METHOD FOR ASSOCIATING WITH EXPRESSION LEVEL OF AKR1C3 ENZYME VIA CONTENT OF PROSTAGLANDIN, AND USE OF SCREENING FOR DRUG ADMINISTRATION

Abstract

Described is a method for associating with the expression level of an AKR1C3 enzyme via the content of prostaglandin, and the use of screening for drug administration. In particular, the content of prostaglandin is measured to associate with the expression level of the AKR1C3 enzyme in a biological sample; and the change in the content and the change rate of the content of prostaglandin before and after administering interfering drugs are measured to associate with the expression level of the AKR1C3 enzyme in the biological sample.

Claims

1. A method for measuring the expression level of AKR1C3 enzyme, comprising measuring the content of PGF2α and/or PGD2 and/or PGH2 to associate with the expression level of AKR1C3 enzyme in a biological sample, or comprising measuring the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of an interfering drug to associate with the expression level of AKR1C3 enzyme in a biological sample, or comprising measuring the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of an interfering drug to associate with the expression level of AKR1C3 enzyme in a biological sample.

2. (canceled)

3. (canceled)

4. The method for measuring according to claim 1, wherein the method comprises measuring the content of PGF2α and/or PGD2 and/or PGH2 to associate with the expression level of AKR1C3 enzyme in a biological sample, wherein the association refers to: when the content of PGF2α is in the range of A1, the expression level of AKR1C3 enzyme in the biological sample is high; when the content of PGF2α is in the range of B1, the expression level of AKR1C3 enzyme in the biological sample is medium; when the content of PGF2α is in the range of C1, the expression level of AKR1C3 enzyme in the biological sample is low; and/or when the content of PGD2 and/or PGH2 is in the range of A2, the expression level of AKR1C3 enzyme in the biological sample is low; when the content of PGD2 and/or PGH2 is in the range of B2, the expression level of AKR1C3 enzyme in the biological sample is medium; when the content of PGD2 and/or PGH2 is in the range of C2, the expression level of AKR1C3 enzyme in the biological sample is high; wherein the minimum value in the range of A1 is greater than or equal to the maximum value in the range of B1, the minimum value in the range B1 is greater than or equal to the maximum value in the range of C1, the minimum value in the range of A2 is greater than or equal to the maximum value in the range of B2, and the minimum value in the range of B2 is greater than or equal to the maximum value in the range of C2.

5. The method for measuring according to claim 1, wherein the method comprises measuring the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of an interfering drug to associate with the expression level of AKR1C3 enzyme in a biological sample, wherein the association refers to: when the content change of PGF2α is in the range of a1, the expression level of AKR1C3 enzyme in biological sample is high; when the content change of PGF2α is in the range of b1, the expression level of AKR1C3 enzyme in biological sample is medium; when the content change of PGF2α is in the range of c1, the expression level of AKR1C3 enzyme in biological sample is low; and/or when the content change of PGD2 and/or PGH2 is in the range of a2, the expression level of AKR1C3 enzyme in biological sample is low; when the content change of PGD2 and/or PGH2 is in the range of b2, the expression level of AKR1C3 enzyme in biological sample is medium; when the content change of PGD2 and/or PGH2 is in the range of c2, the expression level of AKR1C3 enzyme in biological sample is high; wherein the minimum value in the range of a1 is greater than or equal to the maximum value in the range of b1, the minimum value in the range of b1 is greater than or equal to the maximum value in the range of c1; the minimum value in the range of a2 is greater than or equal to the maximum value in the range of b2, and the minimum value in the of range b2 is greater than or equal to the maximum value in the range of c2.

6. The method for measuring according to claim 1, wherein the method comprises measuring the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of an interfering drug to associate with the expression level of AKR1C3 enzyme in a biological sample, wherein the association refers to: when the content change rate of PGF2α is in the range of α1, the expression level of AKR1C3 enzyme in biological sample is high; when the content change rate of PGF2α is in the range of β1, the expression level of AKR1C3 enzyme in biological sample is medium; when the content change rate of PGF2α is in the range of γ1, the expression level of AKR1C3 enzyme in biological sample is low; and/or when the content change rate of PGD2 and/or PGH2 is in the range of α2, the expression level of AKR1C3 enzyme in biological sample is low; when the content change rate of PGD2 and/or PGH2 is in the range of β2, the expression level of AKR1C3 enzyme in biological sample is medium; when the content change rate of PGD2 and/or PGH2 is in the range of γ2, the expression level of AKR1C3 enzyme in biological sample is high; wherein the minimum value in the range of α1 is greater than or equal to the maximum value in the range of β1, the minimum value in the range of β1 is greater than or equal to the maximum value in the range of γ1, the minimum value in the range of α2 is greater than or equal to the maximum value in the range β2, and the minimum value in the range of β2 is greater than or equal to the maximum value in the range of γ2.

7. The method for measuring according to claim 1, wherein the interference drug is an AKR1C3 enzyme inhibitor or an AKR1C3 enzyme agonist.

8. The method for measuring according to claim 7, wherein the AKR1C3 enzyme inhibitor is ##STR00032## or Indomethacin.

9. The method for measuring according to claim 1, wherein the biological sample is blood or serum.

10. The method for measuring according to claim 1, comprising the following operations: measuring the content of PGF2α and/or PGD2 and/or PGH2 in a living body, a living biological organ or a living biological tissue; administering the interfering drug to the living body, the living biological organ or the living biological tissue; measuring the content of PGF2α and/or PGD2 and/or PGH2 in the living body, the living biological organ or the living biological tissue after the administration of the interfering drug; calculating the content change and the content change rate before and after administration of the interfering drug, and obtaining the expression level of AKR1C3 enzyme in biological sample according to the corresponding relationship.

11. A method for screening and administrating to a patients with cancer, tumor or a disorder caused by cancer or tumor, or a cell proliferative disease, comprising administering an AKR1C3 enzyme-activated anti-cancer prodrug to the patient after the expression level of AKR1C3 enzyme is obtained by using the method for measuring the expression level of AKR1C3 enzyme according to claim 1.

12. (canceled)

13. (canceled)

14. The method for administrating according to claim 11, wherein the AKR1C3 enzyme-activated anti-cancer prodrug contains the compound of structural formula I: ##STR00033## wherein, X.sup.10 is O, S, SO or SO.sub.2; A is C.sub.6-C.sub.10 aryl or substituted aryl, 5-15 membered heteroaryl or substituted heteroaryl or —N═CR.sup.1R.sup.2; wherein R.sup.1 and R.sup.2 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; X, Y and Z are each independently hydrogen, CN, halogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkynyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; R is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; R.sup.13 and R.sup.14 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether, or R.sup.13 and R.sup.14 groups together with the nitrogen atoms to which they are bonded form 5-7 membered heterocycle; T comprises an amino phosphate alkylating agent, i.e., T is -L-D, and includes the following six cases: -D is —P(Z.sup.1)(Z.sup.5—X.sup.5—Y.sup.5).sub.n, Z.sup.1 is O or S, Z.sup.5 is N, S or O, X.sup.5 is an optionally substituted ethylidene, Y.sup.5 is a halogen atom or —OSO.sub.2—R.sup.20, R.sup.20 is optionally substituted hydrocarbyl, aryl, cycloalkyl, heterocycyl or heteroaryl, n is 1 or 2, L is selected from —O—, —S—, —OCOO—, —NR.sup.6CO—, —OCO—, —NR.sup.6SO.sub.2—, —OCONR.sup.6—, quaternary ammonium, sulfonate group —OSO.sub.2—; or Z.sup.1 is O or S, Z.sup.5—X.sup.5—Y.sup.5 is an aziridinyl-NCH.sub.2CH.sub.2 moiety; or -L- is —O—, -D is —P(Z.sup.1)(Z.sup.5—X.sup.5—Y.sup.5).sub.n, Z.sup.1 is O or S, Z.sup.5 is N, S or O, X.sup.5 is an optionally substituted ethylidene, Y.sup.5 is a halogen atom or —OSO.sub.2—R.sup.20, R.sup.20 is an optionally substituted hydrocarbyl, aryl, cycloalkyl, heterocyclyl or heteroaryl, and n is 1 or 2; or -L- is —O—, Z.sup.1 is O or S, and Z.sup.5—X.sup.5—Y.sup.5 is an aziridinyl —NCH.sub.2CH.sub.2 moiety; or -L-D is —OP(Z.sup.1)(NR.sup.30CH.sub.2CH.sub.2Cl).sub.2, —OP(Z.sup.1)(NR.sup.30CH.sub.2CH.sub.2Br).sub.2, —OP(Z.sup.1)(NR.sup.30.sub.2)(N(CH.sub.2CH.sub.2X.sup.1).sub.2), —OP(Z.sup.1)(N(CH.sub.2).sub.2).sub.2, or —OP(Z.sup.1)(N(CH.sub.2CH.sub.2Cl).sub.2).sub.2, wherein each R.sup.30 is each independently H, C.sub.1-C.sub.6 hydrocarbyl or two R.sup.30 groups together with the nitrogen atoms to which they are linked form 5-7 membered heterocycle, Z.sup.1 is O or S, X.sup.1 is Cl, Br or —OSO.sub.2Me; or -L-D is —OP(Z.sup.1)(NHCH.sub.2CH.sub.2Cl).sub.2, —OP(Z.sup.1)(NHCH.sub.2CH.sub.2Br).sub.2, —OP(Z.sup.1)(NH.sub.2)(N(CH.sub.2CH.sub.2X.sup.1).sub.2), —OP(Z.sup.1)(N(CH.sub.2).sub.2).sub.2, or —OP(Z.sup.1)(N(CH.sub.2CH.sub.2Cl).sub.2).sub.2, and X.sup.1 is Cl, Br or —OSO.sub.2Me; and the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, and the ether group are substituted or unsubstituted.

15. The method for administrating according to claim 11, wherein the AKR1C3 enzyme-activated anti-cancer prodrug contains a compound having a structural formula selected from the group consisting of: ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##

16. The method for administrating according to claim 11, wherein the AKR1C3 enzyme-activated anti-cancer prodrug contains a compound of structural formula II: ##STR00051## wherein, X.sup.10 is O, S, SO or SO.sub.2; A is C.sub.6-C.sub.10 aryl or substituted aryl, 5-15 membered heteroaryl or substituted heteroaryl or —N═CR.sup.1R.sup.2; wherein R.sup.1 and R.sup.2 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; X, Y and Z are each independently hydrogen, CN, halogeno, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; each R is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, —CONR.sup.13R.sup.14 or —NR.sup.13COR.sup.14; R.sup.13 and R.sup.14 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether, or R.sup.13 and R.sup.14 together with the nitrogen atom to which they are bonded form 5-7 membered heterocyclyl; L.sup.1 and D are as defined in the description, and the specific definitions are as follows: L.sup.1 is selected from: ##STR00052## wherein R.sup.40 and R.sup.41 are independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl; R.sup.42 is a C.sub.2-C.sub.3 alkylene or heteroalkylene optionally substituted with 1-3 C.sub.1-C.sub.6 alkyl; V(−) is any anion, preferably a pharmaceutically acceptable anion, and D is a moiety that makes D-OH an anti-cancer drug, wherein OH is an aliphatic hydroxyl or phenolic hydroxyl; in other words, D is a group after hydroxyl of the anti-cancer drug D-OH is removed; or L.sup.1 is ##STR00053## wherein R.sup.40 is as defined above, R.sup.43 is hydrogen or together with D forms heterocycle, and the phenyl moiety is optionally substituted; and D is a moiety that makes D-NR.sup.43H an anti-cancer drug; in other words, D is a group after amino or amine of the anti-cancer drug D-NR.sup.43H is removed; or L.sup.1 is bond, —O—C(R.sup.40R.sup.41)—, —O—C(R.sup.40R.sup.41)—NR.sup.40R.sup.41(+)—C(R.sup.40R.sup.41)— or ##STR00054## wherein R.sup.40, R.sup.41 and V are as defined above; and D is an anti-cancer drug containing a primary or secondary amine, wherein the primary or secondary amine is bonded to L.sup.1; and the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, and the ether group are substituted or unsubstituted.

17. The method for administrating according to claim 16, wherein in the compound of structural formula II contained in the AKR1C3 enzyme-activated anti-cancer prodrug, D-OH is selected from the following anti-cancer drugs containing an —OH group: gemcitabine, estramusting, pudnimnstine, chlorozotocin, ranimustine, mannomustine, mitobronitol, dibromodulcitol, aclacinomycins, anthramycin, bleomycin, carubicin, carzinophilin, chromomycin, actinomycin D, daunorubicin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, fludarabine, ancitabine, azacitidine, 6-azauridine, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, L-asparaginase, pulmozyme, aceglatone, elliptinium acetate, etoglucid, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoxantrone, mopidamol, pentostatin, pirarubicin, podophyllinic acid, sizofiran, paclitaxel, teniposide, tenuazonic acid, vinblastine, vincristine; D-NR.sup.43H is selected from the following anti-cancer drugs: erlotinib, meturedepa, uredepa, imatinib, trimethylolomelamine, gefitinib, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, actinomycin, anthramycin, bleomycin, actinomycin C, carubicin, carzinophilin, actinomycin D, peplomycin, puromycin, streptozocin, ubenimex, zinostatin, denopterin, pteropterin, trimetrexate, 6-mercaptopurine, thiamiprine, thioguanine, 6-azauridine, carmofur, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L-asparaginase, pulmozyme, amsacrine, bisantrene, demecolcine, diaziquone, elliptinium acetate, flutamide, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, mitoxantrone, nitracrine, pentostatin, phenamet, 2-ethylhydrazide, procarbazine, razoxane, erlotonib, urethane, vinblastine, vincristine; the anti-cancer drugs containing a tertiary or secondary nitrogen atom is selected from altretamine, triethylenemelamine, chlorambuci, chlornaphazine, estramustine, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, pipobroman, actinomycin, anthramycin, carzinophilin, actinomycin D, nogalamycin, porfiromycin, puromycin, streptozocin, tubercidin, fludarabine, ancitabine, azacitidine, cytarabine, dideoxyuridine, enocitabine, floxuridine, L-asparaginase, pulmozyme, aldophosphamide glycoside, bestrabucil, diaziquone, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, mitoguazone, mopidamol, nitracrine, pentostatin, phenamet, razoxane, spirogermanium, tamoxifen, triaziquone, 2,2′,2″-trichlorotriethylamine, vinblastine, vincristine.

18. The method for administrating according to claim 11, wherein the AKR1C3 enzyme-activated anti-cancer prodrug contains a compound with a structural formula selected from the group consisting of: ##STR00055##

19. An assembly for measuring the expression level of AKR1C3 enzyme, comprising: a component that contacts and reacts with the biological sample, and quantitatively or semi-quantitatively associates with the content of PGF2α and/or PGD2 and/or PGH2 in the biological sample according to the signal of the reaction; a control comparison component that is used to compare the signal of reaction to comparatively compare and obtain the expression level of AKR1C3 enzyme corresponding to the content of PGF2α and/or PGD2 and/or PGH2, the content change of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interfering drug, or the content change rate of PGF2α and/or PGD2 and/or PGH2 before and after administration of the interfering drug in the biological sample.

20. An administration device, comprising: the assembly for measuring the expression level of AKR1C3 enzyme according to claim 19; an administration assembly, which contains an AKR1C3 enzyme-activated anti-cancer prodrug.

21. The method for administrating according to claim 11, wherein an AKR1C3 enzyme-activated anti-cancer prodrug is administered to the patient when the level of enzyme: a) is high in the patient or b) is high or medium in the patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0193] FIG. 1 is a schematic diagram of metabolism principle of DNA alkylated anti-cancer prodrugs targeting Aldo-keto reductase 1C3 (AKR1C3).

[0194] FIG. 2 is a schematic diagram showing the effect of AKR1C3 on metabolism of prostaglandin.

[0195] FIG. 3 is a schematic diagram showing the effect of AKR1C3 inhibitors on metabolism of prostaglandin.

[0196] FIG. 4 is a schematic diagram showing the effect of AKR1C3 agonists on metabolism of prostaglandin.

DETAILED DESCRIPTION OF THE INVENTION

[0197] The present invention will be described below with reference to specific examples. Those skilled in the art could understand that these examples are only used for describing the invention and do not in any way limit its scope.

[0198] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials of the drugs, the reagents and the like used in the following examples are all commercially available products unless otherwise specified.

[0199] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from the definitions of the terms as generally understood in the art.

[0200] All numerical designations, e.g., pH, temperature, time, concentration, and weight, including ranges of each thereof, are approximations that typically may be varied (+) or (−) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”. Reagents described herein are exemplary and equivalents of such may be known in the art.

[0201] “C.sub.x-C.sub.y” or “C.sub.x-y” before a group refers to a range of the number of carbon atoms that are present in that group. For example, C.sub.1-C.sub.6 alkyl refers to an alkyl group having at least 1 and up to 6 carbon atoms.

[0202] “Alkoxy” refers to —O-Alkyl.

[0203] “Amino” refers to NR.sup.pR.sup.q wherein R.sup.p and R.sup.q independently are hydrogen or C.sub.1-C.sub.6 alkyl, or R.sup.p and R.sup.q together with the nitrogen atom to which they are bonded form a 4-15 membered heterocycle.

[0204] “Aryl” refers to an aromatic group having carbon atoms and containing no ring heteroatoms and having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). For multiple ring systems, including fused, bridged, and spiro ring systems having aromatic and non-aromatic rings that have no ring heteroatoms, the term “aryl” or “Ar” applies when the point of attachment is at an aromatic carbon atom (e.g., 5,6,7,8 tetrahydronaphthalene-2-yl is an aryl group, as its point of attachment is at the 2-position of the aromatic phenyl ring).

[0205] According to specific embodiments of the present application, C.sub.6-C.sub.10 aryl can be phenyl, naphthyl and various substituted phenyl or naphthyl.

[0206] “Heteroaryl” (Heterocyclic aryl) refers to an aromatic group of from 1 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur and includes single ring (e.g. imidazolyl-2-yl and imidazol-5-yl) and multiple ring systems (e.g. imidazopyridyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl). For multiple ring systems, including fused, bridged, and spiro ring systems having aromatic and non-aromatic rings, the term “heteroaryl” applies if there is at least one ring heteroatom, and the point of attachment is at an atom of an aromatic ring (e.g. 1,2,3,4-tetrahydroquinolin-6-yl and 5,6,7,8-tetrahydroquinolin-3-yl). In some embodiments, the nitrogen and/or the sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N.fwdarw.O), sulfinyl, or sulfonyl moieties. The term heteroaryl or 5-15 membered heteroaryl includes, but is not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazopyridyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl.

[0207] “Alkyl” refers to a monovalent saturated aliphatic hydrocarbyl group having carbon atom(s) and, in some embodiments, from 1 to 6 carbon atoms. “C.sub.x-y alkyl” refers to an alkyl group having from x to y carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH.sub.3—), ethyl (CH.sub.3CH.sub.2—), n-propyl (CH.sub.3CH.sub.2CH.sub.2—), isopropyl ((CH.sub.3).sub.2CH—), n-butyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2—), isobutyl ((CH.sub.3).sub.2CHCH.sub.2—), sec-butyl ((CH.sub.3)(CH.sub.3CH.sub.2)CH—), tert-butyl ((CH.sub.3).sub.3C—), n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2—), and neopentyl ((CH.sub.3).sub.3CCH.sub.2—).

[0208] “Cycloalkyl” refers to a saturated or partially saturated cyclic group with more than 3 carbon atoms and no ring heteroatoms and having a single ring or multiple rings including fused, bridged, and spiro ring systems. For multiple ring systems having aromatic and non-aromatic rings that have no ring heteroatoms, the term “cycloalkyl” applies when the point of attachment is at a non-aromatic carbon atom (e.g. 5,6,7,8-tetrahydronaphthalene-5-yl). The term “cycloalkyl” or “C.sub.3-C.sub.8 cycloalkyl” includes cycloalkenyl groups. Examples of cycloalkyl groups or C.sub.3-C.sub.8 cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl.

[0209] “Heterocyclic” or “heterocycle” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated or partially saturated cyclic group having carbon atom(s) and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen and includes single ring and multiple ring systems including fused, bridged, and spiro ring systems. For multiple ring systems having aromatic and/or non-aromatic rings, term “heterocyclic”, or “heterocycle”, or “heterocycloalkyl”, or “heterocyclyl” applies when there is at least one ring heteroatom, and the point of attachment is at an atom of a non-aromatic ring (e.g. 1,2,3,4-tetrahydroquinoline-3-yl, 5,6,7,8-tetrahydroquinoline-6-yl, and decahydroquinolin-6-yl). In some embodiments, the heterocyclic groups herein are 3-15 membered, 4-14 membered, 5-13 membered, 7-12 membered, or 5-7 membered heterocycles. In some other embodiments, the heterocycles contain 4 heteroatoms. In some other embodiments, the heterocycles contain 3 heteroatoms. In another embodiment, the heterocycles contain up to 2 heteroatoms. In some embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, sulfinyl, or sulfonyl moieties. Heterocyclyl includes, but is not limited to, tetrahydropyranyl, piperidinyl, N-methylpiperidin-3-yl, piperazinyl, N-methylpyrrolidin-3-yl, 3-pyrrolidinyl, 2-pyrrolidon-1-yl, morpholinyl, and pyrrolidinyl. A prefix indicating the number of carbon atoms (e.g., C.sub.3-10) refers to the total number of carbon atoms in the portion of the heterocyclyl group exclusive of the number of heteroatoms. A divalent heterocyclic group will have the appropriately adjusted hydrogen content.

[0210] “Ether” refers to a C.sub.1-C.sub.6 alkyl group substituted with 1-3 C.sub.1-C.sub.6 alkoxy groups, wherein alkoxy refers to —O-alkyl.

[0211] “Halo” or “Halogen” refers to one or more of fluoro, chloro, bromo, and iodo.

[0212] “Alkenyl” refers to a linear or branched hydrocarbyl group having carbon atoms and in some embodiments from 2 to 6 carbon atoms or 2 to 4 carbon atoms and having at least 1 site of vinyl unsaturation (>C═<). For example, C.sub.x-y alkenyl refers to an alkenyl group having from x to y carbon atoms and is meant to include, for example, ethenyl, propenyl, 1,3-butadienyl, and the like.

[0213] “Alkynyl” refers to a linear monovalent hydrocarbon group or a branched monovalent hydrocarbon group having more than 2 carbon atoms and in some embodiments from 2 to 6 carbon atoms or 2 to 4 carbon atoms and containing at least one triple bond. The term “alkynyl” is also meant to include those hydrocarbyl groups having one triple bond and one double bond. For example, C.sub.2-6 alkynyl includes ethynyl, propynyl, and the like.

[0214] “Phosphoramidate alkylating agent” refers to an alkylating agent comprising one or more Z.sup.5—X.sup.5—Y.sup.5 moieties bonded to an —O—P(Z1) moiety, where Z.sup.5 is a heteroatom such as nitrogen, sulfur or oxygen, X.sup.5 is optionally substituted ethylene, Y.sup.5 is halo or another leaving group, or Z.sup.5—X.sup.5—Y.sup.5 together form an aziridinyl (NCH.sub.2CH.sub.2) moiety, and Z.sup.1 is defined as above. Such an alkylating agent can react with DNA or another nucleic acid or protein. In some instances, an alkylating agent can be cross-linked with DNA.

[0215] The group may be substituted with one or more substituents, e.g., 1, 2, 3, 4 or 5 substituents. Preferably, the substituents are selected from the group consisting of oxo, halo, —CN, NO.sub.2, —N.sub.2+, —CO.sub.2R.sup.100, —OR.sup.100, —SR.sup.100, —SOR.sup.100, —SO.sub.2R.sup.100, —NR.sup.100, —SO.sub.2R.sup.100, —NR.sup.101R.sup.102, —CONR.sup.101R.sup.102, —SO.sub.2NR.sup.101R.sup.102, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, —CR.sup.100═C(R.sup.100).sub.2, —CCR.sup.100, C.sub.3-C.sub.10 cycloalkyl, C.sub.3-C.sub.10 heterocyclyl, C.sub.6-C.sub.12 aryl and C.sub.2-C.sub.12 heteroaryl, or a divalent substituent such as —O—(CH.sub.2)—O—, —O—(CH.sub.2).sub.2—O—, and, 1-4 substituted methyl groups, wherein each R.sup.100, R.sup.101, and R.sup.102 independently is hydrogen or C.sub.1-C.sub.8 alkyl; C.sub.3-C.sub.12 cycloalkyl; C.sub.3-C.sub.10 heterocyclyl; C.sub.6-C.sub.12 aryl; or C.sub.2-C.sub.12 heteroaryl; or R.sup.100 and R.sup.102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocycle; wherein each alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 halo, 1-3 C.sub.1-C.sub.6 alkyl, 1-3 C.sub.1-C.sub.6 haloalkyl or 1-3 C.sub.1-C.sub.6 alkoxy groups. Preferably, the substituents are selected from the group consisting of chloro, fluoro, —OCH.sub.3, methyl, ethyl, iso-propyl, cyclopropyl, —CO.sub.2H and salts and C.sub.1-C.sub.6 alkyl esters thereof, CONMe.sub.2, CONHMe, CONH.sub.2, —SO.sub.2Me, —SO.sub.2NH.sub.2, —SO.sub.2NMe.sub.2, —SO.sub.2NHMe, —NHSO.sub.2Me, —NHSO.sub.2CF.sub.3, —NHSO.sub.2CH.sub.2C1, —NH.sub.2, —OCF.sub.3, —CF.sub.3 and —OCHF.sub.2.

[0216] “Alkylene” refers to a divalent saturated aliphatic hydrocarbyl group having carbon atom(s) and, in some embodiments, from 1 to 6 carbon atoms, and an alkyl group further losing one H atom. “C.sub.u-v alkylene” refers to alkylene groups having from u to v carbon atoms. The alkylene group includes branched and straight chain hydrocarbyl groups. For example, “C.sub.1-6 alkylene” includes methylene, ethylene, propylene, 2-methylpropylene, pentylene, and the like.

[0217] “Heteroalkylene” refers to an alkylene wherein a chain carbon atom is replaced with a heteroatom such as O, S, N, or P, or a heteroatom containing a substituent.

[0218] The “drug” regarding D herein includes without limitation, gemcitibine, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, trimethylmelamine, chlorambucil, chlomaphazine, estramustine, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carubicin, carzinophilin, chromomycin, actinomycin D, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, defofamide, demecolcine, diaziquone, elfomithine, elliptinium acetate, etoglucid, flutamide, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, erlotonib, teniposide, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, urethane, vinblastine, and vincristine.

[0219] “Administering” or “administration of” a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administered to a patient by a medical professional or may be self-administered, and/or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is to administer the drug to the patient.

[0220] “Cancer” refers to leukemias, lymphomas, carcinomas, and other malignant tumors, including solid tumors, of potentially unlimited growth that can expand locally by invasion and spread throughout the body by metastasis. Examples of cancers include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. Certain other examples of cancers include, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, poor cervical intraepithelial differentiation and in situ carcinoma, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforma, hairy-cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary epithelial carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteo sarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythermia vera, primary brain tumor, small-cell lung tumor, squamous cell carcinoma of both ulcerating and papillary type, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, reticulum cell sarcoma, and Wilm's tumor.

[0221] “Patient” and “subject” are used interchangeably to refer to a mammal in need of treatment for cancer. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain embodiments, a “patient” or “subject” may refer to a non-human mammal used in screening, characterizing, and evaluating drugs and therapies, such as, a non-human primate, a dog, cat, rabbit, pig, mouse or a rat.

[0222] “Prodrug” refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is made such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.

[0223] The patient in the present application refers to a patient suffering from a disease or disorder, complication associated with the AKR1C3 enzyme and its corresponding gene, or further defined as a cancer or tumor corresponding to the cytotoxic prodrug activated by the AKR1C3 enzyme or disorder or cell proliferative disease caused by the cancer or tumor.

[0224] “Solid tumor” refers to solid tumors including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph node, pancreas, prostate, skin and soft tissue (sarcoma).

[0225] “Therapeutically effective amount” of a drug refers to an amount of a drug that, when administered to a patient with cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer in the patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0226] “Treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms of cancer; diminishment of extent of disease; delay or slowing of disease progression; alleviation, palliation, or stabilization of the disease state; or other beneficial results. Treatment of cancer may, in some cases, result in partial response or stable disease.

[0227] “Tumor cells” refers to tumor cells of any appropriate species, e.g., mammalian such as murine, canine, feline, equine or human.

[0228] The above description of embodiments of the present invention does not limit the present invention. Those skilled in the art can make various modifications and changes according to the present invention, and any modification and change within the spirit of the present invention shall be covered in the scope of the claims appended to the present invention.

[0229] Since the present invention is based on the following three invention applications: Application No. PCT/US2016/021581, publication No. WO2016/145092 corresponding to Chinese application No. 2016800150788, publication No. Application No. PCT/US2016/025665, publication No. WO2016/061342 corresponding to Chinese application No. 2016800200132, publication No. CN108136214A; and

[0230] Application No. PCT/US2016/062114, publication No. WO2017/087428 corresponding to Chinese application No. 2016800446081, publication No. CN108290911A, the above-mentioned three applications are incorporated into the text of the present application for this purpose.

[0231] Further, the AKR1C3 inhibitors mentioned in the present invention are for this purpose also incorporated into the present application by reference to the following applications:

TABLE-US-00002 Publication No. Patent application title application Ser. No. US20130116277A1 Aldo-keto reductase subfamily 1c3 (akr1c3) U.S. 13/607,633 inhibitors US20180305305A1 2-beta-naphthyl-acetic acid analogs as akr1c3 U.S. 15/769,565 inhibitors and methods of using same US20160159731A1 Bifunctional akr1c3 inhibitors/androgen receptor U.S. 14/993,742 modulators and methods of use thereof US20140107085A1 Bifunctional akr1c3 inhibitors/androgen receptor U.S. 14/050,937 modulators and methods of use thereof US20170260226A1 3-nitrogen or sulphur substituted U.S. 15/510,348 oestra-1,3,5(10),16-tetraene akr1c3 inhibitors US20160303082A1 Indomethacin analogs for the treatment of U.S. 15/132,937 castrate-resistant prostate cancer US20180271835A1 Indomethacin analogs for the treatment of U.S. 15/899,171 castrate-resistant prostate cancer US20140371261A1 Indomethacin analogs for the treatment of U.S. 14/352,421 castrate-resistant prostate cancer US20140249119A1 Estra-1,3,5(10),16-tetraene-3-carboxamide U.S. 14/348,645 derivatives, processes for their preparation, pharmaceutical preparations comprising them and their use for preparing medicaments US20150210734A1 3-substituted estra-1,3,5(10),16-tetraene U.S. 14/414,386 derivatives, methods for the production thereof, pharmaceutical preparations containing same, and use thereof for the production of medicaments US20160024142A1 Estra-1,3,5(10),16-tetraene-3-carboxamides for U.S. 14/770,444 inhibition of 17.beta.-hydroxysteroid dehydrogenase (akr1c3) US20090275608A1 Methods of diagnosing and treating parp-mediated U.S. 12/322,551 diseases US20170342082A1 [8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl] U.S. 15/596,383 (1h-1,2,3-triazol-4-yl)methanones US20180319807A2 [8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl] U.S. 15/596,383 (1h-1,2,3-triazol-4-yl)methanones CN201811133143.4 A dihydropyranopyrazole compound, preparation CN109305972A method and application thereof

[0232] With regard to the AKR1C3 inhibitors mentioned in the present invention, it has been reported in the following literatures (Higaki, Y, Usami, et al. Selective and potent inhibitors of human 20a-hydroxysteroid dehydrogenase (AKR1C1) that metabolizes neurosteroids derived from progesterone[J]. CHEMICOBIOLOGICAL INTERACTIONS, 2003, 503-513; Bydal P, Luu—The V, Labrie F, et al. Steroidal lactones as inhibitors of 17beta-hydroxysteroid dehydrogenase type 5: chemical synthesis, enzyme inhibitory activity, and assessment of estrogenic and androgenic activities. [J]. European Journal of Medicinal Chemistry, 2009, 44(2):632-644; Skarydova L, Wsol V, Zivna L, et al. AKR1C3 as a potential target for the inhibitory effect of dietary flavonoids[J]. Chemico-biological interactions, 2010, 178:138-144; Byrns M C, Steckelbroeck S, Penning T M. An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies. [J]. Biochemical Pharmacology, 2008, 75(2):484-493; Bauman, D. R. Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo-keto reductase isoforms: potential antineoplastic agents that work independently of cyclooxygenase isozymes.[J]. Molecular Pharmacology, 2005, 67(1):60-68; Penning, T, M, et al. Inhibition of a major NAD(P)-linked oxidoreductase from rat liver cytosol by steroidal and nonsteroidal anti-inflammatory agents and by prostaglandins.[J]. Proceedings of the National Academy of Sciences, 1983, 8:4504-4508; Davies N J, Hayden R E, Simpson P J, et al. AKR1C isoforms represent a novel cellular target for jasmonates alongside their mitochondrial-mediated effects.[J]. Cancer Research, 2009, 69(11):4769-75; Brozic P, Golob B, Gomboc N, et al. Cinnamic acids as new inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (AKR1C3).[J]. Molecular & Cellular Endocrinology, 2006, 248(1-2):233-235; Yining Zhao, Xuehua Zheng, Hong Zhang, et al. In vitro inhibition of AKR1Cs by sulphonylureas and the structural basis.[J]. Chemico-Biological Interactions, 2015, 240:310-315) that some commercially available drugs have an inhibitory effect on AKR1C3, and these drugs are also inhibitors of the AKR1C3 enzyme:

[0233] Glycyrrhetinic acid, Glycyrrhizinate and salt or glycoside thereof, Ursodeoxycholic acid, medroxyprogesterone acetate (MPA), Estradiol, Hexestrol, Bethamethasone, cortisone, Prednisone, methylprednisolone, triamcinolone, Hydrocortisone, Dexamethasone, Spironolactone, Brotizolam, Estazolam, Flunitrazepam, Flurazepam, meclonazepan, Lormetazepam, Midazolam, nimetazepam, Nitrazepam, Temazepam, triazolam, alprazolam, Bromazepam, Chlordiazepoxide, clobazam, clonazepam, Delorazepam, diazepam, fludiazepam, Halazepam, Lorazepam, Medazepam, nordazepam, oxazepam, Prazepam, Cloxazolam, Phenolphthalein, Ipriflavone, Flavoxate Hydrochloride, Purified Micronised Flavonoid Fraction, Seabuckthorn flavone, silibinin, efloxate, Quercetin, Luteolin, Aspirin, Sodium salicylate, Paracetamol, Naproxen, Nabumetone, Diclofenac, Ibuprofen, rofecoxib, Celecoxib, Aceclofenac, Diflunisal, Etodolac, Fenoprofen, Flurbiprofen, ketoprofen, suprofen, Tiaprofenic Acid, Ketorolac, Zomepirac, Mefenamic acid, Flufenamic acid, Meclofenamic Acid, Meloxicam, Oxaprozin, Piroxicam, Tenoxicam, Lornoxicam, Sasapyrine, Sulindac, Tolmetin, Phenacetin, Loxoprofen Sodium, Aminopyrine, Metamizole Sodium, Sudoxicam, Phenylbutazone, Oxyphenbutazone, Chlorpropamide, Tolbutamide, Gliclazide, Glibenclamide, Gliquidone, Glipizide, and Glimepiride.

[0234] The following are specific tests and examples of the present invention.

[0235] The following tests will disclose experimental data of the applicant for proving relevant conclusions and relevant experimental facts of the present application, and the applicant hereby declares that the rights of the following experimental data belong to the applicant.

[0236] The compound

##STR00030##

(i.e., the racemic isomer of compound AST-3424, the S-configuration of which is AST-3424

##STR00031##

has AKR1C3 enzyme inhibitory activity.

[0237] In Vitro Activity Inhibition Experiment of the Compounds on AKR1C3

[0238] Experimental apparatus: Waters Acquity I Class UPLC Ultra Performance Liquid Chromatograph equipped with a Xevo G2-XS Q T of HRMS Quadrupole Time-of-flight High-Resolution Mass Spectrometer

[0239] Buffers and Materials:

[0240] 1. PBS phosphate buffered saline solution,

[0241] 2. PBS phosphate buffered saline solution of 20 mM NADPH

[0242] 3. PBS phosphate buffered saline solution of 250 μg/mL AKR1C3

[0243] 4. 50% MeOH/H.sub.2O solution of 250 μM AST-3424

[0244] 5. 50% MeOH/H.sub.2O solution of 250 μM progesterone

[0245] 6. 100% acetonitrile solution of 1 μg/mL propranolol

Experimental Procedures

[0246] Step 1, the reaction mixtures were put into Eppendorf tubes (microcentrifuge tubes) in quadruplicate (n=4) according to the table below and mixed gently.

TABLE-US-00003 Negative controls Samples Materials (μL) (μL) PBS 68 58 NADPH (20 mM) 10 10 AKR1C3 (250 μg/mL) 10 10 AST-3424 (250 μM) 0 10

[0247] Step 2, the above mixtures were pre-incubated in duplicate at 37° C. for 30 minutes and 60 minutes.

[0248] Step 3, another 10 μL of PBS phosphate buffered saline solution of 20 mM NADPH and 2 μL of 50% MeOH/H.sub.2O solution of 250 μM progesterone were added to each Eppendorf tube and mixed gently.

[0249] Step 4, 50 μL of the mixture in the above step was immediately transferred to 100 μL of 100% acetonitrile solution of 1 μg/mL propranolol (internal standard (IS)).

[0250] Step 5, the remaining samples were incubated at 37° C. for 30 minutes, and 100 μL of 100% acetonitrile solution of 1 μg/mL propranolol (internal standard (IS)) was added.

[0251] Step 6, 100 μL of reagent water was added to all the samples, vortexed at 1,100 rpm for 5 minutes, and centrifuged at 15,000 rpm for 10 minutes at room temperature.

[0252] Step 7, all the samples were loaded on LC/MS to determine the content of reduced progesterone, namely 20α-dihydroprogesterone.

[0253] The test conditions for the LC-MS apparatus are shown below.

TABLE-US-00004 Items Conditions Apparatus: Waters Acquity I Class Liquid Chromatograph Chromatographic Acquity UPLC BEHC18 Chromatographic Column column: (50*2.1 mm, 1.7 μm) Flow rate: 0.4 mL/min Injection volume: 3 μL The composition of A: 0.1% (V/V) formic acid aqueous solution the mobile phase: B: 0.1% (V/V) formic acid acetonitrile solution The temperature of 40° C. the column oven: Detector: Quadrupole Time-of-flight Mass Spectrometer Q-TOF MS

[0254] The gradient of the liquid phase elution

TABLE-US-00005 Time (min) A (%) B (%) 0.00 90.0 0.0 1.5 5.0 95.0 2.00 5.0 95.0 2.30 90.0 10.0 3.00 90.0 10.0

[0255] Parameters of the Quadrupole Time-of-flight Mass Spectrometer

TABLE-US-00006 Items Parameters Capillary kV 2.5 Sampling Cone V 40 Source temperature (° C.) 100 Cone Gas (L/h) 50 Desolvation Gas (L/h) 600 Interface type ES electron impact, Positive Analyser mode Sensitivity Scan range 50-800 m/z

[0256] Step 9, the reduced progesterone (20α-dihydroprogesterone) is calculated: the peak area of the reduced progesterone, namely 20α-dihydroprogesterone and propranolol, in each sample was determined by LC/MS. The peak area ratios of reduced progesterone to propranolol (i.e., the ratios in the above table) were calculated, and when the time is 0 the ratio is set to 0%.

AKR1C3 activity (%)=[(the amount of reduced progesterone after normalization of the sample) 30 min−(the amount of reduced progesterone after normalization of the sample) 0 min]/[(the amount of reduced progesterone after normalization of the negative control group) 30 min−(the amount of reduced progesterone after normalization of the negative control group) 0 min]*100.

[0257] The AKR1C3 activity results in the above table were calculated according to the above formula.

Experimental Results

[0258]

TABLE-US-00007 The peak areas of the liquid phase Reduced AKR1C3 Duration Progesterone progesterone Propranolol activity Incubation Reaction AST-3424 Values Averages Values Averages Ratios* Values Averages % Negative 30 min 0 Absent 36658 36835 150 158 0.0034 51141 46879 0 control 37011 166 42616 groups 60 min 35244 35125 87 83 0.0019 42794 43650 0 35006 79 44506 30 min 30 min 28675 26228 5616 6617 0.1506 43589 43935 100 23780 7617 44280 60 min 25917 26492 6747 6051 0.1343 44258 45067 100 27067 5354 45876 Samples 30 min 0 154017 37107 37438 70 72 0.0017 43247 43436 0 154843 37769 74 43625 60 min 108351 38050 37989 58 64 0.0015 43907 43697 0 107508 37928 70 43487 30 min 30 min 104748 35187 35210 344 331 0.0074 45657 45051 3.9 110630 35234 319 44445 60 min 52296 32636 32520 622 616 0.0137 45395 45101 9.2 51309 32404 610 44807

[0259] Analysis and Summary of the Experimental Results

[0260] Table: The effect of AST-3424 on AKR1C3 activity

TABLE-US-00008 % AKR1C3 activity The time of 0 μM 25 μM incubation (min) AST-3424 AST-3424 30 100% 3.9% 60 100% 9.2%

[0261] The activity was 92.4% when the test value of indometacin was at a concentration of 5 um/L.

[0262] The effect of AST-3424 on the production of reduced progesterone: The aforementioned in vitro experiment proves that after pre-incubation for 30 minutes and 60 minutes, AST-3424 at a concentration of 25 μM basically inhibited AKR1C3 activity: compared with the negative control groups, the production of the reduced progesterone, namely 20α-dihydroprogesterone, was reduced to 3.9% and 9.2%, respectively, proving that the compound AST-3424 are inhibitors of AKR1C3 enzyme.

[0263] Content Change Experiment of Prostaglandin in Blood of Cynomolgus Monkeys Before and After Administration of AKR1C3 Inhibitor AST-3424

[0264] An experiment with three cynomolgus monkeys was performed according to the table below.

TABLE-US-00009 Administration The concentration The dosage of of the solution of The volume of Amount Test the test substance the test substance administration Mode of Collected Group Male substance (mg/kg) (mg/mL) (mL/kg) administration Sample 1 3(No. 101/ AST3424 1/Compound 0.2 5 Intravenous Plasma/ 102/103) infusion serum

[0265] Four male cynomolgus monkeys were purchased from Guangxi Xiongsen Primate Development and Experiment Co., Ltd., and all of them were healthy cynomolgus monkeys that had passed the physical examination and have no abnormalities. Among them, three cynomolgus monkeys were used in the experiment of administration and the remaining one was used for preparing blank plasma.

[0266] Before administration, and 6, 24, 48 and 72 hours after the start of administration, 1 mL of blood was collected from the femoral vein or other suitable vein, and placed in a blood collection tube without anticoagulant. After collected, the blood sample was placed on ice and allowed to stand for 30-60 minutes before being centrifuged at 3,500 rpm for 10 minutes at 2-8° C. to separate the serum. The collected serum was stored at −80° C. before being analyzed.

[0267] Prostaglandin F2 in serum samples was analyzed by conventional ELISA methods. The measurement results are as follows.

[0268] The Concentrations of Prostaglandins E2 and F2 in the Serum after Single Administration to Cynomolgus Monkeys by Intravenous Infusion

TABLE-US-00010 The measured concentration of prostaglandin F2 (pg/ml) Time point 101 102 103 Before change and change change and change change and change administration 1828.51 percentage 310.17 percentage 1125.58 percentage  6 h 165.32 −1663.19, −90.95% 260.80 −49.37, −15.91% 300.65 −824.93, −73.29%  24 h 816.63 −1011.88, −55.34% 3460.25 3150.08, 1000.15% 4208.19 3082.61, +273.87% 48 h 183.73 −1644.78, −89.95% 216.12 −94.05, −30.32% 541.05 −584.53, −51.93%  72 h 441.76 −1386.75, −75.84% 968.01  657.84, +212.09% 1369.67 244.09, +21.68%

[0269] Six hours after administration of 0.58 mg/kg of AST-3424 to cynomolgus monkeys, the content of prostaglandin F2a in all three cynomolgus monkeys decreased, indicating that the process of prostaglandin F2a secretion in cynomolgus monkeys can be inhibited after administration of AST-3424.

[0270] Obviously, it can be found that:

[0271] I: the levels of the content of prostaglandin F2a in blood of the three cynomolgus monkeys before using the interfering drugs have a significant difference, which were very different from each other.

[0272] II: Further, exploration of the content change of prostaglandin F2a in the three cynomolgus monkeys (101/102/103) after using infectious drugs shows that the contents of prostaglandin F2a of them are decreased within 6 hours while they are decreased or increased at 24, 48 and 72 hours after using AKR1C3 inhibitor (AST-3424), and this experimental fact shows that using an interfering drug (inhibitor) against AKR1C3 enzyme activity should be followed by the detection of change of prostaglandin within a suitable period of time, and for the three cynomolgus monkeys used in this experiment, 6 hours were appropriate.

[0273] III: Further, a comprehensive I/II exploration of the three cynomolgus monkeys for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the absolute values of the content change of prostaglandin F2a were also completely different: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change values of prostaglandin F2a levels in their blood after administration of the interfering drug.

[0274] IV: Further, a comprehensive I/II exploration of the three cynomolgus monkeys for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the change ratios of the content change of prostaglandin F2a were also completely different: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change ratios of the content of prostaglandin F2a levels in their blood after administration of the interfering drug.

[0275] In conclusion, a comprehensive exploration of the three cynomolgus monkeys without administration of interfering drugs and for 6 hours after administration of the same amount of interfering drugs in the same intravenous injection, the absolute value/relative change ratios of the content change of prostaglandin F2a was positively correlated with PGF2α in cynomolgus monkeys without administration of interfering drug: the cynomolgus monkeys with highest content levels of prostaglandin F2a in their blood before administration of the interfering drugs also had the highest change value/relative change ratio of the content of prostaglandin F2a levels in their blood after administration of the interfering drug.

[0276] In fact, after using traditional immunohistochemical staining method (IHC) to detect AKR1C3 in the liver tissues obtained from these three Cynomolgus monkeys, it was found that the staining area of No.s 101 and 103 cynomolgus monkeys was higher than that of No. 102 Cynomolgus monkey, i.e. the expression level of AKR1C3 enzyme in No. 101 and 103 Cynomolgus monkeys was higher than that of No. 102 Cynomolgus monkey.

[0277] The above in vitro and in vivo experiments confirm that route 1/2/3 is truly present, the conclusion by measuring the content of PGF2α and/or PGD2 and/or PGH2 or the content change and content change ratio before and after administration of interfering drugs in vitro and then combining with the data statistics can correlate the expression levels of AKR1C3 enzyme in biological samples, that is, it is clinically possible to directly obtain the expression levels of AKR1C3 enzyme of patients by measuring the content of prostaglandin, thereby screening a cancer or tumor patient with an appropriate expression level of AKR1C3 enzyme and administering the AKR1C3 enzyme-activated anti-cancer prodrug disclosed in the present invention to the patient.