US 2010.0086931A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0086931 A1 Mizui et al. (43) Pub. Date: Apr. 8, 2010
(54) METHOD FOR ASSAYING ACTION OF (86). PCT No.: PCT/UP2008/053981 ANTITUMIORAGENT USING DECREASE IN GENE EXPRESSION LEVELAS INDEX S371 (c)(1), (2), (4) Date: Sep. 4, 2009 (75) Inventors: Yoshiharu Mizui, Ibaraki-Ken (JP); Related U.S. Application Data SR s (E. Ri (60) Provisional application No. 60/904,775, filed on Mar. asao Iwata, Ibaraki- en (JP): Kie 5, 2007, provisional application No. 60/960,486, filed Sugano, Ibaraki-Ken (JP): Naoki on Oct. 1, 2007. Toritsuka, Ibaraki-Ken (JP); Taku Yoshida, Ibaraki-Ken (JP); Mai Publication Classification Uesugi, Ibaraki-Ken (JP); Tadashi (51) Int. Cl. Kadowaki, Ibaraki-Ken (JP) CI2O I/68 (2006.01) C07D 407/06 (2006.01) Correspondence Address: C7H 2L/04 (2006.01) FSH & RICHARDSON P.C. C07K 6/00 (2006.01) P.O. BOX 1022 (52) U.S. Cl...... 435/6; 549/271; 536/24.3:536/24.33; MINNEAPOLIS, MN 55440-1022 (US) 530,387.9 (57) ABSTRACT (73)73) AssiAssignee : EisaiS R&D M anagementt U0.,Co. An object of the present invention is to provide a method, a Ltd., Tokyo-To (JP) probe, a primer, an antibody, a reagent, and a kit for assaying an action of a pladienolide derivative to a living Subject. (21) Appl. No.: 12/529,971 According to the present invention, there is provided a method for assaying an action of the pladienolide derivative (22) PCT Filed: Mar. 5, 2008 using a decrease in gene expression level as an index. Patent Application Publication Apr. 8, 2010 Sheet 1 of 3 US 2010/0086931 A1
(A)
HNRPA2B1 ZCCHC6 EDN1 D1 GTF2H1 TRAPPC4 SLC25A19 18SrRNA
mRNA EXPRESSION
(B)
HNRPA2B1 ZCCHC6 EDN1 D GTF2H1 TRAPPC4 SLC25A19 18SrrNA
mRNA EXPRESSION
F. G. 1 Patent Application Publication Apr. 8, 2010 Sheet 2 of 3 US 2010/0086931 A1
(A)
STK17B CDKN1B(p27) D1 ROK3 NUP54 BZW1 DNAJB1 18SrRNA
mRNA EXPRESSION
(B)
STK17B CDKN1B(p27) D1 ROK3 NUP54 BZW DNAJB 18SrRNA
mRNA EXPRESSION
F. G. 2 Patent Application Publication Apr. 8, 2010 Sheet 3 of 3 US 2010/0086931 A1
1.2
EÐNVHONIHEXENOISSE TEMAET\/NHUU-JO
Ø • ·• Ø TRAPPC4. SLC25A19 GTF2H
F. G. 3
EÐNW/HONOÏSSE|HEXE,NI TIE/\ET\/NHUU-JO
TRAPPC4 SLC25A19 GTF2H1 F. G. 4 US 2010/0086931 A1 Apr. 8, 2010
METHOD FOR ASSAYING ACTION OF 2) an optionally Substituted C-2 alkoxy group, ANTITUMIORAGENT USING DECREASE IN 3) an optionally Substituted unsaturated C- alkoxy group, GENE EXPRESSION LEVELAS INDEX 4) an optionally Substituted C-2 aralkyloxy group, 5) an optionally substituted 5- to 14-membered heteroaralky BACKGROUND OF THE INVENTION loxy group, 6) RCO O— wherein R represents 0001 1. Field of the Invention 0008 a) a hydrogen atom, 0002 The present invention relates to a method for assay 0009 b) an optionally substituted C- alkyl group, ing an action of an antitumor agent using a decrease in gene 0010 c) an optionally substituted unsaturated C- alkyl expression level as an index, more particularly, a method for grOup, assaying an action of an antitumor agent using a decrease in 0011 d) an optionally substituted Caryl group, 0012 e) an optionally substituted 5- to 14-membered het the expression level of mRNA or the expression level of a eroaryl group, protein as an index. 0013 f) an optionally substituted Czaralkyl group, 0003 2. Background Art 001.4 g) an optionally substituted 5- to 14-membered het 0004 Pladienolide derivatives are derivatives of natural eroaralkyl group, pladienolide. Since pladienolide exhibits an excellent antitu 00.15 h) an optionally substituted C- alkoxy group, mor activity (Mizui et al., 2004, J. Antibiotics 577: 188-196), 0016 i) an optionally Substituted unsaturated C- alkoxy search for a compound with higher antitumor activity has grOup, been performed to find the pladienolide derivatives 0017 j) an optionally Substituted Caryloxy group or (WO2002/060890 and WO2003/099813). 0018 k) an optionally substituted 5- to 14-membered het eroaryloxy group, SUMMARY OF THE INVENTION 7) R' RRSiO wherein R', R, and R, the same or different, each represents 0005. The present inventors have found that the expression I0019 a) a C- alkyl group or level of mRNA of a certain group of genes decreased and 0020 b) a Caryl group, concomitantly so did proteins encoded by the genes, when a 8) a halogen atom, pladienolide derivative was contacted with a sample obtained 9) RRYN R' wherein R represents from living cells including cancerous cells and peripheral 0021 a) a single bond, blood (PBMC) and whole blood (PBC) of a subject. Without (0022 b) - CO-O-, wishing to be bound by any theory, administration of the 0023 c) - SO. O. , pladienolide derivative may suppress transcription and trans (0024 d) - CS O– or lation of a certain group of genes, thereby decreasing the 0025 e)—CO NRY wherein R^ represents a hydro expression level of the genes. The present invention was made gen atom or an optionally substituted C. alkyl group, pro based on Such findings. vided that each of the leftmost bond in b) to e) is bound to the 0006. It is an object of the present invention to provide a nitrogen atom; and R'' and R', the same or different from method, a probe, a primer, an antibody, a reagent, and a kit for each other and each represents assaying an action of the pladienolide derivative on a living 0026 a) a hydrogen atom, Subject. 0027 b) an optionally substituted C. alkyl group, 0007 According to the present invention, there are pro 0028 c) an optionally substituted unsaturated C- alkyl vided inventions (1) to (19) as follows. grOup, (1) A method for assaying an action of a compound repre 0029 d) an optionally substituted aliphatic C acyl sented by formula (I), a pharmaceutically acceptable salt grOup, thereof, or a Solvate of them to a mammal, which comprises 0030 e) an optionally substituted aromatic C7 is acyl detecting a decrease in gene expression level caused by the grOup, compound represented by formula (I), the pharmaceutically 0031 f) an optionally substituted Caryl group, 0032 g) an optionally substituted 5- to 14-membered het acceptable salt thereof, or the solvate of them: eroaryl group, 0033 h) an optionally substituted C7 aralkyl group, (I) 0034 i) an optionally substituted C- alkylsulfonyl
grOup, 0035 j) an optionally substituted Carylsulfonyl group, 0036 k) an optionally substituted 3- to 14-membered non aromatic heterocyclic group formed by R'' and R' together with the nitrogen atom to which R'' and RY are bound, and the non-aromatic heterocyclic group optionally has substitu ent(s), 0037 l) an optionally substituted 5- to 14-membered het eroaralkyl group, 0038 m) an optionally substituted C. cycloalkyl group O wherein R. R. R7 and R', the same or different, each 0039 n) an optionally substituted 3- to 14-membered non represents aromatic heterocyclic group, 1) a hydroxyl group or an oxo group formed together with the 10) RYSO. O— wherein R^ represents carbon atom to which it is bound, provided that R is limited 0040 a) an optionally substituted C-2 alkyl group, to a hydroxyl group, 0041 b) an optionally substituted Caryl group, US 2010/0086931 A1 Apr. 8, 2010
0.042 c) an optionally substituted C-2 alkoxy group, 0065 (8E, 12E. 14E)-7-((4-Cyclopentylpiperazin-1-yl) 0043 d) an optionally substituted unsaturated C carbonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pen alkoxy group, tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; 0044 e) an optionally substituted Caryloxy group, 0066 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-7-((4-iso 0045 f) an optionally substituted 5- to 14-membered het propylpiperazin-1-yl)carbonyl)oxy-6,10,12,16.20-pen eroaryloxy group, tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; 0046 g) an optionally Substituted C-2 aralkyloxy group 0067 (8E, 12E. 14E)-7-((4-Cycloheptylpiperazin-1-yl) O carbonyl)oxy-3,6,16.21-Tetrahydroxy-6,10,12,16.20 0047 h) an optionally substituted 5- to 14-membered het pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; eroaralkyloxy group, 0068 (8E.12E,14E)-7-(N-(2-(N',N'-Diethylamino) 11) (RYO)PO O— wherein R^ represents ethyl)-N-methylcarbamoyloxy)-3,6,16.21-tetrahydroxy 0.048 a) an optionally substituted C-2 alkyl group, 6,10,12,16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 0049 b) an optionally substituted unsaturated C- alkyl trien-11-olide; grOup, 0069 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-7-((4- 0050 c) an optionally substituted Caryl group, isobutylhomopiperazin-1-yl)carbonyl)oxy-6,10,12,16.20 0051 d) an optionally substituted 5- to 14-membered het pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; eroaryl group, 0070 (8E, 12E. 14E)-7-((4-Ethylhomopiperazin-1-yl)car 0052 e) an optionally substituted Caralkyl group or bonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pen 0053 f) an optionally substituted 5- to 14-membered het tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; eroaralkyl group), 0071 (8E, 12E. 14E)-7-((4-Butylhomopiperazin-1-yl)car 12) (R'R''N), PO O wherein R^' and RY have the bonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pen same meanings as defined above or 13) (RNRVN)(R^O)PO O wherein RN, RN2 and RNS tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; have the same meanings as defined above, provided that a (0072 (8E.12E,14E)-3.16.21-Trihydroxy-6-methoxy-6, compound in which R. R. R7 and R are all hydroxyl 10,12,16.20-pentamethyl-7-((4-methylpiperazin-1-yl)car groups, and a compound in which R, R and R'' are all bonyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; hydroxyl groups and R is an acetoxy group are excluded, (0073 (8E.12E,14E)-3.16.21-Trihydroxy-6-methoxy-6, 0054) R' represents a hydrogenatom or hydroxyl group. 10,12,16.20-pentamethyl-7-((4-(piperidin-1-yl)piperidin (2) The method according to (1), wherein the compound 1-yl)carbonyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11 represented by formula (I) is selected from the group consist olide; ing of (0074 (8E.12E,14E)-3,6,7,21-Tetrahydroxy-6,10,12,16, 0055 (8E.12E,14E)-7-(N-(2-(N',N'-Dimethylamino) 20-pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11 ethyl)-N-methylcarbamoyloxy)-3,6,16.21-tetrahydroxy olide; 6,10,12,16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 (0075 (8E.12E,14E)-7-((4-(2,2-Dimethylpropyl)ho trien-11-olide; mopiperazin-1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy 0056 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-6,10,12,16, 6,10,12,16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 20-pentamethyl-7-((4-methylhomopiperazin-1-yl)carbo trien-11-olide; and nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; (0076 (8E.12E,14E)-3,6,16-Trihydroxy-21-methoxy-6, 0057 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-6,10,12,16, 10,12,16.20-pentamethyl-7-((4-methylpiperazin-1-yl)car 20-pentamethyl-7-((4-methylpiperazin-1-yl)carbonyl) bonyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide. oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; (3) The method according to (1), wherein the detection of the 0058 (8E.12E,14E)-7-((4-Butylpiperazin-1-yl)carbonyl) decrease in the gene expression level comprises the steps of oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pentamethyl 0077 (a) measuring the expression level of mRNA before 18, 19-epoxytricosa-8,12,14-trien-11-olide; and after administration of the compound represented by 0059 (8E.12E,14E)-7-((4-Ethylpiperazin-1-yl)carbonyl) formula (I), the pharmaceutically acceptable salt thereof, or oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pentamethyl the Solvate of them to a mammal; 18, 19-epoxytricosa-8,12,14-trien-11-olide; 0078 (b) comparing, based on the expression level mea 0060 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-6,10,12,16, sured in (a), the expression level of the mRNA before and 20-pentamethyl-7-((4-propylpiperazin-1-yl)carbonyl) after administration of the compound represented by formula oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; (I), the pharmaceutically acceptable salt thereof, or the sol 0061 (8E, 12E. 14E)-7-((4-Cyclohexylpiperazin-1-yl)car vate of them, to determine that the compound represented by bonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pen formula (I), the pharmaceutically acceptable salt thereof, or tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; the solvate of them exerts an action to the mammal when the 0062 (8E, 12E. 14E)-7-((4-(Cyclopropylmethyl)piper expression level of mRNA after the administration decreases. azin-1-yl)carbonyl)oxy-3,6, 16, 21-tetrahydroxy-6,10,12. (4) The method according to (3), wherein the mRNA of which 16.20-pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11 expression level is measured is mRNA of at least one gene olide; selected from the genes listed in Table 1, Table 2, Table 3 and 0063 (8E.12E,14E)-3,6,16.21-Tetrahydroxy-6,10,12,16, Table 4, or a homologous gene thereof. 20-pentamethyl-7-((4-propylhomopiperazin-1-yl)carbo (5) The method according to (4), wherein the gene(s) are nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; selected from TRAPPC4, SLC25A19, GTF2H1, ID1, 0064 (8E, 12E. 14E)-7-((4-(Cyclopropylmethyl)ho ZCCHC6 and EDN1. mopiperazin-1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy (6) The method according to (3), wherein in step (a), the 6,10,12,16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 expression level of mRNA in samples obtained from a subject trien-11-olide; before and after administration of the compound represented US 2010/0086931 A1 Apr. 8, 2010 by formula (I), the pharmaceutically acceptable salt thereof, (18) Use of a probe or primer consisting of a polynucleotide or the Solvate of them, is measured. capable of hybridizing with a polynucleotide consisting of a (7) The method according to (6), wherein the samples nucleotide sequence of at least one gene selected from the obtained from the subject are selected from hemocytes in genes listed in Table 1, Table 2, Table 3, and Table 4, or a peripheral blood, plasma and serum. homologous gene thereof, or a complementary sequence (8) A probe or primer for assaying an action of a compound thereof, for assaying an action of a compound represented by represented by formula (I), a pharmaceutically acceptable formula (I), a pharmaceutically acceptable salt thereof, or a salt thereof, or a solvate of them, which consists of a poly Solvate of them to a mammal. nucleotide capable of hybridizing with a polynucleotide con (19) Use of an antibody against a protein consisting of amino sisting of a nucleotide sequence of at least one gene selected acids encoded by at least one gene selected from the genes from the genes listed in Table 1, Table 2, Table 3 and Table 4, listed in Table 1, Table 2, Table 3, and Table 4, or a homolo or a homologous gene thereof, or a complementary sequence gous gene thereof, or a fragment of the antibody, for assaying thereof. an action of a compound represented by formula (I), a phar (9) The probe or primer according to (8), which is capable of maceutically acceptable salt thereof, or a solvate of them to a detecting a genomic intron region or a part thereof in a gene mammal. listed in Table 1, Table 2, Table 3 or Table 4, or which is I0081. According to the present invention, an action of the capable of detecting a polynucleotide lacking a part of a compound represented by formula (I) to a living body can be genomic exon region in a gene listed in Table 1, Table 2, Table confirmed using the expression level of a gene in a cancerous 3 or Table 4. and normal tissue of a cancer patient or a normal tissue of a (10) A reagent or kit for assaying an action of a compound healthy individual, more specifically, the expression level of represented by formula (I), a pharmaceutically acceptable mRNA or the expression level of a protein as an index. For salt thereof, or a solvate of them to a mammal, which com instance, when the expression level of the gene decreases in prises the probe or the primer according to (8). the cancerous or normal tissue of the cancer patient, more (11) The method according to (1), wherein the detection of the specifically the expression level of mRNA or the expression decrease in the gene expression level comprises the steps of level of the protein decreases, it can be determined that the 0079 (f) measuring the expression level of a protein compound represented by formula (I) exerts the action and before and after administration of the compound represented that thus administration of the drug is no longer required or by formula (I), the pharmaceutically acceptable salt thereof, less amount of the drug should be administrated. Further, when the expression level of the gene does not exhibit a or the solvate of them to a mammal; downtrend in the cancerous or normal tissue of the cancer 0080 (g) comparing, based on the expression level mea patient, more specifically, the expression level of mRNA or sured in (f), the expression level of the protein before and after the expression level of the protein exhibits an uptrend or administration of the compound represented by formula (I), Substantially reach the same amount as before the adminis the pharmaceutically acceptable salt thereof or the solvate of tration, it can be determined that the compound represented them, to determine that the compound represented by formula by formula (I) does not exert the action and further adminis (I), the pharmaceutically acceptable salt thereof, or the sol tration of the drug is required. Hence, according to the inven vate of them exerts an action to the mammal when the expres tion, by monitoring periodically the expression of mRNA or sion level of the protein after the administration decreases. the expression level of the protein, the antitumor agent can be (12) The method according to (11), wherein the protein of administered more effectively to the patient and a minimally which expression level is measured is a protein consisting of required amount of the drug can be administered to the amino acids encoded by a polynucleotide of at least one gene patient. In particular, since the action of the compound rep selected from the genes listed in Table 1, Table 2, Table 3 and resented by formula (I) can be judged by monitoring the Table 4 or a homologous gene thereof. expression level of mRNA and the expression level of the (13) The method according to (11), wherein the protein of protein in samples obtained from the normal tissue Such as which expression level is measured is a protein consisting of blood of the patient, it is an advantage that the action of the amino acids encoded by a polynucleotide of at least one gene compound represented by formula (I) to the living body can selected from TRAPPC4, SLC25A19, GTF2H1, ID1, be readily and reliably assessed. ZCCHC6 and EDN1. (14) The method according to (11), wherein in step (f), the BRIEF DESCRIPTION OF THE DRAWINGS expression level of the protein in the samples obtained from a subject before and after administration of the compound rep I0082 FIG. 1 shows mRNA expression level. A: Results of resented by formula (I) or the pharmaceutically acceptable the samples obtained from the Tempus Blood RNA Tube; B: salt thereof or the solvate of them, is measured. Results of the samples obtained from the PAXgene Blood (15) The method according to (14), wherein the samples RNA Tube. obtained from the subject are selected from hemocytes in I0083 FIG. 2 shows expression level of mRNA in whole peripheral blood, plasma and serum. blood (PBC) samples obtained from human peripheral blood. (16) An antibody againstan protein consisting of amino acids A: Results of the samples obtained from the Tempus Blood encoded by a polynucleotide of at least one gene selected RNA Tube; B: Results of the samples obtained from the from the genes listed in Table 1, Table 2, Table 3 and Table 4 PAXgene Blood RNA Tube. or a homologous gene thereof, or a fragment thereof. I0084 FIG.3 shows results measured expression of mRNA (17) A reagent or kit for assaying an action of a compound (TRAPPC4, SLC25A19, and GTF2H1) in blood of nude represented by formula (I), a pharmaceutically acceptable mice to which human colon carcinoma cells were subcutane salt thereof, or a solvate of them to a mammal, comprising the ously transplanted and the pladienolide derivatives was antibody or the fragment thereof according to (16). administrated. US 2010/0086931 A1 Apr. 8, 2010
0085 FIG. 4 shows results measured expression of mRNA 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-bu (TRAPPC4, SLC25A19, and GTF2H1) in tumor of nude tynyl group, 3-butynyl group or 3-methyl-1-propynyl group. mice to which human colon carcinoma cells were subcutane I0090 The "Caryl group' used in the specification of ously transplanted and the pladienolide derivatives was the present application means an aromatic cyclic hydrocar administrated. bon group having 6 to 14 carbon atoms, and a monocyclic group and condensed rings such as a bicyclic group and a DETAILED DESCRIPTION OF THE INVENTION tricyclic group are included. Examples thereof include phenyl 0.086 All technical terms, scientific terms and terminolo group, indenyl group, 1-naphthyl group, 2-naphthyl group, gies used in the present specification have the same meanings aZulenyl group, heptalenyl group, indacenyl group, acenaph as those that are generally understood by those ordinary thyl group, fluorenyl group, phenalenyl group, phenanthrenyl skilled in the art in the technical fields to which the present group and anthracenyl group; and preferred examples include invention belongs, and are used merely for the purpose of phenyl group, 1-naphthyl group and 2-maphthyl group. explaining a specific embodiment but are not intended to make limitation. The present invention can be carried out in 0091. The “5- to 14-membered heteroaryl group' used in various embodiments as long as not departing from the spirit the specification of the present application means a monocy thereof. All the prior art documents, published publications, clic, bicyclic or tricyclic 5- to 14-membered aromatic hetero patent publications and other patent documents cited in the cyclic group which contains one or more of hetero atoms present specification are incorporated into the present speci selected from the group consisting of nitrogen atom, Sulfur fication as references, and can be used for carrying out the atom and oxygen atom. Preferred examples thereof include present invention. nitrogen-containing aromatic heterocyclic groups such as 0087. The “halogen atom' used in the specification of the pyrrolyl group, pyridyl group, pyridazinyl group, pyrimidinyl present application means a fluorine atom, a chlorine atom, a group, pyrazinyl group, triazolyl group, tetrazolyl group, bromine atom and an iodine atom. Among them, for example, benzotriazolyl group, pyrazolyl group, imidazolyl group, a fluorine atom, a chlorine atom or a bromine atom is pre benzimidazolyl group, indolyl group, isoindolyl group, ferred, of which a fluorine atom or a chlorine atom is pre indolizinyl group, purinyl group, indazolyl group, quinolyl ferred. group, isoquinolyl group, quinolizinyl group, phthalazinyl I0088. The “C. alkyl group' used in the specification of group, naphthyridinyl group, quinoxalinyl group, quinazoli the present application indicates a linear or branched alkyl nyl group, cinnolinyl group, pteridinyl group, imidazotriazi group having 1 to 22 carbon atoms. Such as methyl group, nyl group, pyrazinopyridaZinyl group, acridinyl group. ethyl group, n-propyl group, iso-propyl group, n-butyl group, phenanthridinyl group, carbazolyl group, carbazolinyl group, iso-butyl group, sec-butyl group, tert-butyl group, n-pentyl perimidinyl group, phenanthrolinyl group, phenazinyl group, group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, imidazopyridinyl group, imidazopyrimidinyl group, pyrazol 2,2-dimethylpropyl group, 1-ethylpropyl group, n-hexyl opyridinyl group and pyrazolopyridinyl group; Sulfur-con group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl taining aromatic heterocyclic groups such as thienyl group group, 1-propylpropyl group, 1-methylbutyl group, 2-meth and benzothienyl group; and oxygen-containing aromatic ylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl heterocyclic groups such as furyl group, pyranyl group, group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, cyclopentapyranyl group, benzofuryl group and isobenzofu 2,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl ryl group; aromatic heterocyclic groups containing two or group, 3-methylpentyl group, n-heptyl group, n-octyl group, more different hetero atoms, such as thiazolyl group, isothia n-nonyl group or n-decyl group; preferably a linear or Zolyl group, benzothiazolyl group, benzthiadiazolyl group, branched alkyl group having 1 to 6 carbon atoms, such as phenothiazinyl group, isoxazolyl group, furazanyl group, methyl group, ethyl group, n-propyl group, iso-propyl group, phenoxazinyl group, oxazolyl group, isoxazoyl group, ben n-butyl group, iso-butyl group, sec-butyl group, tert-butyl ZOxazolyl group, oxadiazolyl group, pyrazolooxazolyl group, group or n-pentyl group; and more preferably, for example, imidazothiazolyl group, thienofuranyl group, furopyrrolyl methyl group, ethyl group, propyl group, iso-propyl group, group and pyridoxazinyl group; and preferred examples n-butyl group, iso-butyl group or tert-butyl group. include thienyl group, furyl group, pyridyl group, pyridaZyl 0089. The “unsaturated C- alkyl group' used in the group, pyrimidyl group and pyrazyl group. specification of the present application indicates a linear or 0092. The “3- to 14-membered non-aromatic heterocyclic branched alkenyl group having 2 to 22 carbon atoms or a group' used in the specification of the present application linear or branched alkynyl group having 2 to 22 carbonatoms, indicates a monocyclic, bicyclic or tricyclic 3- to 14-mem Such as vinyl group, allyl group, 1-propenyl group, isoprope bered non-aromatic heterocyclic group which may contain nyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl one or more hetero atoms selected from the group consisting group, 1-butenyl group, 2-butenyl group, 3-butenyl group, of nitrogen atom, Sulfur atom and oxygen atom. Preferred 1-pentenyl group, 1-hexenyl group, 1.3-hexadienyl group, examples thereof include aziridinyl group, aZetidinyl group, 1.5-hexadienyl group, ethynyl group, 1-propynyl group, pyrrolidinyl group, pyrrolyl group, piperidinyl group, piper 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-buty azinyl group, homopiperidinyl group, homopiperazinyl nyl group, 1-ethynyl-2-propynyl group, 2-methyl-3-butynyl group, imidazolyl group, pyrazolidyl group, imidazolidyl group, 1-pentynyl group, 1-hexynyl group, 1.3-hexanediynyl group, morpholyl group, thiomorpholyl group, imidazolinyl group or 1.5-hexanediynyl group. It preferably indicates a group, oxazolinyl group, 2,5-diazabicyclo2.2.1]heptyl linear or branched alkenyl group having 2 to 10 carbonatoms group, 2,5-diazabicyclo2.2.2]octyl group, 3.8-diazabicyclo or a linear or branched alkynyl group having 2 to 10 carbon 3.2.1]octyl group, 1,4-diazabicyclo4.3.0nonyl group, qui atoms, such as vinyl group, allyl group, 1-propenyl group, nuclidyl group, tetrahydrofuran-yl group and tetrahy 2-propenyl group, isopropenyl group, 3-methyl-2-butenyl drothiophen-yl group. The non-aromatic heterocyclic groups group, 3,7-dimethyl-2,6-octadienyl group, ethynyl group, also include groups derived from pyridone ring, and non US 2010/0086931 A1 Apr. 8, 2010 aromatic fused rings (e.g., a group derived from, for example, gyloxy group and 2-butynyloxy group; and preferred phthalimide ring or Succinimide ring). examples include allyloxy group, propargyloxy group and 0093. The “Caralkyl group' used in the specification 2-butynyloxy group. 0099. The “Caryloxy group' used in the specification of the present application means a group corresponding to the of the present application means a group corresponding to the above-defined “C. alkyl group' of which substitutable above-defined "Caryl group' to which end an oxygen moiety is replaced by the above-defined “Caryl group'. atom is bonded. Specific examples thereof include phenyloxy Specific examples thereof include benzyl group, phenethyl group, indenyloxy group, 1-naphthyloxy group, 2-naphthy group, 3-phenylpropyl group, 4-phenylbutyl group, 1-naph loxy group, aZulenyloxy group, heptalenyloxy group, indace thylmethyl group and 2-naphthylmethyl group; and preferred nyloxy group, acenaphthyloxy group, fluorenyloxy group, examples include aralkyl groups having 7 to 10 carbonatoms phenalenyloxy group, phenanthrenyloxy group and anthrace Such as benzyl group and phenethyl group. nyloxy group; and preferred examples include phenyloxy 0094. The “5- to 14-membered heteroaralkyl group' used group, 1-naphthyloxy group and 2-maphthyloxy group. in the specification of the present application means a group 0100. The "Czaralkyloxy group' used in the specifica corresponding to the above-defined "C. alkyl group' of tion of the present application means a group corresponding which substitutable moiety is replaced by the above-defined to the above-defined “C-2 aralkyl group' to which end an “5- to 14-membered heteroaryl group'. Specific examples oxygen atom is bonded. Specific examples thereof include thereof include thienylmethyl group, furylmethyl group, benzyloxy group, phenethyloxy group, 3-phenylpropyloxy pyridylmethyl group, pyridaZylmethyl group, pyrimidylm group, 4-phenylbutyloxy group, 1-naphthylmethyloxy group ethyl group and pyrazylmethyl group; and preferred and 2-maphthylmethyloxy group; and preferred examples examples include thienylmethyl group, furylmethyl group include benzyloxy group. and pyridylmethyl group. 0101 The “5- to 14-membered heteroaralkyloxy group' s 0095. The “C-acycloalkyl group' used in the specifica used in the specification of the present application means a tion of the present application indicates a cycloalkyl group group corresponding to the above-defined "5- to 14-mem having 3 to 14 carbon atoms, and Suitable examples thereof bered heteroaralkyl group' to which end an oxygen atom is include cyclopropyl group, cyclobutyl group, cyclopentyl bonded. Specific examples thereof include thienylmethyloxy group, cyclohexyl group, cycloheptyl group and cyclooctyl group, furylmethyloxy group, pyridylmethyloxy group, group; and preferred examples include cyclopentyl group, pyridaZylmethyloxy group, pyrimidylmethyloxy group and cyclohexyl group, cycloheptyl group and cyclooctyl group. pyrazylmethyloxy group; and preferred examples include 0096. The “C., cycloalkylalkyl group' used in the speci thienylmethyloxy group, furylmethyloxy group and pyridi fication of the present application means a group correspond nylmethyloxy group. ing to the above-defined “C. alkyl group' of which Substi 0102 The “5- to 14-membered heteroaryloxy group' used tutable moiety is replaced by the above-defined “C. in the specification of the present application means a group cycloalkyl group'. Specific examples thereof include cyclo corresponding to the above-defined “5- to 14-membered het propylmethyl group, cyclobutylmethyl group, cyclopentyl eroaryloxy group' to which end an oxygen atom is bonded. methyl group, cyclohexyl methyl group, cycloheptyl methyl Specific examples thereof include pyrrolyloxy group, pyridy group and cyclooctylmethyl group; and preferred example loxy group, pyridazinyloxy group, pyrimidinyloxy group, include cyclopropylmethyl group, cyclobutylmethyl group pyrazinyloxy group, triazolyloxy group, tetrazolyloxy group, and cyclopentylmethyl group. benzotriazolyloxy group, pyrazolyloxy group, imidazoly 0097. The “C. alkoxy group' used in the specification loxy group, benzimidazolyloxy group, indolyloxy group, of the present application means a group corresponding to the isoindolyloxy group, indolizinyloxy group, purinyloxy above-defined "C. alkyl group' to which end an oxygen group, indazolyloxy group, quinolyloxy group, isoquinoly atom is bonded. Suitable examples thereof include methoxy loxy group, quinolizyloxy group, phthalazyloxy group, naph group, ethoxy group, n-propoxy group, iso-propoxy group. n-butoxy group, iso-butoxy group, sec-butoxy group, tert thyridinyloxy group, quinoxalyloxy group quinazolinyloxy butoxy group, n-pentyloxy group, iso-pentyloxy group, sec group, cinnolinyloxy group, pteridinyloxy group, imidazot pentyloxy group, n-hexoxy group, iso-hexoxy group, 1,1- riazinyloxy group, pyrazinopyridazinyloxy group, acridiny dimethylpropyloxy group, 1,2-dimethylpropyloxy group, loxy group, phenazinyloxy group, imidazopyridinyloxy 2,2-dimethylpropoxy group, 1-methyl-2-ethylpropoxy group, imidazopyrimidinyloxy group, pyrazolopyridinyloxy group, 1-ethyl-2-methylpropoxy group, 1,1,2-trimethylpro group, pyrazolopyridinyloxy group, thienyloxy group, ben poxy group, 1.2.2-trimethylpropoxy group, 1,1-dimethylbu Zothienyloxy group, furyloxy group, pyranyloxy group, toxy group, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy cyclopentapyranyloxy group, benzofurlyoxy group, isoben group, 2,3-dimethylbutyloxy group, 1,3-dimethylbutoxy Zofuryloxy group, thiazolyloxy group, isothiazolyloxy group, 2-ethylbutoxy group, 2-methylpentoxy group, 3-me group, benzothiazolyloxy group, benzothiadiazolyloxy thylpentyloxy group and hexyloxy group; and preferred group, phenothiazinyloxy group, isoxazolyloxy group, fura examples include methoxy group, ethoxy group, n-propoxy Zanyloxy group, phenoxazinyloxy group, oxazolyloxy group, group, iso-propoxy group, iso-butoxy group and 2,2-dimeth isoxazolyloxy group, benzoxazolyloxy group, oxadiazoly ylpropyloxy group. loxy group, pyrazolooxazolyloxy group, imidazothiazoly 0098. The “unsaturated C- alkoxy group' used in the loxy group, thienofuranyloxy group, furopyrrolyloxy group specification of the present application means a group corre and pyridoxazinyloxy group; and preferred examples include sponding to the above-defined “unsaturated C- alkyl thienyloxy group, pyridyloxy group, pyrimidyloxy group and group' to which end an oxygen atom is bonded. Suitable examples thereof include vinyloxy group, allyloxy group, pyrazyloxy group. 1-propenyloxy group, 2-propenyloxy group, isopropenyloxy (0103) The “aliphatic C-acyl group' used in the speci group, 2-methyl-1-propenyloxy group, 2-methyl-2-propeny fication of the present application means a group correspond loxy group, 1-butenyloxy group, 2-butenyloxy group, 3-bute ing to the above-defined “C-2 alkyl group' or “unsaturated nyloxy group, 1-pentenyloxy group, 1-hexenyloxy group, C-2 alkyl group' to which end a carbonyl group is bonded. 1.3-hexadienyloxy group, 1,5-hexadienyloxy group, propar Examples thereof include acetyl group, propionyl group, US 2010/0086931 A1 Apr. 8, 2010 butyryl group, iso-butyryl group, Valeryl group, iso-Valeryl 0111. The “C. alkylsulfinyl group' used in the specifi group, pivaloyl group, caproyl group, decanoyl group, lau cation of the present application means a group correspond royl group, myristoyl group, palmitoyl group, Stearoyl group, ing to the above-defined "C. alkyl group' to which end a arachidoyl group, acryloyl group, propiol group, crotonyl sulfinyl group is bonded. Examples thereof include methyl group, iso-crotonyl group, olenoyl group and linolenoyl Sulfinyl group, ethylsulfinyl group, n-propylsulfinyl group group; and preferred examples include aliphatic acyl groups and iso-propylsulfinyl group; and preferred examples include having 2 to 6 carbon atoms, such as acetyl group, propionyl methanesulfinyl group or ethanesulfinyl group. group, butyryl group, iso-butyryl group and acryloyl group. 0112 The “C. alkylsulfonyloxy group' used in the 0104. The “aromatic C-acyl group' used in the speci specification of the present application means a group corre fication of the present application means a group correspond sponding to the above-defined “C. alkylsulfonyl group' to ing to the above-defined "Caryl group' or “5- to 14-mem which end an oxygen atom is bonded. Examples thereof bered heteroaryl group' to which end a carbonyl group is include methylsulfonyloxy group, ethylsulfonyloxy group, bonded. Examples thereof include benzoyl group, 1-naph n-propylsulfonyloxy group and iso-propylsulfonyloxy thoyl group, 2-naphthoyl group, picolinoyl group, nicotinoyl group; and preferred examples include methylsulfonyloxy group, isonicotinoyl group, furoyl group and thiophenecar group. bonyl group; and preferred examples include benzoyl group, 0113. The substituent of the phrase “an optionally substi picolinoyl group, nicotinoyl group and isonicotinoyl group. tuted used in the specification of the present application may 0105. The “C. alkylsulfonyl group' used in the speci be one or more groups selected from: fication of the present application means a group correspond (1) a halogen atom, ing to the above-defined “C-2 alkyl group' to which a Sul fonyl group is bound. Specific examples thereof include (2) a hydroxyl group, methylsulfonyl group, ethylsulfonyl group, n-propylsulfonyl (3) a thiol group, group and iso-propylsulfonyl group; and preferred examples (4) a nitro group, include methylsulfonyl group. (5) a nitroso group, 0106 The “Carylsulfonyl group' used in the specifi (6) a cyano group, cation of the present application means a group correspond (7) a carboxyl group, ing to the above-defined "Caryl group' to which a Sulfo (8) a hydroxysulfonyl group, nyl group is bound. Specific examples thereof include benzenesulfonyl group, 1-naphthalenesulfonyl group and (9) a amino group, 2-naphthalenesulfonyl group; and preferred examples (10) a C- alkyl group (for example, methyl group, ethyl include benzenesulfonyl group. group, n-propyl group, iso-propyl group, n-butyl group, iso 0107 The “aliphatic C- acyloxy group' used in the butyl group, sec-butyl group and tert-butyl group), specification of the present application means a group corre (11) an unsaturated C- alkyl group (for example, vinyl sponding to the above-defined “aliphatic C-acyl group' to group, allyl group, l-propenyl group, 2-propenyl group, iso which end an oxygen atom is bonded. Specific examples propenyl group, ethynyl group, 1-propynyl group, 2-propy thereof include acetoxy group, propionyloxy group and acry nyl group, 1-butynyl group, 2-butynyl group and 2-butynyl loxy group; and preferred examples include acetoxy group group). and propionyloxy group. (12) a Caryl group (for example, phenyl group, 1-naph 0108. The “C. alkoxycarbonyl group' used in the speci thyl group and 2-maphthyl group), fication of the present application means a group correspond (13) a 5- to 14-membered heteroaryl group (for example, ing to the above-defined “Calkoxy group' to which end a thienyl group, furyl group, pyridyl group, pyridaZyl group, carbonyl group is bonded. Examples thereof include meth pyrimidyl group and pyrazyl group), oxycarbonyl group, ethoxycarbonyl group, n-propoxycarbo (14) a 3- to 14-membered non-aromatic heterocyclic group nyl group, iso-propoxycarbonyl group, n-butoxycarbonyl (for example, aziridinyl group, aZetidyl group, pyrrolidinyl group, iso-butoxycarbonyl group, sec-butoxycarbonyl group group, pyrrolyl group, piperidinyl group, piperazinyl group. and tert-butoxycarbonyl group; and preferred examples homopiperidinyl group, homopiperazinyl groups, imidazolyl include ethoxycarbonyl group, iso-propoxycarbonyl group group, pyrazolidyl group, imidazolidyl group, morpholyl and tert-butoxycarbonyl group. group, thiomorpholyl group, imidazolinyl group, oxazolinyl 0109 The “unsaturated Calkoxycarbonyl group' used group and quinuclidyl group), in the specification of the present application means a group corresponding to the above-defined “unsaturated C (15) a C- a cycloalkyl group (for example, cyclopropyl alkoxy group' to which end a carbonyl group is bonded. group, cyclobutyl group, cyclopentyl group, cyclohexyl Examples thereof include vinyloxycarbonyl group, allyloxy group, cycloheptyl group and cyclooctyl group), carbonyl group, 1-propenyloxycarbonyl group, isopropeny (16) a C- alkoxy group (for example, methoxy group, loxycarbonyl group, propargyloxycarbonyl group and 2-bu ethoxy group, n-propoxy group, iso-propoxy group, sec-pro tynyloxycarbonyl group; and preferred examples include poxy group, n-butoxy group, iso-butoxy group and tert-bu allyloxycarbonyl group. toxy group), 0110. The "Calkylthio group”used in the specification (17) an unsaturated C- alkoxy group (for example, viny of the present application means a group corresponding to the loxy group, allyloxy group, 1-propenyloxy group, 2-prope above-defined “Calkyl group' to which end a sulfur atom nyloxy group, isopropenyloxy group, ethynyloxy group, is bonded. Examples thereof include methylthio group, eth 1-propynyloxy group, 2-propynyloxy group, 1-butynyloxy ylthio group, n-propylthio group and iso-propylthio group; group and 2-butynyloxy group), and preferred examples include methylthio group or ethylthio (18) a Caryloxy group (for example, phenyloxy group, group. 1-naphthyloxy group and 2-maphthyloxy group), US 2010/0086931 A1 Apr. 8, 2010
(19) a C-2 aralkyloxy group (for example, benzyloxy group, compound can include certain isomers drawn from the phenethyloxy group, 3-phenylpropyloxy group, 4-phenylbu chemical formula. The present invention can include all iso tyloxy group, 1-naphthylmethyloxy group and 2-maphthylm mers and mixtures of the isomers such as a geometric isomer ethyloxy group), which is generated from the configuration of the compound, (20) a 5- to 14-membered heteroaralkyloxy group (for an optical isomer based on an asymmetric carbon, a rotamer, example, thienylmethyloxy group, furylmethyloxy group, pyridylmethyloxy group, pyridaZylmethyloxy group, pyrim a stereoisomer and a tautomer. The present invention is not idylmethyloxy group and pyrazylmethyloxy group), limited to the expediential description of the chemical for (21) a 5- to 14-membered heteroaryloxy group (for example, mula, and can include either of isomers or a mixture thereof. thienyloxy group, furyloxy group, pyridyloxy group, Accordingly, when the compound according to the present pyridaZyloxy group, pyrimidyloxy group and pyrazyloxy invention has an asymmetric carbon in the molecule, and its group). optically active substance and racemate exist, any one is (22) an aliphatic C-acyl group (for example, acetyl group, included. Further, when polymorphic crystals exist, the crys propionyl group, butyryl group, iso-butyryl group, Valeryl tal form according to the present invention is not specifically group, iso-Valeryl group, pivalyl group, caproyl group. limited to one form, and any one of the crystal forms may be decanoyl group, lauroyl group, myristoyl group, palmitoyl single or a mixture of the crystal forms. group, Stearoyl group, arachidoyl group, acryl group, propiol 0115 The “pharmaceutically acceptable salt” used in the group, crotonyl group, iso-crotonyl group, olenoyl group and present invention is not particularly restricted as long as it can linolenoyl group), forma salt with the compound represented by formula (I), and (23) an aromatic C7 is acyl group (for example, benzoyl is pharmaceutically acceptable. Preferred examples thereof group, 1-naphthoyl group and 2-maphthoyl group), include halide hydroacid salt such as hydrochloric acid salt, (24) an aliphatic C-2 acyloxy group (for example, acetoxy hydrobromic acid salt, hydroiodic acid salt; inorganic acid group, propionyloxy group and acryloxy group), salt Such as Sulphic acid salt, nitric acid salt, perchloric acid (25) a C- alkoxycarbonyl group (for example, methoxycar salt, phosphoric acid salt, carbonic acid salt, bicarbonic acid bonyl group, ethoxycarbonyl group, n-propoxycarbonyl salt; organic carboxylic acid salt Such as acetic acid salt, group, iso-propoxycarbonyl group, n-butoxycarbonyl group, trifluoroacetic acid salt, maleic acid salt, tartaric acid salt, iso-butoxycarbonyl group, sec-butoxycarbonyl group and fumaric acid salt, citric acid salt; organic Sulfonic acid salt tert-butoxycarbonyl group), Such as methanesulfonic acid salt, trifluoro methanesulfonic (26) an unsaturated C-2 alkoxycarbonyl group (for example, acid salt, ethanesulfonic acid salt, benzenesulfonic acid salt, vinyloxycarbonyl group, allyloxycarbonyl group, 1-propeny toluenesulfonic acid salt, camphorsulfonic acid salt; amino loxycarbonyl group, 2-propenyloxycarbonyl group, isopro acid salt Such as aspartic acid salt, glutamic acid salt, quater penyloxycarbonyl group, propargyloxycarbonyl group and nary amine salt; alkaline metal salt Such as Sodium salt, potas 2-butynyloxycarbonyl group), sium salt; and alkaline earth metal salt Such as magnesium (27) a C- alkylthio group (for example, methylthio group, salt, calcium salt. ethylthio group, n-propylthio group and iso-propylthio 0116. The “solvate” used in the present invention is not group). particularly restricted as long as it can form a Solvate with the (28) a C- alkylsulfinyl group (for example, methylsulfinyl compound represented by formula (I) or the salt thereof, and group, ethylsulfinyl group, n-propylsulfinyl group and iso is pharmaceutically acceptable. Preferred examples include propylsulfinyl group), hydrate, alcoholate Such as ethanolate, and etherate. (29) a C- alkylsulfonyl group (for example, methylsulfonyl 0117 The present invention also includes a metabolite group, ethaylsulfonyl group, n-propanesulfonyl group and generated by degradation of the compound represented by iso-propanesulfonyl group), formula (I) within a living body, as well as a prodrug of the (30) a Carylsulfonyl group (for example, benzenesulfo compound represented by formula (I) and the salt thereof. nyl group, 1-naphthalenesulfonyl group and 2-maphthalene The “prodrug used herein means an inert substance to which Sulfonyl group), “an active moiety of a drug” (meaning "drug corresponding (31) a C- alkylsulfonyloxy group (for example, methylsul to a prodrug) has been chemically modified, for the purpose fonyloxy group, ethylsulfonyloxy group, n-propylsulfony of improvement of bioavailability and reduction of a side loxy group and iso-propanesulfonyloxy group), effect. After absorbed, it is metabolized into the active moiety (32) carbamoyl group, and in vivo and exerts an action. Accordingly, the term “prodrug (33) formyl group. refers to any compound having a lower intrinsic activity than Among them, a preferred example is an amino group, a C the corresponding "drug, which is, once administrated to a alkyl group, an unsaturated C2-22 alkyl group, a C-14 aryl biological system, converted into the "drug” substance via a group, a 5- to 14-membered heteroaryl group, a 3- to spontaneous chemical reaction, enzyme catalyzed reaction or 14-membered non-aromatic heterocyclic group and a C metabolic reaction. Examples of Such prodrugs include vari cycloalkyl group, and a more preferred example is an amino ous prodrugs, for example, compounds produced by acyla group, a C- alkyl group, a 3- to 14-membered nonaromatic tion, alkylation, phosphorylation, boration, carbonation, heterocyclic group and a C-acycloalkyl group. The above esterification, amidation, or urethanation of a functional mentioned (9) amino group and (31) carbamoyl group as the group Such as an amino, hydroxyl, or carboxyl group in the substituent in “an optionally substituted” may each be further compound represented by formula (I). However, it should be Substituted with one or two of a C- alkyl group, an unsat noted that the exemplified prodrugs are not comprehensive urated C-22 alkyl group or a C-14 aryl group. but are merely typical, and other conventional various pro 0114. In the present specification, the chemical formula of drugs can be prepared by a conventional method by a person the compound according to the present invention is illustrated having ordinary skill in the art from the compound repre as a planimetric chemical formula for convenience but the sented by formula (I). US 2010/0086931 A1 Apr. 8, 2010
0118 When the compound represented by formula (I) is these methods are well-known, and the required devices and administrated to a mammal in the method according to the apparatuses therefor are commercially available. Moreover, present invention, the compound represented by formula (I) in Examples below, an example in which the expression level may be formulated by known methods. Conventional carriers of mRNA is measured with these methods will be described. are used for formulations, and the pharmaceutical products Those skilled in the art can measure the expression level of are prepared by conventional methods. Namely, when a Solid mRNA with no difficulties using the Northern blot method, formulation for oral use is prepared, a filler is added to the the dot blot method, the RT-PCR method, and the microarray. main drug, and if necessary, a binder, a disintegrant, a lubri In the measurement of the expression level of mRNA in step cant, a colorant, a flavoring agent and the like are added (a), preferably, the microarray can be used. thereto, and then tablets, coated tablets, granules, powders, capsules and the like are prepared by conventional methods. 0.124 For the measurement of the expression level of It is needless to say that Sugar coating, gelatin coating or mRNA in step (a), a probe and a primer which consist of a Suitable coating may be conducted on the tablet and granule, polynucleotide capable of hybridizing with nucleotide if necessary. When the compounds are formulated as an injec sequences of genes listed in Table 1, Table 2, Table 3, and tion, a pH regulator, a buffer, a stabilizer, a solubilizer and the Table 4 and homologous genes thereof or their complemen like are added to the main drug, if necessary, to prepare an tary sequences can be employed as a detection marker. Subcutaneous, intramuscular, intra-articular or intravenous 0.125. Any probe and primer according to the present injection according to conventional procedures. When the invention may be employed as long as it can detect the expres compound represented by formula (I) is administered as a sion of mRNA (including a part thereof) of the genes listed in therapeutic or preventive agent for various diseases, it may be Table 1, Table 2, Table 3, and Table 4 or homologous genes orally administered as tablets, powders, granules, capsules, thereof. The probe and the primer according to the present syrups and the like, and may be parenterally administered as invention refers to a polymer consisting of bases or base pairs a spray, a Suppository, an injection, a topical preparation oran such as deoxyribonucleic acid (DNA) or ribonucleic acid infusion. Although the dose remarkably varies according to (RNA). It has been known that double stranded cDNA can be the severity of symptom, age, the kind of disease etc., used in tissue in situ hybridization, and the probe and the approximately 1 mg to 100 mg per day for an adult is admin primer according to the present invention include Such double istered in general at one time or several times per day. stranded cDNA. Particularly preferred RNA probe and 0119. When the expression level of mRNA of the genes primer for detecting RNA in tissue include RNA probes (ribo listed in Table 1, Table 2, Table 3, and Table 4 or homologous probe). genes thereofanda protein encoded by the genes is monitored 0.126 The probe and the primer according to the present in the method according to the present invention, the expres invention includes a probe or a primer which consists of a sion level may be preferably monitored before administration polynucleotide comprising a nucleotide sequence of at least of the compound represented by formula (I), followed by 10, preferably at least 15, more preferably at least 20, further another monitoring at 3, 6, 8, 24, or 48 hours after the admin preferably at least 25 continuous nucleotides, of the genes istration. In a preferred embodiment, follow-up monitoring of listed in Table 1, Table 2, Table 3, and Table 4, and homolo the expression level is carried out at the earliest three hours gous genes thereof, or complementary sequences thereof as after the administration of the compound represented by for well as all mutated polynucleotide sequences thereof. The mula (I). probe and the primer according to the present invention 0120. Upon implementing the method according to the include a probe or a primer which consists of a polynucleotide present invention the decrease in the gene expression level comprising a nucleotide sequence of 10 to 50 or 10 to 30 can be detected using a decrease in the expression level of continuous nucleotides, 15 to 50 or 15 to 30 continuous mRNA or a decrease in the protein expression level associ nucleotides, 20 to 50 or 20 to 30 continuous nucleotides, 25 to ated with the decrease in the mRNA expression level as an 50 or 25 to 30 continuous nucleotides, of the genes listed in index. Table 1, Table 2, Table 3, and Table 4, and homologous genes 0121 According to a first aspect of the invention, a method thereof or mutated polynucleotide sequence thereof. for assaying the action of the compound represented by for I0127. The probe and the primer according to the present mula (I), using the decrease in the expression level of mRNA invention may beat least 10 bases in length, preferably at least as an index (invention (3)) is provided. 15 bases in length, more preferably at least 20 bases in length, 0122. In step (a), cancer tissue or normal tissue such as further preferably at least 25 bases in length. The probe and hemocytes in peripheral blood, platelets, and serum can be the primer according to the present invention may also be 10 taken from the mammal subjected to the assay and mRNA to 50 bases or 10 to 30 bases in length, 15 to 50 bases or 15 to samples can be prepared from the obtained samples to quan 30 bases in length, 20 to 50 bases or 20 to 30 bases in length, tify the expression level of mRNA. Preparation of mRNA is 25 to 50 bases or 25 to 30 bases in length. well known (for example, “Molecular Cloning, A Laboratory I0128. According to the preferred embodiment of the probe Manual 3 ed.” (Cold Spring Harbor Press (2001)), and and the primer according to the present invention, there are required devices, instruments, and reagents therefor are com provided the probe and the primer having 15 to 30 bases in mercially available. Hence those skilled in the art may pre length for assaying the action of the compound represented pare mRNA with no difficulties using the devices, appara by formula (I) to the mammals, which consist of a polynucle tuses, and reagents as needed. otide comprising at least 10, preferably at least 15, more 0123. Measurement of the expression level of mRNA in preferably at least 20, further preferably at least 25 continu step (a) can be performed with any method selected from a ous nucleotides of a polynucleotide sequence of the genes Northern blot method, a dot blot method, an RT-PCR method, listed in Table 1, Table 2, Table 3, and Table 4 or homologous and a microarray (preferably, Human Genome U133 plus 2.0 genes thereof, as well as all mutated sequences thereof, and array). The principles of these methods and how to carry out which are capable of hybridizing with a polynucleotide US 2010/0086931 A1 Apr. 8, 2010
sequence of the genes listed in Table 1, Table 2, Table 3, and more, particularly preferably 98% or more, and most prefer Table 4 or homologous genes thereof. ably 99% or more homology to the target polynucleotide 0129. According to a preferred aspect of the probe and the when the homology is calculated by a homology search Soft primer according to the present invention, there are provided ware, such as FASTA, BLAST, Smith-Waterman (Meth. a probe and a primer which are capable of hybridizing with a Enzym., 164, 765, (1988)), using default parameters. Further, more distinct region within a nucleotide sequence of the hybridization “under stringent conditions' can be performed, genes listed in Table 1, Table 2, Table 3, and Table 4 or for example, by a method of carrying out the reaction at a homologous genes thereof. The above probe and primerallow temperature of 40°C. to 70° C., preferably at a temperature of more precise detection of the action of the compound repre 60° C. to 65°C., in a hybridization buffer solution generally sented by formula (I) to the mammal. used by those skilled in the art, and carrying out washing in a 0130. The probe according to the present invention can be washing solution at a salt concentration of 15 to 300 mmol/L, chemically synthesized based on the nucleotide sequence of preferably at 15 to 60 mmol/L. The temperature and salt the gene subjected to be detected. Preparation of the probes is concentration can be appropriately adjusted depending on the well known and can be carried out in accordance with, for length of the probe to be used. Further the hybridized product example, “Molecular Cloning, A Laboratory Manual 2" ed.” can be washed under conditions in 0.2x or 2xSSC and 0.1% (Cold Spring Harbor Press (1989)), “Current Protocols in SDS at a temperature of 20° C. to 68° C. Whether stringent Molecular Biology” (John Wiley & Sons (19874997)). (high Stringency) or mild (low stringency) conditions is used 0131 The primers according to the present invention can depends on a difference in salt concentrations and tempera be used as a primer set comprising of two or more types of the tures while the washing process. In cases where the difference primers. in hybridizing depends on the salt concentration, the washing 0132) The primer and the primer set according to the process can be carried out in 0.2xSSC and 0.1% SDS as a present invention can be used as a primer and a primer set in stringent washing buffer (high Stringency wash buffer) and accordance with a conventional method in a known method 2xSSC and 0.1% SDS as a mild washing buffer (low strin for detecting the target gene using a nucleic acid amplification gency wash buffer). Also, in cases where the difference in method such as a PCR method, a RT-PCR method, a real-time hybridizing depends on the temperature, the washing process PCR method, and an in situ PCR method. may be carried out at 68°C. for stringent conditions, at 42°C. 0133. The primer set according to the present invention for medium (moderate stringency) conditions, or at room can be selected Such that the nucleotide sequence of the target temperature (20-25°C.) for mild conditions, but 0.2xSSC and gene can be amplified with the nucleic acid amplification 0.1% SDS are used in all the cases. method such as the PCR method. The nucleic acid amplifi 0.138. When pre-hybridization is carried out, it is carried cation method is well known and selection of the primer pair out under the same condition as in hybridization, but pre therein is obvious for those skilled in the art. For example, in (preliminary)-washing is not necessarily carried out under the the PCR method, the primers can be selected such that one of same condition as in hybridization. two primers (a primer pair) undergoes base pairing with the 0.139. The “homologous gene' used in the specification of plus strand of the double stranded DNA of the gene subjected the present application means a gene encoding a protein func to be detected whereas the other of the primers undergoes tionally equivalent to a protein encoded by a certain gene. base pairing with the minus strand of the double stranded Whether it is “functionally equivalent” or not can be deter DNA, as well as an extending strand extended with one mined by evaluating if the protein has functions equivalent to primer can be paired with the other primer. In a LAMP biological phenomena or functions related to the expression method (WO00/28082), three regions from the 3' terminus of the gene. Such a gene encoding the functionally equivalent termed F3c, F2c and F1 c, and three regions from the 5' ter minus termed B1, B2 and B3 are respectively defined for the protein includes not only the so-called homologous gene but gene subjected to be detected. Four types of primers can be also a gene with polymorphism and a gene having a mutation designed using these six regions. without affecting the function. 0134. The primer according to the present invention may 0140 Examples of the homologous gene include genes be chemically synthesized based on the nucleotide sequence which have a nucleotide sequence of a certain gene wherein of the gene subjected to be detected. Preparation of the primer one or more (preferably one to several, or 1, 2, 3 or 4) nucle is well known and can be carried out in accordance with, for otides are inserted, substituted or deleted, or added to one or example, “Molecular Cloning, A Laboratory Manual 2" ed.” both termini, and which encode the functionally equivalent (Cold Spring Harbor Press (1989)), “Current Protocols in protein. Molecular Biology” (John Wiley & Sons (1987-1997)). 0141 Examples of the homologous gene also include 0135 Table 1, Table 2, Table 3, and Table 4 describe infor genes which encode an amino acid sequence encoded by a mation specifying the genes and homologous genes thereof certain gene wherein one or more amino acids are inserted, listed in these tables. Accordingly those skilled in the art can substituted, or deleted, or added to one or both termini (modi obtain information on the nucleotide sequence of the Subject fied amino acid sequence), and which encode the functionally gene to be detected based on the information described in equivalent protein. Table 1, Table 2, Table 3, and Table 4 to design the probe and 0.142 "One or more amino acids are inserted, substituted, primer based thereon. or deleted, or added to one or both termini” used in the 0136. In addition, the genes listed in Table 1 and Table 2 specification of the present application means that a modifi are known genes and probes and primers for detecting them cation is made by a known technical method such as a site are commercially available individually or as a detection kit specific mutagenesis method or by Substitution of several or detection array. amino acids as in naturally occurring mutation. 0137 The term “to hybridize' used in the specification of 0143. The “modified amino acid sequence' used in the the present application means to hybridize with a target poly specification of the present application can be an amino acid nucleotide under stringent conditions. A specific example of sequence wherein, for example, 1 to 30 amino acids, prefer the polynucleotide which hybridizes under stringent condi ably 1 to 20 amino acids, more preferably 1 to 9 amino acids, tions includes a polynucleotide having at least 70% or more, further preferably 1 to 5 amino acids, particularly preferably preferably 80% or more, more preferably 85% or more, fur 1 to 2 amino acids have been inserted, substituted, or deleted, ther preferably 90% or more, further more preferably 95% or or added to one or both termini. Preferably, the modified US 2010/0086931 A1 Apr. 8, 2010
amino acid sequence may be an amino acid sequence having cedures of these methods are well known and devices and one or more (preferably one or several or 1, 2, 3, or 4) con instruments necessary for carrying out the methods are com servative substitutions. mercially available. Those skilled in the art may therefore 0144. The term “conservative substitution” is used herein measure the expression level of mRNA with no difficulties to mean that one or more amino acid residues are substituted using the fluorescent antibody method, the enzyme immu with other chemically similar amino acid residues, so as not to noassay (ELISA) method, the radioimmunoassay (RIA) substantially modify the functions of a protein. Examples of method, the Western blot method and the immunostaining Such conservative Substitution include a case where a certain (immunohistochemistry) method. In the measurement of the hydrophobic residue is substituted with another hydrophobic expression level of the protein in step (f), the enzyme immu residue and a case where a certain polar residue is substituted noassay (ELISA) method, the Western blot method, the with another polar residue having the same electric charge. immunostaining (immunohistochemistry) method and a Such functionally similaramino acids that can be used in Such mass spectrometry method can be used. Substitution are known as every amino acid types in the 0151. In the measurement of the protein in step (f), an present technical field. Specific examples of a nonpolar (hy antibody against a protein encoded by the genes listed Table drophobic) amino acid include alanine, Valine, isoleucine, 1, Table 2, Table 3, and Table 4 and homologous genes leucine, proline, tryptophan, phenylalanine, and methionine. thereof, and a fragment thereof can be used as a detection Examples of a polar (neutral) amino acid include glycine, marker. serine, threonine, tyrosine, glutamine, asparagine, and cys 0152 Any protein may be employed for obtaining the teine. Examples of a (basic) amino acid having a positive antibody according to the present invention as long as it has charge include arginine, histidine, and lysine. Examples of an antigenicity. A protein having an amino acid sequence of the (acidic) amino acid having a negative charge include aspartic protein wherein one or more amino acids are deleted, acid and glutamic acid. inserted, Substituted or added can be used as the antigen for 0145. In cases where the first aspect of the present inven the protein. It is known that Such a protein maintains the same tion is carried out using a cancer tissue and its Surrounding biological activity as the original protein (Mark et al. (1984) tissue as assay samples, the action of the compound repre Proc. Natl. Acad.Sci. USA 81:5662-6; Zoller and Smith (1982) sented by formula (I) can be assayed preferably by using the Nucleic Acids Res. 10:6487-500; Wang et al. (1984) Science expression level of mRNA of the genes listed in Table 1 or 224:1431-3; Dalbadie-McFarland et al. (1982) Proc. Natl. Ac homologous genes thereof as an index. Measurement of the ad.Sci. USA 79:6409-13). A technique to delete, insert, sub expression level of the mRNA in cases where the cancer stitute or add one or more amino acids while maintaining the tissue and the Surrounding tissue are used as the samples, the antigenicity of the original protein is known. For example, microarray can be preferably used. Such a protein may be obtained by preparing and properly 0146 In cases where the first aspect of the present inven expressing a polynucleotide encoding a protein by site tion is carried out using peripheral blood and whole blood as directed mutagenesis technique (Molecular Cloning, A Labo the assay samples, the action of the compound represented by ratory Manual 2nd ed., Cold Spring Harbor Press (1989); formula (I) can be assayed preferably by using the expression Current Protocols in Molecular Biology, John Wiley & Sons, level of mRNA of the genes listed in Table 2, Table 3, and (1987-1997) Section 8.1-8.5; Hashimoto-Goto et a? 41995) Table 4, or homologous genes thereof as an index. Measure Gene 152:271-5; Kinkel (1985) Proc. Natl. Acad.Sci. USA ment of the expression level of the mRNA of the genes listed 82:488-92: Kramer and Fritz (1987) Method. Enzymol. 154: in Table 2, Table 3, and Table 4, or homologous genes thereof 350-67; Kunkel (1988) Method. Enzymol.85:2763-6). in cases where the peripheral blood and whole blood are used 0153. The antibody according to the present invention as the samples, the microarray can be preferably used. includes an antibody having specificity against a part of the 0147 According to a second aspect of the present inven protein. That is, the protein for obtaining the antibody accord tion, a method for assaying the action of the compound rep ing to the present invention includes a polypeptide having the resented by formula (I), using a decrease in the expression full length amino acid sequence of the protein as well as a level of a protein associated with a decrease in the mRNA fragment thereofhaving at least six amino acid residues (for expression level as an index (invention (11)) is provided. example, not less than 6, 8, 10, 12 or 15 amino acid residues). 0148. The protein measured in step (f) is a protein encoded A preferred fragment is a polypeptide fragment Such as an by the genes listed Table 1, Table 2, Table 3, and Table 4, or amino terminus and a carboxyl terminus of the protein. An homologous genes thereof. Table 1, Table 2, Table 3, and antigen determination site of the polypeptide can be predicted Table 4 describe how to obtain sequence information of the by a method analyzing the hydrophobicity/hydrophilicity of genes listed in these tables. the amino acid sequence of the protein (Kyte-Doolittle (1982) 0149. In step (f), samples are taken from a cancertissue or J. Mol. Biol. 157:105-22), and a method analyzing a second normal tissue Such as hemocytes in peripheral blood, plasma, ary structure (Chou-Fasman (1978) Ann. Rev. Biochem. and serum from a mammal Subjected to the assay. Measure 47:251-76) and further confirmed by a computer program ment of the expression level of the protein may be carried out (Anal. Biochem. 151:540-6 (1985)) or a technique such as with the collected samples as they are or a protein extracted PEPSCAN analysis (patent application publication therefrom. Extraction of the protein is well known (for JP60500684T) involving the synthesis of a short peptide to example, Campa, M. J. et al. Cancer Res. 63, 1652-1656, confirm the antigenicity. 2003), and devices, instruments, and reagents necessary for 0154 Table 1, Table 2, Table 3, and Table 4 describe infor carrying out the extraction are commercially available. Hence mation specifying the genes listed in these Tables and those skilled in the art may extract the protein with no diffi homologous genes thereof. Accordingly, those skilled in the culties using Such the commercially available devices, appa art can obtain information on an amino acid encoded by the ratuses, and reagents as needed. subject gene to be detected based on the information 0150. The measurement of the expression level of the pro described in Table 1, Table 2, Table 3, and Table 4, and can tein in step (f) can be carried out with a method selected from design and obtain an antibody based thereon. a fluorescent antibody method, an enzyme immunoassay 0.155. In addition, the genes listed in Table 1, Table 2, (ELISA) method, a radioimmunoassay (RIA) method, a Table 3, and Table 4 are known genes and an antibody for Western blot method and an immunostaining (immunohis detecting a protein encoded thereby is commercially avail tochemistry) method. The principle and implementation pro able individually or as a detection kit or detection array. US 2010/0086931 A1 Apr. 8, 2010
0156 The antibody according to the present invention 0.165. Using such wells, cloning by, for example, a limit may be obtained with a method known those skilled in the art ing dilution method can be further carried out to obtain a (for example, "Current Protocols in Molecular Biology’ clone. Selection and breeding of the hybridoma is usually (John Wiley & Sons (1987), Antibodies: A Laboratory carried out culture medium for mammalian cells (such as Manual, Ed. Harlow and David Lane, Cold Spring Harbor RPMI1640) containing 10-20% bovine fetus serum and Laboratory (1988)). Supplemented with HAT (hypoxanthine, aminopterin, and 0157. The antibody according to the present invention thymidine). The clone obtained in Such a way is intraperito includes a polyclonal antibody, a monoclonal antibody, a neally transplanted into a SCID mouse previously adminis chimeric antibody, a single chain antibody (scEv), a human trated with pristine. Ten to fourteen days later, ascites con ized antibody and a multispecific antibody. Also, the frag taining the monoclonal antibody at a high concentration is ment of the antibody according to the present invention obtained, which ascites can be used as a raw material for includes an antibody fragment such as Fab, Fab', F(ab'), Fc. antibody purification. Also the clone may be cultured and the and Fv. obtained culture may be used as a raw material for antibody 0158 For a polyclonal antibody, blood can be taken from purification a mammal sensitized with an antigen and blood serum can be 0166 Any purification method may be used for purifying isolated with known procedures from the blood to yield blood the monoclonal antibody as long as it is a known method for serum containing the polyclonal antibody. As needed, a frac purifying an immunoglobulin. The purification can be readily tion containing the polyclonal antibody can further be iso accomplished by, for example, an ammonium Sulfate frac lated from this blood serum. tionation method, a PEG fractionation method, an ethanol fractionation method, and use of an anion exchanger, as well 0159 For a monoclonal antibody, antibody-producing as means such as affinity chromatography using the protein. cells are taken from spleen or lympho node of a mammal 0.167 Purification of the polyclonal antibody from serum sensitized with the above-mentioned antigen, and then can be carried out in the same manner. undergo cell fusion with myeloma cell. The resultant hybri 0.168. In cases where the procedure in the second aspect doma is subjected to cloning and the antibody was recovered according to the present invention is carried out by using the from the culture thereof to yield the monoclonal antibody. cancertissue and its Surrounding tissue as assay samples, the 0160 A fragment of the protein can be used as an immu action of the compound represented by formula (I) can pref nogen. Alternatively, the synthesized one based on the amino erably be assayed by using the expression level of the protein acid sequence of the protein can be used. The antigen can be (s) encoded by the genes listed in Table 1 or homologous used as a complex with a carrier protein. A variety of con genes thereofas an index. In cases where the cancertissue and densing agents can be used for preparation of the complex the Surrounding tissue are used as the samples, the measure between the antigen and the carrier protein, which condens ment of the expression level of the proteins can preferably be ing agents include glutaraldehyde, carbodiimide, and male employed with the enzyme immunoassay (ELISA) method, imide active ester. The carrier protein may be a usually used the Western blot technique, the immunostaining (immunohis one such as bovine serum albumin, thyroglobulin, and tochemistry) method and the mass spectrometry method. hemocyanin. A procedure for coupling at a rate (volume) of 1 0169. In cases where the procedure in the second aspect of time to 5 times is usually employed. the present invention using peripheral blood or whole blood 0161 Examples of the animal immunized include mice, as assay Samples, the action of the compound represented by rats, rabbits, guinea pigs, hamsters. An example of a method formula (I) can preferably be assayed by using the expression of inoculation is subcutaneous, intramuscular or intraperito level of the protein(s) encoded by the genes listed in Table 2, neal administration. The administration may be done in com Table 3, and Table 4 or homologous genes thereofas an index. bination with Freund's complete adjuvant and Freund's In cases where peripheral blood or whole blood are used as incomplete adjuvant, and usually once every two to five the samples, the measurement of the expression level of the weeks. proteins can preferably be employed with the enzyme immu 0162 The antibody-producing cells obtained from the noassay (ELISA) method, the Western blot method, the spleen or lymph node of the animal immunized undergo cell immunostaining (immunohistochemistry) method and the fusion with myeloma cells, and is isolated as hybridoma. As mass spectrometry method. the myeloma cells, cells derived from mouse, rat, Homo sapi 0170 The samples obtained from a subject refer to tissues, ens and etc. are used. It is preferred that antibody-producing cells, body fluids, and the like which are obtained from the cell be derived from the same species. Yet there are cases Subject. Specific examples include biopsy, blood (including where the cell fusion can be carried out between different hemocytes, plasma, and serum), urine, tissue samples Such as species. curettage tissue (buccal scrapes) of oral cavity, and tumor 0163 Procedures for the cell fusion may be carried out cells (cells from tumors of breast, lung, stomach, head and with a known method, in accordance with, for example, neck, colorectum, kidney, pancreas, uterus, liver, urinary Nature, 256, 495, 1975. Examples of fusion accelerator bladder, endometrium, and prostate, as well as hemocytes of include polyethylene glycols and Sendai virus. The cell leukemia patients or of lymphocytes). fusion can be usually carried out by using about 20 to 50% of concentration of polyethylene glycols (average molecular EXAMPLES weight 1000 to 4000); at a temperature of 20 to 40° C., preferably 30 to 37°C.; at a ratio in number of cells between 0171 The present invention is described in more detail by antibody production cells and myeloma of usually about 1:1 the examples below. The followings are illustrative of the invention and by no means intended to limit the invention to to 10:1, and for about 1 to 10 minutes. the embodiments described herein. 0164. Various immunochemical methods can be employed for Screening the antibody-producing hybridoma. Examples thereof include ELISA method using a microtiter Example 1 plate coated with the protein, EIA method using a microtiter Analysis with Microarray plate coated with an anti-immunoglobulin antibody, immune blot method using a nitrocellulose blotting membrane after 0172 Microarray analysis (Human Genome U133 plus electrophoresis of samples containing the protein. 2.0 array: Affymetrix) was carried out using RNA purified US 2010/0086931 A1 Apr. 8, 2010
from human colon carcinoma cell strain Wir treated with sity at a gene level normalized with log signal intensity at a 2.8nM and 14 nM of (8E.12E. 14E)-7-((4-Cycloheptylpiper probe level was obtained. By subtracting the mean between azin-1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16. control 1 and control 3 from the mean between log signal 20-pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide intensity 1 and log signal intensity 3 for each case, the log (hereinafter also referred to as “compound A') for six hours. signal intensity was converted into a log signal ratio to the control. A t-test was conducted between each log signal inten (0173 WiDr cells were first suspended in RPMI1640 sity and control, and candidate genes were narrowed down in medium (containing 10% FBS, penicillin, and streptomycin) the basis of a p-value, a log signal ratio, and a signal value. and seeded in a 10 cm dish (2x10 cells/dish). After an over Specifically, genes showing that the P value was 5% or less; night culture in an incubator with 5% carbon dioxide gas at the expression level in the untreated (control) is 100 or more 37°C., the medium was changed with medium containing 2.8 by signal value; and the expression level when treated with nM and 14 nM of compound A or containing vehicle alone. both 2.8 nM and 14 nM of compound A is, compared to the After cultured additional six hours, TRI reagent (SIGMA) control, 25% or less, were picked out. The software used is was added to cells, and the cells were harvested. RNA was R2.2.1 (http://www.r-project.org/), affy package 1.8.1 (http:// purified in accordance with the protocol of TRI reagent, fol www.bioconductor.org). lowed by further purification with RNeasy (QIAGEN). 0176 Genes showing that, by the treatment with com Absorbance at 260 nm was measured to quantify an amount pound A, the P value was 5% or less; the expression level in of RNA. the untreated (control) is 100 or more by signal value; and the 0.174 Subsequently, single stranded cDNA synthesis, expression level when treated with both 2.8nM and 14 nM of double stranded cDNA synthesis, biotin-labeled cFNA syn compound A is, compared to the control, 25% or less, and thesis, and cRNA fragmentation in order were carried out results thereof were shown in Table 1. Probe ID used in Table from 5-10 ug (WilDr 10 ug, PBMC 5ug) of total RNA from 1 represents Human Genome U133 Plus 2.0 Array Probeset the control cells and the treated cells, by using One-Cycle ID. In Table 1, for each gene evaluated, gene name (Gene Target Labeling and Control Reagents (Affymetrix). cRNA Name), abbreviated name (Gene Symbol), accession number probe was used for hybridization with Human Genome U133 (Accession), alias name (Synonym), Probe set number in Plus 2.0 Array (Affymetrix). The array was then washed and Human Genome U133 Plus 2.0 Array (Human Genome U133 stained, and luminescence intensity was measured by a scan Plus 2.0 Array Probeset ID), the expression level upon the six-hour treatment with 2.8nM of E7107 (2.8nM (6 h)), and (0175. The RMA method was applied to 33 of cel files the expression level upon the six-hour treatment with 14 nM obtained in the microarray experiment and log signal inten of E7107 (14 nM (6 h)) were respectively shown.
TABLE 1. Gene Name Gene Symbol Accession Synonym death-associated protein kinase 1 DAPK1 NM OO4938 DAPK//DKFZp781IO35 WD repeat domain 67 WDR67 NM 145647 G85, MGC104222. MGC126773, MGC138159, MGC21654 unc-50 homolog (C. elegans) UNCSO NM 014044 DKFZp564G0222/GMH1// HSD23, UNCL FURP; ; hCMH1p fibroblast growth factor 19 FGF19 NM OO5117 Transcribed locus, moderately similar to XP 518244.1 MCM10 minichromosome maintenance deficient 10 MCM10 NM 018518, NM 182751 CNA43, MGC126776, (S. cerevisiae) PRO2249 hypothetical protein LOC51315 LOCS1315 FKSG44 gene FKSG44 NM 03.1904 FLJ32216, MGC31785 Iron-responsive element binding protein 2 IREB2 NM 004136 ACO3, FLJ23381, IRP2, IRP2AD chromosome 7 open reading frame 23 C7orf23 NM O24315 MGC417S, MM-TRAG nucleoporin like 1 NUPL1 NM OO10O8564? KIAAO)410, PRO2463 NM OO100.8565, NM 014089 RAP1 interacting factor homolog (yeast) RIF1 NM 0181.51 DKFZp781N1478// replication factor C (activator 1) 4,37kDa RFC4 NM OO2916, NM 181573 A1, MGC27291, RFC37 tRNA splicing endonuclease 2 homolog TSEN2 NM O25265 MGC2776, MGC4440, (S. cerevisiae) SEN2, SEN2L CDC6 cell division cycle 6 homolog (S. cerevisiae) CDC6 NM OO1254 CDC1.8L, HSCDC18, HSCDC6 Fanconi anemia, complementation group L. FANCL NM 018062 FLJ10335, PHF9F POG jagunal homolog 1 (Drosophila) JAGN1 NM 032492 FLJ14602A GLOO9 chromosome 1 open reading frame 63 C1orf53 NM 020317 DJ46SN24.2.1. NPDO14f. RP3-46SN24.4 hypothetical protein LOC124512 LOC124512 XM 497558, XM 94.0962, NM 001080510 nuclear receptor Subfamily 2, group C, member 1 NR2C1 NM OO 1032287// TR2. TR2-11 NM OO3297 chromosome 1 open reading frame 79 C1orf79 XM 378848, XM 930434, RP4-669K10.5 XM 934.809 XM 934810, XM 934811, XM 94.0389. XM 94.4565, XM 94.4566 US 2010/0086931 A1 Apr. 8, 2010 13
TABLE 1-continued pleckstrin homology domain containing, family A PLEKHAS NM O19012 FLJ10667, FLJ31492, member 5 KIAA1686. PEPP2 EH domain binding protein 1 EHBP1 NM O15252 KIAAO903, NACSIN hypothetical protein FLJ20628 FLU20628 NM O17910 DKFZp564I2178 Zinc finger, MIZ-type containing 1 ZMIZ1 NM O2O338 FLJ13541 KIAA1224ff. MIZ/RAI17//Zimp10// ZIMP10 polymerase (DNA-directed), delta 3, accessory POLD3 NM OO6591 KIAAO039, MGC119642. Subunit MGC119643, P66. P68 Transcribed locus, strongly similar to NP 689873.1 claudin domain containing 1 CLDND1 NM 001040184f. C3orf24, GENX-3745, NM 001040181. MGC111162, MGC3316, NM 001040182. MGC9861 NM OO1040183f. NM OO1040199. NM OO1040200. NM O19895 chromosome 7 open reading frame 36 C7orf6 NM O2O192 tubulin, gamma complex associated protein 3 TUBGCP3 NM OO6322 KIAAO859 KIAAO859 NM OO1007239, NM O14955, NM O15935 sideroflexin 1 NM O22754 ATPase, H+ transporting, lysosomalVO subunit a2 NM O12463 chromosome 15 open reading frame 40 C15 orf20 NM 144597 WW domain binding protein 4 (formin binding WBP4. NM OO7187 protein 21) Transcribed locus retinoblastoma binding protein 6 RBBP6 NM O06910, NM 018703, NM O32626 meiotic nuclear divisions 1 homolog (S. cerevisiae) MIND1 NMO32117 ecotropic viral integration site 1 EVI1 NM O05241
RNA binding motif, single stranded interacting RBMS1 NM OO2897, NM O16836. protein 1 NM O16839 Zinc finger, CCHC domain containing 6 ZCCHC6 NM O24617 kinesin family member 21A KIF21A NM O17641
polymerase (RNA) III (DNA directed) polypeptide E POLR3E NM O18119 (80 kD) synaptotagmin-like 2 SYTL2 NM O32379, NM 032943, CHR11SYT KIAA1597, NM 206927, MGC102768. SGA72M, NM 206928, NM 206929, NM 206930 CDC7 cell division cycle 7 (S. cerevisiae) CDC7 NM OO3503 CDC7L1, HSCDC7, Hsk1 if MGC117361, MGC126237, MGC126238, CDC7 Zinc finger protein 518 ZNF518 NM O14803 DKFZ781O2147, MGC125707, MGC125710 Transcribed locus KIAAO776 KIAAO776 NM O15323 RP3-393D12.1 RAD54 homolog B (S. cerevisiae) RADS4B XM OO1126364f. FSBP XM OO1126393/. NM O12415 chromosome 12 open reading frame 32 C12Orf32 NM O31.465 HKMT1188, MGC13204 KIAA1387 protein SMEK2 NM 020463 FLFL2, FLJ31474ff KIAA1387 FPSY2. Smk1 STAM binding protein-like 1 STAMEBPL1 ALMalpha/AMSH-FP// AMSH-LP, FLJ31524ff KIAA1373 ba99019.2 SNF2 histone linker PHDRING helicase SHPRH NM OO1042683, FLJ27258, FLJ3.7625, NM 173082 FLJ45O12, FLJ90837, KIAA2023, MGC134886. bAS4SIS.2
US 2010/0086931 A1 Apr. 8, 2010 15
TABLE 1-continued etratricopeptide repeat domain 4f chromosome TTC4 NM 004623 MGC5097 open reading frame 175 nucleolar complex associated 3 homolog NM O224.51 AD24, C10orf117. FAD24. (S. cerevisiae) FLJ1282O microtubule associated serine/threonine kinase MASTL NM 032844 FLJ14813, RP11-85G18.2. ike THC2 chromosome 6 open reading frame 136 NM 145029 MGC15854 urry homolog (Drosophila) FRY NM O23037 13CDNA73, 214K23.2ff C13orf14, CGOO3, bA2O7N42, ba37E23.1 KIAAO323 KIAAO323 NM O15299 ribosomal protein S6 kinase, 52 kDa, polypeptide 1 RPS6KC1 NM 012424 RPK118 humS6PKh1 Zinc finger CCCH-type, antiviral 1-like ZC3HAV1L, NM O80660 C7orf59, MGC14289 origin recognition complex, Subunit 5-like (yeast) ORCSL NM OO2553, NM 181747 ORCS, ORC5P, ORC5T PAX interacting (with transcription-activation PAXIP1 NM O07349 CAGF28, CAGF29, domain) protein 1 FLJ41049. PACIP1, PAXIP1L PTIP TNRC2 ADP-ribosylhydrolase like 2 ADPRHL2 NM O1782.5 ARH3, FLJ2O446, J66SN4.2 caspase recruitment domain family, member 10 CARD10 NM O14550 BIMP1. CARMA3, MGC142219 phosphoinositide-3-kinase, class 3 PIK3C3 NM OO2647 MGC61518//Vps34 methyltransferase like 3 METTL3 NM O19852 M6A, MGC4336, MTA7Off Spo8 chromosome 20 open reading frame 112 C20orf112 NM 080616 transmembrane protein 177 TMEM177 NM O30577 MGC10993 grainyhead-like 1 (Drosophila) GRHL1 NM O14552, NM 198182 LBP-32. LBP32, MGR. TFCP2L2 FCH domain only 2 FCHO2 NM 138782 Niemann-Pick disease, type C1 NPC1 NMOOO271 NPC ATPase, Ca++ transporting, cardiac muscle, slow ATP2A2 NM OO1681, NM 170665 ATP2B. DAR. DD, twitch 2 MGC45367, SERCA2 cancer Susceptibility candidate 5 CASCS NM 144508, NM 170589 AF15Q14//D40 ?/ Transcribed locus, strongly similar to NP 002194.1 integrin alpha 2 precursor CDNA FLJ34214 fis, clone FCBBF3021807 protein phosphatase 1B (formerly 2C), magnesium NM OO1033556// MGC21657. PP2C-beta-XI. dependent, beta isoform NM OO1033557 PP2CB, PP2CBETA, NM OO2706, NM 177968, PPC2BETAX NM 177969 PHD finger protein 20-like 1 NM O16O18, NM O32205, CGI-72, MGC64923 NM 198513 single-strand-selective monofunctional uracil-DNA SMUG1 NM O14311 FDG, HMUDGif glycosylase 1 MGC10437O, UNG3 integrator complex subunit 7 INTS7 NM O15434 C1orf73, DKFZP434B168, NTT elongation factor RNA polymerase II-like 3 ELL3 NM O25165 FLT22637 Solute carrier family 17 (anion sugar transporter), SLC17AS NM 012434 AST, FLJ22227, member 5 FLU23268, ISSD, NSD, SD, SIALIN SLASD, SLD DnaJ (Hsp40) homolog, subfamily C, member 16 DNAJC16 NM O15291 DKFZp686G1298// DKFZp686NO387// DKFZP7811547, KIAAO962 CAP-GLY domain containing linker protein 1 CLIP1 NM OO2956, NM 198240 CLIP, iCLIP-17Of CLIP170, CYLN1, MGC131604, RSN WD repeat domain 26 WDR26 NM O25160 FL21016, MIP2 solute carrier family 25 (mitochondrial SLC25A19 NM O21734 DNC, MCPHA, MUP1 deoxynucleotide carrier), member 19 tripartite motif-containing 31 TRIM31 NM OO7028, NM 052816 jub, ajuba homolog (Xenopus laevis) JUB NM O32876, NM 198086 Ajuba/MGC15563 excision repair cross-complementing rodent repair ERCC3 NM OOO122 BTF2. GTF2HARAD25, deficiency, complementation group 3 TFIIH XPB phosphatase and actin regulator 4 PHACTR4 NM OO10481837/ DKFZp686LO7205// NM O23923 FLJ13171, MGC2O618, MGC341.86, SRP11 442N24 A.1 US 2010/0086931 A1 Apr. 8, 2010 16
TABLE 1-continued
Human Genome U133 Plus 2.0 Array 14 nM Gene Name Probeset ID (6 h) (6 h) death-associated protein kinase 1 203139 at O.09 WD repeat domain 67 214061 at O.09 unc-50 homolog (C. elegans) 203583 at O. O fibroblast growth factor 19 223761 at O. O Transcribed locus, moderately similar to 235046 at O. 1 XP 518244.1 MCM10 minichromosome maintenance deficient 10 220651 s at O. 1 (S. cerevisiae) hypothetical protein LOC51315 2183.03 X at FKSG44 gene 227964 at ron-responsive element binding protein 2 225892 at chromosome 7 open reading frame 23 204215 at nucleoporin like 1 223984 S at RAP1 interacting factor homolog (yeast) 226821 at replication factor C (activator 1) 4,37kDa 204023 at RNA splicing endonuclease 2 homolog 219581 at (S. cerevisiae) CDC6 cell division cycle 6 homolog (S. cerevisiae) 203967 at O.08 Fanconi anemia, complementation group L. 218397 at O.21 jagunal homolog 1 (Drosophila) 223104 at O.09 chromosome 1 open reading frame 63 209006 s at O.19 hypothetical protein LOC124512 225808 at O16 nuclear receptor Subfamily 2, group C, member 1 204791 at O.13 chromosome 1 open reading frame 79 223774 at O.14 pleckstrin homology domain containing, family A 220952 s at O.O6 member 5 EH domain binding protein 1 212653 s at O.OS hypothetical protein FLJ20628 221229 s at O.25 Zinc finger, MIZ-type containing 1 212124 at O.OS polymerase (DNA-directed), delta 3, accessory 212836 at O.09 Subunit Transcribed locus, strongly similar to NP 6898.73.1 228.638 at claudin domain containing 1 208925 at O chromosome 7 open reading frame 36 2234.33 at tubulin, gamma complex associated protein 3 203690 at KIAAO859 2124.05 s at sideroflexin 1 230069 at g ATPase, H+ transporting, lysosomal VO subunita2 205704 S at chromosome 15 open reading frame 40 1552310 at WW domain binding protein 4 (formin binding 203598 s at protein 21) Transcribed locus 230.098 a retinoblastoma binding protein 6 212783 a meiotic nuclear divisions 1 homolog (S. cerevisiae) 223700 a ecotropic viral integration site 1 226,420 a RNA binding motif, single stranded interacting 209868 s at protein 1 Zinc finger, CCHC domain containing 6 220933 s at kinesin family member 21A 226003 a polymerase (RNA) III (DNA directed) polypeptide E 222490 a (80 kD) synaptotagmin-like 2 232914 S at CDC7 cell division cycle 7 (S. cerevisiae) 204510 a Zinc finger protein 518 204291 a Transcribed locus 230177 a 2 KIAAO776 212633 a RAD54 homolog B (S. cerevisiae) 219494 a chromosome 12 open reading frame 32 225837 a KIAA1387 protein 226230 a. STAM binding protein-like 1 227.606 s at SNF2 histone linker PHDRING helicase 226366 a F-box and leucine-rich repeat protein 5 209004 S at Syntaxin binding protein 3 203310 a amily with sequence similarity 86, member C 65585 at bromodomain PHD finger transcription factor 23290.9 s at ATPase, Ca++ transporting, plasma membrane 1 212930 a. potassium voltage-gated channel, Isk-related 227647 a amily, member 3 chromosome 7 open reading frame 49 220949 s at O.20 chromosome 1 open reading frame 82 222893 s at O.20 kinesin family member 2C 211519 s at O.20 kelch-like 12 (Drosophila) 225,068 at O.20 US 2010/0086931 A1 Apr. 8, 2010 17
TABLE 1-continued tensin 3 217853 a O.20 chromosome 5 open reading frame 28 219029 a O.20 myosin head domain containing 1 225947 a O.20 intersex-like (Drosophila) 225708 a O.20 O 4 nuclear receptor coactivator 5 225145 a. O.20 makorin, ring finger protein, 1 201285 a O.20 armadillo repeat containing 8 2034.86 s at O.20 RNA binding motif protein 17 224780 a O.2 nucleoporin like 2 204003 s at O.2 myeloid lymphoid or mixed-lineage leukemia 2 2275.27 a O.2 O lipase, endothelial 219181 a O.2 coiled-coil domain containing 14 225,017 a O.2 thyroid hormone receptor interactor 4 203732 a O.2 histone deacetylase 4 204225 a. O.2 TAF4 RNA polymerase II, TATA box binding protein 213090 s. at O.2 (TBP)-associated factor, 135 kDa Zinc finger with KRAB and SCAN domains 1 214670 a 235436 a 228106 a Transcribed locus 235609 a peptidylprolyl isomerase domain and WD repeat 213483 a containing 1 polycomb group ring finger 1 210023 s at O.2 RALBP1 associated Eps domain containing 1 224366 s at O.22 chromosome 17 open reading frame 80 223351 a O.22 KIN, antigenic determinant of recA protein homolog 205664 a O.22 (mouse) PMS1 postmeiotic segregation increased 1 213677 S at O.22 O. 7 (S. cerevisiae) E2F transcription factor 7 228033 a O.22 methyltransferase like 4 219698 s at O.22 ninein (GSK3B interacting protein) 225921 a O.22 angelhomolog 2 (Drosophila) 221825 a. O.22 cyclin E2 205034 a O.22 Full-length cDNA clone CSODL007YI24 of B cells 226648 a O.22 (Ramos cell line) Cot 25-normalized of Homo Sapiens (human) solute carrier family 37 (glycerol-3-phosphate 218928 s at O.22 transporter), member 1 etratricopeptide repeat domain 4f chromosome 46167 at O.22 open reading frame 175 nucleolar complex associated 3 homolog 218889 a O.23 (S. cerevisiae) microtubule associated serine/threonine kinase 228468 a O.23 O. 1 ike chromosome 6 open reading frame 136 2274.55 a. O.23 furry homolog (Drosophila) 204072 s at O.23 KIAAO323 212355 a. O.23 ribosomal protein S6 kinase, 52 kDa, polypeptide 1 218909 a O.23 2 Zinc finger CCCH-type, antiviral 1-like 228280 a O.23 origin recognition complex, Subunit 5-like (yeast) 204957 a O.23 PAX interacting (with transcription-activation 212825 a. O.23 domain) protein 1 ADP-ribosylhydrolase like 2 23097 a O.23 caspase recruitment domain family, member 10 0026 s at O.23 phosphoinositide-3-kinase, class 3 04297 a O.23 methyltransferase like 3 O9265 S at O.24 chromosome 20 open reading frame 112 25224 a O.24 transmembrane protein 177 8897 a O.24 grainyhead-like 1 (Drosophila) 22830 a. O.24 FCH domain only 2 28220 a O.24 Niemann-Pick disease, type C1 O2679 a O.24 ATPase, Ca++ transporting, cardiac muscle, slow 2361 s at O.24 twitch 2 cancer Susceptibility candidate 5 228323 a O.24 Transcribed locus, strongly similar to NP 002194.1 227314 a O.24 9. s integrin alpha 2 precursor CDNA FLJ34214 fis, clone FCBBF3021807 227.087 a O.24 protein phosphatase 1B (formerly 2C), magnesium 209296 a O.24 dependent, beta isoform PHD finger protein 20-like 1 226942 a. O.24 single-strand-selective monofunctional uracil-DNA 218685 S at O.24 glycosylase 1 integrator complex subunit 7 222250 s at O.24 O.20 elongation factor RNA polymerase II-like 3 219518 S at O.24 O.20 Solute carrier family 17 (anion sugar transporter), 223441 at O.24 O.18 US 2010/0086931 A1 Apr. 8, 2010 18
TABLE 1-continued member 5 DnaJ (Hsp40) homolog, subfamily C, member 16 212908 at O.24 O.15 CAP-GLY domain containing linker protein 1 201975 at O.24 O.12 WD repeat domain 26 218107 at O.24 O16 solute carrier family 25 (mitochondrial 223222 at O.25 O.22 deoxynucleotide carrier), member 19 tripartite motif-containing 31 210159 s at O.25 O.21 jub, ajuba homolog (Xenopus laevis) 225806 at O.25 O.09 excision repair cross-complementing rodent repair 202176 at O.25 O.22 deficiency, complementation group 3 phosphatase and actin regulator 4 226823 at O.25 O.15
Example 2 was purified in accordance with the protocol of TRI reagent. RNA was further purified using RNeasy (QIAGEN). Absor Analysis in PBMC (1) bance at 260 nm was measured to quantify an amount of 0177 Since it is not easy to obtain a cancer tissue clini RNA. cally, the measurement of the marker in hemocytes readily 0182 (3) Analysis of Microarray Data in PBMC obtainable in peripheral blood would be more useful. Specifi 0183. A cel file for each sample was obtained by using cally blood was taken from a normal individual (volunteer) GeneChip Operating Software Ver.1.2 (Affymetrix). The and peripheral blood mononuclear cells were purified and RMA method was applied to ten of the cel files obtained in treated with compound A, followed by measurement of the microarray experiment. Log signal intensity at a gene change in expression of the gene (mRNA) with the microar level was obtained by normalization of that at a probe level. ray (Human Genome 0133 plus 2.0 array: Affymetrix). By Subtracting a control value from log signal intensity for (0178 (1) Isolation of Peripheral Blood Mononuclear each case, the log signal intensity was converted into a log Cells (PBMC) signal ratio to the control. Genes were narrowed down in the (0179 Ficoll-Paque PLUS solution (Amersham, 17-1440 basis of a log signal ratio, and a signal value. Specifically, 02) was slowly added to the blood taken (with heparin added) among genes showing that the P value was 5% or less; the from a healthy individual to formalayer underneath the blood expression level in the untreated (control) is 100 or more by (15 ml of Ficoll-Paque PLUS solution was added to 25 ml of signal value; and the expression level when treated with 10 blood). After the mixture was centrifuged at 1500 rpm for 30 nM of compound Aboth for two and four hours decreased by min, the upperpart containing platelets was removed and then 50% or more, genes of which expression level decreased to a layer containing mononuclear cells was transferred to 25% or less upon the two-hour treatment were picked out. The another tube. The cells were suspended in PBS and centri software used is R2.2.1 (http://www.r-project.org/), affy fuged at 1500 rpm for 5 min, followed by removal of the package 1.8.1 (http://www.bioconductor.org). Supernatant. After these steps were repeated twice, the cells 0.184 Among genes showing, upon the treatment with were again suspended in RPMI1640 (containing 10% FBS). compound A, that the P value was 5% or less; the expression The number of the cells was then counted. level in the untreated (control) is 100 or more by signal value: 0180 (2) Preparation of RNA of PBMC and the expression level when treated with 10 nM of com 0181. The cells were suspended in the medium to 5x10 pound A both for two and four hours decreased by 50% or cells/ml and plated 1 ml per well of a 24-well plate. Immedi more, genes of which expression level decreased to 25% or ately, 111 ul of compound A (ten times more concentrated less upon the two-hour treatment, and results thereof were than the final concentrations) was added to the well (four shown in Table 2. For each gene evaluated, gene name (Gene wells per each concentration tested). The cells were then Name), abbreviated name (Gene Symbol), accession number cultured in an incubator with 5% carbon dioxide gas at 37°C. (Accession), alias name (Synonym), Probe set number in Two or four hours later, the supernatant was collected and Human Genome U133 Plus 2.0 Array (Human Genome U133 centrifuged at 1500 rpm for five minutes. TRI reagent Plus 2.0 Array Probeset ID), the expression level upon the (SIGMA) (1 ml) was added to each well of the plate from two-hour treatment with E7107 (E7107 (2h)), and the expres which the supernatant was removed to harvest the cells, sion level upon the four-hour treatment with E7107 (E7107 (4 which was added to the centrifuged pellet to dissolve. RNA h)) were respectively shown in Table 2.
TABLE 2 Gene Name Gene Symbol Accession Synonym
oxidised low density OLR1 NM OO2543 CLEC8A LOX1, SCARE1 lipoprotein (lectin-like) receptor 1 chromosome 15 open reading C15Crfa3 NM 032413, NM 197955 FL22645, FOAP-11, frame 48 MGC32925. NMES1 formyl peptide receptor-like 2 FPRL2 NM 002030 FML2 HUMAN FMLPY, FPRH1, FPRH2, RMLP-R-I nuclear receptor interacting NRIP3 NM O2O645 C11orf14, NY-SAR-105 protein 3 US 2010/0086931 A1 Apr. 8, 2010 19
TABLE 2-continued C-type lectin domain family 5, CLEC5A NM 013252 member A colony stimulating factor 2 CSF2RA XM 001133962, NM 172248. receptor, alpha, low-affinity NM OO6140, NM 17224.5/ (granulocyte-macrophage) NM 172246, NM 172247/ NM 172249 tensin 1 TNS1 NM 022648 Zinc finger, CCHC domain ZCCHC6 NM O24617 containing 6
Solute carrier family 43, SLC43A2 NM 152346 member 2 trafficking protein particle TRAPPC4 NM 016146 CGI-104; HSPC172, PTDOO9, complex 4 SBDNFTRS23 general transcription factor GTF2H1 NM 005316 BTF2. TFIIH IH, polypeptide 1, 62 kDa solute carrier family 25 SLC25A19 NM 021734 DNC, MCPHA, MUP1 (mitochondrial deoxynucleotide carrier), member 19 G protein-coupled receptor 84 GPR84 NM O2O370 EX33, GPCR4 heparanase HPSE NM OO6665 HPA HPR1, HPSE1, HSE1 tumor necrosis factor, alpha- TNFAIP6 NM 0071.15 TSG-6 induced protein 6 Ras association (RalGDSAF- RAPH1 NM O25252, NM 203365, ALS2CR18, ALS2CR9 6) and pleckstrin homology NM 213589 KIAA1681, FLPD, PREL2. domains 1 RMO1 RalGDSAF-6 chromosome 13 open reading C13orf51 NM 153218 DKFZp686D11119/FLJ38725 rame 31 WD repeat and SOCS box- WSB1 NM O15626, NM 134265 SWIP1, WSB-1 containing 1 hypothetical protein LOCS1315 LOCS1315 cathepsin L1 CTSL1 NM OO1912, NM 145918 CATL CTSL FLJ31037, MEP AHA1, activator of heat shock AHSA2 NM 152392 DKFZp564C236//Heh1 90 kDa protein ATPase homolog 2 (yeast) guanine nucleotide binding GNA1S NM 002068 GNA16 protein (G protein), alpha 15 (Gd class) Transcribed locus chemokine (C-C motif) CCRL2, NM OO3965 receptor-like 2 LOC642312 retinoblastoma binding protein RBBP6 NM OO6910, NM 018703/ 6 NM 032626 Heterogeneous nuclear HNRPA2B1 NM 002137, NM O31243 ribonucleoprotein A2, B1 docking protein 3 DOK3 NM O24872 FLJ22570, FLJ39939 NLR family, pyrin domain NLRP12 NM 033297, NM 144687 CLR19.3 Monarch1, NALP12?. containing 12 PAN6, PYPAF7, RNO. RNO2 membrane-spanning 4- MS4A7 NM 021201, NM 206938, 4SPAN2, CD2OL4, CFFM4, domains, subfamily A, member NM 206939, NM 206940 MGC22368, MS4A8 non-SMC element 4 homolog NSMCE4A NM O17615 C10orf36, FLJ2OOO3, NSE4Aff A (S. cerevisiae) RP11-SOOG22.3, basOOG22, bASOOG22.3 IQ motif containing G IQCG NM O32263 DKFZp434B227/FLJ11667// FLJ23571 ataxin 1 ATXN1 NM OOO332 ATX1, D6SS04E SCA1 dehydrogenase/reductase DHRS9 NM O05771, NM 1992.04 3alpha-HSD/RDH15//RDHL// (SDR family) member 9 RETSDR8
Human Genome U133 Plus 2.0 Array E7107 (2h) E7107 (4 h) Gene Name Probeset ID 10 nM 100 nM 10 nM 100 nM
oxidised low density 21.0004 at O.OS O.O1 O.O2 O.OO lipoprotein (lectin-like) receptor 1 chromosome 15 open reading 223484 at O.O6 O.O2 O.O1 O.O1 frame 48 US 2010/0086931 A1 Apr. 8, 2010 20
TABLE 2-continued formyl peptide receptor-like 2 230422 at O.06 0.05 O.04 O.O2 nuclear receptor interacting 219557 s at O.O7 0.11 O.29 O.24 protein 3 C-type lectin domain family 5, 219890 at O.O8 0.08 O.OS O.O1 member A colony stimulating factor 2 210340 S at O.1O O.O8 OO6 O.10 receptor, alpha, low-affinity (granulocyte-macrophage) tensin 1 221748 s at O.1O 0.09 O.09 O.O6 Zinc finger, CCHC domain 220933 s at O.17 O.14 O.08 O.O6 containing 6 Solute carrier family 43, 228918 a O.17 O.17 O.08 O.08 member 2 trafficking protein particle 21795.9 s at O.18 0.08 O.O7 O.04 complex 4 general transcription factor 202451 a O.18 O.O7 O.O7 O.O2 IIH, polypeptide 1, 62 kDa solute carrier family 25 223222 a. O.18 0.17 O.2O O.18 (mitochondrial deoxynucleotide carrier), member 19 G protein-coupled receptor 84 223767 a O.18. O.19 O.2O O.38 heparanase 222881 a O.18 0.13 O.29 O.11 tumor necrosis factor, alpha- 206026 s at O.19 O.O7 O.O6 O.O2 induced protein 6 Ras association (RalGDSAF- 225188 a O.19 O.OS 0.18 O.10 6) and pleckstrin homology domains 1 chromosome 13 open reading 228937 a O.19 O.04 O.11 O.O6 rame 31 WD repeat and SOCS box- 201294 is at O.2O O.29 O.17 O.25 containing 1 hypothetical protein 233329 s at O.21 O. 11 O.13 O.O6 LOCS1315 cathepsin L1 202087 s at O.21 O.2O O.13 O.13 AHA1, activator of heat shock 226665 at O.21 O.2O O.15 O.23 90 kDa protein ATPase homolog 2 (yeast) guanine nucleotide binding 205349 at O.22 O. 11 O.15 O.15 protein (G protein), alpha 15 (Gd class) Transcribed locus 230098 at O.22 O.O8 O.14 O.08 chemokine (C-C motif) 211434 S at O.22 O.22 O.3S O.29 receptor-like 2 retinoblastoma binding protein 212781 at O.23 O.12 O.17 O.17 6 Heterogeneous nuclear 225107 at O.23 O.O7 O.2O O.10 ribonucleoprotein A2, B1 docking protein 3 223553 S at O.23 O.30 O.26 O.35 NLR family, pyrin domain 1554952 S at O.23 O.24 O.26 O.20 containing 12 membrane-spanning 4- 224358 s at O.24 O.22 O.04 O.O3 domains, subfamily A, member non-SMC element 4 homolog 228506 at O.24 O.19 O.18 O.19 A (S. cerevisiae) IQ motif containing G 235347 at O.24 O.O8 O.23 O.OS ataxin 1 242230 at O.25 0.25 O.24 O.13 dehydrogenase/reductase 223952 x at O.25 0.18 O.31 O.12 (SDR family) member 9
Example 3 0186 RNA samples were first prepared in accordance with the steps described in Example 2 (1) and (2). Analysis in PBMC (2) 0187 Subsequently RNA was prepared to 30 ng/ul and 0185. Since it is not easy to obtain a cancer tissue clini cDNA was synthesized by using TaqMan Reverse Transcrip cally, the measurement of the marker in hemocytes readily tion Reagents (Applied Biosystems). The expression level of obtainable in peripheral blood would be more useful. Specifi SLC25A19, TRAPPC4, GTF2H1, Id1, EDN1, ZCCHC6, cally peripheral blood was taken from three normal individu HNRPA2B1, and HSPA9B was amplified with TaqMan Gene als (volunteers) and peripheral blood mononuclear cells were Expression Assays (catalog numbers below) (Applied Bio purified and treated with compound A, followed by measure systems) as a probe by using TaqMan Universal PCR Master ment of change in expression of the gene (mRNA) by qPCR. Mix (Applied Biosystems). In cases where a decrease in the expression of mRNA is used 0188 Catalog number as a marker, the genes in Table 3 below, as a representative (0189 SLC25A19 (HsO0222265 m1) example, were found to be usable. (0190. TRAPPC4 (HsO0211691 m1) US 2010/0086931 A1 Apr. 8, 2010 21
(0191 GTF2H1 (HsO0366525 g1) 0201 (2) Preparation of RNA with PAXgene Blood RNA (0192 ID1 (HsO0357821A1) Tube (QIAGEN) (0193 EDN1 (HsO0174961 m1) 0202 Human peripheral blood was collected in the tube (0194 ZCCHC6 (HsO0612265 m1) (2.5 ml/tube), combined with Stabilizing Reagent, and left to (0195 HNRPA2B1 (HsO0242600 m1) stand overnight at room temperature. In accordance with the (0196. HSPA9B (HsO02698.18 m1) protocol of the Tube, RNA was purified with PAXgene Blood 0.197 Reagents were prepared in accordance with the pro RNA Kit (QIAGEN). Absorbance at 260 nm was measured to tocol, and measurement was carried out with ABI7900 (Ap quantify an amount of RNA. plied Biosystems). Measured values were corrected by using 0203 (3) Quantification of the Expression Level of the expression level of 18S rRNA (Hs99999901 s1, Applied mRNA Biosystems) as an internal control. The expression level of 0204 RNA was prepared to 30 ng/ul and cDNA was syn each gene was calculated with the expression level in the cells thesized by using High Capacity cDNA Reverse Transcrip untreated with compound Aas 1. Table 3 shows genes whose tion Kits (Applied BioSystems). A probe corresponding expression level in PBMC is 25% or less and their results. In respectively to SLC25A19, TRAPPC4, GTF2H1, ID1, Table 3, for each gene evaluated, abbreviated name (Gene EDN1, ZCCHC6, HNRPA2B1 and 18S rRNA was purchased Symbol), accession number (Accession), alias name (Syn from TaqMan Gene Expression Assays (described above). onym), gene name (Gene Name), and the expression level Reagents were prepared in accordance with the protocol of upon treatment with varied concentration of E7107 (E7107 TaqMan Universal PCR Master Mix (Applied Biosystems) (nM)) were respectively shown. and the expression level of mRNA was measured with
TABLE 3
Gene E7107 (M
Symbol Accession Synonym Gene Name O 3 10 30 SLC25A19 NM 021734 DNC, MCPHA, MUP1 solute carrier family 25 (mitochondrial 1 0.11 0.02 0.01 deoxynucleotide carrier), member 19 TRAPPC4 NM 018146 CGI-104; HSPC172, PTDOO9, SBDNF. trafficking protein particle complex 4 1 O.12 O.O2 O.O1 TRS23 GTF2H1 NM 005316 BTF2 TFIIH general transcription factor IIH, 1 O.22 O.O7 O.OS polypeptide 1, 62 kDa ID1 NM 002165, ID inhibitor of DNA binding 1, dominant 1 O.S O.15 O.O9 NM 181353 negative helix-loop-helix protein EDN1 NM OO1955 ET1 endothelin 1 1 0.45 0.2 O.11 ZCCHC8 NM O24617 DKFZp666B142//DKFZp686C11112// Zinc finger, CCHC domain containing 3 1 0.49 0.19 0.13 DKFZp686F119//DKFZp686I1269/PAPD6 HNRPA2B1 NM 002137 HNRNPA2, HNRNPB1. HNRPA2, HNRPB1?. heterogeneous nuclear ribonucleoprotein 1 0.48 0.29 0.24 NM 031243 RNPA2, SNRPB1 A2B1 HSPA9 NM 004134 CSA GRP7S, HSPA9B, MGC4SOO, MOT, heat shock 70 kDa protein 9 (mortalin) 1 0.39 0.26 0.17 MOT2, MTHSP7S, PBP74, not-2
Example 4 ABIT900 (Applied Biosystems). As shown in A (Tempus Analysis in PBC Blood RNA Tube) and B (PAXgene Blood RNA Tube) in FIG. 1 and FIG. 2, it was demonstrated that gene expression 0198 Since fractionation of hemocytes is required for could sufficiently be confirmed in RNA obtained by both employing PBMC, use of PBMC in a clinical test is compli methods. cated. If change of mRNA is confirmed in whole blood (PBC), such a test would be clinically more useful. However, Example 5 since it is difficult to culture PBC, it is not easy to monitor the change of mRNA in vitro. If expression of mRNA in PBC, Analysis in a Nude Mouse like in PBMC, was confirmed, the change of mRNA can be monitored. Accordingly, in regard to the genes of Table 2 and 0205 (1) Administration of the pladienolide derivative to Table 3 confirmed to function as the marker in PBMC nude mice subcutaneously transplanted with WiLDr human (SLC25A19, TRAPPC4, GTF2H1, ID1, EDN1, ZCCHC6, colon carcinoma cells and HNRPA2B1), it is examined whether the expression of 0206 Nude mice (BALB/cAJcl-nu/nu, 6 weeks old, mRNA can be detected for RNA obtained by the same RNA female) were purchased from CLEA Japan, Inc. After an purification method as in the clinical setting (Tempus, PAX acclimated period of a week, the mice were subcutaneously gene). transplanted WiLDr cells suspended in Hanks’ Balanced Salt (0199 (1) Preparation of RNA with Tempus Blood RNA Solution (GIBCO) (5x10 cells per mouse). Two weeks after Tube (Applied Biosystems) the transplantation, when tumor Volume was confirmed to 0200 Human peripheral blood was collected in the tube (3 grow to more than 200 mm, the mice were administrated ml/tube) and combined with Stabilizing Reagent. In accor with compound A (30 mg/kg) in a single dose via tail vein dance with the protocol of the Tube, RNA was purified by the injection. RNA Blood-DNA Method in ABI 6100 PrepStation (Applied 0207 (2) Blood Collection and Isolation of the Tumor Biosystems). Absorbance at 260 nm was measured to quan 0208. At the time point of 30 minutes, 1 hour, 2 hours, 4 tify an amount of RNA. hours, and 8 hours, after the administration of compound A. US 2010/0086931 A1 Apr. 8, 2010 22 two mice per each time point were put down by euthanasia peripheral blood was taken from three normal individuals with CO. Whole blood (with heparin added) was taken from (volunteers) and peripheral mononuclear cells were purified abdominal aorta of each mouse, TRIZol LS Reagent (Invitro and treated with (8E.12E. 14E)-7-((4-Cycloheptylpiperazin gen) was added thereto, and the mixture was stored at -20°C. 1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20 After removed, the tumor was stored -20°C. in RNA later pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide for (Ambion). six hours, followed by measurement of change in expression 0209 (3) RNA Preparation of the gene by a microarray (Human Exon 1.0 ST Array: 0210 RNA preparation from blood was carried out in Affymetrix). In cases where a change in the expression of the accordance with the protocol of TRIZol LS Reagent (Invitro gene is used as a marker, the genes in Table 4 below, as a gen). The obtained RNA was further purified with RNeasy representative example, were found to be usable. (QIAGEN). The tumor treated with RNA later (Ambion) was 0224 (1) Isolation of Peripheral Blood Mononuclear placed in TRI reagent (SIGMA) and grinded by a homog Cells (PBMC) enizer. RNA was purified in accordance with the protocol of 0225. Ficoll-Paque PLUS solution (Amersham, 17-1440 TRI reagent. The obtained RNA was further purified using 02) was slowly added to the blood taken (with heparin added) RNeasy (QIAGEN). Absorbance at 260 nm was measured to from healthy individuals to form a layer underneath the blood quantify an amount of each RNA. (15 ml of Ficoll-Paque PLUS solution was added to 25 ml of 0211 (4) Measurement of mRNA Expression Level in blood). After the mixture was centrifuged at 1500 rpm for 30 Blood min, the upperpart containing platelets was removed and then 0212 RNA was prepared to 100 ng/ul and cDNA was a layer containing mononuclear cells was transferred to synthesized using TaqMan Reverse Transcription Reagents another tube. The cells were suspended in PBS and centri (Applied Biosystems). For the expression level of mouse fuged at 1500 rpm for 5 min, followed by removal of the TRAPPC4, mouse SLC25A19, and mouse GTF2H1, the Supernatant. After these steps were repeated twice, the cells amplification was carried out by using TaqMan Universal were again suspended in RPMI1640 (containing 10% FBS, PCR Master Mix (Applied Biosystems) with TaqMan Gene Expression Assays (catalog numbers below) (Applied Bio penicillin, and streptomycin) and the number of the cells was systems) as a probe. Reagents were prepared in accordance then counted. with the protocol and the expression level was measured with 0226 (2) Preparation of Total RNA ABIT900 (Applied Biosystems). Measured values were cor 0227 PBMC was suspended in RPMI1640 medium (con rected using the expression level of 18S rRNA (Hs99999901 taining 10% FBS, penicillin, and streptomycin) to 5x10 S1, Applied Biosystems) as an internal control to be calcu cells/ml and 1 ml of the suspension was plated per well of a lated the expression level of each gene with the expression 24-well plate. Immediately, 111 ul of 10 times-concentrated level in the group of mice untreated with compound A. (8E.12E,14E)-7-((4-Cycloheptylpiperazin-1-yl)carbonyl) Results were shown in FIG. 3. oxy-3,6, 16.21-tetrahydroxy-6,10,12,16.20-pentamethyl-18, 0213 Catalog number 19-epoxytricosa-8,12,14-trien-11-olide was added to the 0214 SLC25A19 (Mm01252059 g1) well (six wells per each concentration tested). The cells were 0215 TRAPPC4 (Mm00445555 ml) then cultured in an incubator with 5% carbon dioxide gas at 0216 GTF2H1 (Mm00500417 m1) 37° C. Three hours later, the supernatant was collected and 0217 (5) Measurement of mRNA in Tumor centrifuged at 1500 rpm for five minutes. TRI reagent 0218 RNA was prepared to 100 ng/ul and cDNA was (SIGMA) (1 ml) was added to each well of the plate from synthesized using TaqMan Reverse Transcription Reagents which the supernatant was removed to harvest the cells, (Applied Biosystems). For the expression level of human which was added to the centrifuged pellet to dissolve. RNA TRAPPC4, human SLC25A19, human GTF2H1, amplifica was purified in accordance with the protocol of TRI reagent. tion was carried out by using TaqMan Universal PCR Master RNA was further purified using RNeasy (QIAGEN) and, in Mix(Applied Biosystems) with TaqMan Gene Expression Assays (catalog numbers below) (Applied BioSystems) as a accordance with the protocol, DNase I was added to the probe. Reagents were prepared in accordance with the proto samples during the procedure. Absorbance at 260 nm was col and the expression level was measured with ABI/900 measured to quantify an amount of RNA. (Applied Biosystems). The measurement was corrected using 0228 (3) Analysis of Microarray Data in PBMC the expression level of 18S rRNA (Hs99999901 s1, Applied 0229 Ribosomal RNA was removed from 1 g of each Biosystems) as an internal control to be calculated the expres RNA by using RiboMinus Human/Mouse Transcriptome Iso sion level of each gene with the expression level in the group lation Kit (Invitrogen). Single stranded cDNA synthesis, of mice treated without compound A. Results were shown in double stranded cDNA synthesis, cRNA synthesis, second FIG. 4. single stranded cDNA synthesis, cDNA fragmentation, and 0219 Catalog number cDNA labeling in order were carried out for total RNA from 0220 SLC25A19 (HsO0222265 m1) which ribosomal RNA was removed, using GeneChip Whole 0221) TRAPPC4 (HsO0211691 m1) Transcript Sense Target Labeling and Control Reagents (Af 0222 GTF2H1 (HsO0366525 g1) fymetrix) to make a cDNA probe. Subsequently, the cDNA probe was used for hybridization with Human Exon 1.0 ST Example 6 Array (Affymetrix). The array was washed and stained, and luminescence intensity was measured by a scanner. Analysis in PBMC (2) 0230. The expression level of the gene was quantified by 0223 Since it is not easy to obtain a cancer tissue clini using Expression Console Ver. 1.0 (Affymetrix) with Sum cally, the measurement of the marker in hemocytes readily marization Method and Normalization Method being set to obtainable in peripheral blood would be more useful. It was “Median polish as used in RMA and “None”, respectively. then examined whether the marker gene obtained in cancer 0231 Genes showing the change in the expression of the cells could change in peripheral mononuclear cells in a simi gene in both groups with the 10 nM treatment and the 30 nM lar manner as observed in the cancer cells. Specifically treatment is, compared to that in the treated group, less than US 2010/0086931 A1 Apr. 8, 2010 23
25%; the expression level of the gene in the untreated group is (Gene Name), abbreviated name (Gene Symbol), accession more than 100; both P values oft-test for the 10 nM treated number (Accession), alias name (Synonym), transcription group and the untreated group, and for the 30 nM treated cluster number in Human Exon ST 1.0 Array (Trascript Clus group and the untreated group are less than 5% were picked ter ID), and change in expression level (Fold Change) were out. (Table 4). In Table 4, for the gene evaluated, gene name respectively shown.
TABLE 4
Human Exon ST 1.0 Transcript Fold Change Gene Name Gene Symbol Accession Synonym Cluster ID 10 nM 30 nM reticulocalbin 1, EF-hand calcium binding domain RCN1 NM OO2901 FLJ37041, PIG2Off 3325503 O.09 O.17 RCAL RCN oxidized low density lipoprotein (lectin-like) OLR1 NM OO2543 CLEC8A LOX1 if 3444043 O.09 O.04 receptor 1 SCARE1 cathepsin L1 CTSL1 NM OO1912, CATL, CTSL 3178147 O.09 O.09 NM 145918 FLJ31037, MEP tumor necrosis factor (ligand) Superfamily, TNFSF15 NM OO5118 MGC129934, 3222128 O.10 O.O7 member 15 MGC129935, TL1, TL1A VEGIF VEGI192A kynureninase (L-kynurenine hydrolase) KYNU NM 001032998 - 2508520 O.10 O.10 NM OO3937 phospholipase A2, group VII (platelet-activating PLA2G7 NM 005084 LDL-PLA2, PAFAH 2955827 O.12 O.15 factor acetylhydrolase, plasma) Solute carrier family 7, (cationic amino acid SLC7A11 NM O14331 CCBR1, xCT 2.786322 O.13 O.O6 transporter, y- system) member 11 kynurenine 3-monooxygenase (kynurenine 3- KMO NM O03679 J317G-22.1 2388O85 O.13 O.13 hydroxylase) nuclear receptor interacting protein 3 NRIP3 NM O2O645 C11orf14. NY-SAR- 33621.59 O.15 O.13 05 SLAM family member 7 SLAMF7 NM 021181 9A. CD319, 23632O2 O.15 O.14 CRACC. CS1 dedicator of cytokinesis 4 DOCK4 NM 014705 FLJ34238; KIAAO716, 3068097 O16 O.14 MGC134911, MGC134912 solute carrier family 43, member 3 SLC43A3 NM 014096 DKFZp752A227// 3373845 O.17 O.14 NM O17611 EEG1, FOAP-13, NM 199329 PRO1659, SEEEG-1 tumor necrosis factor, alpha-induced TNFAIP6 NM 007115 TSG-6 2510464 O.18 O.19 protein 6 CD274 molecule CD274 NM O14143 B7-EHF B7EH1 31 61082 O.19 O.18 MGC142294, MGC142296, PD-L1, PDCD1L1 PDCD1LG1, PDL1 TNF receptor-associated factor 1 TRAF NM O05658 EBI6, MGC:1O3S3 3223.738 O.19 O.17 glycerol kinase GK NM OOO167// GK1, GKD 3972.929 O.19 O.21 NM 203391 STEAP family member 4 STEAP4 NM 024636 DKFZp666D049// 3060332 O.20 O.22 FLU23153, STAMP2, TIARP, TNFAIP9 leukocyte-associated immunoglobulin-like LAIR NM OO2287/. CD3OS, LAIR-1 387O824 O.21 O.14 receptor 1 NM O21706 ADP-ribosylation factor GTPase activating ARFGAP3 NM 014570 ARFGAP1 396.2587 O.21 O.14 protein 3 solute carrier family 41, member 2 SLC41A2 NM O32148 DKFZP434KO427. 34.6918O O.21 O.17 MGC12S330, MGG12S331, SLO41A1-L1 chromosome 13 open reading frame 31 C13orf51 NM 153218 DKFZp686D11119// 34876OO O.21 O.19 FLJ38725 solute carrier family 37 (glycerol-3- SLC37A2 NM 198277 FLOO171, 33S4443 O.21 O.23 phosphate transporter), member 2 MGC71430/?pp.11662 solute carrier family 43, member 2 SLC43A2 NM 152346 FLJ23848, LAT4, 3740367 O.22 O.18 MGC3468O purinergic receptor P2X, ligand-gated ion P2RX4 NM OO2560 P2X4, P2X4R 3434760 O.22 O.22 channel, 4 trafficking protein particle complex 4 TRAPPC4 NM O16146 CGI-104, HSPC172, 3351775 O.23 O.21 PTDOO9, SBDNA, TRS23 US 2010/0086931 A1 Apr. 8, 2010 24
TABLE 4-continued
Human Exon ST 10 Transcript Fold Change Gene Name Gene Symbol Accession Synonym Cluster ID 10 nM 30 nM phosphoinositide-3-kinase, regulatory PIK3RS NM 014308 F73OO381SRiki, 374468O O.23 O.21 subunit 5, p101 FOAP-2, P101-PI3K Sema domain, transmembrane domain (TM), and SEMA6B NM 02O241. SEM-SEMA-Y, 3846860 O.23 O.22 cytoplasmic domain, (Semaphorin) 6B NM 032108, SEMA-VIIB, SEMAN NM 133327 Sema VIb, semaZ endoglin (Osler-Rendu-Weber syndrome 1) ENG NM OOO118 CD1 OS, END, . 3226097 O.24 O.20 FLJ41744, HHT1 if ORW ORW1
1. The method according to claim 1, wherein the antitumor 8) a halogen atom, agent is a compound represented by formula (I), a pharma 9) NY'NYN R' wherein R represents ceutically acceptable salt thereof, or a solvate of them: a) a single bond, b) —CO O—, c) —SO O—, (I) d) —CS O— or e) –CO NRY - wherein R^ represents a hydrogen atom or an optionally substituted C. alkyl group, provided that each of the leftmost bond in b) to e) is bound to the nitrogen atom; and R'' and R', the same or different from each other and each represents a) a hydrogen atom, b) an optionally Substituted C alkyl group, c) an optionally Substituted unsaturated C-2 alkyl grOup, d) an optionally substituted aliphatic C-acyl group. wherein R. R. R7 and R', the same or different, each e) an optionally substituted aromatic C7 is acyl group, represents f) an optionally Substituted Caryl group, 1) a hydroxyl group or an oxo group formed together with g) an optionally substituted 5- to 14-membered het the carbonatom to which it is bound, provided that Ris eroaryl group, limited to a hydroxyl group, h) an optionally substituted Czaralkyl group, 2) an optionally Substituted C-2 alkoxy group, i) an optionally Substituted C-2 alkylsulfonyl group, 3) an optionally substituted unsaturated C- alkoxy j) an optionally Substituted Carylsulfonyl group, grOup, k) an optionally substituted 3- to 14-membered non 4) an optionally Substituted C-2 aralkyloxy group, aromatic heterocyclic group formed by R'' and R' 5) an optionally substituted 5- to 14-membered het together with the nitrogenatom to which R'' and R' eroaralkyloxy group, are bound, and the non-aromatic heterocyclic group 6) RCO O— wherein R represents optionally has Substituent(s), a) a hydrogen atom, 1) an optionally substituted 5- to 14-membered het b) an optionally substituted C- alkyl group, eroaralkyl group, c) an optionally Substituted unsaturated C-2 alkyl m) an optionally Substituted C-acycloalkyl group or grOup, n) an optionally substituted 3- to 14-membered non d) an optionally Substituted Caryl group, aromatic heterocyclic group, e) an optionally substituted 5- to 14-membered het eroaryl group, 10) RYSO. O— wherein R^* represents f) an optionally Substituted C7 aralkyl group, a) an optionally Substituted C- alkyl group, g) an optionally substituted 5- to 14-membered het b) an optionally Substituted Caryl group, eroaralkyl group, c) an optionally substituted Calkoxy group, h) an optionally Substituted C-2 alkoxy group, d) an optionally Substituted unsaturated C- alkoxy i) an optionally Substituted unsaturated C- alkoxy grOup, grOup, e) an optionally substituted Caryloxy group, j) an optionally substituted Caryloxy group or f) an optionally substituted 5- to 14-membered het k) an optionally substituted 5- to 14-membered het eroaryloxy group, eroaryloxy group, g) an optionally substituted Czaralkyloxy group or 7) R'R''RSiO wherein R', R, and R, the same or h) an optionally substituted 5- to 14-membered het different, each represents eroaralkyloxy group, a) a C- alkyl group or 11) (RYO)PO-O wherein RY represents b) a C-14 aryl group, a) an optionally Substituted C- alkyl group, US 2010/0086931 A1 Apr. 8, 2010
b) an optionally substituted unsaturated C- alkyl (8E.12E,14E)-7-(N-(2-(N',N'-Diethylamino)ethyl)-N- grOup, methylcarbamoyloxy)-3,6,16.21-tetrahydroxy-6,10,12. c) an optionally substituted Caryl group, 16.20-pentamethyl-18, 19-epoxytricosa-8,12,14-trien d) an optionally substituted 5- to 14-membered het 11-olide; eroaryl group, (8E.12E,14E)-3,6, 16.21-Tetrahydroxy-7-((4-isobutylho mopiperazin-1-yl)carbonyl)oxy-6,10,12,16.20-pen e) an optionally substituted C-2 aralkyl group or tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; f) an optionally substituted 5- to 14-membered het (8E.12E,14E)-7-((4-Ethylhomopiperazin-1-yl)carbonyl) eroaralkyl group), oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pentam 12) (R'R''N), PO-O wherein R^' and R^ have the ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; same meanings as defined above or (8E.12E,14E)-7-((4-Butylhomopiperazin-1-yl)carbonyl) 13) (R'R''N)(RYO)PO O wherein R^, R^* and oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20-pentam RY have the same meanings as defined above, provided ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; that a compound in which R. R. R. and R'' are all (8E.12E,14E)-3, 16.21-Trihydroxy-6-methoxy-6,10,12. hydroxyl groups, and a compound in which R, R and 16.20-pentamethyl-7-(4-methylpiperazin-1-yl)carbo R" are all hydroxyl groups and R is an acetoxy group nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; are excluded, (8E.12E,14E)-3, 16.21-Trihydroxy-6-methoxy-6,10,12. R'K represents a hydrogen atom or hydroxyl group. 16.20-pentamethyl-7-44-(piperidin-1-yl)piperidin-1- 2. The method according to claim 1, wherein the antitumor yl)carbonyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11 agent is selected from the group consisting of: olide; (8E.12E, 14E)-7-(N-(2-(N',N'-Dimethylamino)ethyl)-N- (8E.12E,14E)-3,6,7,21-Tetrahydroxy-6,10,12,16.20-pen methylcarbamoyloxy)-3,6,16.21-tetrahydroxy-6,10,12. tamethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; 16.20-pentamethyl-18, 19-epoxytricosa-8,12,14-trien (8E.12E,14E)-7-((4-(2,2-Dimethylpropyl)homopiper 11-olide; azin-1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy-6.10, (8E.12E, 14E)-3,6,16.21-Tetrahydroxy-6,10,12,16.20 12, 16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 trien-11-olide; and pentamethyl-7-((4-methylhomopiperazin-1-yl)carbo (8E.12E,14E)-3,6, 16-Trihydroxy-21-methoxy-6,10,12. nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; 16.20-pentamethyl-7-(4-methylpiperazin-1-yl)carbo (8E.12E, 14E)-3,6,16.21-Tetrahydroxy-6,10,12,16.20 nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide. pentamethyl-7-((4-methylpiperazin-1-yl)carbonyl) 3. The method according to claim 1, wherein the detection oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; of the decrease in the gene expression level comprises the (8E.12E. 14E)-7-((4-Butylpiperazin-1-yl)carbonyl)oxy-3, steps of: 6, 16.21-tetrahydroxy-6,10,12,16.20-pentamethyl-18, (a) measuring the expression level of mRNA before and 19-epoxytricosa-8,12,14-trien-11-olide; after administration of the antitumor agent to a mammal; (8E.12E. 14E)-7-((4-Ethylpiperazin-1-yl)carbonyl)oxy-3, (b) comparing, based on the expression level measured in 6, 16.21-tetrahydroxy-6,10,12,16.20-pentamethyl-18, (a), the expression level of the mRNA before and after 19-epoxytricosa-8,12,14-trien-11-olide; administration of the antitumor agent to determine that (8E.12E, 14E)-3,6,16.21-Tetrahydroxy-6,10,12,16.20 the antitumor agent exerts an action to the mammal pentamethyl-7-((4-propylpiperazin-1-yl)carbonyl)oxy when the expression level of mRNA after the adminis 18, 19-epoxytricosa-8,12,14-trien-11-olide; tration decreases. (8E.12E. 14E)-7-((4-Cyclohexylpiperazin-1-yl)carbonyl) 4. The method according to claim3, wherein the mRNA of oxy-3,6, 16.21-tetrahydroxy-6,10,12,16.20-pentam which expression level is measured is mRNA of at least one ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; gene selected from the genes listed in Table 1, Table 2, Table (8E.12E. 14E)-7-((4-(Cyclopropylmethyl)piperazin-1-yl) 3 and Table 4, or a homologous gene thereof. carbonyl)oxy-3,6,16.21-tetrahydroxy-6,10,12,16.20 5. The method according to claim 4, wherein the gene(s) pentamethyl-18, 19-epoxytricosa-8,12,14-trien-11 are selected from TRAPPC4, SLC25A19, GTF2H1, ID1, olide; ZCCHC6 and EDN1. (8E.12E, 14E)-3,6,16.21-Tetrahydroxy-6,10,12,16.20 6. The method according to claim3, wherein in step (a), the pentamethyl-7-((4-propylhomopiperazin-1-yl)carbo expression level of mRNA in samples obtained from a subject nyl)oxy-18, 19-epoxytricosa-8,12,14-trien-11-olide; before and after administration of the antitumor agent is mea (8E.12E,14E)-7-((4-(Cyclopropylmethyl)homopiper Sured. azin-1-yl)carbonyl)oxy-3,6,16.21-tetrahydroxy-6.10, 7. The method according to claim 6, wherein the samples obtained from the subject are selected from hemocytes in 12, 16.20-pentamethyl-18, 19-epoxytricosa-8,12,14 peripheral blood, plasma and serum. trien-11-olide; 8. A probe or primer for assaying an action of a compound (8E.12E. 14E)-7-((4-Cyclopentylpiperazin-1-yl)carbonyl) represented by formula (I), a pharmaceutically acceptable oxy-3,6, 16.21-tetrahydroxy-6,10,12,16.20-pentam salt thereof, or a Solvate of them to a mammal, which consists ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; of a polynucleotide capable of hybridizing with a polynucle (8E.12E. 14E)-3,6, 16.21-Tetrahydroxy-7-((4-isopropy otide consisting of a nucleotide sequence of at least one gene lpiperazin-1-yl)carbonyl)oxy-6,10,12,16.20-pentam selected from the genes listed in Table 1, Table 2, Table 3 and ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; Table 4, or a homologous gene thereof, or a complementary (8E.12E. 14E)-7-((4-Cycloheptylpiperazin-1-yl)carbonyl) sequence thereof. oxy-3,6, 16.21-Tetrahydroxy-6,10,12,16.20-pentam 9. The probe or primer according to claim 8, which is ethyl-18, 19-epoxytricosa-8,12,14-trien-11-olide; capable of detecting a genomic intron region or a part thereof US 2010/0086931 A1 Apr. 8, 2010 26 in a gene listed in Table 1, Table 2, Table 3 or Table 4, or which 13. The method according to claim 11, wherein the protein is capable of detecting a polynucleotide lacking a part of a of which expression level is measured is a protein consisting genomic exon region in a gene listed in Table 1, Table 2, Table of amino acids encoded by a polynucleotide of at least one 3 or Table 4. gene selected from TRAPPC4, SLC25A19, GTF2H1, ID1, 10. A reagent or kit for assaying an action of a compound ZCCHC6 and EDN1. represented by formula (I), a pharmaceutically acceptable 14. The method according to claim 11, wherein in step (f), salt thereof, or a solvate of them to a mammal, which com the expression level of the protein in the samples obtained prises the probe or the primer according to claim 8. from a subject before and after administration of the antitu mor agent is measured. 11. The method according to claim 1, wherein the detection 15. The method according to claim 14, wherein the samples of the decrease in the gene expression level comprises the obtained from the subject are selected from hemocytes in steps of: peripheral blood, plasma and serum. (f) measuring the expression level of a protein before and 16. An antibody against an protein consisting of amino after administration of the antitumor agent to a mammal; acids encoded by a polynucleotide of at least one gene (g) comparing, based on the expression level measured in selected from the genes listed in Table 1, Table 2, Table 3 and (f), the expression level of the protein before and after Table 4 or a homologous gene thereof, or a fragment thereof. administration of the antitumor agent to determine that 17. A reagent or kit for assaying an action of a compound the antitumor agent exerts an action to the mammal represented by formula (I), a pharmaceutically acceptable when the expression level of the protein after the admin salt thereof, or a solvate of them to a mammal, comprising the istration decreases. antibody or the fragment thereof according to claim 16. 12. The method according to claim 11, wherein the protein 18. A method for assaying an action of an antitumor agent of which expression level is measured is a protein consisting to a mammal, which comprises detecting a decrease in gene of amino acids encoded by a polynucleotide of at least one expression level caused by the antitumor agent. gene selected from the genes listed in Table 1, Table 2, Table 3 and Table 4 or a homologous gene thereof. c c c c c