TWI651321B - Acrylic derivative, process for producing the same and medical use thereof - Google Patents

Abstract

The present invention relates to an acrylic derivative represented by the formula (I), a stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, a process for producing the same, a pharmaceutical composition containing the same, and the like Therapeutic agents, particularly as a selective estrogen receptor down-regulator (SERD), wherein R ¹ , R ² , R ³ , R ⁴ in the formula (I) are as defined in the specification.

Description

Acrylic derivative, preparation method thereof and use thereof in medicine

The present invention belongs to the field of medicine, and in particular relates to a novel acrylic derivative, a stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, a process for preparing the same, a pharmaceutical composition containing the same, and a treatment thereof The use of an agent, in particular as an estrogen receptor antagonist or an estrogen receptor alpha downregulator.

The estrogen receptor (ER) is a ligand-initiated transcriptional regulatory protein that mediates the induction of various biological effects through interaction with endogenous estrogens, including endogenous estrogens including 17β-estradiol and Estrone. ER has been found to have two subtypes of estrogen receptor alpha (ER alpha, ESR1 and NR3A) and estrogen receptor beta (ER beta, ESR2 and NR3b). Estrogen receptor alpha and estrogen receptor beta are members of the steroid hormone receptor, which is a member of the nuclear receptor family. Similar to the mechanism of nuclear receptors, ERα is composed of six functional domains (named AF), which is a transcription factor initiated by a ligand, because it binds to a specific ligand, including endogenous estrogen 17β. Estradiol (E2) binds to the genomic sequence to form a complex, an estrogen receptor response element, and a co-regulatory factor to regulate transcription of the targeted gene. The ERα gene is located at 6q25.1 and encodes the 595A protein, resulting in different subtypes depending on the cleavage site and the starting point of transcriptional transcription. In addition to the DNA binding domain (domain C) and the ligand binding domain (domain E), the receptor also contains an N-terminal (A/B) domain, a hinge region (D, which links the C and E domains), and a carbon terminus ( F domain). The C and E domains of ERα and ERβ are consistent, and the A/B, D and F domains are less consistent. Both receptors are involved in the regulation and growth of the female reproductive tract, and also play an important role in the central nervous system, cardiovascular and vascular systems, and bone metabolism. The binding of estrogen to the receptor can lead to a variety of cellular changes, and its regulatory mechanisms can be divided into two pathways: the genomic and non-genomic pathways. The genomic pathway of ER vector transduction includes the formation of estrogen receptor dimer, binding to the ERE in the estrogen regulatory gene promoter, mediation of the aggregation of other regulatory proteins to the promoter, and finally the mRNA level of the gene. Raise or lower. In the non-genomic pathway of estrogen-mediated transduction, estrogen can react with estrogen-binding proteins present in or adjacent to the cell membrane of ERs, even cell membranes without ERs. Estrogen's cellular responses through non-genomic pathways can increase intracellular calcium and NO levels, as well as the initiation of a variety of intracellular kinases, including MAPK, PI3K, PKA, and PKC, which initiates phosphorylation of nER.

The expression of ER and/or progesterone receptors in approximately 70% of breast cancer patients indicates that the growth of this tumor cell is hormone-dependent, and the growth of other tumors such as ovarian cancer and endometrial cancer is also dependent on ERα. The treatment of these diseases can inhibit ER signaling by various means, including antagonizing the binding of ligands and ER, antagonizing or downregulating ERα, and blocking the synthesis of estrogen. At the same time, ERα and ERβ are expressed in endocrine tumors such as adrenal cortical tumors, pancreatic cancer, prostate cancer and thyroid cancer, digestive system tumors such as colon cancer, esophageal cancer, liver cancer and pancreatic cancer, and lung cancer. Although the above treatments play a role in ER-positive tumor patients, they also develop resistance. Recently, ESR1 mutations may be one of the causes of drug resistance in patients with metastatic ER-positive breast cancer. (Toy et al., Nat . Genetic 2013, 45: 1439-1445; Li, S. et al Cell Rep. 4, 1116-1130 (2013)). However, among the possible drug resistance mechanisms, tumor growth has ER-dependent activity, so a mechanism for selectively down-regulating ERα provides a better method for blocking early, metastatic and resistant ERα activity mediation. Medicinal cancer.

A number of drugs have been disclosed that can be used as selective estrogen receptor downregulator (degrader, SERD), including GDC-0810 and GDC-0927 of Gene Tec, respectively, in clinical phase II and Clinical Phase I; Azlikon's AZD-9496, in Phase I, also discloses a series of patent applications for SERD, including WO2011156518, WO2012037410, WO2015082990, and the like. However, the estrogen receptor down-regulation in the prior art is still not satisfactory, and in order to obtain better effects and avoid the mechanism of resistance, it is still necessary to study and develop a new estrogen receptor alpha down-regulator.

It is an object of the present invention to provide an acrylic derivative having estrogen receptor antagonism which is different from the prior art structure.

Thus, according to a first aspect of the invention, the invention provides a compound of the formula (I), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof:

Wherein: R ¹ and R ² are each independently selected from a hydrogen atom or a halogen, wherein said halogen is preferably F; and R ^{3 is} selected from an alkyl group, wherein said alkyl group is further substituted by one or more halogen substituents. Substituting; R ⁴ are each independently selected from the group consisting of halogen, alkyl, alkoxy, trifluoromethyl, cyano, -C(O)NR ⁵ R ⁶ , -C(O)R ⁷ , -SO ₂ R ⁷ , -C(O)OR ⁷ or -NR ⁵ C(O)R ⁶ , wherein said alkyl or alkoxy group is further further selected from one or more selected from the group consisting of halogen, -C(O)NR ⁵ R ⁶ , -C (O) R a substituted _{^{7, -SO 2 R 7, -C}} (O) oR 7 or -NR ⁵ C (O) R ⁶ group is substituted; R ⁵ is selected from a hydrogen atom or an alkyl group; R ⁶ is selected from Or a hydrogen atom, an alkyl group, a cycloalkyl group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, aryl group or heteroaryl group is further further selected from one or more selected from the group consisting of a hydroxyl group, a halogen, an alkyl halide Base, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR ⁸ R ⁹ , -C(O)NR ⁸ R ⁹ , -C(O Substituting a substituent of R ¹⁰ , -SO ₂ R ¹⁰ , -C(O)OR ¹⁰ or -NR ⁸ C(O)R ⁹ ; or R ⁵ , R ⁶ together with the atom to which they are attached form 4-8 Metacyclic heterocyclic group, wherein The heterocyclic group is optionally further selected from one or more selected from the group consisting of alkyl, halogen, hydroxy, cyano, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR ⁸ R ⁹ , ^{-C (O) NR 8 R 9} , -C (O) R 10, -SO 2 R 10, -C (O) oR 10 , or -NR ⁸ C (O) R ⁹ is substituted with a substituent; R ⁷ is selected from From a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group is optionally further one or more Selected from hydroxy, halogen, haloalkyl, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR ⁸ R ⁹ , -C(O)NR ⁸ Substituents of R ⁹ , -C(O)R ¹⁰ , -SO ₂ R ¹⁰ , -C(O)OR ¹⁰ or -NR ⁸ C(O)R ⁹ are substituted; R ⁸ , R ⁹ and R ¹⁰ are each independently Is selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group is optionally further one or a plurality of selected from the group consisting of hydroxyl, halogen, haloalkyl, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid or carboxylic acid esters Substituted with a substituent; and n is 2, 3 or 4.

In another embodiment of the present invention, the compound of the formula (I), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, which is represented by the formula (II) a compound, a stereoisomer, a tautomer thereof, or a pharmaceutically acceptable salt thereof:

Wherein: R ¹ , R ² , R ³ , R ⁴ and n are as defined in the formula (I).

In a preferred embodiment of the invention, the compound of the formula (I) or (II), or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein: the alkane The group is preferably a C ₁ -C ₁₀ alkyl group, more preferably a C ₁ -C ₆ alkyl group, most preferably a C ₁ -C ₃ alkyl group; the alkoxy group is preferably a C ₁ -C ₁₀ alkoxy group, More preferably, it is a C ₁ -C ₆ alkoxy group, most preferably a C ₁ -C ₃ alkoxy group; the cycloalkyl group is preferably a C ₃ -C ₁₂ cycloalkyl group, more preferably a C ₃ -C ₈ ring The alkyl group is most preferably a C ₃ -C ₆ cycloalkyl group; the heterocyclic group is preferably a C ₃ -C ₁₀ heterocyclic group, more preferably a C ₅ -C ₇ monocyclic heterocyclic group or a C ₇ -C ₁₀ bicyclic or tricyclic heterocyclic group; said aryl group is preferably a C ₆ -C ₁₀ aryl group, more preferably a phenyl group and a naphthyl group; said heteroaryl group is preferably a 5- to 10-membered heteroaryl group, More preferably, it is a 5- to 6-membered monocyclic heteroaryl group or a 9- to 10-membered bicyclic heteroaryl group.

In one embodiment of the invention, the compound of the formula (I) or (II), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are each independently It is selected from halogen, wherein the halogen is preferably F.

In one embodiment of the invention, the compound of the formula (I) or (II), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ³ is C ₁ -C ₄ The alkyl group, preferably an isopropyl group, wherein the alkyl group is further substituted by one or more substituents selected from F, Cl, Br or I, preferably F or Cl, more preferably F.

In one embodiment of the invention, the compound of the formula (I) or (II), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein each R ^{4 is} independently selected from C _1- C ₄ alkyl, halogen, alkoxy, trifluoromethyl or cyano.

In one embodiment of the invention, the compound of the formula (I) or (II), a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein: R ¹ and R ² are each independently Is selected from halogen, wherein said halogen is preferably F; R ^{3 is} selected from halogen-substituted C ₁ -C ₄ alkyl, preferably F-substituted C ₁ -C ₄ alkyl; more preferably fluoroisopropyl; ⁴ is C ₁ -C ₄ alkyl, halogen, alkoxy, trifluoromethyl or cyano.

Exemplary compounds of the invention include, but are not limited to, the compounds described in Table 1:

Or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a process for the preparation of a compound of the formula (I) or a salt thereof, comprising the steps of:

The compound of the formula (Ia) is reacted with the formula ( 1b ) in the presence of palladium acetate and tri-o-tolylphosphine under basic conditions to give a compound of the formula ( IB) ; a compound of the formula (Ic) and a formula (I) Id) a compound which is reacted under basic conditions to give a compound of the formula (IA) ; a compound of the formula (IA) is reacted with a compound of the formula (IB) under acidic conditions, and further ester-hydrolyzed to give a compound of the formula (I) ; X is a halogen, preferably bromine; R ^a is an alkyl group; R ^b is a leaving group, preferably a halogen and a sulfonate group, more preferably a Br or mesylate group; R ¹ to R ⁴ and n The definition is as described in the general formula (I).

Another aspect of the present invention provides a process for the preparation of a compound of the formula (II) or a salt thereof, comprising the steps of:

The compound of the formula (IIa) is reacted with a compound of the formula (Id) under basic conditions to give a compound of the formula (IIA) ; the compound of the formula (IIA) is reacted with a compound of the formula (IB) under acidic conditions, and further ester hydrolysis is carried out. To give a compound of the formula (II) ; wherein: R ^a is an alkyl group; R ^b is a leaving group, preferably a halogen and a sulfonate group, more preferably a Br or mesylate group; R ¹ , R ² R ³ , R ⁴ and n are as defined in the general formula (I).

In the above production method, the reagent for providing acidic conditions is an inorganic acid or an organic acid, the inorganic acid is preferably hydrochloric acid, sulfuric acid or phosphoric acid, more preferably hydrochloric acid; and the organic acid is preferably derived from formic acid or acetic acid, more preferably acetic acid.

In the above preparation method, the basic condition is provided by organic hydrazine or inorganic hydrazine, and the organic hydrazine is preferably selected from the group consisting of diisopropylethylamine, diisopropylamine, pyridine, triethylamine, acridine, N-methylpyridazine, 4-di The methotrexate is more preferably diisopropylamine and triethylamine; the inorganic hydrazine is preferably selected from the group consisting of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, more preferably sodium carbonate and sodium hydroxide.

Furthermore, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of the formula (I) or (II) or a stereoisomer, tautomer thereof or pharmaceutically acceptable thereof Salts, and pharmaceutically acceptable carriers, excipients or combinations thereof. The composition optionally further comprises an antioxidant or a metal chelating agent.

The present invention provides a compound of the formula (I) or (II) or a stereoisomer, tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, which is prepared in the preparation for the treatment of estrogen Use of a medicament for receptor-mediated disease, wherein the disease is preferably cancer, wherein the cancer is preferably breast cancer and gynecological cancer, wherein the gynecological cancer is preferably ovarian cancer and endometrial cancer. Wherein the estrogen receptor is preferably an estrogen receptor alpha.

The present invention provides a compound of the formula (I) or (II) or a stereoisomer, tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a selective estrogen receptor The use in the formulation is preferably an estrogen receptor alpha downregulation.

The present invention provides a compound of the formula (I) or (II), or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, which may be combined with one or more other suitable The antitumor drug is used in combination to treat an estrogen receptor vector-mediated disease, wherein the disease is preferably cancer, wherein the cancer is preferably a breast cancer or a gynecological cancer, wherein the gynecological cancer is preferably ovarian cancer. Or endometrial cancer, wherein the estrogen receptor is preferably an estrogen receptor alpha, wherein the other antitumor drugs include alkylating agents, antimetabolites, natural products having antitumor activity and derivatives thereof , cytotoxic drugs or block immune cell migration drugs.

Suitable other antineoplastic agents include alkylating agents (but not limited to nitrogen mustard, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes) such as uraramustine, nitrogen Mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, terpene bromide, quetiapine, busulfan, carmustine, lomustine, streptavidin, Dacarbazine and temozolomide.

Suitable other anti-tumor drugs include, for example, antimetabolites (including but not limited to folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, fluorouracil , cytarabine, 6-mercaptopurine, 6-thiouracil, fludarabine phosphate, pentastatin and gemcitabine.

Other suitable anti-tumor drugs include, for example, certain natural products having antitumor activity and derivatives thereof (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, etc.), such as vinca, vinca, vinca Amine, bleomycin, actinomycin D, daunorubicin, doxorubicin, epirubicin, idarubicin, cytarabine, paclitaxel, pucamycin, deoxycholic acid, Mitomycin-C, L-aspartate, interferon (especially IFN-a), etoposide and teniposide.

Other suitable anti-tumor drugs include cytotoxic drugs including noviben, CPT-11, anastrozole, letrozole, capecitabine and droloxifene, topoisomerase. Inhibitors, procarbazine, mitoxantrone, platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutics; folinic acid; tegafur and hematopoietic growth factors Be applicable.

In addition, other suitable anti-tumor drugs include antibody therapeutic drugs such as trastuzumab, antibodies to costimulatory molecules such as CTLA-4, 4-1BB and PD-1 or cytokine antibodies. (IL-10, IGF-β, etc.); drugs that block immune cell migration, such as chemokine receptor antagonists, including CCR2 and CCR4; and drugs that enhance the immune system, such as adjuvants or adoptive T cell transfers; Anti-cancer vaccines, including dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

The present invention provides a method for selectively downregulating an estrogen receptor, which comprises a compound of the formula (I) or (II) or a stereoisomer, tautomer thereof or a pharmaceutically acceptable salt thereof, or The pharmaceutical composition is contacted with an estrogen receptor, preferably an estrogen receptor alpha.

The invention further provides a method of treating a disease transduced by an estrogen receptor vector comprising administering to a subject in need thereof a compound of the formula (I) or (II) or a stereoisomer thereof, tautomerism thereof Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof. In the method of treatment, the estrogen receptor is preferably an estrogen receptor alpha; the disease transduced by the estrogen receptor vector is preferably a cancer, wherein the cancer is preferably breast cancer and gynecological cancer, Gynecological cancers are preferably ovarian cancer and endometrial cancer.

The present invention also relates to a compound of the formula (I) or (II), or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the treatment of estrogen receptor vector transduction Use of the disease, wherein the disease is cancer, wherein the cancer is preferably breast cancer or gynecological cancer, wherein the gynecological cancer is preferably ovarian cancer or endometrial cancer, wherein the estrogen is affected by The body is preferably an estrogen receptor alpha.

A safe and effective method of administering most chemotherapeutic drugs (anti-tumor drugs) known to those skilled in the art. In addition, their dosing standards have been discussed in the standard literature, such as the "physicians desk reference" (PDR, eg1996 edition, medical Economics Company, Montvale, NJ), the method of administration of various chemotherapeutic drugs, the disclosure content This is incorporated herein by reference.

Unless otherwise stated, some of the terms used in the specification and claims of the present invention are defined as follows:

When "alkyl" as a group or part of a group is meant to include C ₁ -C ₂₀ linear or branched aliphatic hydrocarbon group with a chain. It is preferably a C ₁ -C ₁₀ alkyl group, more preferably a C ₁ -C ₆ alkyl group, and most preferably a C ₁ -C ₃ alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1, 1-di Methylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1 -ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl Butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl Wait. The alkyl group can be substituted or unsubstituted.

"Cycloalkyl" means a saturated or partially saturated monocyclic, fused, bridged, and spiro carbon ring, ie, includes a monocyclic cycloalkyl, a fused cycloalkyl, a bridged cycloalkyl, and a spirocycloalkyl. It is preferably a C ₃ -C ₁₂ cycloalkyl group, more preferably a C ₃ -C ₈ cycloalkyl group, and most preferably a C ₃ -C ₆ cycloalkyl group. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene The alkenyl group, the cyclooctyl group and the like are preferably a cyclopropyl group or a cyclohexenyl group.

"Spirocycloalkyl" means a polycyclic group of 5 to 18 members, two or more cyclic structures, and a single ring sharing a carbon atom (referred to as a spiro atom), and the ring contains one or more A double bond, but none of the rings have a fully conjugated π-electron aromatic system. It is preferably 6 to 14 members, more preferably 7 to 10 members. The spirocycloalkyl group is classified into a monospiro, a spiro- or a spirocycloalkyl group, preferably a mono- and bi-spirocycloalkyl group, preferably 4 yuan/5 yuan, 4, depending on the number of common spiro atoms between the rings. Yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan. Non-limiting examples of "spirocycloalkyl" include, but are not limited to, spiro[4.5]decyl, spiro[4.4]decyl, spiro[3.5]decyl, spiro[2.4]heptyl.

"Fused cycloalkyl" means 5 to 18 members, an all-carbon polycyclic group containing two or more cyclic structures that share a carbon atom with each other, and one or more rings may contain one or more double bonds, However, none of the rings have a fully conjugated π-electron aromatic system, preferably 6 to 12 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyl group, preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicycloalkyl group. Non-limiting examples of "fused cycloalkyl" include, but are not limited to, bicyclo[3.1.0]hexyl, bicyclo[3.2.0]hept-1-enyl, bicyclo[3.2.0]heptyl, ten Hydronaphthyl or tetradecafluorophenanyl.

"Bridge cycloalkyl" means 5 to 18 members, containing two or more cyclic structures, sharing two all-carbon polycyclic groups that are not directly bonded to each other, and one or more rings may contain one or A plurality of double bonds, but none of the rings have a fully conjugated π-electron aromatic system, preferably 6 to 12 members, more preferably 7 to 10 members. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, and more preferably a bicyclic ring or a tricyclic ring. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to: (1s, 4s)-bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, (1s,5s)-bicyclic [3.3.1] Mercapto, bicyclo [2.2.2] octyl, (1r, 5r)-bicyclo[3.3.2] fluorenyl.

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the parent The ring to be joined together is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, and the like. The cycloalkyl group can be optionally substituted or unsubstituted.

"Heterocyclyl", "heterocyclic" or "heterocyclic" are used interchangeably herein to refer to a non-aromatic heterocyclic group wherein one or more of the ring-forming atoms are heteroatoms such as oxygen, The nitrogen, sulfur atom and the like include a monocyclic ring, a fused ring, a bridged ring and a spiro ring, that is, a monocyclic heterocyclic group, a bridged heterocyclic group, a fused heterocyclic group and a spiroheterocyclic group. It preferably has a 5- to 7-membered monocyclic ring or a 7- to 10-membered double- or tricyclic ring which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, acridinyl, 2-oxo- Acridinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, pyridazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl and pyridazinyl. The heterocyclic group may be substituted or unsubstituted.

"Spiroheterocyclyl" means a polycyclic group of 5 to 18 members, two or more cyclic structures, and a single ring sharing one atom with each other, and the ring contains one or more double bonds, but no An aromatic system having a fully conjugated π-electron, wherein one or more ring atoms are selected from the group consisting of nitrogen, oxygen or S(O) _m (where m is selected from 0, 1 or 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. The spiroheterocyclyl group is classified into a monospiroheterocyclic group, a dispiroheterocyclic group or a polyspirocyclic group according to the number of shared spiro atoms between the ring and the ring, and is preferably a monospiroheterocyclic group and a dispiroheterocyclic group. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6-membered monospiroheterocyclic group. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to, 1,7-dioxaspiro[4.5]fluorenyl, 2-oxa-7-azaspiro[4.4]decyl, 7-oxa Spiro[3.5]decyl and 5-oxaspiro[2.4]heptyl.

"Fused heterocyclic group" means an all-carbon polycyclic group containing two or more cyclic structures that share a pair of atoms with each other, and one or more rings may contain one or more double bonds, but none of the rings have complete A conjugated π-electron aromatic system in which one or more ring atoms are selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to, octahydropyrrolo[3,4-c]pyrrolyl, octahydro-1H-isoindenyl, 3-azabicyclo[3.1.0 Hexyl, octahydrobenzo[b][1,4]dioxine.

"Bridge heterocyclyl" means 5 to 14 members, 5 to 18 members, containing two or more cyclic structures, sharing two polycyclic groups which are not directly connected to each other, and one or more rings may be used. An aromatic system containing one or more double bonds, but none of which has a fully conjugated π-electron, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2) Of the heteroatoms, the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, and more preferably a bicyclic ring or a tricyclic ring. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to, 2-azabicyclo[2.2.1]heptyl, 2-azabicyclo[2.2.2]octyl and 2-azabicyclo [3.3.2] 癸基.

The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring wherein the ring to which the parent structure is attached is a heterocyclic group. The heterocyclic group may be optionally substituted or unsubstituted.

"Aryl" means a carbocyclic aromatic system containing one or two rings wherein the rings may be joined together in a fused manner. The term "aryl" includes aryl groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably, the aryl group is a C ₆ -C ₁₀ aryl group, more preferably the aryl group is a phenyl group and a naphthyl group, and most preferably a phenyl group. The aryl group can be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the parent structure is attached to an aryl ring, non-limiting examples include, but are not limited to:

"Heteroaryl" means an aromatic 5 to 6 membered monocyclic or 9 to 10 membered bicyclic ring which may contain from 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1,2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl , oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodiazepine Oxacyclopentenyl, benzimidazolyl, fluorenyl, isodecyl, 1,3-dioxo-isoindenyl, quinolyl, oxazolyl, benzisothiazolyl, benzo Oxazolyl and benzoisoxazolyl. Heteroaryl groups can be substituted or unsubstituted. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples include, but are not limited to:

"Alkoxy" means a group of (alkyl-O-). Among them, the alkyl group is defined in the relevant definition herein. Alkoxy groups of C ₁ -C ₆ are preferred. Examples thereof include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"Hydroxy" refers to an -OH group.

"Halogen" means fluoro, chloro, bromo and iodo, preferably chloro, bromo and iodo.

"Amino" means -NH ₂ .

"Cyano" means -CN.

"Nitro" means -NO ₂ .

"Benzyl" refers to -CH ₂ - phenyl.

"Carboxy" refers to -C(O)OH.

"Carboxylic acid ester group" means -C(O)O(alkyl) or (cycloalkyl) wherein alkyl, cycloalkyl are as defined above.

"Sulfonate group" means -S(O) ₂ O(alkyl) or (cycloalkyl) wherein alkyl, cycloalkyl are as defined above.

"Substituted" refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.

As used herein, "substituted" or "substituted", unless otherwise indicated, may be substituted by one or more groups selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane. Sulfur, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -NR ⁵ R ⁶ , -C(O)NR ⁵ R ⁶ , -C(O)R ⁷ , -SO ₂ R ⁷ , —C(O)OR ⁷ or —NR ⁵ C(O)R ⁶ , wherein R ^{5 is} selected from a hydrogen atom or an alkyl group; and R ^{6 is} selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, aryl or heteroaryl group is further further selected from one or more selected from the group consisting of hydroxyl, halogen, haloalkyl, nitro, cyano, alkyl, alkoxy , cycloalkyl, heterocyclic, aryl, heteroaryl, -NR ⁸ R ⁹ , -C(O)NR ⁸ R ⁹ , -C(O)R ¹⁰ , -SO ₂ R ¹⁰ , -C(O ) oR ^10, or -NR ⁸ C (O) R ⁹ is substituted with a substituent; Alternatively, a 4 to 8-membered heterocyclic group together with R ^5, R ⁶ atoms connected thereto, wherein Said heterocyclic group optionally further substituted with one or more substituents selected from alkyl, halo, hydroxy, cyano, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR ⁸ R ^9, - Substituted by a substituent of C(O)NR ⁸ R ⁹ , -C(O)R ¹⁰ , -SO ₂ R ¹⁰ , -C(O)OR ¹⁰ or -NR ⁸ C(O)R ⁹ ; wherein R ⁷ Selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group is optionally further one or more One selected from the group consisting of hydroxyl, halogen, haloalkyl, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR ⁸ R ⁹ , -C(O)NR Substituted by a substituent of ⁸ R ⁹ , -C(O)R ¹⁰ , -SO ₂ R ¹⁰ , -C(O)OR ¹⁰ or -NR ⁸ C(O)R ⁹ ; wherein R ⁸ , R ⁹ and R ¹⁰ each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group is optionally further One or more selected from the group consisting of hydroxyl, halogen, haloalkyl, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxy Unsubstituted or substituted carboxylic acid ester group.

"Pharmaceutically acceptable salt" refers to certain salts of the above compounds which retain their original biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salt of the compound of the present invention may be a metal salt, an amine salt formed with a suitable acid, a metal salt preferably an alkali metal or an alkaline earth metal salt, and suitable acids include inorganic acids and organic acids such as acetic acid, benzenesulfonic acid, Benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionethane, lactic acid, malic acid, maleic acid, mandelic acid, Sulfonic acid, nitric acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. Particularly preferred are hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid, and most preferred is the hydrochloride salt.

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and Shape agent. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

Figure 1 is a graph showing the tumor growth inhibition rate of the compound of Example 4 and AZD-9496 in breast cancer MCF-7 tumor-bearing mice in Test Example 7.

The invention is further described in the following examples, which are not to be construed as limiting the scope of the invention.

Preparation example

The following examples give the preparation of representative compounds of the present invention and related structural identification data.

^{The 1} H NMR spectrum was measured using a Bruker instrument (400 MHz) and the chemical shift was expressed in ppm. The internal standard (0.00 ppm) of tetramethyl decane was used. ¹ H NMR representation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If a coupling constant is provided, its unit is Hz.

Mass spectrometry was measured by LC/MS, and the ionization method was ESI or APCI.

Thin layer chromatography tantalum sheet using Yantai Yellow Sea HSGF254 or Qingdao GF254 tannin sheet, thin layer chromatography (TLC)

The specification of the silicone sheet used is 0.15mm~0.2mm, and the specification for thin layer chromatography separation and purification is 0.4mm~0.5mm.

Column chromatography was performed using Yantai Huanghai Silicone 200-300 mesh gelatin as a carrier.

In the following examples, all temperatures are in degrees Celsius unless otherwise indicated; unless otherwise indicated, the various starting materials and reagents are either commercially available or synthesized according to known methods, and the commercially available starting materials and reagents are not directly purified. Use; unless otherwise indicated, commercial suppliers include, but are not limited to, Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Guangzan Chemical Technology Co., Ltd., Jingyan Chemical Technology Co., Ltd. and Shanghai Changfeng Biotechnology Co., Ltd. .

CD ₃ OD: Deuterated methanol.

CDCl ₃ : deuterated chloroform.

DMSO-d ₆ : deuterated dimethyl hydrazine.

An argon atmosphere was supplied from the reaction flask to an argon balloon of approximately 1 L volume.

If not specifically stated in the examples, the solution in the reaction means an aqueous solution.

Example 1

( E )-3-(3,5-Difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetrahydro -1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

first step

( E )-3-(3,5-Difluoro-4-methylnonylphenyl) acrylate

4-Bromo-2,6-difluorobenzaldehyde 1a (6.5 g, 75.0 mmol, prepared according to the method disclosed in WO2014191726), triethylamine (21.0 ml, 150.0), palladium acetate (840 mg, 3.75 mmol) and three O-tolylphosphine (1.96 g, 7.50 mmol) was dissolved in 100 mL of dimethylformamide, and methyl acrylate 1b (10.0 mL, 112.5 mmol) was added with stirring, and the reaction mixture was heated to 100 ° C for 20 hours. The reaction solution was cooled, and the solvent was evaporated to dryness. EtOAc (EtOAc) (EtOAc) Drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, and the obtained residue was further purified by chromatography (eluent: cyclohexane and ethyl acetate) to give ( E )-3-(3, Methyl 5-difluoro-4-methylindenyl) acrylate 1c (10.0 g, yellow solid), yield: 59%.

MS m/z (ESI): 227.2 [M+1]

Second step

2-fluoro-2-methylpropan-1-ol

Methyl 2-fluoro-2-methylpropanoate 1d (24.0 g, 200.0 mmol) was dissolved in 500 mL of diethyl ether, cooled to 0 ° C, and lithium aluminum hydride (9.10 g, 240.0 mmol) was added portionwise for 1.5 hours. After the addition was completed, the reaction was carried out at 0 ° C for 1.5 hours. The reaction solution was sequentially added with 9 mL of water, 9 mL of sodium hydroxide solution (15%) and 18 mL of water, and the mixture was stirred for 15 minutes, dried over anhydrous magnesium sulfate (30.0 g), stirred for 15 minutes, filtered, and the filter cake was washed with diethyl ether. The filtrate was concentrated under reduced pressure to give 2-fluoro-2-methylpropan-1-ol 1e (12.6 g, colorless liquid), yield: 68%.

Second step

2-fluoro-2-methylpropyl trifluoromethanesulfonate

2-Fluoro-2-methylpropan-1-ol 1e (12.6 g, 137.0 mmol) was dissolved in 295 mL of dichloromethane (dried with calcium hydride), cooled to -10 ° C, and trifluoromethanesulfonic anhydride was added dropwise. 24.3 mL, 144.0 mmol) and pyridine (13.2 mL, 164.0 mmol) were then reacted at 0 ° C for 1.5 h. 2-methylpropyl trifluoromethanesulfonate successively with 2N hydrochloric acid solution (200mL x2) and water (200mL x2) to the reaction mixture, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give 2-fluoro 1f (18.2 g, red oil), yield: 59%.

third step

(Z)-4-methyl-3-(2-nitroprop-1-en-1-yl)-1H-indole

1-methyl-1H-indole-3-carbaldehyde 1 g (1.0 g, 6.3 mmol), 14.7 mL of nitroethane, ammonium acetate (243 mg, 3.15 mmol) was dissolved in 6.3 mL of acetic acid under argon atmosphere. The reaction was carried out at 110 ° C for 3 hours. The reaction mixture was concentrated under reduced pressure. EtOAc (EtOAc)EtOAc. -Nitroprop-1-en-1-yl-1H-indole 1h (1.0 g, yellow solid), yield: 73.5%.

MS m/z (ESI): 217.0 [M+1]

the fourth step

1-(4-methyl-1H-indol-3-yl)propan-2-amine

Lithium aluminum hydride (0.70 g, 18.5 mmol) was dissolved in 24 mL of anhydrous tetrahydrofuran, and (Z)-4-methyl-3-(2-nitroprop-1-en-1-yl)- 1H-吲哚1h (1.0 g, 4.6 mmol) 6 mL of a solution in tetrahydrofuran, then heated to reflux for 6 hours. Under ice-cooling, the reaction mixture was gradually added with 0.7 mL of water, 0.7 mL of sodium hydroxide solution (15%), and 1.4 mL of water, and the mixture was dried, filtered, filtered, and evaporated. -Methyl-1H-indol-3-yl)propan-2-amine 1i (728 mg, off-white solid), yield: 84%.

MS m/z (ESI): 189.0 [M+1]

the fifth step

2-fluoro-2-methyl-N-(1-(4-methyl-1H-indol-3-yl)propan-2-yl)propan-1-amine

1-(4-Methyl-1H-indol-3-yl)propan-2-amine 1i (258 mg, 1.37 mmol), 2-fluoro-2-methylpropyltrifluoromethanesulfonate under argon The ester 1f (369 mg, 1.64 mmol) was dissolved in diisopropylethylamine (0.34 mL, 2.05 mmol) in 3 mL of 1,4-dioxane. The mixture was heated to 105 ° C for 10 hours, cooled to room temperature and concentrated under reduced pressure. The solvent was removed, and the mixture was washed with EtOAc (EtOAc) (EtOAc) (eluent: dichloromethane: methanol system) to give 2-fluoro-2-methyl-N-(1-(4-methyl-1H-indol-3-yl)propan-2-yl) 1-amine 1j (679 mg, brown oil), yield: 67%.

MS m/z (ESI): 263.0 [M+1]

Step 6

( E )-3-(3,5-Difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetrahydro- Methyl 1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate

Under argon, 2-fluoro-2-methyl-N-(1-(4-methyl-1H-indol-3-yl)propan-2-yl)propan-1-amine 1j (527 mg, 2.0 mmol), methyl ( E )-3-(3,5-difluoro-4-carbamidophenyl)acrylate 1c (454 mg, 2.0 mmol) and acetic acid (0.492 mL, 4.0 mmol) were dissolved in 5 mL of toluene. The reaction was carried out at 80 ° C for 10 hours, cooled to room temperature, and concentrated under reduced pressure. The obtained residue was purified and purified by eluent column chromatography (eluent: petroleum ether: ethyl acetate) to afford ( E )-3- (3,5-Difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetrahydro-1H-pyrrolo[3 , 4-b]Indol-1-yl)phenyl)methyl acrylate 1k (773 mg, red oil), yield: 82%.

MS m/z (ESI): 471.0 [M+1]

Seventh step

( E )-3-(3,5-Difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetrahydro- 1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

( E )-3-(3,5-Difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetrahydro Methyl-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 1k (773 mg, 1.64 mmol) in 15 mL of a mixed solution of tetrahydrofuran and methanol (V/V=2/1) Slowly add 2.2mL of 7.5M sodium hydroxide solution, react at room temperature for 12 hours, adjust the pH of the reaction solution with 2N hydrochloric acid solution, remove the solvent under reduced pressure, adjust the pH of the solution with 2M hydrochloric acid, stratify, use the water phase. The organic layer was extracted with EtOAc (EtOAc) (EtOAc) , ( E )-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,5-dimethyl-2,3,4,9-tetra Hydrogen-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid 1 (260 mg, pale yellow solid), yield: 35%.

MS m/z (ESI): 455.0 [M+1]

^{1 H NMR (400MHz, DMSO-} d6) δ = 1.04-1.13 (m, 5 H) 1.17 (d, J = 9.03Hz, 3 H) 1.24 (s, 2 H) 2.29-2.42 (m, 1 H) 2.58 (s, 3 H) 2.77-2.92 (m, 2 H) 3.15 (d, J = 11.29 Hz, 1 H) 3.46 (d, J = 6.02 Hz, 1 H) 5.22 (s, 1 H) 6.62-6.70 ( m, 2 H) 6.84 (t, J = 7.65 Hz, 1 H) 6.98 (d, J = 8.03 Hz, 1 H) 7.45 (d, J = 10.54 Hz, 2 H) 7.53 (d, J = 16.06 Hz, 1 H) 10.50 (s, 1 H).

Example 2

( E )-3-(3,5-Difluoro-4-(7-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Hydrogen-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

first step

(Z)-6-fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole

6-Fluoro-1H-indole-3-carbaldehyde 2a (1.0 g, 6.1 mmol), 14.2 mL of nitroethane, ammonium acetate (235 mg, 3.05 mmol) was dissolved in 6.0 mL of acetic acid under argon atmosphere, 110 The reaction was carried out at ° C for 6 hours. The reaction mixture was concentrated under reduced pressure. EtOAc (EtOAc m. 6-fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole 2b (1.31 g, brown solid), yield: 98%.

Second step

1-(6-fluoro-1H-indol-3-yl)propan-2-amine

Lithium aluminum hydride (0.95 g, 23.6 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and (Z)-6-fluoro-3-(2-nitroprop-1-en-1-yl)- 1H-吲哚2b (1.31 g, 5.9 mmol) was dissolved in 10 mL of tetrahydrofuran and then heated to reflux for 6 hours. The reaction mixture was successively added with 0.95 mL of water, 0.95 mL of sodium hydroxide solution (15%), and 1.8 mL of water, and the mixture was stirred. The residue was stirred with anhydrous magnesium sulfate (3.0 g) for 15 min, filtered and concentrated under reduced -Fluoro-1H-indol-3-ylpropan-2-amine 2 c (1.20 g, brown oil), yield: 100%.

MS m/z (ESI): 193.0 [M+1]

third step

2-fluoro-N-(1-(6-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine

1-(6-Fluoro-1H-indol-3-yl)propan-2-amine 2c (1.20 g, 6.20 mmol), 2-fluoro-2-methylpropyltrifluoromethanesulfonate under argon The ester 1f (1.67g, 7.44mmol) was dissolved in diisopropylethylamine (1.50mL, 9.30mmol) in 12mL of 1,4 dioxane, heated to 105 ° C for 10 hours, cooled to room temperature, decompression After concentration, the obtained residue was further separated and purified by silica gel column chromatography (eluent: dichloromethane: methanol) to afford 2-fluoro-N-(1-(6-fluoro-1H-indol-3-yl) Propane-2-yl)-2-methylpropan-1-amine 2d (1.65 g, brown oil), yield: 100%.

MS m/z (ESI): 267.0 [M+1]

the fourth step

( E )-3-(3,5-Difluoro-4-(7-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Methyl-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate

2-Fluoro-N-(1-(6-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine 2d (1.65 g, under argon) 6.20 mmol), methyl ( E )-3-(3,5-difluoro-4-methylindenyl) acrylate 1c (1.27 g, 5.63 mmol) and acetic acid (0.645 mL, 11.3 mmol) dissolved in 10 mL toluene The reaction was carried out at 80 ° C for 5 hours, cooled to room temperature, and concentrated under reduced pressure. The obtained residue was further purified and purified by eluent column chromatography (eluent: petroleum ether: ethyl acetate) to give ( E )-3 -(3,5-difluoro-4-(7-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrole Methyl [3,4-b]indol-1-yl)phenyl) 2e (1.00 g, off-white solid), yield: 38.5%.

MS m/z (ESI): 474.9 [M+1]

the fifth step

( E )-3-(3,5-Difluoro-4-(7-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Hydrogen-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

( E )-3-(3,5-Difluoro-4-(7-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9- Methyl tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 2e (1.00 g, 2.10 mmol) was dissolved in 19.5 mL of tetrahydrofuran and methanol (V/V = 2/1) The mixed solution, slowly add 2.8mL of 7.5M sodium hydroxide solution, react at room temperature for 3 hours, adjust the pH of the reaction solution with 2N hydrochloric acid solution = 6~7, remove the solvent under reduced pressure, add 10 mL of water and 20 mL of ethyl acetate. The solution was adjusted to pH = 2 with 2M hydrochloric acid, and the layers were separated, and the aqueous phase was extracted with ethyl acetate (15 mL), and the organic phase was combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Further separation and purification (eluent: petroleum ether: ethyl acetate system) to obtain ( E )-3-(3,5-difluoro-4-(7-fluoro-2-(2-fluoro-2-) Propyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid 2 (180 mg, light yellow Solid), Yield: 18.6%.

MS m/z (ESI): 460.9 [M+1]

^{1 H NMR (400MHz, DMSO-} d 6) δ = 12.60 (br.s., 1 H), 10.71 (s, 1 H), 7.54 (d, J = 16.1Hz, 1 H), 7.46 (d, J =10.5 Hz, 2 H), 7.39 (dd, J = 5.6, 8.4 Hz, 1 H), 6.95 (dd, J = 2.0, 10.0 Hz, 1 H), 6.85-6.77 (m, 1 H), 6.67 ( d, J = 15.8 Hz, 1 H), 5.19 (s, 1 H), 3.49 (d, J = 5.5 Hz, 1 H), 2.93 - 2.80 (m, 2 H), 2.56 (dd, J = 4.4, 15.4 Hz, 1 H), 2.40-2.27 (m, 1 H), 1.24-1.16 (m, 3 H), 1.16-1.08 (m, 3 H), 1.04 (d, J = 6.5 Hz, 3 H).

Example 3

( E )-3-(3,5-Difluoro-4-(6-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Hydrogen-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

first step

(Z)-5-fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole

5-Fluoro-1H-indole-3-carbaldehyde 3a (1.0 g, 6.1 mmol), 14.2 mL of nitroethane, ammonium acetate (235 mg, 3.05 mmol) was dissolved in 6.0 mL of acetic acid under argon atmosphere, 110 The reaction was carried out at ° C for 6 hours. The reaction mixture was concentrated with EtOAc EtOAc EtOAc. (Z)-5-Fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole 3b (1.30 g, brown solid).

Second step

1-(5-fluoro-1H-indol-3-yl)propan-2-amine

Lithium aluminum hydride (0.90 g, 23.6 mmol) was dissolved in 30 mL of anhydrous tetrahydrofuran, and (Z)-5-fluoro-3-(2-nitroprop-1-en-1-yl)- 1H-吲哚3b (1.30 g, 5.9 mmol) was dissolved in 10 mL of tetrahydrofuran, then heated to reflux for 6 hours. The reaction solution was successively added with 0.90 mL of water, 0.90 mL of sodium hydroxide solution (15%), and 1.8 mL of water to quench the reaction, and anhydrous magnesium sulfate (3.0 g) was stirred for 15 minutes, filtered, and concentrated under reduced pressure to give 1-(5) -Fluoro-1H-indol-3-ylpropan-2-amine 3c (1.18 g, brown oil), yield: 100%.

MS m/z (ESI): 193.0 [M+1]

third step

2-fluoro-N-(1-(5-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine

1-(5-Fluoro-1H-indol-3-yl)propan-2-amine 3c (1.18 g, 6.20 mmol), 2-fluoro-2-methylpropyltrifluoromethanesulfonate under argon The ester 1f (1.67g, 7.44mmol) was dissolved in diisopropylethylamine (1.50mL, 9.30mmol) in 12mL of 1,4 dioxane, heated to 105 ° C for 10 hours, cooled to room temperature, decompression After concentration, the obtained residue was further separated and purified by silica gel column chromatography (eluent: dichloromethane: methanol) to afford 2-fluoro-N-(1-(5-fluoro-1H-indol-3-yl) Propane-2-yl)-2-methylpropan-1-amine 3d (1.155 g, brown oil), yield: 70%.

MS m/z (ESI): 267.0 [M+1]

the fourth step

( E )-3-(3,5-Difluoro-4-(6-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Methyl-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate

2-Fluoro-N-(1-(5-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine 3d (1.155 g, under argon) 4.34 mmol), methyl ( E )-3-(3,5-difluoro-4-carbamidophenyl)acrylate 1c (0.98 g, 5.63 mmol) and acetic acid (0.52 g, 8.68 mmol) dissolved in 10 mL toluene The reaction was carried out at 85 ° C for 5 hours, cooled to room temperature, and concentrated under reduced pressure. The obtained residue was purified and purified by eluent column chromatography (eluent: petroleum ether: ethyl acetate) to afford ( E )-3 -(3,5-difluoro-4-(6-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrole [3,4-b]Indol-1-yl)phenyl) acrylate 3e (0.95 g, yellow foamy solid), yield: 47.5%.

MS m/z (ESI): 474.9 [M+1]

the fifth step

( E )-3-(3,5-Difluoro-4-(6-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra Hydrogen-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

( E )-3-(3,5-Difluoro-4-(6-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9- Methyl tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 3e (0.95 g, 2.00 mmol) was dissolved in 19.5 mL of tetrahydrofuran and methanol (V/V=2/1 The mixed solution was slowly added with 2.7 mL of 7.5 M sodium hydroxide solution, and reacted at room temperature for 2 hours. The pH of the reaction solution was adjusted to 6-7 with 2N hydrochloric acid solution, and the solvent was concentrated under reduced pressure to add 10 mL of water and 20 mL of ethyl acetate. The solution was adjusted to pH = 2 with 2M hydrochloric acid, and the layers were separated, and the aqueous phase was extracted with ethyl acetate (15 mL), and the organic phase was combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Further separation and purification (eluent: petroleum ether: ethyl acetate system) to obtain ( E )-3-(3,5-difluoro-4-(6-fluoro-2-(2-fluoro-2-) Propyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid 3 (320 mg, pale yellow solid ), yield: 34.4%.

MS m/z (ESI): 460.9 [M+1]

^{1 H NMR (400MHz, DMSO-} d 6) δ = 12.61 (br.s., 1 H), 10.70 (s, 1 H), 7.54 (d, J = 16.1Hz, 1 H), 7.47 (d, J =10.5 Hz, 2 H), 7.19-7.14 (m, 2 H), 6.87-6.80 (m, 1 H), 6.67 (d, J = 16.1 Hz, 1 H), 5.21 (s, 1 H), 3.50 (d, J = 5.3 Hz, 1 H), 2.93 - 2.81 (m, 2 H), 2.57 (d, J = 4.3 Hz, 1 H), 2.41-2.27 (m, 1 H), 1.24-1.16 (m) , 3 H), 1.16.10.08 (m, 3 H), 1.04 (d, J = 6.5 Hz, 3 H).

Example 4

( E )-3-(3,5-Difluoro-4-((1 R ,3 R )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2 ,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

first step

(Z)-4-fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole

4-Fluoro-1H-indole-3-carbaldehyde 4a (1.0 g, 6.1 mmol), 14.2 mL of nitroethane, ammonium acetate (235 mg, 3.05 mmol) was dissolved in 6.0 mL of acetic acid under argon, 110 The reaction was carried out at ° C for 6 hours. The reaction mixture was concentrated with EtOAc EtOAc EtOAc. (Z)-4-Fluoro-3-(2-nitroprop-1-en-1-yl)-1H-indole 4b (1.374 g, brown solid). Yield: 100%.

Second step

1-(4-fluoro-1H-indol-3-yl)propan-2-amine

Lithium aluminum hydride (0.95 g, 25.0 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and (Z)-4-fluoro-3-(2-nitroprop-1-en-1-yl)-1H was slowly added in an ice bath. - 4b (1.374 g, 6.20 mmol) was dissolved in 10 mL of tetrahydrofuran and then heated to reflux for 6 hours. The reaction solution was successively added with 0.95 mL of water, 0.95 mL of sodium hydroxide solution (15%), and 19 mL of water, and the mixture was stirred for 15 minutes, and the mixture was stirred for 15 minutes, filtered, and the filter cake was washed with tetrahydrofuran (5 mL x 3). The filtrate was concentrated under reduced pressure to give 1-(4-fluoro-1H-indol-3-yl)propan-2-amine 4c (1.20 g, brown oil).

MS m/z (ESI): 193.0 [M+1]

third step

2-fluoro-N-(1-(4-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine

1-(4-Fluoro-1H-indol-3-yl)propan-2-amine 4c (1.20 g, 6.20 mmol), 2-fluoro-2-methylpropyltrifluoromethanesulfonate under argon The ester 1f (1.67g, 7.44mmol) was dissolved in diisopropylethylamine (1.50mL, 9.30mmol) in 12mL of 1,4 dioxane, heated to 105 ° C for 10 hours, cooled to room temperature, decompression The mixture was concentrated, washed with ethyl acetate (20 mL), EtOAc (EtOAc) Deprotection: dichloromethane: methanol system), 2-fluoro-N-(1-(4-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1- Amine 4d (1.07 g, brown oil), yield: 65%.

MS m/z (ESI): 267.0 [M+1]

the fourth step

( E )-3-(3,5-Difluoro-4-((1 R ,3 R )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2 ,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl) acrylate

( E )-3-(3,5-Difluoro-4-((1 S ,3 S )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2 ,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl) acrylate

2-Fluoro-N-(1-(4-fluoro-1H-indol-3-yl)propan-2-yl)-2-methylpropan-1-amine 4d (1.07 g, under argon) 4.00 mmol), methyl ( E )-3-(3,5-difluoro-4-carbamidophenyl)acrylate 1c (0.90 g, 4.00 mmol) and acetic acid (0.46 mL, 8.00 mmol) dissolved in 9 mL toluene The reaction was carried out at 85 ° C for 7 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The obtained residue was purified and purified by chromatography (eluent: petroleum ether: ethyl acetate). A large amount of solid was precipitated, filtered, and the filter cake was washed with a small amount of ice-cooled dichloromethane, and the filter cake was dried to give ( E )-3-(3,5-difluoro-4-(5-fluoro-2-(2-fluoro-) Methyl 2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate (0.68 g, white solid), yield: 36%, resolved by preparative equipment and chiral column chiral isomer by critical fluid chromatography (SFC) method (chiral column ChiralCel OJ, 250 × 30 mm ID 50 mL / min; Mobile phase A: CO2; B: methanol), ( E )-3-(3,5-difluoro-4-((1 R ,3 R )-5-fluoro-2-(2-fluoro-2-) Methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 4e (325.68 mg , white solid) and ( E )-3-(3,5-difluoro-4-((1 S ,3 S )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3 Methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 4f (313.33 mg, white solid).

MS m/z (ESI): 474.9 [M+1]

the fifth step

( E )-3-(3,5-Difluoro-4-((1 R ,3 R )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2 ,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylic acid

( E )-3-(3,5-Difluoro-4-((1 R ,3 R )-5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl- Methyl 2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indol-1-yl)phenyl)acrylate 4e (325.68 mg, 0.686 mmol) was dissolved in 6 mL of tetrahydrofuran and methanol ( V/V=2/1) mixed solution, slowly add 0.9mL of 7.5M sodium hydroxide solution, react at room temperature for 1 hour, adjust the pH of the reaction solution with 2N hydrochloric acid solution = 6~7, remove the solvent under reduced pressure, add 5mL Water and 10 mL of ethyl acetate, the solution was adjusted to pH 2 with 2M hydrochloric acid, and the mixture was separated, and the aqueous phase was extracted with ethyl acetate (5 mL×2). The residue was further separated and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate) to give ( E )-3-(3,5-difluoro-4-((1 R ,3 R ) -5-fluoro-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrrolo[3,4-b]indole- 1-(1)phenyl)acrylic acid 4 (295 mg, pale yellow solid), yield: 93%.

MS m/z (ESI): 460.9 [M+1]

^{1 H NMR (400MHz, DMSO-} d 6) δ = 10.92 (br.s., 1 H), 7.54 (d, J = 16.1Hz, 1 H), 7.47 (d, J = 10.5Hz, 2 H), 7.05-6.99 (m, 1 H), 6.98-6.91 (m, 1 H), 6.73-6.64 (m, 2 H), 5.22 (br.s., 1 H), 3.47 (d, J = 5.0 Hz, 1 H), 3.15-2.99 (m, 1 H), 2.93-2.81 (m, 1 H), 2.71 (dd, J = 4.1, 15.2 Hz, 1 H), 2.41-2.29 (m, 1 H), 1.18 (br.s., 3 H), 1.16.10.09 (m, 3 H), 1.07 (d, J = 6.5 Hz, 3 H).

Biological evaluation

Test Example 1 Estrogen Receptor ERα Coordination Analysis

Control fluorescence resonance energy transfer (TR-FRET) on the competitive complexation between estrogen receptor ERα ligand compound and scope isolated using the present invention when evaluated Lantha Screen ^TM. The fluorophore (Fluormone ES2, product number P2645) and the recombinant human estrogen receptor ERα ligand domain (product number PV4543) used in the TR-FRET coordination evaluation technique were purchased from Invitrogen. The design principle of this test is as follows. The estrogen receptor ERα-LBD (GST) and the fluorophore-containing ligand form a receptor/fluorescence complex, and then the Tb-labeled anti-GST antibody is added. PV3551), indirect fluorescent labeling of the receptor by GST linkage to the receptor, by detecting between the chromophore (Tb-anti-GST antibody) and the fluorescent ligand (Fluormone ES2) on the fluorescently labeled ERa Attenuation of the TR-FRET effect is used to evaluate the competitive coordination ability between the test compound and the fluorescent ligand and the receptor. The above compounds were tested on the synthesized compounds using the following sample preparation and test methods. The instrument used was a Beckman Coulter BioPAPTR FRD microfluidic instrument.

(1) 120 nL of the test compound was ultrasonically dispersed into a black small-capacity 384-well assay plate.

(2) A 1x ER α -LBD/Tb-anti-GST antibody complex was prepared in a buffer of ES2 and cultured therein for 20 minutes.

(3) The test was carried out by adding 1x ES2 to the above ER α -LBD/Tb-anti-GST antibody complex solution.

(4) ER α -LBD prepared in Step 3 / Tb- anti-GST antibody complex was added to a solution of 12 μ L assay plate wells.

(5) The analysis plate was stored in the dark and incubated at room temperature for one hour.

(6) The emission light of the 490 nm and 520 nm bands was detected by BMG Phera STAR under excitation light of 337 nm.

Various concentrations (10 mM, 0.1 mM, 1 μM, and 10 nM) of test compound prepared on the microplate were transferred to the assay plate using Labcyte Echo550. 120 nL of DMSO solution of each test compound was added to the wells of the assay plate and tested at twelve different concentrations (100, 29.17, 10.42, 2.083, 1, 0.292, 0.104, 0.02083, 0.01, 0.0029, 0.00104, 0.001 μM). ). The obtained TR-FRET raw fluorescence test data was obtained by means of data processing software such as Origin or Genedata. Each compound employed inhibition concentration IC ₅₀ characterized competing ligand effects of test compounds to the estrogen receptor ERα. The IC ₅₀ represents the concentration of the test compound estimated when the coordination of the fluorophore (ES2) with the estrogen receptor is reduced by 50%, and the IC ₅₀ determined is shown in Table 2.

Test Example 2 ERα down-regulation test of MCF-7 cells

The down-regulation of the ERα protein level by the preferred compounds of the invention was evaluated by immunofluorescence analysis in human breast cancer cell line MCF-7. MCF-7 cells are used in the recovery experiment from frozen cells (about 5x10 ⁶ th) over use. The MCF-7 frozen cell strain (Sigma D5921) purchased from Sigma was stored in DMEM medium containing 2 mM L-glutamic acid. 5% (v/v) Charcoal/Dextran-treated bovine serum embryo cells were added to the resuscitated MCF-7 cells, and the cell concentration was determined using a Coulter Counter. The cells used for the test were diluted to 3.75 x 10 ⁴ /mL with the culture medium, and 40 uL/well of the above cell liquid was transferred to a black transparent bottom 384-well plate, and then the well plate was cultured at 37 ° C, 5% CO ₂ overnight. A 10 mM test compound solution was used for the preparation of different concentrations of test solutions (10 mM, 0.1 mM, 1 [mu]M, 0.01 [mu]M). To various concentrations of test compound and MCF-7 cell culture medium (40 μL), 20 μL of 11.1% (v/v) aqueous formaldehyde solution (phosphoric acid PBS buffer) was added, and finally the concentration of formaldehyde in the solution was 3.7% (v/). v). After the cells were fixed at room temperature for 20 minutes, they were washed twice with 250 μL of PBS/Proclin, and then 40 μL of PBS/Proclin was added, followed by chilling at 4 °C. Immunostaining analysis of proteins was performed using an automated AutoElisa kit. From each plate wells Pipette PBS / Proclin was then added 40μL of 0.5% for cell permeabilization process ^{Tween PBS TM 20 (v / v} ) . After one hour, plates were coated with 250μL of PBS / 0.05% Tween 20 / Proclin washed three times, added ERα rabbit monoclonal antibody (Thermofisher) of the PBS 20μL ^{/ Tween TM 20/3% (} w / v) BSA solution (1: 1000). Analysis plates incubated overnight at 4 ℃, with 250uL of PBS / Tween ^TM 20 / Proclin washed three times, and then 20μL added to each well goat anti-rabbit IgG AlexaFluor 594 or goat anti-rabbit IgG AlexaFluor 488 antibody (containing Hoechst stain ) in ^{PBS / Tween TM 20/3%} (w / v) BSA solution (1: 5000), the system incubated at room temperature for one hour. Analysis plate with 250μL of PBS / 0.05% (v / v ) Tween TM 20 / Proclin washed three times with PBS solution was added 20μL of the orifice plate stored at 4 ℃ protected from light. The level of estrogen receptor ERa in MCF-7 cells was calculated by measuring the fluorescence emission intensity of the two emission bands of the 594 nm (24 hour time point) and 488 nm (5 hour time point) of the well plate by Cellomics Arrayscan. The average fluorescence emission intensity of each cell is positively correlated with the ERα receptor level of the cell. The obtained original fluorescent test data is obtained by means of data processing software such as Origin or Genedata. We characterized IC ₅₀ concentration of test compound downregulation of the estrogen receptor ERα by semi inhibition, IC ₅₀ refers to the fluorescence emission intensity was reduced by 50% when the concentration of the test compound the maximum mean fluorescence intensity, measured IC ₅₀ As shown in table 2.

Table 2 Estrogen receptor ERα coordination analysis test results and ERα down-regulation test results

Remarks: IC ₅₀ range 0.1nM A 10nM.

Conclusion: The preferred compounds of the present invention are capable of better coordination with estrogen receptors and have a better down-regulation effect on ERα.

Test Example 3 Competitive binding analysis of estrogen receptor ERα with preferred compounds of the invention

The present invention utilizes Fluorescece polarization ^TM (FP) competition fluorescence polarization technique between estrogen receptor ERα ligand compound and give scope to evaluate binding.

The PolarScreen ER Alpha competitor Assay kit used in the FP coordination evaluation technique was purchased from Invitrogen (Cat. No. A15883) and consisted mainly of fluorophore (Fluormone ES2, Cat. No. P2645) and recombinant human estrogen receptor ERα ligand. Domain (Cat. No. A15674).

The design principle of this test is as follows. The estrogen receptor ERα forms a receptor/fluorescence complex with the fluorophore-containing ligand Fluormone ES2, which generates a highly polarized light signal value (ΔmP) after excitation. Attenuation of ΔmP to evaluate the competitive binding ability of the test compound and the fluorescent ligand to the receptor.

The test compound was subjected to the above test using the following sample preparation and test methods: (1) A 10 mM test compound solution was diluted with DMSO in a Labcyte 384-well plate (Cat. No. P-05525) (the first concentration was 33.33 μM, 3 fold dilution, 10 concentrations); (2) Echo550 transferred 60nL to black low volume 384 well assay plate (purchased from Corning, Product Number 3676); (3) was diluted in ES2 Screening Buffer in ERα and Fluormone ^TM ES2, final concentration: ^{ERα = 150nM, Fluormone TM ES2 =} 4.5nM, mix gently, Apricot 20μL transferred to a 384 well assay plate; (4) the sealing plate membrane seal plate, incubated for 60 min in the dark 25 ℃; (5) Envision read fluorescence polarization Value(mP); The obtained mP value was processed by Graphpad Prism 6.0 to obtain a fitting curve.

Semi inhibitory concentration IC ₅₀ characterized competing test compounds to the estrogen receptor ERα binding effect. The IC ₅₀ represents the concentration of the test compound calculated when the binding of the fluorophore (ES2) to the estrogen receptor is decreased by 50%, and the IC ₅₀ determined is shown in Table 3.

Conclusion: Preferred compounds of the invention have a better affinity for the estrogen receptor.

Test Example 4 A preferred compound of the present invention down-regulates the amount of ERα protein in MCF-7 cells.

The downregulation of the ERα protein level by the preferred compounds of the present invention was evaluated by immunofluorescence analysis in human breast cancer cell line MCF-7.

The MCF-7 cells used in the experiment (purchased from ATCC, item number HTB-22) were frozen in the presence of 20% fetal bovine serum (FBS, purchased from GIBCO, Cat. No. 10099-141) and 10% DMSO (purchased from Solarbio, item number D8371). DMEM (purchased from GIBCO, Cat. No. 11995-063) in the culture solution. Frozen cells (about 2x10 ⁶ th) after resuscitation containing 10% FBS and 1% Pen / Step (purchased from Gibco, Order number 15140-122) DMEM culture solution cultured, passaged twice for the experiments. The cells were resuspended in DMEM containing 2% Activated charcoal (purchased from Sigma, Cat. No. C3345) and 1% Pen/Step in DMEM. Cell counts (purchased from Invitrogen, model C10281) were used to determine cell concentration and then cultured by test. The solution was diluted to 3.75×10 ⁴ cells/mL, and 40 μL/well of the above cell suspension was transferred to a black-bottomed 384-well plate (purchased from BD, Cat. No. 356663) at 37 ° C, 5% CO ₂ cell incubator. Cultivate overnight. Dilute a 10 mM test compound solution in a Labcyte 384-well plate with DMSO (3 mM diluted 300-fold to obtain 33.33 μM as the first concentration, 3 times dilution, 10 concentrations), then Echo550 (purchased from Echo, model number) 550) Transfer 120 nL to 40 μL of cell culture medium, the final concentration of DMSO was 0.3%, the first concentration of the test compound was 100 nM, and the cells were centrifuged at 1000 rpm for 1 min at room temperature, and the cells were cultured at 37 ° C in a 5% CO ₂ cell incubator overnight. The medium in the cell culture plate was aspirated with Apricot (purchased from Apricot Designs, model PPA-384-E), the cells were washed once with 40 μL of PBS, and PBS was aspirated, and 40 μL of a 3.7% (v/v) aqueous formaldehyde solution was purchased (purchased from Sigma). , Item No. F1635, diluted with PBS), the cells were fixed at 25 ° C for 20 minutes; after aspirating the aqueous formaldehyde solution, the cells were washed once with 40 μL of PBS, and 40 μL of a final concentration of 0.5% Tween-20 (diluted with PBS) was permeated at 25 ° C for 1 h; After permeate, the cells were washed once with PBS-T (PBS containing 0.05% Tween-20), and 25 μL of ER antibody (purchased from CST, Cat. No. 8644S) was added (1:1000, containing 1% in PBS-T). Milk diluted), incubate at 25 ° C for 1.5 h; PBS-T was immersed for 3 times, and a secondary antibody (purchased from ThermoFisher, Cat. No. A-11008) was added (1:1000, diluted with 1% milk in PBS) 2 μg /mL of Hochest 33342 (purchased from Molecular Probe, item number H3570), incubation at 25 ° C for 40 min in the dark; PBS-T dipping the cells 3 times, PBS dipping the cells 2 times, finally adding 40 μL of PBS and reading the ERα protein signal with Acumen Ratio of values to nuclear signal values (relative to fluorescence intensity), fitting the software with Graphpad Prism 6.0 curve.

The half-inhibitory concentration IC _{50 was} used to characterize the down-regulation of the estrogen receptor ERα by the test compound. The IC ₅₀ refers to the concentration of the test compound when the relative fluorescence intensity of the ERα protein is reduced to 50% of the average maximum relative fluorescence intensity. _{50 is} shown in Table 4.

Conclusion: The preferred compounds of the invention have a good down-regulation effect on the amount of ERα protein.

Test Example 5 Test of ERα Functional Activity Down-regulation in MCF-7 Cells by Preferred Compounds of the Invention

The downregulation of the functional activity of the ERα protein by the preferred compounds of the present invention was evaluated by immunofluorescence analysis in human breast cancer cell line MCF-7.

The MCF-7 cells used in the experiment were frozen in DMEM medium containing 20% fetal bovine serum and 10% DMSO. Frozen cells (approximately 2×10 ⁶ cells) were cultured in DMEM medium containing 10% FBS and 1% Pen/Step after resuscitation, and passaged twice for use in experiments. The cells were resuspended in DMEM containing 10% (v/v) Activated charcoal-treated FBS and 1% Pen/Step, and the cell concentration was measured by a cell counter and diluted to 3.75×10 ⁴ cells/mL with the test medium. 40 μL/well of the above cell suspension was pipetted into a black-bottomed 384-well plate and cultured overnight at 37 ° C in a 5% CO ₂ cell incubator. The stock solution of 20 mM Estradiol was diluted with DMSO in Labcyte 384-well plates to 33.33 nM, and transferred to 120 μL to 40 μL of cell culture medium with Echo550. The final concentration of Estradiol was 0.1 nM, and the final concentration of DMSO was 0.3% at 37 ° C, 5%. Incubate for 30 min in a CO ₂ cell incubator; dilute 10 mM test compound solution (first concentration of 33.33 μM, 3 fold dilution, 10 concentrations) in a Labcyte 384-well plate with DMSO, then use Echo 550 (purchase from Echo) , Model 550) Transfer 120nL to 40μL cell culture medium, the final concentration of DMSO is 0.6%, the first concentration of test compound is 100nM, centrifuge at 1000 rpm for 1min at room temperature, then place the cells in 37°C, 5% CO ₂ cell incubator. Cultivate overnight. Apricot was used to aspirate the medium in the cell culture plate, and the cells were washed once with 40 μL of PBS. After aspirating the PBS, 40 μL of a 3.7% (v/v) aqueous solution of formaldehyde was added, and the cells were fixed at 25 ° C for 20 minutes; the aqueous formaldehyde solution was aspirated and then immersed in 40 μL of PBS. Wash the cells once, add 40 μL of 0.5% Tween-20 (diluted in PBS) at 25 ° C for 1 h; aspirate the permeate, then dilute the cells once with PBS-T, add 25 μL of PR antibody dilution (1:2500, with PBS). -T contains 1% milk dilution), incubate at 25 °C for 1.5h; PBS-T immersed the cells 3 times, added secondary antibody (diluent (1:1000, diluted with 1% milk in PBS) 2μg/mL Hochest 33342, incubate for 40 min at 25 °C; PBS-T was immersed for 3 times, PBS was immersed twice, and finally 40 μL of PBS was added to read the ratio of ERα protein signal to nuclear signal value (relative fluorescence intensity) with Acumen. The software was processed with Graphpad Prism 6.0 to obtain a fitted curve.

The half-inhibitory concentration IC _{50 was} used to characterize the down-regulation of the estrogen receptor ERα by the test compound. The IC ₅₀ refers to the concentration of the test compound when the relative fluorescence intensity of the ERα protein is reduced to 50% of the average maximum relative fluorescence intensity. _{50 is} shown in Table 5.

Conclusion: The preferred compounds of the invention have a good down-regulation effect on the functional activity of the estrogen receptor ERα.

Test Example 6 Inhibition of IC by MCF-7 cells by the compounds of the examples of the present invention ₅₀ Value determination

1 Reagents and consumables

Cell Counting Kit-8 (Cat# CK04-13, Dojindo)

96-well culture plate (Cat# 3599, Corning Costar)

Medium and fetal bovine serum (GIBCO)

Benchtop Microplate Reader SpectraMax M5 Microplate Reader (Molecular Devices)

MCF-7 human breast cancer cell line (purchased at Shanghai Cell Resource Center of Chinese Academy of Sciences)

2 reagent preparation

Preparation of medium: MEM+10%FBS+0.01mg/ml human recombinant insulin

Preparation of the compound: the compound is diluted with DMSO to a final concentration of 10 mM;

3 Experimental steps

(1) Collect logarithmic growth phase cells, count, resuspend the cells with complete medium, adjust the cell concentration to an appropriate concentration (determined according to the cell density optimization test results), inoculate 96-well plates, and add 100 μL of cell suspension per well. The cells were incubated for 24 hours at 37 ° C, 100% relative humidity, 5% CO 2 incubator; (2) The test compound was diluted with the medium to the corresponding concentration of action set, and the cells were added at 25 μL/well. The final concentration of the compound was started from 1 μM, 4 times gradient dilution, 9 concentration points; (3) cells were incubated at 37 ° C, 100% relative humidity, 5% CO ₂ incubator for 72 hours; (4) aspirate medium Add the complete medium containing 10% CCK-8 and incubate in a 37 °C incubator for 1 to 5 hours; (5) Determine the absorbance at 450 nm on a SpectraMax M5 Microplate Reader with gentle shaking, and take the absorbance at 650 nm as the reference. Ratio, calculate the inhibition rate.

4 data processing

The inhibition rate of the drug on tumor cell growth was calculated as follows: tumor cell growth inhibition rate % = [(A _{c -} A _s ) / (A _{c -} A _b )] × 100%

A _s : sample OA (cell + CCK-8 + test compound)

A _c : negative control OA (cell + CCK-8 + DMSO)

A _b : positive control OA (medium + CCK-8 + DMSO)

And using Graphpad Prism 5 software using the formula log (inhibitor) vs.response-variable slope curve for the IC ₅₀ is calculated to be merged Preferred compounds of the invention inhibit ₅₀ values MCF-7 cells IC, such as shown in Table 6.

Conclusion: The preferred compounds of the invention have a significant inhibitory effect on the proliferation of MCF-7 cells.

Test Example 7 Growth inhibition test of preferred compounds of the present invention on MCF-7 tumor-bearing SCID mouse xenografts

1. Purpose

This test was used to evaluate the growth inhibitory effect of oral administration of test substances on MCF-7 tumor-bearing SCID mice for 21 consecutive days.

2. Preparation of test substance

Solvent: 20% PEG400, 80% deionized water;

Preparation of the test substance: Weigh an appropriate amount of the compound of Example 4 and AZD-9496, dissolve it in PEG400 (20%), dissolve it, add 80% of sterilized deionized water, and shake evenly. The test drug is freshly prepared daily before administration.

3. Experimental animals

Varieties and strains: SCID rats, SPF, females, 7-9 weeks old (16-22 grams), 100, purchased from Beijing Huakangkang Biotechnology Co., Ltd., 100 healthy ones for experiments, adapted to the environment Time is 5~7 days.

4. MCF-7 tumor cell culture

MCF-7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Culture in a 37 ° C, 5% CO ₂ incubator. Logarithmic growth phase cells were taken before inoculation, washed with 0.25% trypsin and washed with PBS. The cells were resuspended in serum-free medium and the cell concentration was adjusted to 7.5×10^7 cells/mL (1:1 Matrigel, Extracellular Matrix Proteins, 356234, BD).

5 Animal vaccination and grouping

Each mouse was inoculated with 0.2 mL of cell suspension (1.5 x 10^7 cells/mouse) under sterile conditions on the right side of each mouse. Estrogen was administered subcutaneously after inoculation. When the tumor grows to a volume of about 150-250 mm ³ , mice with similar tumor volume and good shape (the shape is as single spherical as possible, no irregular shape or multiple tumors are gathered together) are 10 in each group.

6 Animal administration and observation

Each group of animals was given a test substance once a day (qd) according to the body weight of the above-mentioned table at a fixed time per day, orally administered (po) for 21 consecutive days, and the animal body weight per day was recorded.

The formation of tumors at the inoculation site of each group of animals was observed. The long diameter (Y) and short diameter (X) of the tumor nodules were measured twice a week using vernier calipers and calculated according to the following formula: Volume of tumor nodules (V): V = ( X ² Y)/2.

Evaluation index of antitumor activity: tumor growth inhibition rate TGI (%), relative tumor growth rate T/C (%).

Tumor growth inhibition rate TGI (%): TGI (%) = (V _c - V _t ) / V _c × 100. Where V _c is the tumor volume of the model control group and V _t is the tumor volume of the test subject group.

Relative tumor volume (RTV): RTV = V _n /V ₀ . Where V ₀ is the tumor volume at the time of group administration, and V _n is the tumor volume at the time of measurement.

Relative tumor growth rate T/C (%): T/C (%) = T _RTV / C _RTV × 100. Among them, T _RTV was the treatment group RTV, and C _RTV was the negative control group RTV.

7 results

The tumor growth inhibition rate (TGI%) of the preferred compound of the present invention against breast cancer MCF-7 tumor-bearing mice is shown in Table 7.

At the dose of 10 mg/kg, the present invention 4 has a significant inhibitory effect on breast cancer MCF-7 within 21 days, and its TGI% is 29.4-80.4, which is superior to AZD-9496.

The tumor growth inhibition rate of the compound of Example 4 and AZD-9496 against breast cancer MCF-7 tumor-bearing mice at a dose of 10 mg/kg is shown in Figure 1. The inhibition rate of the compound of Example 4 on tumor volume is significantly better than that of AZD- 9496.

Claims (13)

A compound of the formula (I): or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof: Wherein: R ¹ and R ² are each independently selected from a hydrogen atom or a halogen; R ^{3 is} selected from an alkyl group, wherein the alkyl group is further substituted with one or more halogens; and R ⁴ are each independently selected from halogen and C. ₁ -C ₆ alkyl; and n is 1, 2, 3 or 4. The compound of claim 1, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, which is a compound of the formula (II) or a stereoisomer, tautomer thereof or Its pharmaceutically acceptable salt: Wherein: R ¹ , R ² , R ³ , R ⁴ and n are as defined in claim 1. The compound of claim 1 or 2, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are F. The compound of claim 1 or 2, or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ³ is C ₁ -C ₄ alkyl, preferably isopropyl, wherein The alkyl group is further substituted by one or more substituents selected from F, Cl, Br or I, preferably F or Cl, more preferably F. The compound of claim 1 or 2, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: R ¹ and R ² are each independently selected from halogen, wherein said halogen is preferably F. R ^{3 is} selected from a halogen-substituted C ₁ -C ₄ alkyl group, preferably an F-substituted C ₁ -C ₄ alkyl group; more preferably a fluoroisopropyl group. The compound of claim 1 or 2, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: A process for the preparation of a compound of the formula (I) according to claim 1, which comprises: The general formula (IA) and the general formula (IB) are reacted under acidic conditions, and further ester hydrolyzed to obtain a compound of the formula (I); wherein: R ^a is an alkyl group; R ¹ , R ² , R ³ , R ⁴ and n The definition is as described in claim 1. A process for the preparation of a compound of the formula (II) according to claim 2, which comprises: The general formula (IIA) and the general formula (IB) are reacted under acidic conditions, and further ester hydrolyzed to obtain a compound of the formula (II); wherein: R ^a is an alkyl group; R ¹ , R ² , R ³ , R ⁴ and n The definition is as described in claim 1. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 6, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof Carriers, excipients or combinations thereof. The pharmaceutical composition according to claim 9, which further comprises an antioxidant or a metal chelating agent. A compound according to any one of claims 1 to 6, or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 9 to 10, which is prepared Use of a medicament for the treatment of a disease transduced by an estrogen receptor vector, wherein the disease is cancer, wherein the cancer is preferably a breast cancer or a gynecological cancer, wherein the gynecological cancer is preferably ovarian cancer or Endometrial cancer, wherein the estrogen receptor is preferably an estrogen receptor alpha. A compound according to any one of claims 1 to 6, or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 9 to 10, which is prepared Use of a selective estrogen receptor down-regulator, wherein the selective estrogen receptor down-regulator is preferably an estrogen receptor alpha down-regulator. A compound according to any one of claims 1 to 6, or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 9 to 10, Or a combination of a plurality of other anti-tumor drugs for the preparation of a medicament for treating a disease transduced by an estrogen receptor, wherein the disease is cancer, wherein the cancer is preferably breast cancer or gynecological cancer, wherein The gynecological cancer is preferably ovarian cancer or endometrial cancer, wherein the estrogen receptor is preferably an estrogen receptor alpha, wherein the other antitumor drugs include an alkylating agent, an antimetabolite, and have antitumor activity. Natural products and their derivatives, cytotoxic drugs or blocking immune cell migration drugs.