WO2021027567A1

WO2021027567A1 - Combination of ep4 receptor antagonist and pd-1 inhibitor for cancer treatment

Info

Publication number: WO2021027567A1
Application number: PCT/CN2020/105282
Authority: WO
Inventors: 章涵堃; 卢伟强; 易正芳; 刘明耀; 杨俊杰; 于薇薇; 彭世鸿; 周文波
Original assignee: 上海宇耀生物科技有限公司
Priority date: 2019-08-15
Filing date: 2020-07-28
Publication date: 2021-02-18
Also published as: CN112390814B; CN110386941A; CN112390814A

Abstract

Provided is a combination of an EP4 receptor antagonist and a PD-1 inhibitor for treating cancer. Specifically, provided is a product combination comprising: (i) a first pharmaceutical composition comprising (a) a first active ingredient, where the first active ingredient comprises an EP4 receptor antagonist and a pharmaceutically acceptable carrier; and (ii) a second pharmaceutical composition comprising (b) a second active ingredient, where the second active ingredient comprises a PD-1 inhibitor and a pharmaceutically acceptable carrier. The first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions or the same pharmaceutical composition. The product combination of the present invention can synergistically treat malignant tumors.

Description

Combination of EP4 receptor antagonist and PD-1 inhibitor for cancer treatment

Technical field

The present invention relates to the field of biomedicine, in particular to the combined use of EP4 receptor antagonists and PD-1 inhibitors for the treatment of cancer.

Background technique

Cancer has become one of the most terrifying killers threatening human life and health. According to the 2018 Global Cancer Report released by the International Agency for Research on Cancer (IARC), 18.1 million new cancer cases were added globally in 2018, and about 9.6 million people will die from Cancer, and the morbidity and mortality are still rising year by year. According to the report, my country's cancer incidence and death rate are the highest in the world, of which the number of new cases in my country accounts for 3.804 million and the number of deaths accounts for 2.296 million.

Following surgery, radiation therapy and chemotherapy drugs, the development of tumor immunotherapy represented by immune checkpoint inhibitors has promoted revolutionary changes in cancer treatment.

However, the current immune checkpoint inhibitors have many shortcomings in the treatment of cancer, especially malignant tumors, such as low objective response rate to advanced solid tumors (generally, objective remission rate <30%); induce a variety of lethal side effects , Including colitis, neurotoxicity, pneumonia, hepatitis, pituitary inflammation, nephritis, adrenitis and heart disease. These factors cause the most deaths and are the most fatal side effects.

Therefore, there is an urgent need in the art to develop drugs that can effectively treat cancer, especially malignant tumors.

Summary of the invention

The purpose of the present invention is to develop drugs that can effectively treat cancer, especially malignant tumors.

The first aspect of the present invention provides a product portfolio including:

(i) a first pharmaceutical composition, said first pharmaceutical composition containing (a) a first active ingredient, said first active ingredient being an EP4 receptor antagonist, and a pharmaceutically acceptable carrier; and

(ii) A second pharmaceutical composition, the second pharmaceutical composition containing (b) a second active ingredient, the second active ingredient is a PD-1 inhibitor, and a pharmaceutically acceptable carrier;

Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.

In another preferred embodiment, the EP4 receptor antagonist is a compound represented by formula I or a pharmaceutically acceptable salt or hydrate thereof:

among them,

with

Each is independently selected from the following group: C3-C6 carbocyclic ring, C6-C12 aryl group, five-membered or six-membered heteroaromatic ring containing one or more O, N, S atoms, wherein

with

May be optionally substituted by 1-3 R ⁵ groups;

Is a substituted or unsubstituted ring selected from the following group: C4-C7 carbocyclic ring, 4-7 membered saturated heterocyclic ring, benzene ring, 4-7 membered unsaturated heterocyclic ring (including heteroaromatic ring), wherein, the The heterocyclic ring has one or more heteroatoms selected from the following group: O, S or NR ⁶ ; the ring can be a monocyclic, bicyclic, spiro or bridged ring;

X is a group selected from the following group: -O-, -S-, -N(R ⁷ )-;

Y is none, or a group selected from the following group: -CH ₂ -, -O-, -S-, -SO-, -SO ₂ -, -N(R ⁸ )-;

B ₁ and B ₂ are each independently a group selected from the group consisting of none, C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene; preferably, the B1 And B2 are independently selected from the following group: -(CH ₂ ) _n -; wherein n=0, 1, 2, 3 or 4, -CH=CH-, -CH=CH-CH ₂ -, -CH ₂ -CH =CH-, -CH=CH-CH ₂ -CH ₂ -, -CH ₂ -CH=CH-CH ₂ -, -CH ₂ -CH ₂ -CH=CH-; -C≡C-, -C≡C _{_{-CH 2 -, - CH 2 -C≡C}} -, - C≡C-CH 2 -CH 2 -, - CH 2 -C≡C-CH 2 -, - CH 2 -CH 2 -C≡C-; And B ₁ , B ₂ and Y are not all at the same time;

R ¹ is one or more groups selected from the following group: H, C1-C6 alkyl (preferably C1-C4 alkyl), halogen, nitro, -N(R ⁹ )(R ¹⁰ ), -OH , -CN, C1-C6 haloalkyl (preferably C1-C4 haloalkyl, more preferably difluoromethyl, trifluoromethyl), C1-C6 alkoxy (preferably C1-C4 alkoxy, More preferably methoxy, ethoxy), C1-C6 haloalkoxy (preferably C1-C4 haloalkoxy, more preferably difluoromethoxy, trifluoromethoxy), =O;

R ² and R ³ are each independently selected from: H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or R ² , R ³ and the carbon atom to which they are connected together form 3 To a 6-membered ring, the ring is a carbocyclic ring or a 3- to 6-membered heterocyclic ring having 1-3 heteroatoms selected from the group consisting of O, S or N (R ¹¹ );

R ^{4 is} selected from any one of the following groups: -COOR ¹² (preferably -COOH, -COOCH ₃ , -COOCH ₂ CH ₃ , -COOCH ₂ CH ₂ CH ₃ , -COOCH(CH ₃ ) ₂ ), C( O)-N(Ra)(Rb), cyano group, tetrazolium group, phosphoric acid group, sulfonic acid group; wherein, Ra is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted Substituted C1-C6 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, and substituted or unsubstituted C1-C6 alkoxy; Rb is selected from the group consisting of H, -OH, -NH ₂ , substituted or Unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, and substituted or unsubstituted C1-C6 alkoxy;

Each R ⁵ and R ¹² are each independently selected from: H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxy (preferably methoxy, ethyl Oxy);

R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently selected from: H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C3-C6 halo ring Alkyl, C6-C10 aryl, five-membered or six-membered heterocyclic aromatic group,

Unless otherwise specified, one or more hydrogen atoms on the substituent group is substituted by a substituent selected from the following group: F, Cl, Br, I, hydroxyl, methyl, ethyl, isopropyl, methoxy Group, ethoxy, trifluoromethyl, difluoromethoxy, trifluoromethoxy, nitro, -CN, oxo (=O);

R ¹³ and R ¹⁴ are each independently selected from: H, C1-C6 alkyl (preferably methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, pentyl, hexyl), C1- C6 alkoxy, C6-C10 aryl, C1-C6 alkylene, -C6-C10 aryl.

In another preferred embodiment, the formula (I)

Has the structure shown in the following formula:

Wherein, J, K, and L are each independently selected from the following group: -CH ₂ -, -CH(CH ₃ )-, -CH(CH ₂ CH ₃ )-, -C(CH ₃ ) ₂ -,

-O-,

-NR ¹⁶ -;

Wherein, R ¹⁵ and R ¹⁶ are each independently selected from the following group: H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl,

or

Has the structure shown in the following formula:

Wherein, M, N, P, and Q are each independently selected from -CH ₂ -, -CH(CH ₃ )-, -CH(CH ₂ CH ₃ )-, -C(CH ₃ ) ₂ -,

-O-,

-NR ¹⁶ -;

or

Has the structure shown in the following formula:

Wherein, R, S, T, U, and V are each independently selected from the following group: -CH ₂ -, -CH(CH ₃ )-, -CH(CH ₂ CH ₃ )-, -C(CH ₃ ) _2- ,

-O-,

-NR ¹⁶ -.

In another preferred example, M, N, P and Q form a cyclic structure, or between M and Q, between M and P, or between N and Q, form a bridged ring structure composed of 0-4 carbon atoms.

In another preferred example, R, S, T, U and V form a ring structure, or any two of R, S, T, U and V form a bridged ring structure composed of 0-4 carbon atoms .

In another preferred example, X is -S-.

In another preferred example, R ^{4 is} selected from any one of the following groups: -COOR ¹² (preferably -COOH, -COOCH ₃ , -COOCH ₂ CH ₃ , -COOCH ₂ CH ₂ CH ₃ , -COOCH (CH ₃ ) ₂ ), C(O)-N(Ra)(Rb), cyano group, tetrazolium group, phosphoric acid group, sulfonic acid group; wherein, Ra is selected from the following group: H, substituted or unsubstituted C1- C4 alkyl, substituted or unsubstituted C1-C4 haloalkyl, substituted or unsubstituted C3-C5 cycloalkyl, and substituted or unsubstituted C1-C4 alkoxy; Rb is selected from the following group: H, -OH , -NH ₂ , substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 haloalkyl, substituted or unsubstituted C3-C5 cycloalkyl, and substituted or unsubstituted C1-C4 alkoxy base;

In another preferred example,

with

Each is independently selected from the following group: C3-C6 carbocyclic ring, C6-C10 aromatic ring, five-membered or six-membered heteroaromatic ring containing one or more O, N, S atoms, wherein

with

May be optionally substituted with 1-3 R ⁵ groups.

In another preferred embodiment, the halogen includes F, Cl, Br or I. In another preferred example,

with

Each is independently selected from the following group: benzene ring, ten-membered aromatic ring, five-membered or six-membered heteroaromatic ring containing one or more O, N, S atoms, wherein

with

May be optionally substituted with 1-3 R ⁵ groups.

In another preferred example,

It is a substituted or unsubstituted C5-C7 carbocyclic ring, or a substituted or unsubstituted 6-membered oxygen heterocyclic ring.

In another preferred embodiment, the EP4 receptor antagonist is selected from the following group:

(S)-4-(1-(2-((4-fluorophenyl)ethynyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide) Ethyl) benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,6-dihydro-4H-cyclopenta(b)thiophene-3-carboxamide)ethyl )benzoic acid;

4-((1S)-1-(6-ethyl-2-((4-fluorophenyl)ethynyl)-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxamide Yl)ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,6,7,8-tetrahydro-4H-cycloheptano(b)thiophene-3-carboxamide )Ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c] Pyran-3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,6,7,8-tetrahydro-4H-cycloheptano(b)thiophen-3-carboxamide)ethyl )benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,6-dihydro-4H-cyclopenta(b)thiophene-3-carboxamide)ethyl)benzoic acid ；

(S)-4-(1-(2-(4-Fluorophenethyl)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran- 3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-(Trifluoromethyl)phenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-methyl (Amide) ethyl) benzoic acid;

(S)-4-(1-(2-(3-(Trifluoromethyl)phenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-methyl (Amide) ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluorophenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Formic acid

(S)-4-(1-(2-(4-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(S)-4-(1-(2-(3-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(R)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Formic acid

(R)-4-(1-(2-(4-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(R)-4-(1-(2-(3-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

4-((2-(4-fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzoic acid;

(S)-4-(1-(2-((4-(Trifluoromethyl)benzyl)amino)-5,7-dihydro-4H-thieno[2,3-c]pyran-3 -Formamide) ethyl) benzoic acid;

(S)-4-(1-(2-(4-methoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(4-chlorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluoro-4-methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3-chlorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(3,4-Difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(4-methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl基)benzoic acid;

((2-(3-(Trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzoic acid;

4-((2-(4-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamide)methyl)cyclohexane-1-carboxy Acid (racemate);

(1-(2-(4-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)cyclopropyl)benzoic acid;

(S)-4-(1-(2-(3,5-Difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(3-Methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl基)benzoic acid;

3-((2-(3-(Trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzene Formic acid

4-((2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl) ring Hexane-1-carboxylic acid;

4-(1-(2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)cyclopropane基)benzoic acid;

(S)-4-(1-(2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(2-((6-methoxynaphthalen-2-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran- 3-formylamino)ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-6,7-dihydro-4H-thieno[3,2-c]pyran-3-carboxamido )Ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-6,7-dihydro-4H-thieno[3,2-c]pyran-3-carboxamido)ethyl )benzoic acid;

(S)-4-(1-(2-((3,5-Dimethoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran- 3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(2-((3-methoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3,5-Dimethoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3-methoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(2-(6-methoxynaphthalen-2-yl)ethyl)-4,7-dihydro-5H-thieno[2,3-c]pyridine Pyran-3-formylamino)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Methyl formate

(S)-2-(4-Fluorobenzyl)-N-(1-(4-(methoxyamino)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamide;

(S)-4-(1-(2-(4-methylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-ethylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl )benzoic acid;

(S)-2-(4-fluorobenzyl)-N-(1-(4-(methoxy(methyl)carbamoyl)phenyl)ethyl)-4,7-dihydro-5H- Thieno[2,3-C]pyran-3-carboxamide;

(S)-2-(4-Fluorobenzyl)-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamide;

(S)-N-(1-(4-cyanophenyl)ethyl)-2-(4-fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyridine Pyran-3-carboxamide;

(S)-2-(4-fluorobenzyl)-N-(1-(4-(hydrazinecarbonyl)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3-c ]Pyran-3-carboxamide;

(S)-4-(1-(6-(tert-butoxycarbonyl)-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[2,3-c] (Pyridine-3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(6-Acetyl-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-methyl Amido)ethyl)benzoic acid.

In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.

In another preferred example, the weight ratio of the component (i) to the component (ii) is 1-200:1-100, preferably, 1-100:1-50, more preferably 10- 50: 1-10.

In another preferred embodiment, the content of the EP4 receptor antagonist in the product combination is 1%-99%, preferably, 10%-90%, more preferably, 50%-90%.

In another preferred example, the content of the PD-1 inhibitor in the product combination is 1% to 99%, preferably, 1% to 60%, more preferably, 1% to 30%.

In another preferred example, in the product combination, the component (i) and the component (ii) account for 0.01-99.99 wt% of the total weight of the product combination, preferably 0.1-90 wt%, more preferably Ground 1-80wt%.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection dosage form and an oral dosage form.

In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

The second aspect of the present invention provides a composition, which comprises:

(i) EP4 receptor antagonist;

(ii) PD-1 inhibitor; and

(iii) A pharmaceutically acceptable carrier.

In another preferred example, in the composition, the component (i) and component (ii) account for 0.01-99.99 wt% of the total weight of the kit, preferably 0.1-90 wt%, more preferably Ground 1-80wt%.

In another preferred embodiment, the composition also includes other drugs for treating malignant tumors.

In another preferred embodiment, the malignant tumor is selected from the group consisting of liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, leukemia, lymphoma, ovarian cancer, stomach cancer, bladder cancer, and kidney cancer , Oral cancer, melanoma, esophageal cancer, lymphoma, cervical cancer, or a combination thereof.

In another preferred example, the malignant tumor highly expresses PD-1.

In another preferred embodiment, the malignant tumor has a low expression of PD-1.

In another preferred example, the malignant tumor highly expresses PD-L1.

In another preferred embodiment, the malignant tumor has low PD-L1 expression.

In another preferred embodiment, the other drugs for the treatment of malignant tumors are selected from the group consisting of CTLA4 antibody, PD-L1 antibody, nimustine, carmustine, cyclic ammonium phosphate, mustard, and deoxyfluuria Glycosides, 5-fluorouracil, 6-mercaptopurine, thioguanine, cytarabine, gemcitabine, carmofur, hydroxyurea, methotrexate, ufodine, amcitabine, actinomycin D, doxorubicin Bicin, daunorubicin, epirubicin, mitomycin, irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, taxotere, topotecan, vincristine, Niposide, Etoposide, Atamestane, Anastrozole, Amluminide, Letrozole, Formestane, Metasterone, Carboplatin, Cisplatin, Dacarbazine, Oxaliplatin, Le Sadine, coplatin oxa, mitoxantrone, procarbazine, gefitinib, erlotinib, cetuximab, Herceptin, imatinib, rituximab, voribrate Nota, Ceritinib, Crizotinib, Icotinib, Sorafenib, Dacomitinib, Apatinib, Sunitinib, Abxixib, Bevacizumab, West Tuximab, Panitumumab, Regotinib, or a combination thereof.

The third aspect of the present invention provides a medicine kit including:

(a1) The first container, and the EP4 receptor antagonist in the first container, or a drug containing the EP4 receptor antagonist;

(b1) The second container, and the PD-1 inhibitor in the second container, or the drug containing the PD-1 inhibitor.

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, the medicine in the first container is a single preparation containing an EP4 receptor antagonist.

In another preferred embodiment, the medicine in the second container is a unilateral preparation containing a PD-1 inhibitor.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit also contains instructions.

In another preferred example, the description records one or more descriptions selected from the following group:

(a) Combination of EP4 receptor antagonists and PD-1 inhibitors to synergistically treat malignant tumors;

(b) EP4 receptor antagonists have the function of relieving chronic inflammation and pain mediated by the PGE2-EP4 signaling pathway.

The fourth aspect of the present invention provides the use of a combination comprising an EP4 receptor antagonist and a PD-1 inhibitor for preparing a pharmaceutical composition or a kit, and the pharmaceutical composition or a kit is used for Treat malignant tumors.

In another preferred example, the malignant tumor highly expresses PD-1.

In another preferred example, the malignant tumor highly expresses PD-L1.

In another preferred embodiment, the malignant tumor has low PD-L1 expression.

In another preferred embodiment, the action concentration of the EP4 receptor antagonist is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

In another preferred example, the concentration of the PD-1 inhibitor is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

In another preferred embodiment, the pharmaceutical composition or kit includes (a) an EP4 receptor antagonist and a PD-1 inhibitor; and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, in the pharmaceutical composition or kit, the EP4 receptor antagonist and PD-1 inhibitor account for 0.01-99.99 wt% of the total weight of the pharmaceutical composition or kit, Preferably 0.1-90wt%, more preferably 1-80wt%.

In another preferred embodiment, the pharmaceutical composition or kit further includes other drugs for treating malignant tumors.

In another preferred embodiment, the other drugs for the treatment of malignant tumors are selected from the group consisting of nimustine, carmustine, cyclic ammonium phosphate, mustard, deoxyfluridine, 5-fluorouracil, 6- Mercaptopurine, thioguanine, cytarabine, gemcitabine, carmofur, hydroxyurea, methotrexate, ufodine, amcitabine, actinomycin D, doxorubicin, daunorubicin, Epirubicin, Mitomycin, Irinotecan, Harringtonine, Hydrocamptothecin, Vinorelbine, Taxotere, Topotecan, Vincristine, Teniposide, Etoposide, Atamestane, Anastrozole, Aluminide, Letrozole, Formestane, Metasterone, Carboplatin, Cisplatin, Dacarbazine, Oxaliplatin, Loxadine, Coplatin, Mitoxantrone, Procarbazine, Gefitinib, Erlotinib, Cetuximab, Herceptin, Imatinib, Rituximab, Vorinostat, Ceritinib, Crizotinib, icotinib, sorafenib, dacomitinib, apatinib, sunitinib, abaxixib, bevacizumab, cetuximab, panitumumab Anti-, regrotinib, or a combination thereof.

The fifth aspect of the present invention provides a method for treating malignant tumors, including:

Administration of EP4 receptor antagonists and PD-1 inhibitors, or the product combination according to the first aspect of the present invention or the composition according to the second aspect of the present invention or the kit according to the third aspect of the present invention to a subject in need .

In another preferred example, the subject includes a human or non-human mammal suffering from a malignant tumor.

In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

In another preferred embodiment, the administration dose of the EP4 receptor antagonist is 1-250 mg/kg body weight, preferably 1-200 mg/kg body weight, and most preferably 1-100 mg/kg body weight.

In another preferred example, the administration dose of the PD-1 inhibitor is 0.1-100 mg/kg body weight, preferably 1-50 mg/kg body weight, and most preferably 1-10 mg/kg body weight.

In another preferred embodiment, the application frequency of the EP4 receptor antagonist is 1 to 5 times/day, preferably 1 to 2 times/day.

In another preferred embodiment, the administration time of the EP4 receptor antagonist is 1 to 2000 days, preferably 1 to 700 days, and most preferably 1 to 365 days.

In another preferred embodiment, the frequency of administration of the PD-1 inhibitor is 0.1-4 per week per time, preferably once every two weeks.

In another preferred example, the administration time of the PD-1 inhibitor is 1-2000 days, preferably 1-700 days, and most preferably 1-365 days.

In another preferred embodiment, the EP4 receptor antagonist and PD-1 inhibitor are administered simultaneously or sequentially.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the inhibitory results of compound YJ114 and PD-1 in combination with a mouse CT26 colon cancer tumor model:

A represents the volume change of subcutaneous tumor bearing mice; B represents the weight change of mice; C represents the tumor weight distribution on the 20th day; D represents the survival period of mice two weeks after administration.

Figure 2 shows the inhibitory results of compound YJ114 and PD-1 in combination with mouse MC38 colon cancer tumor model:

A represents the volume change of subcutaneous tumor bearing mice; B represents the weight change of mice; C represents the tumor weight distribution on the 20th day.

Figure 3 shows the inhibitory results of compound YJ114 and PD-1 in combination with AOM/DSS model induced mouse orthotopic colon cancer tumor model:

A represents the construction of mouse orthotopic tumor model and dosing plan; B represents the distribution and size of tumors in the colon; C represents the distribution of the number of tumors in each mouse; D represents the length of the colorectal.

Figure 4 shows the inhibitory results of compound YJ114 and PD-1 in combination with mouse RM-1 prostate cancer model:

A represents the volume change of subcutaneous tumor bearing mice; B represents the tumor size distribution; C represents the tumor weight distribution on day 17; D represents the weight change of mice.

Figure 5 shows the inhibitory effect of compound YJ114 in combination with PD-1 on mouse MFC gastric cancer model:

A represents the change of subcutaneous tumor-bearing mouse volume; B represents the tumor elimination rate; C represents the mouse survival curve obtained by using the tumor volume exceeding 2000 mm ³ as the mouse death criterion; D represents the weight change of the mouse.

detailed description

Through extensive and in-depth research and screening a large number of compounds, the present inventors unexpectedly discovered for the first time that the combination of the EP4 receptor antagonist and PD-1 inhibitor of the present invention can effectively treat malignant tumors and has a synergistic effect. On this basis, the inventor completed the present invention.

Group definition

In the present invention, the term "C3-C6 carbocyclic ring" or the term "C4-C7 carbocyclic ring" refers to a saturated or unsaturated ring composed of 3 to 6 carbon atoms or 4 to 7 carbon atoms, including monocyclic rings , Bicyclic, spiro or bridged ring, such as 6-membered alicyclic ring.

As used herein, the term "C6-C12 aromatic ring" refers to a monovalent aromatic carbocyclic group of 6 to 12 carbon atoms, which has a single ring (such as phenyl) or a condensed ring (such as naphthyl or anthracenyl) if The connection point is on the aromatic carbon atom, and the fused ring may be non-aromatic (such as 2-benzoxazolone, 2H-1,4-benzoxazine-3(4H)-keto-7-yl, etc.). The substituted or unsubstituted C6-C12 aromatic ring is selected from the following group: ortho-substituted phenyl, meta-substituted phenyl, and para-substituted phenyl. Preferred aryl groups include phenyl and naphthyl. The term includes substituted or unsubstituted forms, where the substituents are as defined above. The substituent of the substituted phenyl group is selected from the following group: halogen, hydroxy, methyl, ethyl, isopropyl, tert-butyl, methoxy, ethoxy, tert-butoxy, trifluoromethyl, or Its combination. As used herein, the term "C6-C10 aromatic ring" refers to a monovalent aromatic carbocyclic group of 6 to 10 carbon atoms, which has a single ring (such as phenyl) or a condensed ring (such as naphthyl or anthracenyl) if The connection point is on the aromatic carbon atom, and the fused ring may be non-aromatic (such as 2-benzoxazolone, 2H-1,4-benzoxazine-3(4H)-keto-7-yl, etc.). The substituted or unsubstituted C6-C10 aromatic ring is selected from the following group: ortho-substituted phenyl, meta-substituted phenyl, and para-substituted phenyl. Preferred aryl groups include phenyl and naphthyl. The term includes substituted or unsubstituted forms, where the substituents are as defined above. The substituent of the substituted phenyl group is selected from the following group: halogen, hydroxy, methyl, ethyl, isopropyl, tert-butyl, methoxy, ethoxy, tert-butoxy, trifluoromethyl, or Its combination.

The term "five- or six-membered heteroaromatic ring" refers to a 5- to 6-membered aromatic ring having one or more heteroatoms selected from nitrogen, oxygen or sulfur, for example, pyridine, pyrimidine, thiazole, isothiazole, furan , Thiophene, pyrrole.

The term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, including, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl , Sec-butyl, tert-butyl, pentyl and hexyl groups; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

The term "C1-C4 alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms, including, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl , Sec-butyl, tert-butyl, etc.

The term "C1-C6 alkylene" refers to a linear or branched alkylene group having 1 to 6 carbon atoms, including but not limited to methylene, ethylene, propylene, isopropylene, Butylene, isobutylene, sec-butylene, tert-butylene, pentylene and hexylene, etc.; preferably ethylene, propylene, isopropylene, butylene, isobutylene, ethylene Sec-butyl and tert-butylene.

The term "C2-C6 alkenylene" refers to a straight or branched chain alkenylene group having 2 to 6 carbon atoms containing a double bond, including but not limited to vinylene, propenylene, butenylene , Isobutenylene, pentenylene and hexenylene, etc.

The term "C2-C6 alkynylene" refers to a straight-chain or branched alkynylene group with 2 to 6 carbon atoms containing a triple bond, and includes, without limitation, ethynylene, propynylene, butynylene Group, isobutynylene, pentynylene and hexynylene, etc.

The term "C ₃ -C ₆ cycloalkyl" refers to a cyclic alkyl group having 3 to 6 carbon atoms in the ring, and includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "C ₃ -C ₅ cycloalkyl" refers to a cyclic alkyl group having 3 to 5 carbon atoms in the ring, and includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "C6-C10 aryl group" refers to an aromatic ring group having 6 to 10 carbon atoms that does not contain a hetero atom in the ring, such as a phenyl group and the like.

The terms "C1-C6 haloalkyl" and "C1-6 halocycloalkyl" refer to a group in which the hydrogen on an alkyl group or a cycloalkyl group is replaced by one or more halogen atoms, and includes, without limitation, -CHF ₂ , Chlorocyclopropyl, etc.

The terms "C1-C4 haloalkyl" and "C1-4 halocycloalkyl" refer to a group in which the hydrogen on an alkyl group or a cycloalkyl group is replaced by one or more halogen atoms, including without limitation -CHF ₂ , Chlorocyclopropyl, etc.

The term "C1-C6 alkoxy group" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, and includes, without limitation, methoxy, ethoxy, propoxy, and isopropoxy And butoxy and so on.

The term "C1-C4 alkoxy group" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, and isopropoxy And butoxy and so on.

The term "halogenated C1-C6 alkoxy group" refers to a group in which the hydrogen on the alkoxy group is replaced by one or more halogen atoms.

The term "halogenated C1-C4 alkoxy group" refers to a group in which the hydrogen on the alkoxy group is replaced by one or more halogen atoms.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

The compounds of the present invention may contain one or more asymmetric centers, and therefore appear as racemates, racemic mixtures, single enantiomers, diastereomeric compounds, and single diastereomers. The asymmetric centers that can exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible mixtures of optical isomers and diastereomers and pure or partially pure compounds are included within the scope of the present invention. The present invention includes all isomeric forms of the compounds.

PD-1/PD-L1 signaling pathway

The PD-1/PD-L1 signaling pathway is a key signaling pathway that negatively regulates T cell activation. When tumor killer T cells infiltrate the tumor tissue, the activated T cells can secrete interferon-γ (TNF-γ) and other cells These cytokines induce PD-L1 expression on macrophages, T, B cells and tumor cells through a series of signal transduction processes, and then interact with PD-1 receptors on T cells to cause T cell Inactivation and apoptosis promote the occurrence and development of tumor cells. PD-1/PD-L1 pathway inhibitors can block the binding of PD-1 and PD-L1 and block negative regulatory signals, thereby restoring the killing ability of tumor-killing T cells, thereby enhancing the immune response to tumor cells.

Prostaglandin E2 (Prostaglandin E ₂ , PGE ₂ )

Prostaglandin E2 (Prostaglandin E ₂ , PGE ₂ ) is the most abundant and widely distributed prostaglandin subtype in the human body. It participates in the regulation of many physiological and pathological processes including inflammation, pain, kidney function, cardiovascular system, lung function, and cancer. PGE ₂ binds to four different subtypes of G protein-coupled receptors EP1, EP2, EP3, EP4 (also known as PTGER1, PTGER2, PTGER2 and PTGER4) on the cell surface in an autocrine or paracrine manner, and these G Protein coupled receptor subtypes exert their biological effects by coupling different G proteins to activate different downstream signaling pathways. Although both EP2 and EP4 receptor subtypes mediate downstream signals by coupling with G _s protein, their amino acid sequences have only 31% homology and there are certain structural differences, so their main physiological functions are similar. Place, but there are also many differences.

Active ingredient

As used herein, the "EP4 receptor antagonist", "active ingredient of the present invention", and "compound of formula I" are used interchangeably, and all refer to those that can be used in combination with PD-1 inhibitors to synergistically treat malignant tumors. Effective compound

among them,

with

May be optionally substituted by 1-3 R ⁵ groups;

X is a group selected from the following group: -O-, -S-, -N(R ⁷ )-;

R ¹ is one or more groups selected from the following group: H, C1-C6 alkyl (preferably C1-C4 alkyl), halogen, nitro, -N(R ⁹ )(R ¹⁰ ), -OH , -CN, C1-C6 haloalkyl (preferably C1-C4 haloalkyl, more preferably difluoromethyl, trifluoromethyl), C1-C6 alkoxy (C1-C4 alkoxy, more preferably Methoxy, ethoxy), C1-C6 haloalkoxy (preferably C1-C4 haloalkoxy, more preferably difluoromethoxy, trifluoromethoxy), =O;

In a preferred embodiment, the compound of the present invention is selected from the following group:

A particularly preferred class of compounds of formula I is selected from the following group:

YJ114, YJ115, YJ116, YJ120, YJ122, YJ123, YJ124, YJ125, YJ126, YJ127, YJ128, YJ129, YJ130, YJ131.

In the present invention, a pharmaceutically acceptable salt of the compound of formula I is also included. The term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention and an acid or a base suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are the salts of the compounds of this invention with acids. Acids suitable for salt formation include but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

Composition and method of application

As used herein, the term "composition" includes (a1) a first active ingredient, the first active ingredient being an EP4 receptor antagonist; and (a2) a second active ingredient, the second active ingredient being PD-1 Inhibitor; and (b) a pharmaceutically acceptable carrier. In addition, the composition includes a pharmaceutical composition, a food composition or a health care product composition.

Generally, the active ingredient of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective dosage, for example, about 1 microgram-10 mg/kg body weight per day. Preferably, the dosage of the EP4 receptor antagonist can be: 0.1-2000 mg per day for adults, preferably 1-300 mg/day . The dosage of PD-1 inhibitor may be 0.1-2000 mg every two weeks for adults, preferably 1-300 mg/two weeks. As a co-treatment drug for malignant tumors, it can be made into oral and non-oral preparations. Oral administration can be made into common dosage forms such as tablets, powders, granules, capsules, etc. The excipients used can be one or more of starch, lactose, sucrose, mannose, and hydroxymethyl cellulose. The disintegrant can be one or more of potato starch and hydroxymethyl cellulose. The binder can be one or more of gum arabic, corn starch, gelatin, dextrin and the like. In addition to the above dosage forms, oral preparations can also be made into emulsions, syrups and the like.

Non-oral preparations can be made into injections, which can be made into injections with water for injection, physiological saline, and glucose water, or a certain proportion of ethanol, propanol, ethylene glycol, etc. can be added to it. In addition, it can also be made into common dosage forms such as nasal drops, sprays, rectal suppositories, and rectal retention enema.

In addition, the active ingredient of the present invention is also particularly suitable for other malignant tumor treatment drugs (such as nimustine, carmustine, cyclic ammonium phosphate, mustard, deoxyfluridine, 5-fluorouracil, 6-mercapto Purine, thioguanine, cytarabine, gemcitabine, carmofur, hydroxyurea, methotrexate, ufodine, amcitabine, actinomycin D, doxorubicin, daunorubicin, table Rubicin, mitomycin, irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, taxotere, topotecan, vincristine, teniposide, etoposide, a Tametan, anastrozole, aminoglutamin, letrozole, formestane, metgestrol, carboplatin, cisplatin, dacarbazine, oxaliplatin, leroxadine, coplatin oxa, rice Toxantrone, Procarbazine, Gefitinib, Erlotinib, Cetuximab, Herceptin, Imatinib, Rituximab, Vorinostat, Ceritinib, Gram Zoltinib, Icotinib, Sorafenib, Dacomitinib, Apatinib, Sunitinib, Abxixib, Bevacizumab, Cetuximab, Panitumumab , Regotinib, etc.) to more effectively inhibit malignant tumors.

Pill box

The present invention also provides a medicine box, which contains:

In a preferred embodiment, the first container and the second container are the same or different containers.

The preparation containing an EP4 receptor antagonist may be a unit dosage form containing an EP4 receptor antagonist, and the preparation containing a PD-1 inhibitor may be a unit dosage form containing a PD-1 inhibitor.

As used herein, the term "unit dosage form" refers to the preparation of a composition into a dosage form required for single administration for the convenience of taking, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained release Agent.

In another preferred embodiment, the instructions describe the following method of use:

(I) Simultaneously administer a preparation containing an EP4 receptor antagonist and a preparation containing a PD-1 inhibitor to a subject in need; and optionally

(II) Repeat steps (I)-(II).

The preparation of the present invention can be taken three times a day to once every twenty days, or once every ten days in a sustained-release manner. The preferred way is to take it once a day, because it facilitates the patient's adherence, thereby significantly improving the patient's compliance with medication.

When taking, the total daily dose in most cases should be lower (or equal to or slightly greater than) the daily daily dose of each single drug in most cases. Of course, the effective dose of the active ingredient used can vary depending on the mode of administration and the expected dose. The severity of the disease to be treated will vary.

The main advantages of the present invention include:

The present invention finds for the first time that the combined use of the EP4 receptor antagonist of the present invention and PD-1 antibody has a significantly better inhibitory effect on malignant tumor models than that of PD-1 in the single-agent group. It was detected in multiple mouse tumor models The PD-1 single-agent group can inhibit tumor growth to a certain extent, but with the extension of the treatment time, the tumor volume continues to increase, while the tumor volume in the combined drug group gradually shrinks or even subsides, achieving complete remission and greatly prolonging the tumor. The survival period of the mouse has no effect on the weight of the mouse, indicating that the combination of the EP4 receptor antagonist and PD-1 inhibitor of the present invention has achieved a significant synergistic anti-tumor effect, and has no obvious toxic and side effects on the mouse.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, the materials and reagents in the specification of the present invention are all commercially available products.

For the preparation process of the EP4 receptor antagonist YJ101-YJ131 of the present invention, see Chinese Patent Application CN201711206672.8. The preparation process of EP4 receptor antagonist YJ132-YJ155 is further illustrated in conjunction with the examples.

The preparation process of an exemplary compound YJ132-YJ155 of formula I is shown in Table 1 below:

Table 1

Example 1-1, (S)-4-(1-(2-(3,5-difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran- Preparation of 3-carboxamido) ethyl) benzoic acid (YJ132)

Tetrahydro-4H-pyran-4-one (2.00g, 20.0mmol), ethyl cyanoacetate (2.50g, 22.0mmol) and sulfur (704mg, 22.0mmol) were dissolved in 30.0mL ethanol, and then added to the solution Morpholine (1.74g, 20.0mmol) was added to it, and the mixture was stirred overnight at 50°C. The reaction was detected by TLC. After the reaction, the reaction solution was extracted with ethyl acetate and water, the upper organic phase was evaporated to dryness, and column chromatography was purified to obtain a pale yellow solid, namely 2-amino-5,7-dihydro-4H -Thieno[2,3-c]pyran-3-carboxylic acid ethyl ester (4.29 g, yield 94%). Dissolve 2-amino-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid ethyl ester (900mg, 4.0mmol) in 1.5M HCl (20.0mL) at room temperature After stirring for 20 min under ice bath conditions, the temperature was lowered to 0° C. NaNO ₂ (414 mg, 6.0 mmol) was added to the solution, and the reaction solution was stirred under ice bath conditions for 30 min. Then KI (1.66g, 10.0mmol) was added to the reaction solution in portions, and the reaction was continued at 0°C for 45 minutes. After the completion of the reaction, the reaction solution was extracted with ethyl acetate and water, the organic phase was evaporated to dryness, and column chromatography was purified to obtain a pale yellow solid, namely 2-iodo-5,7-dihydro-4H-thieno[2,3- c] Ethyl pyran-3-carboxylate (324 mg, yield 24%). A 1.6M hexane solution of n-butyllithium was added to 10.0mL of ether at a temperature of -78°C, and then 2-iodo-5,7-dihydro-4H-thieno[2,3-c ] Ethyl pyran-3-carboxylate (200mg, 0.62mmol) was added to the solution in portions at -78℃, keeping the temperature constant, stirring for 1.5h, and then adding 3,5-difluorobenzaldehyde (97mg, 0.68mmol) was added dropwise to the solution, then stirred at -78°C for 1h, then heated to 0°C, and continued to stir at 0°C for 1h. After the completion of the reaction, 10.0 mL of saturated aqueous ammonium chloride solution was added to the reaction solution, and then extracted with ethyl acetate, and the upper organic phase was evaporated to dryness. The column chromatography was purified to obtain a pale yellow oily liquid, and then the obtained product was heated at 0°C. Add to 10.0mL of dichloromethane to dissolve, keep the temperature constant, then quickly add triethylsilane (0.41mL, 2.48mmol) to the solution, then add trifluoroacetic acid (0.47mL, 6.20mmol) dropwise Into the solution. After the reaction solution was stirred and reacted at 0°C for 30 minutes, the solvent was evaporated to dryness, then dissolved with chloroform, and washed with 5% NaHCO ₃ aqueous solution. Finally, the organic phase was taken, washed with saturated NaCl solution and evaporated to dryness, and purified by column chromatography to obtain a white solid, namely 2-(3,5-difluorobenzyl)-4,7-dihydro-5H-thieno[2, 3-c] Ethyl pyran-3-carboxylate (188 mg, 91% yield in two steps). Ethyl 2-(3,5-difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylate (85mg, 0.25mmol), 3.0mL THF, 3.0mL methanol, 1.0mL water and lithium hydroxide monohydrate (21mg, 0.5mmol) were mixed together, and the reaction was stirred at 68°C for 3h. After the reaction was completed, the reaction solution was adjusted to acidic with 2M HCl, and then used Extract with ethyl acetate and water, evaporate the organic phase, and purify by column chromatography to obtain a white solid, namely 2-(3,5-difluorobenzyl)-4,7-dihydro-5H-thieno[2,3- c] Pyran-3-carboxylic acid (72 mg, yield 96%). The 2-(3,5-difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid (68mg, 0.22mmol), (S)- Methyl 4-(1-aminoethyl)benzoate (48mg, 0.26mmol), HATU (137mg, 0.36mmol) and DIEA (65mg, 0.50mmol) were dissolved in 2.0mL DMF and stirred at room temperature for 6h. After the reaction was completed The reaction solution was extracted with ethyl acetate and water, the upper organic phase was evaporated to dryness, and purified by column chromatography to obtain a white solid, namely (S)-4-(1-(2-(3,5-difluorobenzyl) -5,7-Dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzoic acid methyl ester (65 mg, yield 62%). (S)-4-(1-(2-(3,5-Difluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide) Ethyl) methyl benzoate (65mg, 0.13mmol) was dissolved in a solution consisting of 3.0mL THF, 3.0mL methanol, 1.0mL water, and then lithium hydroxide monohydrate (10mg, 0.24mmol) was added, and the reaction solution was heated at 68 The reaction was stirred at ℃ for 3h. After the reaction, the reaction solution was adjusted to acidic with 2M HCl, extracted with water and ethyl acetate, the organic phase was evaporated to dryness, and column chromatography was purified to obtain a white solid, which is the final product YJ132 (38mg, yield Rate 61%). 1H NMR(500MHz,DMSO-d ₆ )δ12.86(s,1H),8.66(d,J=7.4Hz,1H),7.90(d,J=7.3Hz,2H),7.80–7.53(m,1H ),7.47(d,J=7.5Hz,2H), 7.31–7.09(m,1H), 7.03–6.86(m,1H), 5.24–5.10(m,1H), 4.66(d,J=15.6Hz, 2H), 4.16-3.91 (m, 2H), 3.80 (d, J = 31.1Hz, 2H), 2.63 (s, 2H), 1.41 (d, 3H).

Example 1-2, (S)-4-(1-(2-(3-methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3 -Carboxamido) ethyl) benzoic acid (YJ133) preparation

Using the same reaction route as the preparation of compound YJ132, replacing 3,5-difluorobenzaldehyde with 3-methoxybenzaldehyde, compound YJ133 was finally obtained (the yield of the last step was 90%). ¹ H NMR(500MHz,DMSO-d ₆ )δ12.84(s,1H), 8.75(d,J=8.0Hz,1H), 7.94–7.85(m,2H), 7.51–7.43(m,2H), 7.20–7.11(m,1H), 6.80–6.67(m,3H), 5.17(p,J=7.1Hz,1H), 4.63(s,2H), 3.86–3.78(m,2H), 3.69(s, 3H), 2.62(s, 2H), 1.43(d, J=7.1Hz, 3H).

Example 1-3, 3-((2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamide (Yl)methyl)benzoic acid (YJ134) preparation

Using the same reaction route as the preparation of compound YJ132, 3,5-difluorobenzaldehyde was replaced with 3-trifluoromethylbenzaldehyde, and (S)-4-(1-aminoethyl)benzoic acid methyl ester was replaced with Methyl 3-(aminomethyl)benzoate finally yielded compound YJ134 (the yield of the last step was 92%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.94 (s, 1H), 8.85 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.61 (s, 1H), 7.69-7.32 ( m,6H), 4.73(s,2H), 4.51--4.41(m,2H), 4.31(s,2H), 3.81--3.70(m,2H), 2.62(s,2H).

Example 1-4, 4-((2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamide (Yl)methyl)cyclohexane-1-carboxylic acid (YJ135)

Using the same reaction route as the preparation of compound YJ134, the 3-(aminomethyl)benzoic acid methyl ester was replaced with 4-aminomethyl-cyclohexanecarboxylic acid methyl ester, and finally compound YJ135 was obtained (the yield of the last reaction was 89 %). ¹ H NMR(500MHz,DMSO-d ₆ )δ12.20(s,1H), 8.20–8.13(m,1H), 7.61(s,1H), 7.58(d,J=6.6Hz,1H), 7.56– 7.51 (m, 2H), 4.64 (s, 2H), 4.27 (s, 2H), 3.83 (t, J = 5.5 Hz, 2H), 3.05 (t, J = 6.2 Hz, 2H), 2.63 (d, J = 6.0Hz, 2H), 1.90-1.83 (m, 2H), 1.77-1.68 (m, 2H), 1.29-1.17 (m, 4H), 0.97-0.89 (m, 2H).

Example 1-5, 4-(1-(2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3- Preparation of carboxamido)cyclopropyl)benzoic acid (YJ136)

Using the same reaction route as the preparation of compound YJ134, replace methyl 3-(aminomethyl)benzoate with methyl 4-(1-aminocyclopropyl)benzoate to finally obtain compound YJ136 (the yield of the last step of the reaction) Is 95%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.77 (s, 1H), 8.97 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.60 (s, 2H), 7.58-7.50 ( m,2H),7.23(d,J=8.1Hz,2H),4.66(s,2H),4.31(s,2H),3.88–3.82(m,2H),2.69(s,2H),1.33–1.26 (m,4H).

Example 1-6, (S)-4-(1-(2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H- Preparation of Thieno[2,3-c]pyran-3-carboxamido)ethyl)benzoic acid (YJ137)

Take 2-iodo-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxylic acid ethyl ester (160mg, 0.48mmol), 10% Pd/C (5mg, 0.048mmol) ), PPh ₃ (5mg, 0.02mmol), CuI (9mg, 0.048mmol) and triethylamine (0.13mL, 0.93mmol) were added to 10.0mL of ethanol, the mixture was stirred under nitrogen protection for 15min, and then added to the reaction solution 5-acetylene-2(1H)-pyridone (85mg, 0.71mmol) was added to the mixture, and the reaction was stirred for 12h at 60℃. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, the organic phase was evaporated and the column was used Purified by chromatography to obtain a white solid, namely 2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3 -c] Ethyl pyran-3-carboxylate (80 mg, yield 51%). Add 2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxy Ethyl acid (80mg, 0.25mmol), 3.0mL THF, 3.0mL methanol, 1.0mL water and lithium hydroxide monohydrate (21mg, 0.5mmol) were mixed together, stirred at 68℃ for 3h, and used after the reaction The reaction solution was adjusted to acidic with 2M HCl, and then extracted with ethyl acetate and water. The organic phase was evaporated to dryness and purified by column chromatography to obtain a white solid, namely 2-((6-oxo-1,6-dihydropyridine-3) -Yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid (70 mg, yield 93%). Add 2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxy Acid (70mg, 0.23mmol), (S)-4-(1-aminoethyl) methyl benzoate (48mg, 0.26mmol), HATU (137mg, 0.36mmol) and DIEA (65mg, 0.50mmol) were dissolved in 2.0 In mL DMF, stirred at room temperature for 6 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate and water. The upper organic phase was evaporated to dryness and purified by column chromatography to obtain a white solid, namely (S)-4-(1-(2) -((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido ) Ethyl) methyl benzoate (59 mg, yield 55%). Take (S)-4-(1-(2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2, 3-c]pyran-3-carboxamido)ethyl)benzoic acid methyl ester (59mg, 0.13mmol) was dissolved in a solution consisting of 3.0mL THF, 3.0mL methanol, 1.0mL water, and then added monohydrate hydroxide Lithium (10mg, 0.24mmol), the reaction solution was stirred and reacted at 68°C for 3h. After the reaction was completed, the reaction solution was adjusted to acidity with 2M HCl, extracted with water and ethyl acetate, and the organic phase was evaporated to dryness. Purification by column chromatography A white solid was obtained, the final product YJ137 (39 mg, yield 66%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.83 (s, 1H), 12.03 (s, 1H), 8.79 (d, J = 6.9 Hz, 1H), 8.01–7.45 (m, 5H), 7.39– 7.22(m,1H),6.41–6.26(m,1H), 5.17(d,J=15.9Hz,1H), 4.85–4.63(m,2H), 3.98–3.76(m,2H), 2.63(s, 2H), 1.50-1.40 (m, 3H).

Example 1-7, (S)-4-(1-(2-((6-methoxynaphthalen-2-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3 -c) Preparation of pyran-3-carboxamido)ethyl)benzoic acid (YJ138)

Using the same reaction route as the preparation of compound YJ137, replacing 5-ethynyl-2(1H)-pyridone with 2-ethynyl-6-methoxynaphthalene, compound YJ138 was finally obtained (the yield of the last step was 90%) ). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.81 (s, 1H), 8.91 (d, J = 7.8 Hz, 1H), 7.95 (s, 1H), 7.86–7.78 (m, 4H), 7.56 ( d,J=8.3Hz,2H),7.41–7.34(m,2H), 7.24–7.19(m,1H), 5.25–5.17(m,1H), 4.75(s,2H), 3.95–3.82(m, 5H), 2.66(d,J=5.2Hz,2H), 1.48(d,J=7.0Hz,3H).

Example 1-8, (S)-4-(1-(2-((4-fluorophenyl)ethynyl)-6,7-dihydro-4H-thieno[3,2-c]pyran Preparation of -3-carboxamido)ethyl)benzoic acid (YJ139)

Using the same reaction route as the preparation of compound YJ137, 5-acetylene-2(1H)-pyridone was replaced with 1-ethynyl-6-fluorobenzene, and tetrahydropyrone was replaced with tetrahydro-2H-pyran- 3-ketone, compound YJ138 is finally obtained (the yield of the last step is 94%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.88 (s, 1H), 8.80 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1Hz, 2H), 7.46--7.42(m, 2H), 7.26--7.22(m, 2H), 5.17--5.13(m, 1H), 4.58(s, 2H), 3.91--3.86(m, 2H), 2.83 (s,2H),1.45(d,J=7.0Hz,3H).

Example 1-9, (S)-4-(1-(2-(4-fluorophenethyl)-6,7-dihydro-4H-thieno[3,2-c]pyran-3- Preparation of carboxamido) ethyl) benzoic acid (YJ140)

Dissolve YJ139 in absolute ethanol, add 10% Pd/C, and stir for 12 hours under a hydrogen atmosphere. After the reaction is complete, filter with Celite to obtain the filtrate. After removing the solvent, compound YJ140 is obtained (yield of the last step of the reaction) Is 100%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.93 (s, 1H), 8.66 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.47 (d, J = 8.0Hz,2H),7.11–7.02(m,4H),5.17–5.11(m,1H),4.52(s,2H), 3.85(t,J=5.2Hz,2H),3.07–3.00(m,2H) ), 2.78–2.72(m,4H),1.43(d,J=7.0Hz,3H).

Example 1-10, (S)-4-(1-(2-((3,5-dimethoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3 -c) Preparation of pyran-3-carboxamido)ethyl)benzoic acid (YJ141)

Using the same reaction route as the preparation of compound YJ137, replacing 5-acetylene-2(1H)-pyridone with 3,5-dimethoxyphenylacetylene, compound YJ138 was finally obtained (the yield of the last step was 95%) . ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.73 (s, 1H), 8.88 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3Hz, 2H), 6.61–6.57 (m, 3H), 5.18 (p, J = 7.1Hz, 1H), 4.73 (s, 2H), 3.74 (s, 6H), 2.64 (t, J = 5.0Hz, 2H), 2.52-2.50 (m, 3H), 1.46 (d, J=7.0Hz, 3H).

Example 1-11, (S)-4-(1-(2-((3-methoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c] Preparation of pyran-3-carboxamido)ethyl)benzoic acid (YJ142)

Using the same reaction route as the preparation of compound YJ137, 5-acetylene-2(1H)-pyridone was replaced with 3-methoxyphenylacetylene, finally compound YJ138 was obtained (the yield of the last step was 94%). ¹ H NMR(500MHz,DMSO-d ₆ )δ12.79(s,1H), 8.89(d,J=7.9Hz,1H),7.83-7.79(m,2H),7.52(t,J=5.9Hz, 2H),7.31(t,J=7.9Hz,1H),7.03-6.95(m,3H),5.21-5.16(m,1H),4.74(s,2H),3.86(t,J=5.9Hz,2H ), 3.76 (s, 3H), 2.65 (t, J = 5.1 Hz, 2H), 1.46 (d, J = 7.0 Hz, 3H).

Example 1-12, (S)-4-(1-(2-(3,5-dimethoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c] Preparation of pyran-3-carboxamido)ethyl)benzoic acid (YJ143)

Dissolve YJ141 in absolute ethanol, add 10% Pd/C, and stir for 12 hours under a hydrogen atmosphere. After the reaction is complete, the filtrate is obtained by suction filtration with diatomaceous earth. After removing the solvent, compound YJ143 is obtained (the yield of the last reaction) Is 100%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.77 (s, 1H), 8.68 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.7Hz, 2H), 6.32 (s, 3H), 5.22--5.15 (m, 1H), 4.66 (s, 2H), 3.70 (s, 6H), 3.15-3.02 (m, 2H), 2.82-2.48 (m ,6H),1.44(d,J=6.7Hz,3H).

Example 1-13, (S)-4-(1-(2-(3,5-dimethoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c] Preparation of pyran-3-carboxamido)ethyl)benzoic acid (YJ144)

Dissolve YJ142 in absolute ethanol, add 10% Pd/C, and stir for 12 hours under a hydrogen atmosphere. After the reaction is complete, filter with Celite to obtain the filtrate. After removing the solvent, compound YJ144 is obtained (yield of the last step of the reaction) Is 100%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.77 (s, 1H), 8.68 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.7Hz, 2H), 7.22-7.16 (m, 1H), 6.32 (s, 3H), 5.22-5.15 (m, 1H), 4.66 (s, 2H), 3.70 (s, 3H), 3.15-3.02 (m ,2H), 2.82–2.48(m,6H), 1.44(d,J=6.7Hz,3H).

Example 1-14, (S)-4-(1-(2-(2-(6-methoxynaphthalen-2-yl)ethyl)-4,7-dihydro-5H-thieno[2 ,3-c]pyran-3-carboxamido)ethyl)benzoic acid (YJ145)

Dissolve YJ138 in absolute ethanol, add 10% Pd/C, and stir for 12 hours under a hydrogen atmosphere. After the reaction is complete, filter with Celite to obtain the filtrate. After removing the solvent, compound YJ145 is obtained (yield of the last step of the reaction) Is 100%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.73 (s, 1H), 8.70 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 9.1Hz,2H),7.52(d,J=7.8Hz,3H),7.27–7.20(m,2H),7.14–7.10(m,1H), 5.19(p,J=7.1Hz,1H), 4.65( s, 2H), 3.85 (d, J = 9.4 Hz, 4H), 3.84–3.81 (m, 2H), 3.20–3.07 (m, 4H), 2.96–2.88 (m, 2H), 2.62 (d, J = 5.5Hz, 2H), 1.44 (d, J = 7.1Hz, 3H).

Example 1-15, (S)-4-(1-(2-(4-fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-methyl Preparation of (YJ146) of (amide) ethyl) methyl benzoate

The 3,5-difluorobenzaldehyde was replaced with 4-fluorobenzaldehyde, and the compound YJ132 was prepared according to the method for preparing compound YJ132 without the last step of hydrolysis, to obtain compound YJ146 (the yield of the last step was 76%). ¹ H NMR(500MHz,DMSO-d ₆ )δ8.74(d,J=8.0Hz,1H), 7.95-7.91(m,2H), 7.50(d,J=8.3Hz,2H), 7.21-7.16( m,2H),7.09–7.04(m,2H), 5.16(p,J=7.1Hz,1H), 4.63(s,2H), 4.17–4.04(m,3H), 3.85(s,3H), 3.83 -3.80(m,2H),2.62(d,J=3.1Hz,2H),1.43(d,J=7.1Hz,3H).

Example 1-16, (S)-2-(4-fluorobenzyl)-N-(1-(4-(methoxyamino)phenyl)ethyl)-4,7-dihydro-5H- Preparation of Thieno[2,3-c]pyran-3-carboxamide (YJ147)

Compound YJ114, methoxyamine hydrochloride, HTAU, and DIPEA were added to DMF in a ratio of 1:1.1:1.2:3, and stirred at room temperature for 6 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate and water, and the upper layer was taken. The organic phase was evaporated to dryness and purified by column chromatography to obtain compound YJ147 (the yield of the last step was 70%). ¹ H NMR(500MHz,DMSO-d ₆ )δ11.70(s,1H), 8.72(d,J=8.0Hz,1H),7.77–7.68(m,2H),7.45(t,J=8.1Hz, 2H), 7.19(ddd,J=8.7,8.0,4.9Hz,2H),7.10–7.03(m,2H), 5.14(p,J=7.1Hz,1H), 4.63(s,2H), 4.20–4.04 (m, 2H), 3.88–3.78 (m, 2H), 3.70 (s, 3H), 2.61 (dt, J = 16.4, 8.2 Hz, 2H), 1.42 (d, J = 7.1 Hz, 3H).

Example 1-17, (S)-4-(1-(2-(4-methylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3- (Formamide) ethyl) benzoic acid (YJ148) preparation

Replace 3,5-difluorobenzaldehyde with 4-methylbenzaldehyde, and prepare according to the method of preparing compound YJ132 to obtain compound YJ146 (the yield of the last step is 96%). ¹ H NMR(500MHz,DMSO-d ₆ )δ12.84(s,1H), 8.71(d,J=8.0Hz,1H),7.92-7.89(m,2H),7.48(d,J=8.3Hz, 2H), 7.03 (s, 4H), 5.16 (p, J = 7.1 Hz, 1H), 4.62 (s, 2H), 4.14-4.00 (m, 2H), 3.86-3.77 (m, 2H), 2.65-2.58 (m, 2H), 2.25 (d, J = 6.8 Hz, 3H), 1.43 (d, J = 7.1 Hz, 3H).

Example 1-18, (S)-4-(1-(2-(4-ethylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3- (Formamide) ethyl) benzoic acid (YJ149) preparation

Replace 3,5-difluorobenzaldehyde with 4-ethylbenzaldehyde, and prepare according to the method of preparing compound YJ132 to obtain compound YJ146 (the yield of the last step is 96%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.87 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2Hz, 2H), 7.05 (s, 4H), 5.18 (p, J = 7.0 Hz, 1H), 4.63 (s, 2H), 4.17-4.01 (m, 2H), 3.86-3.77 (m, 2H), 3.40(s,5H), 2.57–2.52(m,2H), 1.44(d,J=7.0Hz,3H).

Example 1-19, (S)-2-(4-fluorobenzyl)-N-(1-(4-(methoxy(methyl)carbamoyl)phenyl)ethyl)-4,7 -Dihydro-5H-thieno[2,3-C]pyran-3-carboxamide (YJ150)

Using the same reaction route as the preparation of compound YJ147, the methoxyamine hydrochloride was replaced with N,O-dimethylhydroxylamine hydrochloride, and finally compound YJ150 was obtained (the yield of the last step was 93%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 8.73-8.69 (m, 1H), 7.59-7.54 (m, 2H), 7.45-7.40 (m, 2H), 7.20-7.15 (m, 2H), 7.09 --7.04(m,2H), 5.19–5.12(m,1H), 4.63(s,2H), 4.18–4.04(m,2H), 3.87–3.78(m,2H), 3.53(s,2H), 3.25 (d,J=4.9Hz,2H), 2.66–2.59(m,2H), 2.53-2.48(m,2H), 1.44(d,J=7.1Hz,3H).

Example 1-20, (S)-2-(4-fluorobenzyl)-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-4,7-dihydro-5H- Preparation of Thieno[2,3-c]pyran-3-carboxamide (YJ151)

YJ146 was added to the methanol filtrate of fresh hydroxylamine, and then KOH was added. The reaction was stirred at room temperature for 30 minutes, extracted with saturated ammonium chloride and ethyl acetate, and column chromatography was used to obtain compound YJ151 (the yield of the final reaction was 55%). ¹ H NMR(500MHz,DMSO-d ₆ )δ11.18(s,1H),9.02(s,1H),8.75-8.69(m,1H),7.72(d,J=8.3Hz,2H),7.50- 7.42 (m, 2H), 7.21-7.16 (m, 2H), 7.09-7.03 (m, 2H), 5.18-5.10 (m, 1H), 4.63 (s, 2H), 4.18-4.05 (m, 2H), 3.86–3.78(m,2H),2.53–2.48(m,2H),1.42(t,J=6.1Hz,3H).

Example 1-21, (S)-N-(1-(4-cyanophenyl)ethyl)-2-(4-fluorobenzyl)-4,7-dihydro-5H-thieno[2 ,3-c) Preparation of pyran-3-carboxamide (YJ152)

Using the same reaction route as the preparation of compound YJ146, the methyl (S)-4-(1-aminoethyl)benzoate was replaced with p-cyanobenzylamine to obtain compound YJ152 (the yield of the last step was 79%) ). ¹ H NMR(500MHz,DMSO-d ₆ )δ8.74(d,J=8.0Hz,1H), 7.95-7.91(m,2H), 7.50(d,J=8.3Hz,2H), 7.21-7.16( m,2H),7.09–7.04(m,2H), 5.16(p,J=7.1Hz,1H), 4.63(s,2H), 4.17–4.04(m,3H), 3.83–3.80(m,2H) , 2.62(d,J=3.1Hz,2H),1.43(d,J=7.1Hz,3H).

Example 1-22 (S)-2-(4-fluorobenzyl)-N-(1-(4-(hydrazinecarbonyl)phenyl)ethyl)-4,7-dihydro-5H-thieno Preparation of [2,3-c]pyran-3-carboxamide (YJ153)

Using the same reaction route as the preparation of compound YJ147, the methoxyamine hydrochloride was replaced with hydrazine hydrate, and compound YJ153 was finally obtained (the yield of the last step was 69%). ¹ H NMR(500MHz,DMSO-d ₆ )δ9.72(s,1H), 8.70(d,J=8.1Hz,1H), 7.79(d,J=8.3Hz,2H), 7.43(d,J= 8.2Hz, 2H), 7.20-7.16 (m, 2H), 7.06 (t, J = 8.9 Hz, 2H), 5.18-5.10 (m, 1H), 4.63 (s, 2H), 4.17-4.05 (m, 2H) ), 3.86–3.79(m,2H),2.61(s,2H),1.42(d,J=7.0Hz,3H).

Example 1-23, (S)-4-(1-(6-(tert-butoxycarbonyl)-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[ Preparation of 2,3-c)pyridine-3-carboxamido)ethyl)benzoic acid (YJ154)

Using the same reaction route as the preparation of compound YJ140, tetrahydro-2H-pyran-3-one was replaced with N-tert-butoxycarbonyl-4-piperidone to finally obtain compound YJ154 (the yield of the last step was 94 %). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.80 (s, 1H), 8.67 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.7 Hz, 2H), 7.49 (d, J = 7.8Hz, 2H), 7.12–6.99(m, 4H), 5.20–5.12(m, 1H), 4.66–4.52(m, 2H), 3.67(s, 2H), 3.00(d, J=7.6Hz, 2H ), 2.71 (d, J = 45.0 Hz, 4H), 1.49 (s, 9H), 1.43 (d, J = 6.9 Hz, 3H).

Example 1-24, (S)-4-(1-(6-acetyl-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[2,3-c ]Pyridine-3-carboxamido)ethyl)benzoic acid (YJ155)

Using the same reaction route as the preparation of compound YJ140, tetrahydro-2H-pyran-3-one was replaced with N-acetyl-4-piperidone, finally compound YJ157 was obtained (the yield of the last step was 94%) . ¹ H NMR (500MHz, DMSO-d ₆ ) δ 12.80 (s, 1H), 8.67 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.7 Hz, 2H), 7.49 (d, J = 7.8Hz, 2H), 7.12–6.99(m, 4H), 5.20–5.12(m, 1H), 4.66–4.52(m, 2H), 3.67(s, 2H), 3.00(d, J=7.6Hz, 2H ), 2.71(d,J=45.0Hz,4H),2.52(s,3H),1.43(d,J=6.9Hz,3H).

Example 2 The inhibitory effect of the compound YJ114 of the present invention in combination with PD-1 antibody on the mouse CT26 colon cancer tumor model

1. Mouse CT26 cell colon cancer subcutaneous transplantation model and dosing plan

The mouse colon cancer cell CT26 was cultured and expanded in vitro, and 1×10 ⁶ cells/100 μL was injected subcutaneously on the right side of the back of female Balb/c mice at 6-8 weeks. Then, the length and width of the subcutaneous tumor on the back of the mouse were measured with a vernier caliper, and the tumor volume (mm ³ ) = length (mm) × width (mm) × width (mm) × 0.5 was calculated. After the size of the tumor on the back of the mice reached 100mm ³ -200mm ³ , the mice were randomly divided into four groups: control group, YJ114 single-drug group, PD-1 antibody single-drug group and combination drug group. The dosage of YJ114 is 75mg/kg/d, and the way of administration is oral; the dosage of PD-1 antibody is 50μg/head, twice a week, and the way of administration is abdominal cavity. The administration cycle is 20 days. The tumor size and body weight changes of the mice were measured and recorded every two days.

2. Analysis of experimental results:

The results are shown in Figure 1A-C. Compared with the negative control group, the YJ114 and PD-1 antibody single-drug group can inhibit tumor growth to a certain extent, and the therapeutic effect of the PD-1 antibody group is better than 75mg/kg /d YJ114 single agent group, tumor growth has been greatly relieved. At the same time, the anti-tumor growth effect of the combination drug group was better than the inhibitory effect of PD-1 antibody alone. The tumor volume of 8 out of 12 mice was reduced and disappeared, and complete remission was obtained (control group: 0/ 12; YJ114 single-drug group: 2/12; PD-1 antibody single-drug group: 4/12; YJ114+PD-1 antibody combination group: 8/12), and there is no adverse effect on the weight of mice, indicating The combined use of YJ114 and PD-1 antibody has no obvious side effects on mice.

In order to further explore the long-term therapeutic effect of the combination of YJ114 and PD-1 antibody, CT26 tumor-bearing mice were administered according to the above grouping and administration method for 2 weeks, and the tumor size of the mice was continuously measured and recorded. 2000mm ³ is death, and the survival curve of mice in each group for four months from the start of administration was counted. The results are shown in Figure 1D. Both YJ114 and PD-1 antibody single-drug groups prolonged the life cycle of mice to a certain extent, while the mice in the combined drug group had a longer life span, which was significantly different from other groups. At the end of the statistical time 120 days, 7 mice in the combination drug group were still alive (control group: 0/12; YJ114 single drug group: 1/10; PD-1 antibody single drug group: 2/10; combination drug group: 7/10).

Example 3 The inhibitory effect of the compound YJ114 of the present invention in combination with PD-1 antibody on mouse MC38 colon cancer tumor model

1. Mouse MC38 cell colon cancer subcutaneous transplantation model and dosing plan

The mouse colon cancer cell MC38 was cultured and expanded in vitro, and 1×10 ⁶ cells/100 μL was injected subcutaneously on the right side of the back of female C57BL/6 mice at 6-8 weeks. Then, the length and width of the subcutaneous tumor on the back of the mouse were measured with a vernier caliper, and the tumor volume (mm ³ ) = length (mm) × width (mm) × width (mm) × 0.5 was calculated. After the size of the tumor on the back of the mice reached 100mm ³ -200mm ³ , the mice were randomly divided into four groups: control group, YJ114 single-drug group, PD-1 antibody single-drug group and combination drug group. The dosage of YJ114 is 75mg/kg/d, and the way of administration is oral; the dosage of PD-1 antibody is 50μg/head, twice a week, and the way of administration is abdominal cavity. The administration period is 18 days. The tumor size and body weight changes of the mice were measured and recorded every two days.

2. Analysis of experimental results:

The results are shown in Figure 2A-C. Compared with the negative control group, both YJ114 and PD-1 antibody single-drug group can inhibit tumor growth to a certain extent, and the therapeutic effect of PD-1 antibody single-drug group is similar to that of YJ114 administration. The efficacy of the group is similar. At the same time, the anti-tumor growth effect of the combination of YJ114 and PD-1 antibody is better than that of the two groups of single drugs. The tumor volume of 3 out of 8 mice was reduced and disappeared, and the tumor was completely relieved. The weight of the mice did not have any adverse effects, and the tumors in the other groups did not disappear completely. After the experiment, the tumor tissue weight of each group of mice was weighed, and the statistical results were similar to the tumor volume. The tumor growth inhibitory effect of the combined drug group was the most obvious, and there were no obvious toxic and side effects on mice.

Example 4 The inhibitory effect of the compound YJ114 of the present invention in combination with PD-1 antibody on the mouse orthotopic colon cancer tumor model induced by AOM/DSS model

Chronic inflammation has always been considered as one of the major causes of colorectal cancer and is accompanied by the occurrence and development of colorectal cancer. These inflammatory cytokines such as IL-6, TNF, IL-1β, IL-17 and various immune cells act as fertile soil in the environment of chronic inflammation, accumulating and promoting the development of colorectal cancer. The AOM/DSS model is a model of local ulcerative colitis that is caused by chemical stimulation to damage the intestinal cells of mice, which further develops into a colon cancer model under the action of long-term inducers. Therefore, by constructing an AOM/DSS model, it will be further verified that blocking the PGE ₂ /EP4 signaling pathway by YJ114 can cooperate with immune checkpoint blockers to inhibit the PD-1/PD-L1 signaling pathway, which is more effective and durable for in situ tumor formation and growth. The inhibitory effect.

1. Mouse orthotopic colon cancer model and dosage regimen

As shown in Figure 3A, female mice of C57BL/6 at 8-10 weeks were selected for a one-time intraperitoneal injection of 10 mg/kg AOM. From the sixth day, 2.5% of DSS was given water for 5 days and then replaced with normal drinking water for two weeks. The round contains 2.5% DSS water supply cycle to construct a spontaneous mouse colon cancer model. After the third round of water supply containing 2.5% DSS, we randomly divided the mice into four groups according to the weight of the mice at this time: control group, YJ114 single-drug group, PD-1 antibody single-drug group and combination drug group. The dosage of YJ114 is 75mg/kg/d, and the way of administration is oral; the dosage of PD-1 antibody is 50μg/head, twice a week, and the way of administration is intraperitoneal. The administration period is 35 days. After the end of the dosing cycle, we took the colon of each group of mice and started to observe the formation and growth of tumors in the colon of the mice.

2. Analysis of experimental results:

The results are shown in Figure 3B-D. Both YJ114 and PD-1 antibody single-drug treatment can alleviate the formation of tumors in the colon of mice to a certain extent and reduce the total number of tumors in the colon of mice. Compared with the control group, PD-1 antibody alone has almost no inhibitory effect on tumors larger than 4mm in diameter, while YJ114 single-drug treatment can significantly inhibit the formation of tumors larger than 4mm in diameter. In contrast, the volume of tumors in the colon of mice in the combined drug group was significantly reduced, again showing that YJ114 and PD-1 antibody can produce a good anti-tumor synergistic effect. At the same time, we measured the colon length of the mice and found that there was no significant difference between the groups.

Example 5 The inhibitory effect of the compound YJ114 of the present invention in combination with PD-antibody on the mouse RM-1 prostate cancer model

1. Mouse RM-1 cell prostate cancer subcutaneous transplantation model and dosage regimen

1×10 ⁶ prostate cancer cells RM-1 were injected subcutaneously into the right back of male C57BL/6 mice aged 6-8 weeks. Calculate the tumor volume according to the formula: volume = length × width ² × 0.52. When the tumor grows to about 100mm ³ , it is randomly divided into four groups: control group, YJ114 single-drug group, PD-1 antibody single-drug group and combination drug group. The dose of YJ114 is 150 mg/kg/d, once a day. The administration mode is oral; the administration dose of PD-1 antibody is 100 μg/bottle, twice a week, the administration mode is abdominal cavity, and the administration period is 17 days. Measure and record the changes in tumor length and width twice a week, and record the weight of the mice. After the experiment was over, the mice were killed by anesthesia, the subcutaneous tumor was stripped, weighed and photographed to record the tumor size.

2. Analysis of experimental results:

As shown in Figure 4A, in the RM-1 subcutaneous tumor-bearing animal model, the therapeutic effects of YJ114 and PD1 antibody were tested by single use and combined use, respectively. The tumor growth inhibition rate of PD1 antibody and YJ114 single drug was relatively close. Only about 40%. However, when YJ114 and PD1 antibody are used in combination, the inhibitory effect on tumors is significantly increased, and the effect of almost complete inhibition is achieved, and the combined effect is extremely significant (Figure 4A). The pictures of the peeled tumor (Figure 4B) and the measurement results of the quality of the peeled tumor (Figure 4C) also proved that the combined effect is extremely significant. In addition, the physiological indicators of the mice in each group were normal during the treatment period, and the weight gain was stable (Figure 4D). The results of this experiment proved that YJ114 and PD-1 monoclonal antibody combined treatment significantly inhibited prostate tumor growth.

Using the methods of Examples 2-5, experiments were performed on other compounds of the present invention, and the results showed that other compounds of the present invention have similar activities and effects to YJ114.

Example 6 The inhibitory effect of the compound YJ114 of the present invention in combination with PD-1 antibody on the mouse MFC gastric cancer model

1. Mouse gastric cancer MFC cell subcutaneous transplantation model and dosing plan

2×10 ⁶ mouse gastric cancer MFC cells were injected subcutaneously into the right back of 615 mice aged 6-8 weeks. Calculate the tumor volume according to the formula: volume = length × width ² × 0.52. When the tumor grows to about 100mm ³ , it is randomly divided into four groups: control group, YJ114 single-drug group, PD1 antibody single-drug group and combination drug group. The dose of YJ114 is 150 mg/kg/d, once a day. The method is oral; the dosage of PD1 antibody is 10 μg/bottle, twice a week, the method of administration is intraperitoneal, and the administration period is 27 days. Measure and record the changes in tumor length and width twice a week, and record the weight of the mice. After the experiment was over, the mice were killed by anesthesia, the subcutaneous tumor was stripped, weighed and photographed to record the tumor size.

2. Analysis of experimental results:

As shown in Figure 5A, in the subcutaneous tumor-bearing animal model of MFC gastric cancer, the therapeutic effects of YJ114 and PD1 antibody were tested by single and combined methods. The tumor growth inhibition rate of PD1 antibody and YJ114 single drug was relatively close, reaching Around 80%. And when YJ114 and PD1 antibody are used in combination, the inhibitory effect on tumors is more significant, almost completely inhibiting the growth of tumors (Figure 5A). On the day of the end of the experiment, the tumor elimination rate of each group was counted. As shown in the result of Figure 5B, the tumor elimination rate of the YJ114 single-agent group is about 50%, and the tumor elimination rate of the PD1 single-agent group is about 70%. When the two are combined, the tumor is eliminated 100%. Taking the tumor volume exceeding 2000 mm ³ as the mouse death criterion to obtain the mouse survival curve shown in Figure 5C, it can be seen that the combined treatment significantly prolonged the survival period of the experimental mice, and the survival rate of the mice was 100% at the end of the experiment. During the whole experiment, there was no significant difference in the weight change of the mice in each group (Figure 5D).

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A product portfolio characterized by:

(i) a first pharmaceutical composition, said first pharmaceutical composition containing (a) a first active ingredient, said first active ingredient being an EP4 receptor antagonist, and a pharmaceutically acceptable carrier; and

(ii) A second pharmaceutical composition, the second pharmaceutical composition containing (b) a second active ingredient, the second active ingredient is a PD-1 inhibitor, and a pharmaceutically acceptable carrier;

Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
The product combination of claim 1, wherein the EP4 receptor antagonist is a compound represented by formula I or a pharmaceutically acceptable salt or hydrate thereof:

among them,
Each is independently selected from the following group: C3-C6 carbocyclic ring, C6-C12 aryl group, five-membered or six-membered heteroaromatic ring containing one or more O, N, S atoms, wherein
May be optionally substituted by 1-3 R 5 groups;

Is a substituted or unsubstituted ring selected from the following group: C4-C7 carbocyclic ring, 4-7 membered saturated heterocyclic ring, benzene ring, 4-7 membered unsaturated heterocyclic ring (including heteroaromatic ring), wherein, the The heterocyclic ring has one or more heteroatoms selected from the following group: O, S or NR 6 ; the ring can be a monocyclic, bicyclic, spiro or bridged ring;

X is a group selected from the following group: -O-, -S-, -N(R 7 )-;

Y is none, or a group selected from the following group: -CH 2 -, -O-, -S-, -SO-, -SO 2 -, -N(R 8 )-;

B 1 and B 2 are each independently a group selected from the group consisting of none, C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene; preferably, the B1 And B2 are independently selected from the following group: -(CH 2 ) n -; wherein n=0, 1, 2, 3 or 4, -CH=CH-, -CH=CH-CH 2 -, -CH 2 -CH =CH-, -CH=CH-CH 2 -CH 2 -, -CH 2 -CH=CH-CH 2 -, -CH 2 -CH 2 -CH=CH-; -C≡C-, -C≡C -CH 2 -, - CH 2 -C≡C -, - C≡C-CH 2 -CH 2 -, - CH 2 -C≡C-CH 2 -, - CH 2 -CH 2 -C≡C-; And B 1 , B 2 and Y are not all at the same time;

R 1 is one or more groups selected from the following group: H, C1-C6 alkyl (preferably C1-C4 alkyl), halogen, nitro, -N(R 9 )(R 10 ), -OH , -CN, C1-C6 haloalkyl (preferably C1-C4 haloalkyl, more preferably difluoromethyl, trifluoromethyl), C1-C6 alkoxy (preferably C1-C4 alkoxy, More preferably methoxy, ethoxy), C1-C6 haloalkoxy (preferably C1-C4 haloalkoxy, more preferably difluoromethoxy, trifluoromethoxy), =O;

R 2 and R 3 are each independently selected from: H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or R 2 , R 3 and the carbon atom to which they are connected together form 3 To a 6-membered ring, the ring is a carbocyclic ring or a 3- to 6-membered heterocyclic ring having 1-3 heteroatoms selected from the group consisting of O, S or N (R 11 );

R 4 is selected from any one of the following groups: -COOR 12 (preferably -COOH, -COOCH 3 , -COOCH 2 CH 3 , -COOCH 2 CH 2 CH 3 , -COOCH(CH 3 ) 2 ), C( O)-N(Ra)(Rb), cyano group, tetrazolium group, phosphoric acid group, sulfonic acid group; wherein, Ra is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted Substituted C1-C6 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, and substituted or unsubstituted C1-C6 alkoxy; Rb is selected from the group consisting of H, -OH, -NH 2 , substituted or Unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, and substituted or unsubstituted C1-C6 alkoxy;

Each R 5 and R 12 are each independently selected from: H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxy (preferably methoxy, ethyl Oxy);

R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are each independently selected from: H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C3-C6 halo ring Alkyl, C6-C10 aryl, five-membered or six-membered heterocyclic aromatic group,

Unless otherwise specified, one or more hydrogen atoms on the substituent group is substituted by a substituent selected from the following group: F, Cl, Br, I, hydroxyl, methyl, ethyl, isopropyl, methoxy Group, ethoxy, trifluoromethyl, difluoromethoxy, trifluoromethoxy, nitro, -CN, oxo (=O);

R 13 and R 14 are each independently selected from: H, C1-C6 alkyl (preferably methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, pentyl, hexyl), C1- C6 alkoxy, C6-C10 aryl, C1-C6 alkylene, -C6-C10 aryl.
The use according to claim 1, wherein the EP4 receptor antagonist is selected from the following group:

(S)-4-(1-(2-((4-fluorophenyl)ethynyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide) Ethyl) benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,6-dihydro-4H-cyclopenta(b)thiophene-3-carboxamide)ethyl )benzoic acid;

4-((1S)-1-(6-ethyl-2-((4-fluorophenyl)ethynyl)-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxamide Yl)ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,6,7,8-tetrahydro-4H-cycloheptano(b)thiophene-3-carboxamide )Ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c] Pyran-3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,6,7,8-tetrahydro-4H-cycloheptano(b)thiophen-3-carboxamide)ethyl )benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-5,6-dihydro-4H-cyclopenta(b)thiophene-3-carboxamide)ethyl)benzoic acid ；

(S)-4-(1-(2-(4-Fluorophenethyl)-5,5-dimethyl-5,7-dihydro-4H-thieno[2,3-c]pyran- 3-carboxamide)ethyl)benzoic acid;

(S)-4-(1-(2-(4-(Trifluoromethyl)phenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-methyl (Amide) ethyl) benzoic acid;

(S)-4-(1-(2-(3-(Trifluoromethyl)phenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-methyl (Amide) ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluorophenethyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Formic acid

(S)-4-(1-(2-(4-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(S)-4-(1-(2-(3-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(R)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Formic acid

(R)-4-(1-(2-(4-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

(R)-4-(1-(2-(3-(Trifluoromethyl)benzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide )Ethyl)benzoic acid;

4-((2-(4-fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzoic acid;

(S)-4-(1-(2-((4-(Trifluoromethyl)benzyl)amino)-5,7-dihydro-4H-thieno[2,3-c]pyran-3 -Formamide) ethyl) benzoic acid;

(S)-4-(1-(2-(4-methoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(4-chlorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(3-Fluoro-4-methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3-chlorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl) benzoic acid;

(S)-4-(1-(2-(3,4-Difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(4-methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl基)benzoic acid;

((2-(3-(Trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzoic acid;

4-((2-(4-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamide)methyl)cyclohexane-1-carboxy Acid (racemate);

(1-(2-(4-Fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)cyclopropyl)benzoic acid;

(S)-4-(1-(2-(3,5-Difluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(3-Methoxybenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)ethyl基)benzoic acid;

3-((2-(3-(Trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl)benzene Formic acid

4-((2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)methyl) ring Hexane-1-carboxylic acid;

4-(1-(2-(3-(trifluoromethyl)benzyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido)cyclopropane基)benzoic acid;

(S)-4-(1-(2-((6-oxo-1,6-dihydropyridin-3-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(2-((6-methoxynaphthalen-2-yl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran- 3-formylamino)ethyl)benzoic acid;

(S)-4-(1-(2-((4-Fluorophenyl)ethynyl)-6,7-dihydro-4H-thieno[3,2-c]pyran-3-carboxamido )Ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorophenethyl)-6,7-dihydro-4H-thieno[3,2-c]pyran-3-carboxamido)ethyl )benzoic acid;

(S)-4-(1-(2-((3,5-Dimethoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran- 3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(2-((3-methoxyphenyl)ethynyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3,5-Dimethoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-methyl Amido)ethyl)benzoic acid;

(S)-4-(1-(2-(3-methoxyphenethyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxamido) Ethyl) benzoic acid;

(S)-4-(1-(2-(2-(6-methoxynaphthalen-2-yl)ethyl)-4,7-dihydro-5H-thieno[2,3-c]pyridine Pyran-3-formylamino)ethyl)benzoic acid;

(S)-4-(1-(2-(4-Fluorobenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl)benzene Methyl formate

(S)-2-(4-Fluorobenzyl)-N-(1-(4-(methoxyamino)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamide;

(S)-4-(1-(2-(4-methylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl) benzoic acid;

(S)-4-(1-(2-(4-ethylbenzyl)-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide)ethyl )benzoic acid;

(S)-2-(4-fluorobenzyl)-N-(1-(4-(methoxy(methyl)carbamoyl)phenyl)ethyl)-4,7-dihydro-5H- Thieno[2,3-C]pyran-3-carboxamide;

(S)-2-(4-Fluorobenzyl)-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3 -c]pyran-3-carboxamide;

(S)-N-(1-(4-cyanophenyl)ethyl)-2-(4-fluorobenzyl)-4,7-dihydro-5H-thieno[2,3-c]pyridine Pyran-3-carboxamide;

(S)-2-(4-fluorobenzyl)-N-(1-(4-(hydrazinecarbonyl)phenyl)ethyl)-4,7-dihydro-5H-thieno[2,3-c ]Pyran-3-carboxamide;

(S)-4-(1-(6-(tert-butoxycarbonyl)-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[2,3-c] (Pyridine-3-carboxamido)ethyl)benzoic acid;

(S)-4-(1-(6-Acetyl-2-(4-fluorophenethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-methyl Amido)ethyl)benzoic acid.
The use according to claim 1, wherein the EP4 receptor antagonist is selected from the following group:
The use according to claim 1, wherein the EP4 receptor antagonist is selected from the following group:
A composition, characterized in that the composition comprises:

(i) EP4 receptor antagonist;

(ii) PD-1 inhibitor; and

(iii) A pharmaceutically acceptable carrier.
The composition of claim 6, wherein the composition further comprises other drugs for treating malignant tumors.
The composition of claim 7, wherein the other drugs for the treatment of malignant tumors are selected from the group consisting of nimustine, carmustine, cyclic ammonium phosphate, mustard, deoxyfluridine , 5-Fluorouracil, 6-Mercaptopurine, Thioguanine, Cytarabine, Gemcitabine, Carmofur, Hydroxyurea, Methotrexate, Ufodine, Amcitabine, Actinomycin D, Doxorubicin Star, daunorubicin, epirubicin, mitomycin, irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, taxotere, topotecan, vincristine, tinib Posides, etoposide, atamethan, anastrozole, aminoglutamin, letrozole, formestane, metgestrol, carboplatin, cisplatin, dacarbazine, oxaliplatin, rosa Din, Coplatin, Oxa, Mitoxantrone, Procarbazine, Gefitinib, Erlotinib, Cetuximab, Herceptin, Imatinib, Rituximab, Vorinox Him, ceritinib, crizotinib, icotinib, sorafenib, dacomitinib, apatinib, sunitinib, abcixi, bevacizumab, cetoli Coximab, Panitumumab, Regotinib, or a combination thereof.
A medicine box is characterized in that it comprises:

(a1) The first container, and the EP4 receptor antagonist in the first container, or a drug containing the EP4 receptor antagonist;

(b1) The second container, and the PD-1 inhibitor in the second container, or the drug containing the PD-1 inhibitor.
The use of a combination, characterized in that the combination comprises an EP4 receptor antagonist and a PD-1 inhibitor, and is used for preparing a pharmaceutical composition or a kit for the treatment of malignant tumors .