87





aPhenyl replaced by a thiophene;


bPhenyl replaced by a N-2 pyridine;


cPhenyl replaced by a N-3 pyridine;


dEthylene (C2H4) bridge replaced by an acetylene (—C≡C—) bridge.






Minor structural modifications resulted in a wide difference of antiproliferative activities, 24 being the most potent agent (IC50 range between 0.02 and 0.13 μM) and 23 being the least active agent (IC50 range between 24 and 320 μM), that is a difference by at least 3 orders of magnitude (Tables 2-4).
When considering the non-selective cytostatic compounds, the highest activity has been observed for the cyano-substituted compound 24, being 50-fold more cytostatic than the corresponding —COOEt-containing compound 16b. However, the replacement of the —COO alkyl group on the thiophene by a —C≡N group does not guarantee an improved antiproliferative activity since the cyano-containing compounds 23, 21, 22 and 20 are inferior to their —COOMe-containing counterparts, respectively compounds 15, 2, 3 and 1. Thus this invention relates to several and different compounds with different modifications with different cytostatic activity and tumor cell selectivity.
Evaluation of Cytostatic Activity of Compounds of Table 1a Against Leukemia/Lymphoma/Monocytic Cell Lines.
The compounds were first evaluated against a set of 7 different tumor cell lines derived from T-cell lymphomas (Table 2). Whereas the majority of the individual compounds showed rather narrow variations in their antiproliferative activity range for all these tumor cell lines, there were seven notable exceptions on this rule. These seven compounds (3, 1, 2, 7, 9, 14 and 13) were endowed with moderate middle-micromolar 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 5 tumor cell lines: 0.22 to 3.4 μM). Such a strikingly increased cytostatic activity against those five T-lymphocyte tumor cell lines was not observed for any of the other 2-aminothiophene derivatives (Table 2; FIG. 1A). Common features for these unusually selective compounds are a carboxymethyl or carboxyethyl group at R1 of the thiophene ring, one single methoxy on the aryl part of the molecule and a bridge between aryl and thiophene that consists of 2 carbons (either an ethylene or an acetylene). The compounds were also considerably less cytostatic against the B-lymphoma Raji and Daudi cells, as well as the monocytic U937 and THP-1 cells and the promyelocytic HL-60 cells (Table 2).







TABLE 2





Cytostatic activity (μM) of 2-aminothiophene derivatives against T-cell


lymphoma, B-lymphoma and monocyte-derived cell lines. HL60, human leukemia cells


that can be differentiated into monocytes; U937, human monocytic cells, derived from


acute myeloid leukemia (AML); THP-1, human monocytic cells that can be differentiated


to macrophage cells; Raji, non-Hodgkin leukemia/human B-lymphoma cells; Daudi,


human B-lymphoma cells.























CEM
Molt4/C8
Sup T 1
MT 2
C8166
HUT 78
MT 4





TR 560
0.90 ± 0.43
0.27 ± 0.08
0.58 ± 0.26
 0.57 ± 0.064
 1.3 ± 0.87
21 ± 12
25 ± 12


TR 575
1.5 ± 1.3
1.2 ± 0.6
 1.1 ± 0.73
 0.79 ± 0.092
2.2 ± 0  
36 ± 13
  27 ± 0.71


TR 567
 1.8 ± 0.92
0.81 ± 0.49
1.5 ± 0  
 2.1 ± 0.071
 3.0 ± 0.21
 13 ± 4.2
 14 ± 2.8


TR 583
 1.5 ± 0.28
0.27 ± 0.27
0.95 ± 0.64
 1.8 ± 0.64
 1.6 ± 0.95
39 ± 27
  60 ± 0.71


TR 595
1.7 ± 1.1
0.56 ± 0.57
 1.3 ± 0.21
 0.57 ± 0.078
 2.2 ± 0.35
 38 ± 8.5
 30 ± 4.9


TR 530
0.73 ± 0.62
0.22 ± 0  
 1.5 ± 0.14
 1.5 ± 0.21
 2.8 ± 0.14
 44 ± 6.4
22 ± 15


TR 532
 1.9 ± 0.99
0.34 ± 0  
 1.5 ± 0.071
 1.7 ± 0.14
 3.4 ± 0.071
 22 ± 9.2
 8.5 ± 0.92


TR 525
40 ± 1 
 18 ± 2.1
 86 ± 7.1
 52 ± 5.7
102 ± 16 
117 ± 21 
>100


TR 531
27 ± 10
 31 ± 2.1
28 ± 12
 27 ± 4.2
133 ± 7.8 
108 ± 5.7 
 44 ± 5.7


TR 542
20 ± 4 
6.8 ± 0  
 23 ± 2.1
79 ± 17
143 ± 32 
107 ± 70 
 41 ± 5.7


TR 547
 72 ± 6.4
54 ± 6 
73 ± 13
31 ± 29
122 ± 12 
120 ± 44 
 38 ± 3.5


TR 572
 74 ± 3.5
44 ± 9 
55 ± 30
 12 ± 1.4
179 ± 18 
26 ± 17
40 ± 12


TR 581
55 ± 0 
39 ± 8 
45 ± 31
103 ± 52 
149 ± 14 
152 ± 44 
36 ± 15


TR 582
233 ± 29 
275 ± 89 
>200
≧161
>200
>200
76 ± 35


TR 584
 1.6 ± 0.071
0.42 ± 0.11
0.61 ± 0.26
 4.2 ± 0.79
 1.4 ± 0.76
2.6 ± 2.1
 1.6 ± 0.21


TR 585
8.3 ± 2.4
2.0 ± 0.2
 4.7 ± 0.38
4.75 ± 0.08
 16 ± 6.5
7.1 ± 1.3
 8.1 ± 0.78


TR 586
16 ± 17
1.4 ± 0.8
79 ± 42
6.8 ± 3.7
57 ± 56
 17 ± 8.1
74 ± 37


TR 587
0.76 ± 0.48
1.1 ± 1.0
 0.54 ± 0.007
0.70 ± 0.30
0.82 ± 0.16
0.43 ± 0.24
 1.7 ± 0.59


TR 588
47 ± 25
8.5 ± 3.4
>200
6.65 ± 1.9 
≧200
 18 ± 7.1
75 ± 35


TR 589
0.015 ± 0   
0.058 ± 0.056
0.017 ± 0.012
0.027 ± 0.001
0.021 ± 0.009
0.062 ± 0.039
0.086 ± 0.048


TR 592
20 ± 6 
24 ± 17
 4.1 ± 0.40
8.1 ± 1.2
 25 ± 3.5
26 ± 3.5
36 ± 16


TR 596
 63 ± 7.8
21 ± 4 
99 ± 0 
 48 ± 7.1
≧200
142 ± 83 
>100


TR 597
 50 ± 6.4
47 ± 15
147 ± 66 
38 ± 15
≧200
59 ± 33
>100


TR 599
44 ± 0 
47 ± 16
32 ± 15
37 ± 29
110 ± 31 
 21 ± 1.4
58 ± 21


TR 601
 42 ± 1.4
28 ± 6 
38 ± 25
 14 ± 2.8
102 ± 1.4 
109 ± 82 
50 ± 25



















HL 60
U937
THP-1
Raji
Daudi
L1210






TR 560
4.7 ± 6.4
 40 ± 5.7
36 ± 0 
57 ± 16
46 ± 11 
11 ± 1



TR 575
5.7 ± 7.2
 46 ± 7.1
 38 ± 6.4
 50 ± 3.5
38 ± 2.1
25 ± 0



TR 567
4.0 ± 3.3
  10 ± 0.85
 13 ± 1.4
 19 ± 4.2
 13 ± 0.71
17 ± 5



TR 583
4.9 ± 5.4
 40 ± 2.8
 32 ± 4.9
93 ± 11
61 ± 1.4
248 ± 40



TR 595
6.1 ± 8.6
 42 ± 3.5
 41 ± 2.8
 46 ± 1.4
37 ± 3.5
41 ± 2



TR 530
6.2 ± 8.5
 41 ± 1.4
 44 ± 1.4
 48 ± 1.4
43 ± 2.8
40 ± 2



TR 532
7.0 ± 8.7
 43 ± 1.4
 23 ± 3.5
 39 ± 2.8
43 ± 2.1
 20 ± 10



TR 525
 62 ± 9.9
115 ± 19 
102 ± 2.8 
>100

44 ± 3



TR 531
 17 ± 5.7
 73 ± 3.5
25 ± 19
48 ± 13

41 ± 3



TR 542
 11 ± 2.2
 80 ± 5.7
31 ± 13
55 ± 12

40 ± 0



TR 547
46 ± 25
 80 ± 3.5
 26 ± 2.8
58 ± 13

105 ± 48



TR 572
 11 ± 1.4
 63 ± 7.1
 21 ± 0.7
61 ± 18

 60 ± 12



TR 581
 16 ± 3.5
 82 ± 7.1
69 ± 13
59 ± 11

204 ± 85



TR 582
≧135
>200
124 ± 25 
>100

230 ± 65



TR 584
0.89 ± 0.30
 4.2 ± 0.070
 3.1 ± 0.070
 2.2 ± 0.28

 1.8 ± 0.1



TR 585
 3.5 ± 0.57
3.4 ± 2.8
 3.8 ± 0.60
 13 ± 3.5

15 ± 4



TR 586
 1.9 ± 0.42
 17 ± 2.8
 4.3 ± 0.60
36 ± 21

14 ± 4



TR 587
0.46 ± 0.35
 0.92 ± 0.080
0.71 ± 0.18
 1.8 ± 0.42

 1.2 ± 0.4



TR 588
 3.9 ± 0.50
 42 ± 2.1
 8.4 ± 0.28
39 ± 21

40 ± 6



TR 589
0.024 ± 0.004
0.036 ± 0.001
0.022 ± 0.003
0.063 ± 0.013

 0.064 ± 0.025



TR 592
  17 ± 0.70
20 ± 15
 20 ± 1.4
 41 ± 7.1

34 ± 3



TR 596
73 ± 30
>200
116 ± 49 
76 ± 35

101 ± 28



TR 597
63 ± 15
>200
>200
>100

235 ± 49



TR 599
 18 ± 4.9
 68 ± 0.7
 9.0 ± 0.60
49 ± 13

46 ± 3



TR 601
21 ± 0 
 85 ± 2.8
 25 ± 5.7
45 ± 11

50 ± 5









Evaluation of Cytostatic Activity of Compounds of Table 1a Against a Broad Variety of Tumor Cell Lines, Different from Leukemia/Lymphoma/Monocytic Cell Lines.

A moderate (middle to higher micromolar) cytostatic activity range was observed against a variety of other tumor cell types including osteosarcoma HOS CD4+, human MCF-7 and murine FM3A mammary carcinoma, cervix carcinoma HeLa, colorectal carcinoma HT29, glioma U87 and U87 CD4+.CXCR4+.CCR5+, and non-tumorigenic kidney embryo fibroblast 293T, human embryonic lung fibroblast HEL and bovine aortic endothelial BAEC cells (Table 3; FIG. 1B). The human kidney carcinoma Caki-1 was in general markedly more sensitive to the cytostatic activity of 3, 1, 2, 7, 9, 14 and 13 than the Caki-2 cells.







TABLE 3





Cytostatic activity (μM) of 2-aminothiophene derivatives against a broad variety


of tumor cell lines. HOS, OST TK−, human osteosarcoma cells; MCF-7, human mammary


carcinoma cells; FM3A, murine mammary carcinoma cells; HeLa, human cervix


carcinoma cells; 293T, human embryonic kidney cells; HT19, human colon carcinoma


cells; U87, human glioma cells; Caki-1 and Caki-2, human kidney carcinoma cells; HEL,


human embryonic fibroblast cells; BAEC, bovine artery endothelial cells.























HOS CD4+
OST TK-
MCF-7
FM3A
Hela
Caki-1
Caki-2





TR 560
68 ± 25 
4.7 ± 3.3
 38 ± 2.1
75 ± 36
39 ± 11
1.7 ± 1.1
33 ± 32


TR 575
48 ± 1.4
9.8 ± 7.4
 36 ± 9.2
67 ± 18
 38 ± 2.8
6.5 ± 3.5
 43 ± 4.2


TR 567
23 ± 4.2
7.6 ± 2.4
 22 ± 3.5
  37 ± 0.71
 17 ± 2.1
3.7 ± 1.8
4.5 ± 1.9


TR 583
67 ± 6.4
 10 ± 6.9
79 ± 30
 49 ± 9.9
105 ± 68 
4.5 ± 3.8
≧200


TR 595
50 ± 12 
8.2 ± 6.9
25 ± 11
 50 ± 2.8
36 ± 7 
 2.3 ± 0.14
 45 ± 7.1


TR 530
35 ± 7.1
3.8 ± 1.7
  42 ± 0.71
 40 ± 3.5
 42 ± 5.5
5.3 ± 2.2
88 ± 17


TR 532
25 ± 5.7
5.4 ± 3.3
 36 ± 5.7
 46 ± 6.0
39 ± 0 
4.6 ± 2.1
66 ± 37


TR 525

 79 ± 2.8
 81 ± 4.9
49 ± 6 
39 ± 4 
83 ± 36
159 ± 19 


TR 531

 18 ± 1.4
48 ± 11
49 ± 1 
40 ± 9 
 17 ± 3.5
 17 ± 3.5


TR 542

  41 ± 0.71
 60 ± 0.7
53 ± 7 
45 ± 6 
62 ± 12
16 ± 10


TR 547

 71 ± 7.8
87 ± 21
 86 ± 5.6
 41 ± 7.1
87 ± 21
57 ± 24


TR 572

 25 ± 2.1
119 ± 52 
97 ± 32
63 ± 19
4.4 ± 1.9
9.0 ± 1.4


TR 581

 45 ± 4.2
29 ± 20
275 ± 206
 41 ± 4.9
 39 ± 2.1
28 ± 18


TR 582

134 ± 69 
117 ± 58 
320 ± 14 
168 ± 15 
19 ± 18
≧200


TR 584

0.68 ± 0.33
0.93 ± 0.23
 2.5 ± 0.28
0.83 ± 0.25
2.5 ± 0.6
0.93 ± 0.28


TR 585

3.96 ± 1.56
5.42 ± 0.69
  25 ± 0.71
8.3 ± 1.5
4.6 ± 1.3
4.8 ± 4.5


TR 586

27 ± 18
114 ± 62 
34 ± 23
 21 ± 9.9
 1.9 ± 0.42
28 ± 27


TR 587

0.51 ± 0.42
0.83 ± 0.35
 2.0 ± 0.28
 0.33 ± 0.028
1.4 ± 0.5
3.2 ± 2.4


TR 588

27 ± 18
 26 ± 2.1
67 ± 23
27 ± 28
1.3 ± 0.4
78 ± 69


TR 589

0.024 ± 0.013
 0.026 ± 0.0014
0.050 ± 0.046
0.13 ± 0.06
0.016 ± 0.001
0.042 ± 0.004


TR 592

 17 ± 5.7
27 ± 19
33 ± 9 
11 ± 3 
 24 ± 0.7
 42 ± 2.1


TR 596

135 ± 92 
72 ± 45
210 ± 17 
45 ± 3 
17 ± 13
>200


TR 597

≧111
71 ± 30
164 ± 30 
170 ± 25 
7.7 ± 0.5
>200


TR 599

34 ± 23
 11 ± 8.5
 48 ± 3.5
38 ± 11
57 ± 38
52 ± 17


TR 601

32 ± 18
44 ± 28
59 ± 11
 39 ± 9.2
8.8 ± 3.1
 24 ± 9.2






















U87






293T
HT29
U87
CD4+/CXCR4+/CCR5+
HEL
BAEC






TR 560
33 ± 25
 45 ± 5.7
 58 ± 6.4
52 ± 3.5
52 ± 25
39 ± 8.5



TR 575
45 ± 16
 41 ± 2.8
 50 ± 3.5
73 ± 3.5
38 ± 12
26 ± 4.2



TR 567
22 ± 15
 17 ± 7.8
38 ± 0 
32 ± 2.1
 20 ± 7.8
20 ± 4.2



TR 583
84 ± 23
>100
>100
48 ± 6.4
87 ± 19
44 ± 22 



TR 595
 44 ± 5.7
 30 ± 5.7
 51 ± 2.1
51 ± 4.9
30 ± 13
27 ± 7.1



TR 530
  37 ± 0.71
48 ± 23
 37 ± 2.8
28 ± 3.5
35 ± 13
16 ± 16 



TR 532
  41 ± 0.71
27 ± 13
 38 ± 1.4
37 ± 9.9
 20 ± 8.5
 23 ± 0.71



TR 525
>100
93 ± 11
90 ± 14

 43 ± 9.9
74 ± 26 



TR 531
 58 ± 7.1
 43 ± 2.8
 38 ± 8.5

 25 ± 3.5
31 ± 6.4



TR 542
>100
>100
>100

 43 ± 4.9
>100



TR 547
 58 ± 2.8
 46 ± 3.5
81 ± 27

 44 ± 3.5
82 ± 26 



TR 572
>100
 42 ± 3.5
 71 ± 3.5

34 ± 20
47 ± 3.5



TR 581
 72 ± 4.9
 43 ± 1.4
 42 ± 5.7

 24 ± 2.1
38 ± 2.1



TR 582
>100
>100
>100

  33 ± 0.71
>100



TR 584
1.9 ± 0  
 1.8 ± 0.21
 1.8 ± 0.071

 0.87 ± 0.014
 2.0 ± 0.28



TR 585
  10 ± 0.071
 8.9 ± 0.92
  10 ± 0.78

 7.2 ± 0.49
9.7 ± 1.8 



TR 586
 28 ± 3.5
 34 ± 6.4
>100

>100
38 ± 2.1



TR 587
 1.0 ± 0.11
 1.7 ± 0.14
1.6 ± 1.1

0.27 ± 0.22
0.93 ± 0.67 



TR 588
34 ± 14
 66 ± 6.4
 46 ± 7.8

 30 ± 6.4
45 ± 4.9



TR 589
<0.032
0.046 ± 0.012
0.038 ± 0.008

<0.032
<0.032



TR 592
 30 ± 7.1
 43 ± 4.2
 39 ± 2.8

 35 ± 6.4
31 ± 4.2



TR 596
>100
>100
>100

 43 ± 9.2
>100



TR 597
>100
>100
>100

≧100
>100



TR 599
 52 ± 2.1
 47 ± 2.1
 41 ± 4.9

 46 ± 3.5
52 ± 46 



TR 601
43 ± 16
42 ± 0 
25 ± 0 

 17 ± 5.7
26 ± 7.1









Evaluation of Compounds of Table 1a Against Prostate and Hepatoma Cancer Cell Lines.

However, when the compounds were evaluated for their inhibitory potential against the proliferation of three human hepatoma cell lines (i.e. Huh7, HepG2C3A and PLC/PRF/5) and seven different prostate cancer cell lines, 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 Huh7 cells and an (even more) unusual increased cytostatic potential against several of the prostate tumor cell lines (Table 4; FIG. 1C). Nanomolar cytostatic activity was observed against two different PC3 sources and against LNCaP cells, low micromolar antiproliferative activity was shown for DU145 and 22Rv1 prostate cancer cells, and moderate 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 were preferentially cytostatic to the PC3 and LNCaP cell lines compared to the other prostate tumor cell lines.







TABLE 4





Cytostatic activity (μM) of 2-aminothiophene derivatives against


prostate and hepatoma cancer cell lines





















PC3 (rega)
BPH-1 (UZ)
DU145 (UZ)
PC-3 (UZ)
LNCaP (UZ)





TR 560
 0.19 ± 0.035
60 ± 0 
5.8 ± 2.8
0.34 ± 0.14
0.22 ± 0.042


TR 575
0.24 ± 0.19
 52 ± 1.4
 5.4 ± 0.96
 0.51 ± 0.064
0.35 ± 0.014


TR 567
0.71 ± 0.28
 39 ± 2.8
 11 ± 7.0
 1.4 ± 0.071
0.43 ± 0.14 


TR 583
0.57 ± 0.30
>100
 14 ± 1.2
 1.3 ± 0.071
0.35 ± 0.067


TR 595
0.25 ± 0.27
 46 ± 4.9
 15 ± 8.5
 0.60 ± 0.071
0.38 ± 0.10 


TR 530
0.29 ± 0.14
 41 ± 1.4
9.3 ± 8.4
0.96 ± 0.20
0.26 ± 0.081


TR 532
0.67 ± 0.14
 43 ± 2.8
8.4 ± 1.6
 1.1 ± 0.49
0.34 ± 0.050


TR 525
 13 ± 9.6
 36 ± 1.4
41 ± 24
 20 ± 3.5
 2.3 ± 0.071


TR 531
 25 ± 6.4
39 ± 0 
 34 ± 4.2
  28 ± 0.71
11 ± 5.0 


TR 542
8.6 ± 3.4
 74 ± 4.2
 34 ± 7.1
8.5 ± 3.5
3.8 ± 2.9 


TR 547
38 ± 12
 65 ± 9.2
89 ± 16
 31 ± 5.7
19 ± 10 


TR 572
5.5 ± 4.0
 58 ± 6.4
 18 ± 9.2
4.3 ± 1.7
1.1 ± 0.84


TR 581
 26 ± 8.5
 50 ± 5.7
 35 ± 6.4
 28 ± 4.9
9.7 ± 5.1 


TR 582
69 ± 21
>100
>100
>100
4.8 ± 3.4 


TR 584
0.36 ± 0.18
 1.3 ± 0.071
1.6 ± 0  
0.81 ± 0.42
0.38 ± 0.11 


TR 585
 1.7 ± 0.35
 7.0 ± 0.42
6.4 ± 1.8
 2.6 ± 0.64
2.7 ± 0.95


TR 586
1.8 ± 2.0
59 ± 18
8.3 ± 2.5
 4.2 ± 0.14
0.83 ± 0.35 


TR 587
 0.33 ± 0.099
 0.39 ± 0.007
 1.4 ± 0.21
 0.52 ± 0.007
0.35 ± 0.064


TR 588
3.1 ± 1.2
≧100
 24 ± 4.9
 11 ± 4.2
5.3 ± 4.3 


TR 589
<0.032
<0.032
0.061 ± 0.016
<0.032
<0.032


TR 592
 12 ± 2.1
 28 ± 3.5
 18 ± 2.1
 8.5 ± 0.85
4.1 ± 3.6 


TR 596
8.5 ± 7.8
>100
>100
 8.6 ± 0.21
4.3 ± 3.2 


TR 597
6.3 ± 4.7
>100
60 ± 21
 12 ± 5.5
7.4 ± 0.75


TR 599
24 ± 12
 43 ± 3.5
35 ± 16
 25 ± 5.7
8.7 ± 4.7 


TR 601
23 ± 14
31 ± 0 
 37 ± 2.1
25 ± 0 
21 ± 8.5 
















PNT1A (UZ)
22Rv1 (UZ)
PLC/PRF/5
HepG2C3A
Huh-7





TR 560
36 ± 9.2
3.7 ± 2.2
36 ± 1.4
8.4 ± 2.1
0.95 ± 0.21


TR 575
39 ± 3.5
 9.1 ± 0.35
44 ± 2.1
 10 ± 5.2
 1.7 ± 0.21


TR 567
17 ± 2.8
5.7 ± 2.8
18 ± 4.2
 8.6 ± 0.28
3.4 ± 1.2


TR 583
>100
21 ± 17
62 ± 21 
  13 ± 0.71
 4.6 ± 0.28


TR 595
40 ± 7.1
 22 ± 9.2
28 ± 2.8
 8.1 ± 0.71
 1.3 ± 0.28


TR 530
38 ± 7.8
6.3 ± 2.5
41 ± 6.4
 12 ± 2.1
 1.5 ± 0.28


TR 532
37 ± 9.9
 15 ± 7.9
24 ± 2.1
 6.7 ± 0.35
 1.6 ± 0.14


TR 525
42 ± 5.7
 16 ± 5.5
>100
>100
>100


TR 531
45 ± 1.4
 20 ± 5.1
 21 ± 0.71
 10 ± 5.2
43 ± 12


TR 542
45 ± 0  
  33 ± 0.71
39 ± 5.7
  30 ± 0.71
28 ± 17


TR 547
56 ± 0  
 31 ± 4.2
75 ± 35 
48 ± 16
51 ± 14


TR 572
53 ± 2.1
69 ± 44
42 ± 2.1
 28 ± 8.5
 42 ± 7.8


TR 581
58 ± 13 
 42 ± 7.6
36 ± 4.2
38 ± 27
59 ± 17


TR 582
>100
34 ± 26
49 ± 3.5
≧100
>100


TR 584
0.77 ± 0.31 
 0.31 ± 0.087
 1.3 ± 0.21
4.5 ± 1.7
 2.2 ± 0.49


TR 585
 8.0 ± 0.42
9.4 ± 5.8
 6.7 ± 0.64
7.2 ± 4.0
 8.3 ± 0.78


TR 586
38 ± 14 
 17 ± 8.1
 37 ± 0.71
46 ± 13
>100


TR 587
 0.41 ± 0.028
0.19 ± 0.11
0.26 ± 0.15 
 1.1 ± 0.49
0.71 ± 0.35


TR 588
66 ± 31 
61 ± 34
41 ± 3.5
64 ± 52
 21 ± 7.1


TR 589
<0.032
<0.032
0.041 ± 0.013 
0.29 ± 0.18
<0.16


TR 592
 31 ± 0.71
 14 ± 8.3
25 ± 0  
34 ± 12
34 ± 14


TR 596
>100
77 ± 27
>100
69 ± 45
46 ± 34


TR 597
>100
≧100
>100
>100
78 ± 32


TR 599
46 ± 2.1
28 ± 19
36 ± 2.8
34 ± 22
 52 ± 7.8


TR 601
38 ± 5.7
 11 ± 4.8
 26 ± 0.71
22 ± 18
49 ± 19









Thus, among a series of closely-related 2-aminothiophene analogues seven derivatives have been identified that show a preferential cytostatic activity against several T-cell lymphoma, hepatoma and prostate cancer-derived cell lines, but not against any other type of tumor or non-tumorigenic cell line included in this study.
The unusual selectivity of the compounds for several T-cell lymphoma, hepatoma and prostate cancer cell lines together with the structure-activity relationship (SAR) among the 2-aminothiophenes of this study allow us to deduce some SAR parameters. (i) The presence of a carbon bridge linking the thiophene and the phenyl group seems to be necessary for the cytostatic selectivity. For example, compounds 3, 13 and 14 containing an ethylene or acetylene bridge are selective, while their corresponding derivatives without a bridge (15 and 16a) are not selective anymore against the lymphoma, hepatoma and prostate cancer cell lines. (ii) It was surprising to notice that compound 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 & 14. However, replacing —COO alkyl by —CN annihilates the selectivity (compare compounds 21 with 2, 22 with 3, and 20 with 1). (iii) The presence of a second —COOEt on the thiophene is detrimental for selectivity (compare compound 18 with 13). (iv) When the —COOMe at R1 was replaced by an amide (27), methylamide (25), or the free carboxylic acid (26) the selective cytostatic activity was markedly decreased. (v) When the —NH2 at R0 was replaced by H, NHCH3 or N(CH3)2 activity/selectivity was also markedly affected. (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 at the R3 position (compound 3), selectivity is present, but adding an additional MeO group at R6 (compound 5) annihilates selectivity. (vii) Finally, when the aryl had been replaced by a thiazole (compound 9), selectivity is retained, but not when the aryl had been replaced by a pyridine (compounds 10 and 11).
Example 4
Biological Activity of Further Compounds of this Invention, Compounds of Formula I(b)
Following additional structural features to preserve the cytostatic selectivity could also be derived (Table 1b): (i) the ethylene bridge can be replaced by a thiomethyl (—SCH2—) (TJ22), but not by an oxymethyl (—OCH2—) (TJ25) or aminomethyl (—NHCH2) (TJ51) bridge. Also bridge elongation to 3 atom units is allowed (i.e. a methylthiomethyl (—CH2SCH2—) (TJ57) bridge). In several cases, the MeO group at the aryl moiety can be replaced by a Me group or a H atom (i.e. TJ55, TJ54P, TJ57).







TABLE 1b







2-Amino-3-carboxylic acid ester derivatives with heteroatom substitutions in the bridge part,


and substituent substitutions in the aryl part


(Ib)








 





















IC50 (μM)
















R3
R4
R5
R6
R7
X
(CEM)
(HeLa)





TJ22
H
H
OMe
H
H
—SCH2—
0.62 ± 0.33
58 ± 1 


TJ25
H
H
OMe
H
H
—OCH2—
43 ± 13
68 ± 13


TJ51
H
H
OMe
H
H
—NHCH2—
96 ± 16
78 ± 15


TJ55
H
H
Me
H
H
—SCH2—
1.5 ± 0.3
72 ± 30


TJ54P
H
Me
H
H
H
—SCH2—
4.1 ± 1.7
108 ± 0 


TJ57
H
H
H
H
H
—CH2SCH2—
3.5 ± 1.1
61 ± 38


TJ4
H
H
H
H
H
—SCH2—
1.3 ± 0.6
108 ± 7 


TJ21*
H
H
OMe
H
H
—SCH2—
4.9 ± 1.4
112 ± 2 


TJ54SM*
H
Me
H
H
H
—SCH2—
6.9 ± 0.7
121 ± 13 


TJ86*
H
H
H
H
H
—SCH2—
13 ± 7 
184 ± 6 


TJ113**
—
—
—
—
—
—SCH2—
0.90 ± 0.04
74 ± 6 


TJ117***
—
—
—
—
—
—CH2SCH2—
4.9 ± 0.1
102 ± 6 


TJ130
H
H
OMe
H
H
—CH2SCH2—
3.9 ± 1.4
88 ± 10


TJ118****
—
—
—
—
—
—CH2SCH2—
3.3 ± 0.9
99 ± 5 


TJ127*
H
H
OMe
H
H
—CH2SCH2—
25 ± 8 
111 ± 20 








**Phenyl replaced by a naphthyl.


***Phenyl replaced by a furanyl.


****Phenyl replaced by a thiophene.













TABLE 1c







2-Amino-3-carboxylic acid ester derivatives without the aryl moiety








 










IC50 (μM)
Compound










Y
CEM
HeLa
code









13 ± 5 
97 ± 2 
TJ108


 






0.28 ± 0.10
21
TJ110


 






0.13
81
TJ114


 






6.6 ± 3.2
104 ± 1 
TJ119*


 






0.52 ± 0.37
81 ± 5 
TJ125





*point of attachment to the thiophene;












Example 5
Antimetabolic Activity of T-Cell Lymphoma Selective and Non-Selective Compounds
To reveal initial 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 potential antimetabolic activity. Prototype compound 3 and the other tumor-selective compounds have been evaluated for their antimetabolic activity by measuring their inhibitory effect against incorporation of dThd in DNA, uridine in RNA and leucine in proteins of T-lymphocyte CEM and B-lymphocyte Raji cells (Table 5). None of the compounds showed preferential inhibitory activity against DNA, RNA or protein synthesis. The inhibitory activity of the compounds against macromolecular (DNA, RNA and protein) synthesis in CEM tumor cells were generally far more pronounced for the T-lymphoma-selective compounds (1, 2, 3, 7, 9, 13 and 14) than for the tumor-nonselective compounds. Also, virtually none of the compounds, including the tumor-selective compounds, showed pronounced inhibitory activity against the B-lymphocyte Raji cells (Table 5).







TABLE 5







Inhibitory effect of test compounds against the incorporation


of radiolabeled precursors in cellular macromolecules










IC50a (μM)










Compound
CEM
Raji













[CH3—3H]dThd incorporation










1
3.1 ± 1.0
≧100


2
6.4 ± 4.1
54 ± 8 


3
2.8 ± 1.3
63 ± 34


7
3.1 ± 0.3
 >100


9
3.2 ± 1.2
39 ± 33


13 
11 ± 4 
 >100


14 
2.3 ± 1.0
≧100


5
7.5 ± 5.5
82 ± 6 


12 
68 ± 14
≧100


16a
15 ± 3 
≧100


19 
17 ± 8 
25 ± 16


21 
>100
≧100


24 
>100
≧100










[5-3H]uridine incorporation










1
0.96 ± 0.37
 >100


2
2.6 ± 0.6
68 ± 8 


3
1.1 ± 0.7
47 ± 19


7
1.0 ± 0.5
 >100


9
1.9 ± 0.7
27 ± 14


13 
3.0 ± 0.1
 >100


14 
0.66 ± 0.01
60 ± 57


5
2.4 ± 0.2
≧100


12 
39 ± 32
≧100


16a
12 ± 5 
82 ± 26


19 
31 ± 22
31 ± 19


21 
≧100 
≧100


24 
44 ± 11
90 ± 14










[4,5-3H]leucine incorporation










1
0.61 ± 0.04
84 ± 6 


2
1.4 ± 0.9
45 ± 8 


3
0.19 ± 0.13
52 ± 10


7
1.9 ± 1.6
 >100


9
0.66 ± 0.13
43 ± 7 


13 
1.6 ± 1.1
≧100


14 
0.20 ± 0.07
≧100


5
1.1 ± 0.5
62 ± 1 


12 
23 ± 14
94 ± 7 


16a
6.2 ± 7.6
≧100


19 
17 ± 0 
23 ± 7 


21 
11 ± 8 
≧100


24 
 20 ± 0.16
39 ± 36





a50% Inhibitory concentration or compound concentration required to inhibit incorporation of the radiolabeled precursor into TCA-insoluble material by 50%.






FACS analysis revealed that the compound 3-exposed CEM cells did not selectively cause accumulation of the tumor cells in a specific cell phase (i.e. Mitosis, G1, S or G2) ( ) and thus, seems not to cause selective inhibition of a specific cell function directly related to any of the cell phases. However, 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. 2). 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 was only observed after 12 h in the presence of 100 μM compound 3. Thus, compound 3 seemed to induce caspase-3-related apoptosis in PC-3 cell cultures.
The unusual and surprising cytostatic selectivity was observed for a number of 2-aminothiophene derivatives substituted at the 5-position of the thiophene ring. These compounds showed a preferential anti-proliferative activity against a variety of T-cell lymphoma, hepatoma and prostate cancer cell lines. They do not discriminate for inhibition of DNA, RNA and protein synthesis, and do not specifically lead to an accumulation of the drug-exposed tumor cells in a well-defined cell phase. The prototype compound 3 induced apoptosis in prostate tumor cell cultures.
Example 6
Synthesis of the Exemplified Compounds and their Code
The following compounds (with their code) are generated according to the processes described in the present invention:

    Methyl 2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TR560)
    Methyl 2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate (TR575)
    Methyl 2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate (TR567)
    Methyl 2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate (TR583)
    Methyl 2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate (TR595)
    Ethyl 2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate (TR530)
    Ethyl 2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TR532)
    Ethyl 2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate (TR525)
    Ethyl 2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate (TR531)
    Ethyl 2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate (TR542)
    Ethyl 2-amino-4-ethoxycarbonyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TR547)
    Methyl 2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate (TR572)
    Methyl 2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate (TR581)
    2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene (TR582)
    Methyl 2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate (TR584)
    2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene (TR585)
    2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene (TR586)
    Ethyl 2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate (TR587)
    2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene (TR588)
    2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene (TR589)
    Ethyl 2-amino-4-ethoxycarbonyl 5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate (TR592)
    Methyl 2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate (TR596)
    Methyl 2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate (TR597)
    Methyl 2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate (TR599)
    Methyl 2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate (TR601)
    N-Methyl 2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide (TJ19)
    2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ8)
    2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide (TJ9)
    Methyl 2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ6A)
    Methyl 2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ6B)
    Methyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ7)
    Methyl 2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ22)
    Methyl 2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate (TJ25)
    Methyl 2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate (TJ51)
    Methyl 2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ55)
    Methyl 2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ54P)
    Methyl 2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate (TJ57)
    Methyl 2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ4)
    Methyl 2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ21)
    Methyl 2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ54SM)
    Methyl 2-amino-5-benzylthiophene-3-carboxylate (TJ109)
    Methyl 2-amino-5-(6-methylhept-5-en-2-yl)thiophene-3-carboxylate (TJ108)
    Methyl 2-amino-5-(non-8-enyl)thiophene-3-carboxylate (TJ110)
    Methyl 2-amino-5-heptylthiophene-3-carboxylate (TJ114).
    Methyl 2-phthalimido-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ86)
    Methyl 2-phthalimido-5-(2-(4-methoxybenzyl)sulfanylmethyl)thiophene-3-carboxylate (TJ127)
    Methyl 2-phthalimido-5-(2-(butyl)sulfanylmethyl)thiophene-3-carboxylate (TJ119)
    Methyl 2-amino-5-((naphthalen-2-ylthio)methyl)thiophene-3-carboxylate (TJ113)
    Methyl 2-amino-5-((furan-2-ylmethylthio)methyl)thiophene-3-carboxylate (TJ117)
    Methyl 2-amino-5-((thiophen-2-ylmethylthio)methyl)thiophene-3-carboxylate (TJ118)
    Methyl 2-amino-5-(butylthiomethyl)thiophene-3-carboxylate (TJ125)
    Methyl 2-amino-5-((4-methoxyphenylthio)methyl)thiophene-3-carboxylate (TJ130).


2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carboxylic acid ethyl ester (TR525)




2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carboxylic acid ethyl ester 
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a yellow solid (82% yield) mp 127-129° C. 1H NMR (CDCl3, 200 MHz) δH 7.36 (d, 2H, J=8.8 Hz), 7.09 (s, 1H), 6.87 (d, 2H, J=8.8 Hz), 5.93 (bs, 2H), 4.29 (q, 2H, J=7.0 Hz), 3.81 (s, 3H), 1.36 (t, 3H, J=7.0 Hz). [M+1]278.3 (M+H+, C14H16NO3S requires 278.08).
2-Amino-5-(4-methoxy-phenylethynyl)-thiophene-3-carboxylic acid ethyl ester (TR530)




2-Amino-5-(4-methoxy-phenylethynyl)-thiophene-3-carboxylic acid ethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a yellow solid (89% yield) mp 90-92° C. 1H NMR (CDCl3, 200 MHz) δH 7.36 (d, 2H, J=8.8 Hz), 7.16 (s, 1H), 6.84 (d, 2H, J=8.8 Hz), 6.14 (bs, 2H), 4.26 (q, 2H, J=7.2 Hz), 3.81 (s, 3H), 1.33 (t, 3H, J=7.2 Hz). [M+1]302.02 (M+H+, C16H16NO3S requires 302.08).
2-Amino-5-(2-methoxy-phenyl)-thiophene-3-carboxylic acid ethyl ester (TR531)




2-Amino-5-(2-methoxy-phenyl)-thiophene-3-carboxylic acid ethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a yellow oil (72% yield). 1H NMR (CDCl3, 200 MHz) δH 7.53 (dd, 1H, J=7.2 Hz, J=1.4 Hz), 7.41 (s, 1H), 7.17 (m, 1H), 6.95 (m, 2H), 5.99 (bs, 2H), 4.31 (q, 2H, J=7.0 Hz), 3.89 (s, 3H), 1.37 (t, 3H, J=7.0 Hz). [M]277.9 (M+, C14H15NO3S requires 277.08).
2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester (TR532)




2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a colorless oil (81% yield). 1H NMR (CDCl3, 200 MHz) δH 7.10 (d, 2H, J=8.6 Hz), 6.82 (d, 2H, J=8.6 Hz), 7.64 (s, 1H), 5.81 (bs, 2H), 4.23 (q, 2H, J=7.2 Hz), 3.79 (s, 3H), 2.84 (s, 4H), 1.33 (t, 3H, J=7.2 Hz).
2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester (TR542)




2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a colorless oil (100% yield). 1H NMR (CDCl3, 200 MHz) δH 7.25 (m, 2H), 6.87 (m, 2H), 6.67 (s 1H), 5.80 (bs, 2H), 4.25 (q, 2H, J=7.0 Hz), 3.83 (s, 3H), 2.87 (s, 4H), 1.33 (t, 3H, J=7.0 Hz).
2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3,4-dicarboxylic acid diethyl ester (TR547)




2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3,4-dicarboxylic acid diethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (78% yield). H NMR (CDCl3, 200 MHz) δH 7.06 (d, 2H, J=8.6 Hz), 6.80 (d, 2H, J=8.6 Hz), 5.92 (bs, 2H), 4.23 (m, 4H), 3.76 (s, 3H), 2.83 (m, 4H) 1.29 (m, 6H).
2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester (TR560)




2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester 
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a colorless solid (100% yield). 1H NMR (CDCl3, 200 MHz) 67.09 (d, 2H, J=8.6 Hz), 6.82 (d, 2H, J=8.6 Hz), 6.62 (s 1H), 5.81 (bs, 2H), 3.78 (s, 3H), 3.83 (s, 3H), 2.87 (s, 4H).
2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester (TR567)




2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (87% yield). 1H NMR (CDCl3, 200 MHz) δH 7.15 (m, 2H), 6.88 (m, 2H), 6.66 (s 1H), 5.83 (bs, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 2.87 (s, 4H).
2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carboxylic acid methyl ester (TR572)




2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a yellow solid (80% yield). mp 117-119° C. 1H NMR (CDCl3, 200 MHz) δH 7.35 (d, 2H, J=8.8 Hz), 7.08 (s, 1H), 6.83 (m, 2H), 5.94 (bs, 2H), 4.29 (q, 2H, J=7.0 Hz), 3.81 (s, 6H). [M+1]263.2 (M+H+, C13H14NO3S requires 263.06).
2-Amino-5-phenethyl-thiophene-3-carboxylic acid methyl ester (TR575)




2-Amino-5-phenethyl-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (87% yield). 1H NMR (CDCl3, 200 MHz) δH 7.23 (m, 5H), 6.65 (s 1H), 5.83 (bs, 2H), 3.78 (s, 3H), 2.89 (s, 4H).
2-Amino-5-(2-methoxy-phenyl)-thiophene-3-carboxylic acid methyl ester (TR581)




2-Amino-5-(2-methoxy-phenyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a white solid (67% yield) mp 116-118° C. 1H NMR (CDCl3, 200 MHz) δH 7.51 (dd, 1H, J=6.2 Hz, J=1.4 Hz), 7.39 (s, 1H), 7.17 (m, 1H), 6.95 (m, 2H), 5.94 (bs, 2H), 3.91 (s, 3H), 3.83 (s, 3H). [M+1]263.9 (M+, C13H13NO3S requires 263.06).
2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carbonitrile (TR582)




2-Amino-5-(4-methoxy-phenyl)-thiophene-3-carbonitrile
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a white solid (63% yield) mp 204-205° C. 1H NMR (CDCl3, 200 MHz) δH 7.33 (d, 2H, J=8.8 Hz), 6.88 (d, 2H, J=8.8 Hz), 6.78 (s, 1H), 4.80 (bs, 2H).
2-Amino-5-[2-(4-methoxy-2-methyl-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester (TR583)




2-Amino-5-[2-(4-methoxy-2-methyl-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow solid (71% yield) mp 110-112° C. 1H NMR (CDCl3, 200 MHz) δH 7.06 (d, 1H, J=8.0 Hz), 6.70 (m, 3H), 5.85 (bs, 2H), 3.81 (s, 6H), 2.82 (s, 4H), 2.30 (s, 3H).
2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester (TR584)




2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow solid mp 107-108° C. (77% yield). 1H NMR (CDCl3, 200 MHz) δH 6.71 (m, 4H), 5.80 (bs, 2H), 3.79 (s, 6H), 3.75 (s, 3H), 2.86 (s, 4H).
2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carbonitrile (TR585)




2-Amino-5-[2-(2-methoxy-phenyl)-ethyl]-thiophene-3-carbonitrile
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a brown oil (51% yield). 1H NMR (CDCl3, 200 MHz) δH 7.17 (m, 3H), 6.89 (m, 2H), 6.36 (s 1H), 4.64 (bs, 2H), 3.84 (s, 3H), 2.88 (s, 4H).
2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carbonitrile (TR586)




2-Amino-5-[2-(4-methoxy-phenyl)-ethyl]-thiophene-3-carbonitrile
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a brown solid mp 145-147° C. (70% yield). 1H NMR (CDCl3, 200 MHz) δH 7.08 (d, 2H, J=8.8 Hz), 6.83 (d, 2H, J=8.8 Hz), 6.34 (s 1H), 4.62 (bs, 2H), 3.80 (s, 3H), 2.85 (m, 4H).
2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester (TR587)




2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carboxylic acid ethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (86% yield). 1H NMR (CDCl3, 200 MHz) δH 6.72 (m, 4H), 5.79 (bs, 2H), 4.26 (q, 2H, J=7.2 Hz), 3.79 (s, 3H), 3.75 (s, 3H), 2.86 (s, 3H), 1.34 (t, 3H, J=7.2 Hz).
2-Amino-5-phenethyl-thiophene-3-carbonitrile (TR588)




2-Amino-5-phenethyl-thiophene-3-carbonitrile
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a orange solid mp 127-129° C. (65% yield). 1H NMR (CDCl3, 200 MHz) δH 7.23 (m, 5H), 6.35 (s, 1H), 4.63 (s, 2H), 2.90 (s, 4H).
2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carbonitrile (TR589)




2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3-carbonitrile
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow solid mp 89-91° C. (70% yield). 1H NMR (CDCl3, 200 MHz) δH 6.72 (m, 3H), 6.37 (s, 1H), 4.62 (s, 2H), 3.77 (s, 6H), 2.86 (s, 4H).
2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3,4-dicarboxylic acid diethyl ester (TR592)




2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-thiophene-3,4-dicarboxylic acid diethyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 7/3) to provide a yellow oil (81% yield). 1H NMR (CDCl3, 200 MHz) δH 6.71 (m, 4H), 6.02 (bs, 2H), 4.24 (m, 4H), 3.74 (m, 6H), 2.86 (m, 4H), 1.27 (m, 6H).
2-Amino-5-(2-thiophen-3-yl-ethyl)-thiophene-3-carboxylic acid methyl ester (TR595)




2-Amino-5-(2-thiophen-3-yl-ethyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (79% yield). 1H NMR (CDCl3, 200 MHz) δH 7.26 (m, 1H), 6.96 (m, 2H), 6.65 (s, 1H), 5.82 (bs, 2H), 3.80 (s, 3H), 2.92 (s, 4H).
2-Amino-5-(2-pyridin-2-yl-ethyl)-thiophene-3-carboxylic acid methyl ester (TR596)




2-Amino-5-(2-pyridin-2-yl-ethyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 4/6) to provide a yellow oil (87% yield). 1H NMR (CDCl3, 200 MHz) δH 8.52 (d, 1H, J=3.4 Hz), 7.58 (m, 1H), 7.07 (m, 2H), 6.61 (s, 1H), 5.93 (bs, 2H), 3.76 (s, 3H), 3.03 (s, 4H).
2-Amino-5-(2-pyridin-3-yl-ethyl)-thiophene-3-carboxylic acid methyl ester (TR597)




2-Amino-5-(2-pyridin-3-yl-ethyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 4/6) to provide a white solid, mp 120-122° C. (77% yield). 1H NMR (CDCl3, 200 MHz) δ 8.44 (m, 2H), 7.45 (m, 1H), 7.21 (m, 1H), 6.60 (s, 1H), 5.93 (bs, 2H), 3.77 (s, 3H), 2.88 (s, 4H).
2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-4-methyl-thiophene-3-carboxylic acid methyl ester (TR599)




2-Amino-5-[2-(2,5-dimethoxy-phenyl)-ethyl]-4-methyl-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 3/7) to provide a yellow oil (82% yield). 1H NMR (CDCl3, 200 MHz) δH 6.72 (m, 3H), 5.93 (bs, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H), 2.79 (s, 4H), 2.12 (s, 3H).
2-Amino-5-(2,5-dimethoxy-phenyl)-thiophene-3-carboxylic acid methyl ester (TR601)




2-Amino-5-(2,5-dimethoxy-phenyl)-thiophene-3-carboxylic acid methyl ester
The crude of reaction was purified by flash chromatography (EtOAc/PE, 2/8) to provide a white solid, mp 204-205° C. (63% yield). 1H NMR (CDCl3, 200 MHz) δH 7.39 (s, 1H), 7.08 (d, 1H J=3.0 Hz), 6.9 (s, 1H), 6.86 (s, 1H), 6.76 (m, 1H), 3.84 (s, 9H).
Methyl 2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ6A)
To a solution of 2-aminothiophene TR560 (0.130 g, 0.446 mmol) in THF (2 mL) at 0° C., NaH (0.021 g, 0.932 mmol, 2 equiv) was added followed by the addition of MeI (0.095 g, 0.670 mmol, 1.5 equiv). After stirring the resulting mixture for another 1 h at room temperature (RT), the reaction mixture was added to water (10 mL). CH2Cl2 (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-heptane 90-10) afforded TJ6A (0.075 g, 55%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.86 (Sbr, 4H); 2.97 (d, 3H); 3.76 (s, 3H); 3.79 (s, 3H); 6.66 (s, 1H); 6.83 (d, 2H); 7.10 (d, 2H).
Methyl 2-di methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ6B)
To a solution of 2-aminothiophene TR560 (0.05 g, 0.172 mmol) in THF (2 mL) at 0° C., NaH (0.012 g, 0.515 mmol, 3 equiv) was added followed by the addition of MeI (0.073 g, 0.515 mmol, 3 equiv). After stirring the resulting mixture for another 2 h at RT, the reaction mixture was added to water (10 mL). CH2Cl2 (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-heptane 90-10) afforded TJ6B (0.013 g, 23%) as a semi-solid. 1H NMR (300 MHz, CDCl3) δH 2.88 (Sbr, 4H); 2.96 (d, 6H); 3.79 (s, 6H); 6.82 (d, 2H); 6.90 (s, 1H); 7.10 (d, 2H.
Methyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ7)
To a solution of 2-aminothiophene TR560 (0.135 g, 0.4641 mmol) in MeOH (2 mL), concentrated sulfuric acid (0.12 mL) and ice (0.5 g) was added. When the mixture was cooled to 0° C., saturated sodium nitrite (0.0383 g; 0.555 mmol) aqueous solution was added keeping the temperature below 5° C. After 30 min, 50% H3PO2 (0.122 g; 0.924 mol) in 0.5 mL of water was added dropwise. After stirring the resulting mixture for another 5 h at RT, the reaction mixture was added to water (10 mL). CH2Cl2 (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-heptane 95-5) afforded TJ7 (0.035 g, 27%) as a semi-solid. 1H NMR (300 MHz, CDCl3) δH 2.91 (t, 2H); 3.07 (t, 2H); 3.78 (s, 3H); 3.84 (s, 3H); 6.82 (d, 2H); 7.14 (d, 2H); 7.19 (s, 1H); 7.87 (s, 1H).
2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate (TJ8)
To a solution of 2-aminothiophene TR560 (0.210 g, 0.721 mmol) in MeOH (10 mL) at RT, 1 mL of 10N solution of NaOH is added. After stirring the resulting mixture at reflux temperature for 12 h, the reaction mixture was added to water (5 mL) followed by acidification with 2N solution of HCl. Ethyl acetate (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-MeOH 97-3) afforded TJ8 (0.065 g, 32%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.84 (Sbr, 4H); 3.78 (s, 6H); 6.64 (s, 1H); 6.82 (d, 2H); 7.04 (d, 2H).
2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide (TJ9)
To a mixture of 4-(4-methoxyphenyl)butanal 6 (0.5 g, 2.805 mmol), 2-cyanoacetamide 7 (0.236 g, 2.805 mmol) and sulfur (0.090 g, 2.805 mmol) in DMF (10 mL), NEt3 (0.851 g, 8.451 mmol, 3 equiv) was added. After stirring the resulting mixture at 60° C. for 30 h, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-ethyl acetate 60-40) afforded TJ9 (0.202 g, 26%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.83 (Sbr, 4H); 3.78 (s, 6H); 5.44 (s, 2H); 6.30 (s, 1H); 6.82 (d, 2H); 7.10 (d, 2H).
N-Methyl 2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide (TJ19)
To a mixture of 4-(4-methoxyphenyl)butanal 6 (0.5 g, 2.805 mmol), 2-cyano-N-methylacetamide 8 (0.275 g, 2.805 mmol) and sulfur (0.090 g, 2.805 mmol) in DMF (10 mL), NEt3 (0.851 g, 8.451 mmol, 3 equiv) was added. After stirring the resulting mixture at 60° C. for 30 hrs, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2-ethyl acetate 90-10) afforded TJ19 (0.27 g, 33%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.86 (m, 7H); 3.78 (s, 3H); 5.57 (s, 2H); 6.64 (s, 1H); 6.81 (d, 2H); 7.06 (d, 2H).
Methyl 2-(1,3-dioxoisoindolin-2-yl)-5-methylthiophene-3-carboxylate (14)
To a suspension of compound 13 (5 g, 29.23 mmol) in acetic acid (80 mL) was added phthalic anhydride (6.4 g, 43.24 mmol). After stirring for 30 hrs at reflux, the solvent was evaporated and the residue dissolved in CH2Cl2 (30 mL). The organic solution was washed with a saturated solution of NaHCO3 (50 mL), water (50 mL), dried, and concentrated. Purification by column chromatography (silica, eluent CH2Cl2 afforded 14 (7.7 g, 87%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.51 (s, 3H); 3.69 (s, 3H); 7.19 (s, 1H); 7.80 (m, 2H); 7.87 (m, 2H).
Methyl 5-(bromomethyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carboxylate (15)
To a solution of protected 2-aminothiophene 14 (2.5 g; 8.33 mmol), N-bromosuccinimide (1.55 g; 8.74 mmol) and benzoyl peroxide (20 mg) in CCl4 (50 ml) was heated at reflux temperature for 20 h under argon atmosphere. The CCl4 was evaporated under reduced pressure and the residue was extracted with dichloromethane (3×50 ml). The combined organic phases were washed with a saturated solution of NaHCO3, dried over magnesium sulfate and the solvent was evaporated under reduced pressure Purification by column chromatography (silica, eluent CH2Cl2) afforded 15 (1.9 g, 60%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 3.72 (s, 3H); 4.66 (s, 2H); 7.51 (s, 1H); 7.84 (m, 2H); 7.97 (m, 2H).
Methyl 2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ54SM)
To a mixture of (methyl 5-(bromomethyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carboxylate 15 (0.2 g, 0.5263 mmol) and K2CO3 (0.108 g, 0.7894 mmol, 1.5 equiv) in THF (6 mL) was added 3-methylbenzenethiol (0.065 g, 0.5263 mmol). After stirring the resulting mixture for another 24 h at RT, the reaction mixture was added to water (20 mL). CH2Cl2 (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2) afforded TJ54SM (0.143 g, 64%) as a an off-white solid. 1H NMR (300 MHz, CDCl3) δH 2.33 (s, 3H); 3.68 (s, 3H); 4.24 (s, 2H); 7.06 (m, 1H); 7.19 (d, 2H); 7.27 (d, 1H); 7.80 (m, 2H); 7.96 (m, 3H).
Methyl 2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ21)
To a mixture of (methyl 5-(bromomethyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carboxylate (15) (0.2 g, 0.5263 mmol) and K2CO3 (0.108 g, 0.7894 mmol, 1.5 equiv) in THF (6 mL) was added 4-methoxylbenzenethiol (0.073 g, 0.5263 mmol). After stirring the resulting mixture for another 24 h at RT, the reaction mixture was added to water (20 mL). CH2Cl2 (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2) afforded TJ21 (0.167 g, 72%) as a an off-white solid. 1H NMR (300 MHz, CDCl3) δH 3.68 (s, 3H); 3.79 (s, 3H); 4.13 (s, 2H); 6.85 (d, 2H); 7.18 (s, 1H); 7.37 (d, 2H); 7.81 (m, 2H); 7.96 (m, 2H).
A similar procedure as in the case of TJ54SM and TJ21 were used in the synthesis of TJ86, TJ127 and TJ119.
Methyl 2-phthalimido-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ86)
1H NMR (300 MHz, CDCl3) 3.68 (s, 3H); 4.25 (s, 2H); 7.26 (m, 4H); 7.39 (d, 2H); 7.80 (m, 2H); 7.96 (m, 2H).
Methyl 2-phthalimido-5-(2-(4-methoxybenzyl)sulfanylmethyl)thiophene-3-carboxylate (TJ127)
1H NMR (300 MHz, CDCl3) 3.71 (sb, 5H); 3.72 (s, 2H); 3.80 (s, 3H); 6.80 (d, 2H); 7.24 (m, 3H); 7.81 (m, 2H); 7.96 (m, 2H).
Methyl 2-phthalimido-5-(2-(butyl)sulfanylmethyl)thiophene-3-carboxylate (TJ119)
1H NMR (300 MHz, CDCl3) 0.92 (t, 3H); 1.42 (m, 2H); 1.58 (m, 2H); 2.53 (t, 2H); 3.70 (s, 3H); 3.787 (s, 2H); 7.33 (s, 1H); 7.81 (m, 2H); 7.96 (m, 2H).
A similar procedure as in the case of TJ54SM and TJ21 were used in the synthesis of protected analogues of TJ4, TJ22, TJ25, TJ51, TJ57, TJ55 and TJ54P.
General procedure for the synthesis of TJ22, TJ25, TJ51, TJ57, TJ55, TJ54P, TJ113, TJ117, TJ118, TJ125 and TJ130. To a stirred suspension of the compound having general formula 16 in 5 mL EtOH, methyl hydrazine (1.5 equiv) was added. In the case of thiophenol derivative, the reaction mixture was refluxed for 3 h whereas in the case of p-methoxyphenol or p-Anisidine compound, the reaction mixture was stirred at RT for 2 h. The reaction mixture was then added to water (10 mL). CH2Cl2 (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2) afforded corresponding product as a semi-solid.
Methyl 2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ4)
1H NMR (300 MHz, CDCl3) δH 3.76 (s, 3H); 4.07 (s, 2H); 5.89 (s, 2H); 6.71 (s, 1H); 67.29 (m, 2H); 7.32 (d, 2H).
Methyl 2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate (TJ22)
1H NMR (300 MHz, CDCl3) δH 3.65 (s, 3H); 3.79 (s, 3H); 3.96 (s, 2H); 5.85 (s, 2H); 6.62 (s, 1H); 6.82 (d, 2H); 7.32 (d, 2H).
Methyl 2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate (TJ51)
1H NMR (300 MHz, CDCl3) δH 3.74 (s, 3H); 3.79 (s, 3H); 4.22 (s, 2H); 5.90 (s, 2H); 6.63 (d, 2H); 6.78 (d, 2H); 6.84 (s, 1H).
Methyl 2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate (TJ57)
1H NMR (300 MHz, CDCl3) δH 3.57 (d, 2H); 3.66 (s, 2H); 3.80 (s, 3H); 5.93 (s, 2H); 6.74 (s, 1H); 7.28 (m, 5H).
Methyl 2-amino-5-(2-(4-methyl phenyl)sulfanyl methyl)thiophene-3-carboxylate (TJ55)
1H NMR (300 MHz, CDCl3) δH 2.31 (s, 3H); 376 (s, 3H); 4.03 (s, 2H); 5.88 (s, 2H); 6.68 (s, 1H); 7.07 (d, 2H); 7.24 (d, 2H).
Methyl 2-amino-5-(2-(3-methyl phenyl)sulfanyl methyl)thiophene-3-carboxylate (TJ54P)
1H NMR (300 MHz, CDCl3) δH 2.30 (s, 3H); 3.76 (s, 3H); 4.07 (s, 2H); 5.89 (s, 2H); 6.72 (s, 1H); 7.02 (d, 1H); 7.15 (m, 3H).
Methyl 2-amino-5-((naphthalen-2-ylthio)methyl)thiophene-3-carboxylate (TJ113)
1H NMR (300 MHz, CDCl3) δH 3.71 (s, 3H); 4.16 (s, 2H); 5.88 (s, 2H); 6.73 (s, 1H); 7.42 (m, 3H); 7.76 (d, 2H).
Methyl 2-amino-5-((furan-2-ylmethylthio)methyl)thiophene-3-carboxylate (TJ117)
1H NMR (300 MHz, CDCl3) δH 3.65 (s, 2H); 3.67 (s, 2H); 3.79 (s, 3H); 5.98 (s, 2H); 6.16 (d, 1H); 6.31 (t, 1H); 6.79 (s, 1H); 7.31 (d, 1H).
Methyl 2-amino-5-((thiophen-2-ylmethylthio)methyl)thiophene-3-carboxylate (TJ118)
1H NMR (300 MHz, CDCl3) δH 3.65 (s, 2H); 3.67 (s, 2H); 3.79 (s, 3H); 3.85 (s, 2H); 5.95 (s, 2H); 6.76 (Sb, 1H); 6.91 (Sb, 2H); 7.21 (Sb, 1H).
Methyl 2-amino-5-(butylthiomethyl)thiophene-3-carboxylate (TJ125)
1H NMR (300 MHz, CDCl3) 0.87 (t, 3H); 1.39 (m, 2H);) 1.54 (m, 2H); 2.46 (t, 2H); 3.68 (s, 2H); 3.78 (s, 3H); 5.92 (s, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-((4-methoxyphenylthio)methyl)thiophene-3-carboxylate (TJ130)
1H NMR (300 MHz, CDCl3) 3.58 (s, 2H); 3.61 (s, 2H); 3.79 (s, 6H); 5.94 (s, 2H); 6.73 (s, 1H); 6.84 (d, 2H); 7.20 (d, 2H).
Synthetic route to compounds having general formula 23 is described in Scheme 4 (Example 2), wherein for general formula 23 the values for the following compounds are:

    
    
        TJ21, R1=OMe, R2=H, X=S, 89%
        TJ55, R1=Me, R2=H, X=S, 82%
        TJ54P, R1=H, R2=M, X=S, 93%
        TJ4, R1=H, R2=H, X=CH2S, 86%
        TJ25, R1=OMe, R2=H, X=O, 1%
        TJ51, R1=OMe, R2=H, X=NH, 3%
    
    


Methyl 2-amino-5-benzylthiophene-3-carboxylate (TJ109)
To a mixture of 3-henylpropanal (0.261 g, 1.95 mmol), methyl cyanoacetate (0.192 g, 1.95 mmol) and sulfur (0.062 g, 1.95 mmol) in methanol (10 mL), NEt3 (0.196 g, 1.95 mmol) was added. After stirring the resulting mixture at reflux for 15 hrs, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent heptane-ethyl acetate 50-50) afforded TJ109 (0.385 g, 80%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 3.77 (s, 3H); 3.89 (s, 2H); 5.78 (s, 2H); 6.66 (s, 1H); 7.27 (m, 5H).
Methyl 2-amino-5-(6-methylhept-5-en-2-yl)thiophene-3-carboxylate (TJ108)
To a mixture of citronellal 0.3 g, 1.95 mmol), methyl cyanoacetate (0.192 g, 1.95 mmol) and sulfur (0.062 g, 1.95 mmol) in methanol (10 mL), NEt3 (0.196 g, 1.95 mmol) was added. After stirring the resulting mixture at reflux for 15 hrs, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent heptane-ethyl acetate 50-50) afforded TJ108 (0.38 g, 73%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 1.22 (d, 3H); 1.52 (m, 2H); 1.56 (s, 3H); 1.67 (s, 3H); 1.97 (q, 2H); 2.74 (m, 1H); 3.78 (s, 3H); 5.08 (t, 1H); 5.80 (s, 2H); 6.61 (s, 1H).
Methyl 2-amino-5-(non-8-enyl)thiophene-3-carboxylate (TJ110)
To a mixture of undec-10-enal (0.326 g, 1.95 mmol), methyl cyanoacetate (0.192 g, 1.95 mmol) and sulfur (0.062 g, 1.95 mmol) in methanol (10 mL), NEt3 (0.196 g, 1.95 mmol) was added. After stirring the resulting mixture at reflux for 15 hrs, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent heptane-ethyl acetate 50-50) afforded TJ110 (0.465 g, 85%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 1.30 (m, 8H); 1.58 (m, 2H); 2.04 (q, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.49 (m, 2H); 5.79 (m, 3H); 6.60 (s, 1H).
Methyl 2-amino-5-heptylthiophene-3-carboxylate (TJ114)
To a mixture of nonanal (0.275 g, 1.95 mmol), methyl cyanoacetate (0.192 g, 1.95 mmol) and sulfur (0.062 g, 1.95 mmol) in methanol (10 mL), NEt3 (0.196 g, 1.95 mmol) was added. After stirring the resulting mixture at reflux for 15 hrs, the reaction mixture was added to water (20 mL). Ethyl acetate (20 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent heptane-ethyl acetate 50-50) afforded TJ114 (0.365 g, 73%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δH 0.89 (t, 3H); 1.28 (m, 8H); 1.57 (q, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.77 (sb, 2H); 6.60 (s, 1H).
Example 7
Synthesis of Exemplified Compounds and their Activity
Synthesis
The following compounds (with their code) are generated according to the processes described in the present invention:
General procedure for the synthesis of Methyl 2-amino-5-alkylthiophene-3-carboxylate derivatives (TJ15 to TJ161).
To a mixture of the corresponding aliphatic aldehyde (1.95 mmol), methyl cyanoacetate (0.192 g, 1.95 mmol) and sulfur (0.062 g, 1.95 mmol) in methanol (10 mL), NEt3 (0.196 g, 1.95 mmol) was added. After stirring the resulting mixture at reflux for 15 h, the reaction mixture was added to water (20 mL). Ethyl acetate (40 mL) was added and the organic solution was washed with distilled water (3×20 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent heptane-ethyl acetate 50-50) afforded the corresponding methyl 2-amino-5-alkyllthiophene-3-carboxylate derivative (70%-91%).
Methyl 2-amino-5-methylthiophene-3-carboxylate (TJ15)
1H NMR (300 MHz, CDCl3) δH 2.45 (s, 3H); 3.71 (s, 3H); 5.89 (s, 2H); 7.20 (s, 1H).
Methyl 2-amino-5-ethylthiophene-3-carboxylate (TJ157)
1H NMR (300 MHz, CDCl3) δH 1.22 (t, 3H); 2.60 (q, 2H); 3.79 (s, 3H); 5.82 (sb, 2H); 6.61 (s, 1H).
Methyl 2-amino-5-propylthiophene-3-carboxylate (TJ190)
1H NMR (300 MHz, CDCl3) δH 0.95 (t, 3H); 1.59 (m, 2H); 2.55 (t, 2H); 3.79 (s, 3H); 5.71 (sb, 2H); 6.61 (s, 1H).
Methyl 2-amino-5-(3-chloropropyl)thiophene-3-carboxylate (TJ168D)
1H NMR (300 MHz, CDCl3) δH 2.35 (m, 2H); 2.76 (t, 2H); 3.79 (t, 3H); 4.09 (t, 2H); 5.84 (sb, 2H); 6.67 (s, 1H).
Methyl 2-amino-5-butylthiophene-3-carboxylate (TJ154)
1H NMR (300 MHz, CDCl3) δH 0.91 (t, 3H); 1.36 (m, 2H); 1.56 (m, 2H); 2.57 (t, 2H); 3.78 (s, 3H); 5.80 (sb, 2H); 6.61 (s, 1H).
Methyl 2-amino-5-pentylthiophene-3-carboxylate (TJ156)
1H NMR (300 MHz, CDCl3) δH 0.89 (t, 3H); 1.31 (m, 4H); 1.57 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.80 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-hexylthiophene-3-carboxylate (TJ155)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.29 (m, 4H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.79 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-heptylthiophene-3-carboxylate (TJ191 or TJ114)
1H NMR (300 MHz, CDCl3) δH 0.89 (t, 3H); 1.28 (m, 8H); 1.57 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.77 (Sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-octylthiophene-3-carboxylate (TJ144)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.27 (m, 10H); 1.55 (m, 2H); 2.56 (t, 2H); 3.79 (s, 3H); 5.77 (sb, 2H); 6.60 (s, 1H).
2-amino-5-(non-8-enyl)thiophene-3-carbonitrile (TJ145)
1H NMR (300 MHz, CDCl3) δH 1.30 (m, 8H); 1.56 (m, 2H); 2.04 (q, 2H); 2.57 (t, 2H); 4.62 (m, 2H); 4.95 (m, 2H); 5.82 (m, 1H); 6.36 (s, 1H).
Methyl 2-amino-4-methyl-5-octylthiophene-3-carboxylate (TJ163)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.26 (m, 10H); 1.50 (m, 2H); 2.16 (s, 3H); 2.53 (t, 2H); 3.80 (s, 3H); 5.91 (sb, 2H).
Methyl 2-amino-5-nonylthiophene-3-carboxylate (TJ198)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.27 (m, 12H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.80 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-decylthiophene-3-carboxylate (TJ158)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.26 (m, 14H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.78 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-dodecylthiophene-3-carboxylate (TJ159)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.26 (m, 18H); 1.56 (m, 2H); 2.56 (t, 2H); 3.79 (s, 3H); 5.78 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-tridecylthiophene-3-carboxylate (TJ160)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.26 (m, 20H); 1.56 (m, 2H); 2.56 (t, 2H); 3.79 (s, 3H); 5.78 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-tetradecylthiophene-3-carboxylate (TJ188)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.25 (m, 22H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.79 (sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-pentadecylthiophene-3-carboxylate (TJ153)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.25 (m, 24H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.80 (Sb, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-hexadecylthiophene-3-carboxylate (TJ161)
1H NMR (300 MHz, CDCl3) δH 0.88 (t, 3H); 1.25 (m, 26H); 1.56 (m, 2H); 2.56 (t, 2H); 3.78 (s, 3H); 5.80 (sb, 2H); 6.60 (s, 1H).
General Procedure for the Synthesis of 5-Alkyl or Aryl Thiomethyl-Substituted 2-Amino-3-Carboxymethylthiophene Derivatives (TJ125 to TJ117).
To a stirred suspension of the compound having general formula 16 in 5 mL EtOH, methyl hydrazine (1.5 equiv) was added. After stirring the resulting mixture at reflux for 6 h, the reaction mixture was added to water (20 mL). CH2Cl2 (10 mL) was added and the organic solution was washed with distilled water (3×10 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2) afforded corresponding product as a semi-solid.
Methyl 2-amino-5-(pentylthiomethyl)thiophene-3-carboxylate (TJ172)
1H NMR (300 MHz, CDCl3) 0.89 (t, 3H); 1.31 (m, 4H);) 1.56 (m, 2H) 2.45 (t, 2H); 3.69 (s, 2H); 3.79 (s, 3H); 5.92 (sb, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-(isopentylthiomethyl)thiophene-3-carboxylate (TJ173)
1H NMR (300 MHz, CDCl3) 0.80 (d, 6H) 1.39 (m, 2H) 1.53 (m, 1H) 2.39 (t, 2H); 3.61 (s, 2H); 3.72 (s, 3H); 5.84 (sb, 2H); 6.68 (s, 1H).
Methyl 2-amino-5-(hexylthiomethyl)thiophene-3-carboxylate (TJ171)
1H NMR (300 MHz, CDCl3) 0.88 (t, 3H); 1.26 (m, 6H) 1.55 (m, 2H) 2.46 (t, 2H); 3.69 (s, 2H); 3.79 (s, 3H); 5.90 (sb, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-(heptylthiomethyl)thiophene-3-carboxylate (TJ177)
1H NMR (300 MHz, CDCl3) 0.88 (t, 3H); 1.26 (m, 8H) 1.55 (m, 2H) 2.46 (t, 2H); 3.69 (s, 2H); 3.79 (s, 3H); 5.92 (sb, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-(octylthiomethyl)thiophene-3-carboxylate (TJ178)
1H NMR (300 MHz, CDCl3) 0.88 (t, 3H); 1.26 (m, 10H) 1.55 (m, 2H) 2.45 (t, 2H); 3.69 (s, 2H); 3.79 (s, 3H); 5.92 (sb, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-(cyclopentylthiomethyl)thiophene-3-carboxylate (TJ179)
1H NMR (300 MHz, CDCl3) δH 1.53 (m, 4H); 1.72 (m, 2H); 1.96 (m, 2H); 3.03 (m, 1H); 3.71 (s, 2H); 3.79 (s, 3H); 5.93 (sb, 4H); 6.75 (s, 1H).
Methyl 2-amino-5-(cyclohexylthiomethyl)thiophene-3-carboxylate (D8)
1H NMR (300 MHz, CDCl3) δH 1.27 (m, 2H); 1.29 (m, 2H); 1.59 (m, 2H); 1.74 (m, 2H); 1.92 (m, 2H); 2.62 (m, 1H); 3.72 (s, 2H); 3.79 (s, 3H); 5.89 (Sb, 2H); 6.75 (s, 1H).
Methyl 2-amino-5-(o-tolylthiomethyl)thiophene-3-carboxylate (D3)
1H NMR (300 MHz, CDCl3) δH 2.37 (s, 3H); 3.76 (s, 3H); 4.05 (s, 2H); 5.89 (Sb, 2H); 6.73 (s, 1H); 7.14 (m, 3H); 7.29 (m, 1H).
Methyl 2-amino-5-((2-aminophenylthio)methyl)thiophene-3-carboxylate (TJ122)
1H NMR (300 MHz, CDCl3) δH 3.74 (s, 3H); 3.89 (s, 2H); 4.33 (Sb, 2H); 5.89 (Sb, 2H); 6.59 (s, 1H); 6.67 (m, 2H); 7.17 (t, 1H), 7.27 (d, 1H).
Methyl 2-amino-5-((2-methoxyphenylthio)methyl)thiophene-3-carboxylate (D6)
1H NMR (300 MHz, CDCl3) δH 3.75 (s, 3H); 3.89 (s, 3H); 4.07 (Sb, 2H); 5.87 (s, 2H); 6.71 (s, 1H); 6.87 (m, 2H); 7.22 (m, 1H); 7.29 (m, 1H).
Methyl 2-amino-5-((2-(methoxycarbonyl)phenylthio)methyl)thiophene-3-carboxylate (D7)
1H NMR (300 MHz, CDCl3) δH 3.78 (s, 3H); 3.92 (s, 3H); 4.15 (s, 2H); 5.88 (s, 2H); 6.89 (s, 1H); 7.18 (m, 1H); 7.39 (m, 2H); 7.92 (m, 1H).
3-((5-amino-4-(methoxycarbonyl)thiophen-2-yl)methylthio)benzoic acid (TJ53)
1H NMR (300 MHz, CDCl3) δH 3.68 (s, 3H); 412 (s, 3H); 6.07 (s, 2H); 6.71 (s, 1H); 7.40 (t, 1H); 779 (d, 1H); 7.92 (m, 1H), 8.09 (s, 1H).
Methyl 2-amino-5-((4-fluorophenylthio)methyl)thiophene-3-carboxylate (D10)
1H NMR (300 MHz, CDCl3) δH 3.76 (s, 3H); 4.00 (s, 2H); 5.92 (s, 2H); 6.62 (s, 1H); 6.97 (m, 2H); 7.32 (m, 2H).
Methyl 2-amino-5-((4-chlorophenylthio)methyl)thiophene-3-carboxylate (D4)
1H NMR (300 MHz, CDCl3) δH 3.77 (s, 3H); 4.04 (s, 2H); 5.91 (s, 2H); 6.69 (s, 1H); 7.25 (m, 4H).
Methyl 2-amino-5-((4-nitrophenylthio)methyl)thiophene-3-carboxylate (TJ126)
1H NMR (300 MHz, CDCl3) δH 3.78 (s, 3H); 4.22 (s, 2H); 5.95 (s, 2H); 6.86 (s, 1H); 7.36 (d, 2H); 8.12 (d, 2H).
Methyl 2-amino-5-((4-methylphenylthio)methyl)thiophene-3-carboxylate (TJ55)
1H NMR (300 MHz, CDCl3) δH 2.31 (s, 3H); 3.76 (s, 3H); 4.03 (s, 2H); 5.88 (s, 2H); 6.68 (s, 1H); 7.07 (d, 2H); 7.24 (d, 2H).
Methyl 2-amino-5-((4-ethylphenylthio)methyl)thiophene-3-carboxylate (TJ185)
1H NMR (300 MHz, CDCl3) δH 1.20 (t, 3H); 2.60 (q, 2H); 3.75 (s, 3H); 4.03 (s, 2H); 5.89 (s, 2H); 6.67 (s, 1H); 7.12 (d, 2H); 7.25 (d, 2H).
Methyl 2-amino-5-((4-isopropylphenylthio)methyl)thiophene-3-carboxylate (TJ186)
1H NMR (300 MHz, CDCl3) δH 1.22 (d, 6H); 2.87 (q, 1H); 3.75 (s, 3H); 4.04 (s, 2H); 5.89 (s, 2H); 6.67 (s, 1H); 7.13 (d, 2H); 7.27 (d, 2H).
Methyl 2-amino-5-(phenethylthiomethyl)thiophene-3-carboxylate (TJ187)
1H NMR (300 MHz, CDCl3) δH 2.71 (t, 2H); 2.83 (t, 2H); 3.67 (s, 2H); 3.79 (s, 2H); 5.93 (s, 2H); 6.75 (s, 1H); 7.18 (m, 5H).
Methyl 2-amino-5-((biphenyl-4-ylthio)methyl)thiophene-3-carboxylate (TJ184)
1H NMR (300 MHz, CDCl3) 3.75 (s, 3H); 4.10 (s, 2H); 5.91 (s, 2H); 6.73 (s, 1H); 7.04 (m, 5H); 7.52 (m, 4H).
Methyl 2-amino-5-((pyridin-4-ylthio)methyl)thiophene-3-carboxylate (TJ129)
1H NMR (300 MHz, CDCl3) δH 3.78 (s, 3H); 4.19 (s, 2H); 6.06 (s, 2H); 6.89 (s, 1H); 7.15 (d, 2H); 8.41 (d, 2H).
General Procedure for the Synthesis of TJ170 and TJ169.
To a mixture of TJ168D (50 mg) and K2CO3 (1.0 equiv) in THF (4 mL) was added 4-methoxybenzenethiol or cyclohexanethiol (1.0 equiv). After stirring the resulting mixture for another 24 h at 45° C., the reaction mixture was added to water (10 mL). CH2Cl2 (15 mL) was added and the organic solution was washed with distilled water (3×15 mL), dried over MgSO4, filtered, and then evaporated to dryness to afford the crude product mixture. Purification by column chromatography (silica, eluent CH2Cl2) afforded TJ170 or TJ169 as a semi solid.
Methyl 2-amino-5-(3-(cyclohexylthio)propyl)thiophene-3-carboxylate (TJ170)
1H NMR (300 MHz, CDCl3) δH 1.25 (m, 12H); 1.56 (q, 3H); 2.54 (t, 2H); 3.78 (s, 3H); 5.77 (s, 2H); 6.60 (s, 1H).
Methyl 2-amino-5-(3-(4-methoxyphenylthio)propyl)thiophene-3-carboxylate (TJ169)
1H NMR (300 MHz, CDCl3) δH 1.82 (m, 2H); 2.70 (t, 2H); 2.83 (t, 2H); 3.78 (s, 3H); 3.79 (s, 3H); 5.79 (s, 2H); 6.60 (s, 1H), 6.83 (d, 2H), 7.34 (d, 2H).
Activity and Tumor Cell Selectivity:
The activity and tumor cell selectivity of the compounds are shown in Table 6 and Table 7.







TABLE 6







Tumor cell selectivity of 5-alkyl-substituted 2-aminothiophenes








 
















IC50 (μM)














Code
—X—A
R1
R2
CEM
HeLa
Ratio





TJ15
—CH3
—COOMe
—H
>250
>250
>1<


TJ157
—CH2CH3
—COOMe
—H
  175
  180
1


TJ190
—(CH2)2CH3
—COOMe
—H
     6.9
   61
9


TJ168D
—(CH2)2CH2Cl
—COOMe
—H
   26
  150
6


TJ154
—(CH2)3CH3
—COOMe
—H
     4.6
   77
17 


TJ156
—(CH2)4CH3
—COOMe
—H
     0.98
   83
85 


TJ155
—(CH2)5CH3
—COOMe
—H
     0.20
   73
365 


TJ191
—(CH2)6CH3
—COOMe
—H
     0.13
   81
623 


TJ144
—(CH2)7CH3
—COOMe
—H
     0.27
   86
319 


TJ108
—CH(CH3)CH2CH2CH═C(CH3)2
—COOMe
—H
   13
   97
7


TJ145
—(CH2)7CH═CH2
—C≡N
—H
     0.19
   60
111 


TJ110
—(CH2)7CH═CH2
—COOMe
—H
     0.28
   21
214 


TJ163
—(CH2)7CH3
—COOMe
—CH3
   16
   84
5


TJ198
—(CH2)8CH3
—COOMe
—H
     0.20
  171
855 


TJ158
—(CH2)9CH3
—COOMe
—H
     0.33
   68
206 


TJ159
—(CH2)11CH3
—COOMe
—H
     0.56
  187
334 


TJ160
—(CH2)12CH3
—COOMe
—H
     1.3
>250
>192   


TJ188
—(CH2)13CH3
—COOMe
—H
     1.0
>250
>250   


TJ153
—(CH2)14CH3
—COOMe
—H
     6.1
>250
>41   


TJ161-
—(CH2)15CH3
—COOMe
—H
     6.4
>250
>39   


TJ125
—(CH2S)(CH2)3CH3
—COOMe
—H
     0.52
   81
156 


TJ172
—(CH2S)(CH2)4CH3
—COOMe
—H
     0.83
   73
88 


TJ173
—(CH2S)(CH2)2CH(CH3)2
—COOMe
—H
     0.91
   71
78 


TJ171
—(CH2S)(CH2)5CH3
—COOMe
—H
     7.5
≧250
33 


TJ177
—(CH2S)(CH2)6CH3
—COOMe
—H
     1.5
   71
47 
















TABLE 7







Tumor cell selectivity of 5-arylthiomethyl-substituted 2-amino-3-


carboxymethylthiophenes:








 














IC50 (μM)













Code
Y1
n
CEM
HeLa
Ratio





TJ179




1
1.8 
 80
44


 


D8




1
2.1 
 81
39


 


TJ4




1
1.3 
108
83


 


TJ55




1
1.5 
 72
48


 


D3




1
2  
 71
36


 


TJ54P




1
4.1 
108
26


 


TJ122




1
39   
 46
 1


 


TJ22




1
0.62
 58
93


 


D6




1
17   
 94
 6


 


D7




1
27   
190
 7


 


TJ53




1
124   
 75
≦1


 


D10




1
3.1 
102
33


 


D4




1
1.7 
 42
25


 


TJ126




1
12   
 16
 1


 


TJ55




1
1.5 
 72
48


 


TJ185




1
0.34
 49
144 


 


TJ186




1
0.46
 46
100 


 


TJ130




1
3.9 
 88
23


 


TJ57




1
3.5 
 61
17


 


TJ187




1
1.2 
 74
62


 


TJ184




1
1.2 
 55
46


 


TJ113




1
0.9 
 74
82


 


TJ129




1
111   
 93
 1


 


TJ118




1
3.3 
 99
30


 


TJ117




1
4.9 
102
21


 


TJ170




3
0.57
 55
96


 


TJ169




3
0.97
 59
61









REFERENCES


    1. Romagnoli R, Baraldi P G, Pavani M G, Cruz-Lopez O, Hamel E, Balzarini J, Brognara E, Zuccato C, Gambari R. 2010. Synthesis and cellular pharmacology studies of a series of 2-amino-3-aroyl-4-substituted thiophene derivatives. Med. Chem. 6: 329-343.
    2. Huang Y, Dömling A. 2011. The Gewald multicomponent reaction. Mol. Divers. 15: 3-33.

## Claims

1. A compound having the general formula I:



wherein
A is selected from the group consisting of C1-7 alkyl; C2-7 alkenyl; C2-7 alkynyl; halo C1-7 alkyl; C3-10 cycloalkyl; C1-7 alkoxy; C3-10 cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic; thio C1-7 alkyl; thio C3-10 cycloalkyl; arylthio; arylalkylthio; heterocyclic-substituted alkyl; heterocyclic-substituted alkyloxy; heteroaryl and aryl groups, wherein said heteroaryl or aryl groups are optionally substituted with one or more substituents selected from the group consisting of halogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, nitro, hydroxyl, sulfhydryl, amino, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, azido;
R0 is independently selected from the group consisting of amino; (mono- or di) C1-7 alkylamino; (mono- or di) arylamino; azido; 1H-Isoindole-1,3(2H)-dione,2-yl-;
R1 is independently selected from the group consisting of carboxyC1-7 alkyl; cyano; —COOH; —CONRaRb; —SO2R; —SO2NHR; —SO2NRaRb, —SO2OR; —PO(XR)2; and C3-10 cycloalkyl, wherein R, Ra and Rb are each independently selected from the group consisting of H and C1-7alkyl and C3-10 cycloalkyl;
R2 is independently selected from the group consisting of hydrogen; carboxyC1-7 alkyl, C1-7alkyl; cyano; —CONHMe; —COOH; —CONH2 
X is independently selected from the group consisting of —(CH2)n, —(CH2)m-acetylene, -acetylene-(CH2)m—, —(CH2)m-ethylene, -ethylene-(CH2)m—, —S(CH2)m—, —(CH2)mS—, —O(CH2)m—, —(CH2)mO—, —NH(CH2)m—; —(CH2)mNH—, —(CH2)mS(CH2)p—, —(CH2)mO(CH2)p, wherein m is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein p is 0, 1, 2, 3, 4, 5, 6 or 7, and wherein n is 2, 3, 4, 5, 6 or 7;
and/or a pharmaceutical acceptable addition salt thereof and/or a stereoisomer thereof and/or a solvate thereof;
with the proviso that said compound is not Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile.

2. The compound according to claim 1, wherein A is C1-7 alkyl or C2-7 alkenyl.

3. The compound according to claim 1, wherein A is phenyl or thiophene.

4. The compound according to claim 1, wherein R1 is carboxyC1-7alkyl.

5. The compound according to claim 1, wherein A is an aryl group substituted with 1 or 2 substituents selected from the group consisting of a methoxy group and a methyl group.

6. The compound according to claim 1, wherein X is selected from the group consisting of a —(CH2)2, an ethylene and an acetylene group.

7. The compound according to claim 1, wherein n is 2.

8. The compound according to claim 1, wherein R0 is amino.

9. The compound according to claim 1, wherein R2 is hydrogen.

10. The compound according to claim 1, wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-methoxyphenyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-4-ethoxycarbonyl 5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(4-methoxyphenyl)thiophene-3-carboxylate; Methyl-2-amino-5-((2-methoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(4-methoxyphenyl)thiophene; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(2-methoxyphenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(4-methoxyphenyl)ethyl)thiophene; Ethyl-2-amino-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-3-cyano-5-(2-(phenyl)ethyl)thiophene; 2-Amino-3-cyano-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene; Ethyl-2-amino-4-ethoxycarbonyl-5-(2-(2,5-dimethoxymethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-pyridyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-4-methyl-5-(2-(2,5-dimethoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2,5-dimethoxyphenyl)thiophene-3-carboxylate; N-Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; 2-Amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxamide; Methyl-2-methylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-dimethylamino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)oxymethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxyphenyl)aminomethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)methylsulfanylmethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenyl)sulfanylmethyl)thiophene-3-carboxylate; Methyl-2-phthalimido-5-(2-(4-methoxyphenyl)sulfanylmethyl)thiophene-3-carboxylate; and Methyl-2-phthalimido-5-(2-(3-methylphenyl)sulfanylmethyl)thiophene-3-carboxylate.

11. The compound according to claim 1, wherein the compound is selected from the group consisting of: Methyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(phenylethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-methoxyphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(4-methoxy-2-methylphenyl)ethyl)thiophene-3-carboxylate; Methyl-2-amino-5-(2-(2-thienyl)ethyl)thiophene-3-carboxylate; Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethynyl)thiophene-3-carboxylate; and Ethyl-2-amino-5-(2-(4-methoxyphenyl)ethyl)thiophene-3-carboxylate.

12. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 or a compound selected from
Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; and one or more pharmaceutically acceptable excipients.

13. The pharmaceutical composition according to claim 12, further comprising one or more biologically active drugs being selected from the group consisting of antineoplastic drugs and/or immunosuppressant and/or immunomodulator drugs.

14. A method of prevention or treatment of cancer in a subject, comprising the administration of a therapeutically effective amount of a compound according to claim 1, or a compound selected from
Ethyl 2-amino-5-(3-benzyloxypropyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(4-benzyloxybutyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-pentyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-heptyl-thiophene-3-carboxylate; Ethyl 2-amino-5-methyl-4-(n-pentyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-n-hexyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5(7-acetoxy;n-heptyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-(2-phenylethyl)-thiophene-3-carboxylate; Ethyl 2-amino-5-benzyl-4-methyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-decyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-nonyl-thiophene-3-carboxylate; Ethyl 2-amino-5-n-propyl-thiophene-3-carboxylate; 2-Amino-5-phenylthiophene-3-carbonitile; 2-Amino-5-benzythiophene-3-carbonitrile; 2-Amino-5-isopropylthiophene-3-carbonitrile; 2-Amino-5-(tert-butyl)thiophene-3-carbonitrile; 2-Amino-5-heptylthiophene-3-carbonitrile; 2-Amino-5-butylthiophene-3-carbonitrile; 2-Amino-N-cyclopropyl-5-(methylsulfanylmethyl)thiophene-3-carboxamide; 2-Amino-3-cyano-5-[2-(2′,5′-dimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-3-cyano-5-[2-(3′,4′,5′-trimethoxyphenyl)ethyl]-4-methylthiophene; 2-Amino-4-methyl-5-phenethyl-thiophene-3-carbonitrile; 2-amino-5-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-thiophene-3-carbonitrile; optionally in combination with one or more pharmaceutically acceptable excipients.

15. A process for preparing a compound of formula I, comprising the steps of:
(a) reaction of an enolizable compound (aldehyde or ketone), sulfur and an active methylene containing a cyano group under Gewald conditions;
(b) protection of the 5-amino group as phthalimide via reaction with phthalic anhydride in acidic conditions, and further transformations at the 2-position of the thiophene via first bromination to afford the 2-bromo or 2-bromomethyl derivatives; and then nucleophilic substitution with the aromatic, heterocyclic or lipophilic side chain; or standard Sonogashira cross-coupling reaction with the aromatic, heterocyclic or lipophilic side chain; and
(c) deprotection of the phthalimide function by hydrazine or methylhydrazine to liberate the 5-aminothiophene.

16. A compound selected from the group consisting of:
methyl 2-amino-5-propyl-thiophene-3-carboxylate; methyl 2-amino-5-(3-chloropropyl)thiophene-3-carboxylate; methyl 2-amino-5-butyl-thiophene-3-carboxylate; methyl 2-amino-5-pentyl-thiophene-3-carboxylate; methyl 2-amino-5-hexyl-thiophene-3-carboxylate; methyl 2-amino-5-heptyl-thiophene-3-carboxylate; methyl 2-amino-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-(1,5-dimethylhex-4-enyl)thiophene-3-carboxylate; 2-amino-5-non-8-enyl-thiophene-3-carbonitrile; methyl 2-amino-4-methyl-5-octyl-thiophene-3-carboxylate; methyl 2-amino-5-nonyl-thiophene-3-carboxylate; methyl 2-amino-5-decyl-thiophene-3-carboxylate; methyl 2-amino-5-dodecyl-thiophene-3-carboxylate; methyl 2-amino-5-tridecyl-thiophene-3-carboxylate; methyl 2-amino-5-tetradecyl-thiophene-3-carboxylate; methyl 2-amino-5-pentadecyl-thiophene-3-carboxylate; methyl 2-amino-5-hexadecyl-thiophene-3-carboxylate; methyl 2-amino-5-(butylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(pentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(isopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(hexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(heptylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(octylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclopentylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(cyclohexylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(phenylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(p-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(o-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(m-tolylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(2-aminophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxyphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(2-methoxycarbonylphenyl)thiomethyl]thiophene-3-carboxylate; 3-((5-amino-4-(methoxycarbonyl)thiophen-2-yl)methylthio)benzoic acid; methyl 2-amino-5-[(4-fluorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-chlorophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-nitrophenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-ethylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-isopropylphenyl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(4-methoxyphenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-[(phenyl)methylthiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(phenethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-[(biphenyl-4-yl)thiomethyl]thiophene-3-carboxylate; methyl 2-amino-5-(2-naphthylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(4-pyridylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-thienylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(2-furylmethylthiomethyl)thiophene-3-carboxylate; methyl 2-amino-5-(3-cyclohexylthiopropyl)thiophene-3-carboxylate; methyl 2-amino-5-[3-(4-methoxyphenyl)thiopropyl]thiophene-3-carboxylate.

17. A method of prevention or treatment of cancer in a subject, comprising the administration of a therapeutically effective amount of a compound according to claim 16, optionally in combination with one or more pharmaceutically acceptable excipients.

18. The compound according to claim 1, wherein the compound has general formula Ia:



wherein R0, R1 R2 and n are as defined in claim 1;
R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7 alkyl; C1-7 alkoxy; halogen; amino; and hydroxyl; and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, and azido.

19. The compound according to claim 1, wherein the compound has general formula Ib:



wherein X is as defined in claim 1; and
R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen; C1-7 alkyl; C1-7 alkoxy; halogen; amino; and hydroxyl; and wherein said alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, hydroxyl, sulfhydryl, amino, and azido.

20. The compound according to claim 1, wherein R1 is a carboxymethyl or carboxyethyl group.

21. The method according to claim 14, wherein said cancer is a lymphoma, liver cancer of prostate cancer.

22. The method according to claim 21, wherein said lymphoma is a T-cell lymphoma.

23. The method according to claim 14, wherein said subject is a human.

24. The compound according to claim 1, wherein A is C1-7 alkyl substituted with hydroxyl, amino or sulfhydryl.