阅读说明：本技术 治疗中的p2rx7调节剂 (P2RX7 modulators in therapy ) 是由 瓦莱丽·武雷 利蒂希娅·杜盖 阿林娜·吉纳 热尔曼·奥默林 贝妮迪克塔·居伊·玛丽·里戈 于 2019-03-29 设计创作，主要内容包括：本发明涉及式(I)的化合物、其对映体及其药学上可接受的盐以及其在治疗中的用途,特别是用于预防和/或治疗癌症或炎症性疾病。(The present invention relates to compounds of formula (I), the enantiomers and pharmaceutically acceptable salts thereof and their use in therapy, in particular for the prevention and/or treatment of cancer or inflammatory diseases.)

1. A compound selected from compounds of formula (I), enantiomers thereof and pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

r1 represents C1-C14 alkyl, cycloalkyl, aryl, aralkyl or heteroaryl, wherein the aryl, aralkyl or heteroaryl is not substituted or substituted by at least one group selected from:

-a halogen atom,

-a C1-C6 alkoxy group,

-C1-C6 alkyl, and

-C1-C6 haloalkyl;

and R2 represents an aryl, aralkyl or cycloalkyl group, wherein the aryl or aralkyl group is not substituted or substituted by at least one group selected from halogen atoms,

wherein the compound is different from:

and wherein the compound is such that R1 and R2, when different, represent the following heteroaryl groups:

2. the compound of claim 1, wherein the compound is enantiomer S.

3. The compound according to claim 1 or 2, wherein said cycloalkyl is a cyclic saturated hydrocarbon group comprising from 1 to 12 carbon atoms, and is preferably selected from cyclohexylmethyl, adamantyl and cyclohexyl.

4. A compound according to any one of claims 1 to 3, wherein said aryl group is a monocyclic or polycyclic aromatic hydrocarbon group, which may be optionally substituted by at least one halogen atom, one C1-C6 alkoxy group and/or one C1-C6 haloalkyl group, and is preferably selected from 2-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 2-iodophenyl, 2, 4-dichlorophenyl, 3, 4-dichlorophenyl, 2, 4-dimethoxyphenyl, 3-trifluoromethylphenyl, 3, 5-bis (trifluoromethyl) phenyl and 2,4, 6-trimethylphenyl.

5. The compound according to any one of claims 1 to 4, wherein the aralkyl group is not substituted or substituted by at least one halogen atom, one C1-C6 alkoxy group, one C1-C6 alkyl group or one C1-C6 haloalkyl group, and is preferably selected from benzyl, 2-chlorobenzyl, 2, 4-dichlorobenzyl, 3, 4-dichlorobenzyl and 2, 4-dichlorophenethyl.

6. The compound according to any one of claims 1 to 5, wherein the heteroaryl group is an aryl group substituted on at least one carbon atom of the aromatic ring by a heteroatom, preferably nitrogen; preferably, the heteroaryl group may be unsubstituted or substituted by at least one halogen atom, one C1-C6 alkoxy group and/or one C1-C6 haloalkyl group; and more preferably, the heteroaryl group is 6-chloropyridin-3-yl or thiophen-2-yl.

7. The compound of any one of claims 1 to 6, wherein the compound has the following formula (I'):

wherein R1 is as defined in claim 1.

8. The compound according to any one of claims 1 to 7, wherein the compound is selected from the following compounds and pharmaceutically acceptable salts thereof:

9. a compound according to any one of claims 1 to 8 for use as a medicament.

10. Use of a compound according to any one of claims 1 to 8 in the prevention and/or treatment of inflammatory diseases and/or cancer.

11. The compound for use according to claim 10, wherein the inflammatory disease is a chronic inflammatory disease, preferably selected from rheumatoid arthritis, crohn's disease, inflammatory bowel disease, osteoarthritis, and osteoporosis; and/or the cancer is selected from colon cancer, colorectal cancer, melanoma, breast cancer, thyroid cancer, prostate cancer, ovarian cancer, lung cancer, pancreatic cancer, glioma, cervical cancer, endometrial cancer, head and neck cancer, liver cancer, kidney cancer, skin cancer, gastric cancer, testicular cancer, urothelial cancer, or adrenocortical cancer, and non-solid cancers, such as lymphoma.

12. A product, comprising:

a) at least one compound according to any one of claims 1 to 8, and

b) at least one other therapy selected from the group consisting of,

as a combination product for simultaneous, separate or sequential use in the treatment and/or prevention of cancer.

13. The product according to claim 12, wherein the further therapy b) is selected from an anti-checkpoint antibody, preferably from an anti-PD 1 antibody, more preferably from nivolumab and pembrolizumab, and/or the cancer is lung cancer, preferably Lewis lung cancer.

14. The compound for use according to any one of claims 9 to 13, wherein the compound is a P2RX7 modulator.

15. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 8 in a pharmaceutically acceptable carrier.

16. A P2RX7 modulator, which is a compound for use in the prevention and/or treatment of inflammatory diseases and/or cancer that is capable of inducing cell death through the macropore opening in the presence of ATP.

Technical Field

The present invention relates to compounds of formula (I), the enantiomers and pharmaceutically acceptable salts thereof and their use in therapy, in particular for the prevention and/or treatment of cancer or inflammatory diseases.

Background

Inflammation is a key protective response, and most of the mechanisms linking inflammation to injury repair and regeneration are preserved in evolution (karn, m. and Clevers, h. repair inflammation responsible for tissue regeneration (repair inflammation targets charge of tissue regeneration) Nature,2016.529: p.307-315). Inflammation is a fine-tuned response; the disturbance of this response leads to many chronic degenerative diseases. Epidemiological studies have particularly emphasized the link between chronic inflammation and cancer. In this context, in view of the long-term effectiveness of existing therapies, there is an urgent need to develop new strategies for the treatment of inflammation and the pathologies resulting therefrom.

Death of normal or tumor cells can produce risk-related molecular patterns (DAMPs), such as ATP, which are usually located inside the cell. Purinergic receptors of the P2X family are ATP-gated cation channels formed by three protein monomers. Of these seven receptor members, the receptor P2X7 (encoded by the gene P2RX7) is the only one with the potential to mediate pore formation and ultimately lead to cell death in an extracellular ATP-rich microenvironment. P2RX7 is expressed by immune and non-immune cells.

The potential of P2RX7 to establish a fine-tuned inflammatory response has been highlighted; the ability of P2RX7 antagonists to increase the Incidence of tumors in an in situ Mouse Model of Colitis-related Cancer has also been shown (Cesaro, a. et al, P2X7R activation in intestinal epithelial cells in response to transepithelial migration of neutrophils induces an Amplification loop of the inflammatory process (Amplification loop of the Inflammation process induced by P2X7R activation in intestinal epithelial cells to neutral pH shift), Am J physiological gain therapy activity physiological, 2010.299(1): P. g 32-42; Hofman, P. et al, Genetic Inactivation of Purinergic P2RX7 receptors and Pharmacological inhibition of Inflammation, but Increases the Incidence of tumors in a Mouse Model of Colitis-related Cancer (Genetic pharmacologic and activity RX 2015, Cancer Model of Cancer), fluorescence response of Cancer Receptor expression of Cancer Receptor 7).

In addition, most existing treatments for chronic inflammation or cancer deregulate immune cell responses.

Over The past decade, immunotherapy (anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies) has revolutionized The treatment of melanoma and lung cancer (NLSCC) (Sharma, p. and j.p. allison, The future of immune checkpoint therapy (The future of immune checkpoint therapy), Science,2015.348(6230): p.56-61). These therapies may reverse immune checkpoints to amplify T cell responses and reduce immunosuppression within the Tumor Microenvironment (TME). However, some patients with higher Expression levels of PD-L1 are resistant to treatment (McLaughlin, J. et al, Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small Cell Lung Cancer (Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer), JAMA Oncol,2016.2(1): p.46-54). The antitumor Activity of T lymphocytes is also impaired by myeloid suppressor cells (MDSCs) that are attracted within TME (Anderson, K.G., I.M.Stromnes and P.D.Greenberg, a barrier of the Tumor Microenvironment against T Cell Activity: cases of co-therapy (Obstacles placed by the Tumor Microenvironment to T Cell Activity: A Case for Synergistic therapeutics), Cancer Cell,2017.31(3): p.311-325).

Therefore, there is a need to develop new immunotherapies against all immunosuppressive factors. There is also a need for effective therapies that can reactivate the immunogenicity of tumors and/or can restore the patient's immune system.

Disclosure of Invention

The present invention aims to address these needs: it relates to compounds that are modulators of P2RX7 and that are useful for the treatment of chronic inflammatory diseases and cancer.

Surprisingly, the inventors found that the compounds enhance P2RX7 in the presence of extracellular ATP and are capable of restoring a functional anti-tumor T cell response. Furthermore, the inventors have found that: the compound not only directly acts on tumor cells by improving the immunogenicity of the tumor, but also acts on the microenvironment of the tumor by reducing the infiltration of immunosuppressive cells.

Interestingly, the inventors noted that: the compounds are also effective in reducing the clinical symptoms of inflammation in a murine model of colonic inflammation.

Thus, unexpectedly, the molecules may be capable of treating inflammatory diseases and cancer.

The present invention therefore relates to a compound selected from the group consisting of compounds of formula (I), enantiomers thereof and pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

r1 represents C1-C14 alkyl, cycloalkyl, aryl, aralkyl or heteroaryl, wherein the aryl, aralkyl or heteroaryl is not substituted or substituted by at least one group selected from:

-a halogen atom,

-a C1-C6 alkoxy group,

-C1-C6 alkyl, and

-C1-C6 haloalkyl;

and R2 represents an aryl group, an aralkyl group or a cycloalkyl group, wherein the aryl group or the aralkyl group is not substituted or substituted by at least one group selected from halogen atoms.

Preferably, the compounds of the invention are different from the following compounds:

preferably, the compounds of the invention (formula (I) or (II)) are such that R1 and R2 do not each simultaneously represent unsubstituted phenyl.

Preferably, the compounds of the invention (formula (I) or (II)) are such that R1 and R2 each do not simultaneously represent the following heteroaryl:

the heteroaryl group is a chromonic group.

Preferably, the compounds of the invention are selected from the group consisting of compounds of formula (I'), enantiomers thereof and pharmaceutically acceptable salts thereof,

wherein R1 represents C1-C14 alkyl, cycloalkyl, aryl, aralkyl or heteroaryl, wherein the aryl, aralkyl or heteroaryl is not substituted or substituted by at least one group selected from:

-a halogen atom,

-a C1-C6 alkoxy group,

-C1-C6 alkyl, and

-C1-C6 haloalkyl.

Preferably, the compound of formula (I) is enantiomer (S) having the following formula (II):

wherein R1 and R2 are as defined above.

More preferably, the compounds of the invention have the following formula (II'):

wherein R1 is as defined above.

"pharmaceutically acceptable salt" refers to any salt prepared using inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and the like, or organic acids such as acetic acid, citric acid, maleic acid, succinic acid, tartaric acid, lactic acid, malic acid, oxalic acid, fumaric acid, methanesulfonic acid, and the like.

The halogen atom is preferably selected from F, Br, I and Cl, more preferably Cl.

"C1-C14 alkyl" refers to a linear hydrocarbon group containing 1 to 14 carbon atoms, particularly 1 to 12 carbon atoms, or a branched hydrocarbon group containing 3 to 6 carbon atoms. Examples of C1-C14 alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl and dodecyl, and preferably methyl, n-butyl, n-pentyl, n-hexyl, n-dodecyl, isopropyl or tert-butyl.

Similarly, "C1-C6 alkyl" refers to a straight chain hydrocarbon group containing 1 to 6 carbon atoms or a branched chain hydrocarbon group containing 3 to 6 carbon atoms. Examples of C1-C6 alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and n-hexyl, and preferably methyl, n-butyl, n-pentyl, n-hexyl, isopropyl or tert-butyl.

"C1-C6 alkoxy" refers to-O-alkyl wherein the alkyl moiety is C1-C6 alkyl. Preferably, the C1-C6 alkoxy group is methoxy.

"C1-C6 haloalkyl" refers to a C1-C6 alkyl group having at least one hydrogen atom substituted with one or more halogen atoms.

Preferably, the C1-C6 haloalkyl is trifluoromethyl.

"cycloalkyl" refers to a cyclic saturated hydrocarbon group containing 1 to 12 carbon atoms. Preferably, the cycloalkyl group is selected from cyclohexylmethyl, adamantyl and cyclohexyl.

"aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group which may be optionally substituted. Preferably, aryl is phenyl. It may also be biphenyl. The aryl group may be unsubstituted or substituted by at least one halogen atom, one C1-C6 alkoxy group and/or one C1-C6 haloalkyl group.

Preferred aryl groups are 2-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 2-iodophenyl, 2, 4-dichlorophenyl, 3, 4-dichlorophenyl, 2, 4-dimethoxyphenyl, 3-trifluoromethylphenyl, 3, 5-bis (trifluoromethyl) phenyl or 2,4, 6-trimethylphenyl.

"aralkyl" refers to an aryl group attached to the remainder of formula (I) through an alkyl group. The aralkyl group may be unsubstituted or substituted by at least one halogen atom, one C1-C6 alkoxy group, one C1-C6 alkyl group or one C1-C6 haloalkyl group. Preferably, aralkyl is substituted or unsubstituted benzyl or phenethyl.

Preferably, the aralkyl group is selected from the group consisting of benzyl, 2-chlorobenzyl, 2, 4-dichlorobenzyl, 3, 4-dichlorobenzyl and 2, 4-dichlorophenethyl.

"heteroaryl" means an aryl group in which at least one carbon atom of the aromatic ring is replaced by a heteroatom and which may itself be optionally substituted. The heteroatom may be nitrogen, oxygen, phosphorus or sulfur. Preferably, the heteroatom is nitrogen. Examples of heteroaryl groups include: pyridyl, pyrrolyl, thienyl, furyl, pyrimidinyl, pyrazinyl, triazinyl, imidazolyl, thiazolyl, oxazolyl and isoxazolyl.

Preferably, heteroaryl is pyridyl, such as 2-pyridyl or 3-pyridyl. The heteroaryl group may be unsubstituted or substituted by at least one halogen atom, one C1-C6 alkoxy group and/or one C1-C6 haloalkyl group.

Preferably, heteroaryl is 6-chloropyridin-3-yl or thiophen-2-yl.

More preferably, the compounds of the present invention are selected from the following compounds (i.e., all enantiomers (S)) and pharmaceutically acceptable salts thereof:

(this compound is referred to as HEI2314 in the examples of the present invention)

(this compound is referred to as HEI2328 in the examples of the invention)

(this compound is referred to as HEI2333 in the examples of the invention)

(this compound is referred to as HEI2336 in the examples of the invention)

(this compound is referred to as HEI3090 in the examples of the present invention)

(this compound is referred to as HEI 2347 in the examples of the invention)

(this compound is referred to as HEI2346 in the examples of the invention)

(this compound is referred to as HEI 2269 in the examples of the invention)

(this compound is referred to as HEI 2298 in the examples of the present invention)

(this compound is referred to as HEI 2535 in the examples of the invention)

(this compound is referred to as HEI 2541 in the examples of the present invention)

(this compound is referred to as HEI 2534 in the examples of the invention)

(this compound is referred to as HEI 2542 in the examples of the present invention)

(this compound is referred to as HEI 2537 in the examples of the invention)

(this compound is referred to as HEI 2538 in the examples of the invention)

(this compound is referred to as HEI 2315 in the examples of the present invention)

(this compound is referred to as HEI 2329 in the examples of the invention)

(this compound is referred to as HEI 2339 in the examples of the invention)

(this compound is referred to as HEI 2761 in the examples of the present invention)

(this compound is referred to as HEI 2760 in the examples of the present invention)

(this compound is referred to as HEI 2759 in the examples of the present invention)

(this compound is referred to as HEI 2331 in the examples of the invention)

(this compound is referred to as HEI 2338 in the examples of the invention)

(this compound is referred to as HEI 2337 in the examples of the invention)

(this compound is referred to as HEI 2278 in the examples of the present invention)

(this compound is referred to as HEI 2817 in the examples of the present invention)

(this compound is referred to as HEI 2820 in the examples of the present invention)

(this compound is referred to as HEI2548 in the examples of the present invention)

(this compound is referred to as HEI2773 in the examples of the present invention)

(this compound is referred to as HEI 3127 in the examples of the present invention)

(this compound is referred to as HEI3204 in the examples of the invention)

As shown in the examples, the compounds of the present invention are P2RX7 modulators. They potentiate the effect of P2RX7 in the presence of ATP.

Thus, the present invention further relates to the use of a P2RX7 modulator (preferably a P2RX7 compound) that potentiates the effect of P2RX7 in the presence of ATP for the prevention and/or treatment of inflammatory diseases and/or cancer.

"P2 RX7 modulator" refers to a compound that is capable of inducing cell death in the presence of ATP through the large pore opening. "ATP" itself means ATP (adenosine triphosphate) itself, but also its derivatives, in particular its benzophenone derivatives, such as BzATP (3' -O- (4-benzoyl) benzoylATP). Preferably, the P2RX7 modulator is capable of increasing P2RX7 channel activity (i.e., Ca) following a brief (e.g., 5 to 15 minutes) stimulus²⁺And/or the entry of dyes such as Topro3) and induce cell death through the macroporous opening under longer (e.g., 1h) stimulation. Tests useful for determining whether a given compound is a P2RX7 modulator are presented in fig. 2, fig. 14 and fig. 15, and study 2 of example 2. The test is briefly summarized as follows:

cultured cells that do not express any P2X receptor are transfected with an expression vector encoding the mP2rx7 gene. The opening of the macropores was determined with a probe sufficient to measure cell death (e.g., Topro3 from Invitrogen). In each experiment, cells were treated with or without BzATP (3' -O- (4-benzoyl) benzoylatp) in the presence of the test compound. When indicated, a probe sufficient to measure cell death (e.g., Topro3) was added.

Fluorescence was measured by flow cytometry.

Each compound was tested in the absence or presence of BzATP.

A test compound is a P2RX7 modulator if it significantly increases the opening of the macropores (and thus cell death) in the presence of BzATP compared to BzATP alone and the test compound alone.

For example, compounds of formula (I), particularly compounds of formula (I'), are P2RX7 modulators, as they potentiate the effect of P2RX7 on the macropore in the presence of ATP, causing cell death. Preferably, they enhance P2RX7 on Ca in the presence of ATP²⁺Access and macropore opening.

The human P2RX7 protein is available in Uniprot under accession number Q99572.

Preferably, the compounds of formula (I) according to the invention are agonists of P2RX 7. An "agonist" of P2RX7 refers to a compound that binds to P2RX7 and activates the P2RX7 to generate a biological response. Potency of agonists and their EC₅₀The values are inversely proportional. For a given agonist, EC can be measured by determining the concentration of agonist required to elicit half of the maximal biological response of the agonist₅₀。

Preparation of the Compounds of the invention

The compounds of formula (I) (and (I '), (II')) of the present invention can be prepared by readily performed procedures. The process is as follows and as shown in example 1:

a mixture of a 5-oxopyrrolidine-2-carboxamide derivative or a 5-oxopyrrolidine-3-carboxamide derivative (and in the case of formula (II'), in particular N- (2, 4-dichlorobenzyl) -5-oxopyrrolidine- (2S) -carboxamide) and the corresponding isocyanate in a solvent such as toluene is stirred at reflux under a nitrogen atmosphere for a period of time (e.g. 1 to 24 hours).

The reaction scheme is as follows:

preferably, the reaction scheme to obtain the compound of formula (I') is as follows:

more preferably, the reaction scheme to obtain the compound of formula (II') (i.e. (S) enantiomer) is as follows:

the resulting mixture can then be purified, for example, by direct crystallization in methanol (MeOH) or on a silica column, to give the pure compound.

Therapeutic applications of the Compounds of the invention

As described above, and as shown in the examples, the inventors demonstrate that the compounds of the invention are useful in therapy.

The invention therefore also relates to the use of at least one compound of formula (I), preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof, as a medicament.

In particular, the compounds of formula (I), preferably of formula (I '), (II) or (II'), enantiomers thereof and/or pharmaceutically acceptable salts thereof, are useful for the prevention and/or treatment of inflammatory diseases and/or cancer.

"treatment" refers to the curative treatment of inflammatory diseases or cancer, respectively. Curative treatment is defined as treatment that completely treats (cures) or partially treats (i.e., induces tumor growth stabilization, developmental delay, or regression) inflammatory diseases or cancers, respectively.

The invention also relates to a product comprising:

a) at least one compound selected from the group consisting of compounds of formula (I), preferably compounds of formula (I '), (II) or (II'), enantiomers thereof and pharmaceutically acceptable salts thereof, and

b) at least one further therapy as a combination product for simultaneous, separate or sequential use in the treatment and/or prevention of cancer.

It also relates to the use of at least one compound of formula (I), preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof, in combination or association with at least one other therapy in the prevention and/or treatment of cancer.

It further relates to the use of at least one compound of formula (I) (preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of cancer in a subject treated with at least one other therapy. The term "subject" refers to any subject and generally refers to a patient, particularly a subject undergoing treatment for cancer (e.g., immunotherapy, chemotherapy, and/or radiation therapy).

The invention also relates to the use of a compound selected from at least one compound of formula (I), preferably a compound of formula (I '), (II) or (II'), enantiomers thereof and pharmaceutically acceptable salts thereof, as an adjunct cancer therapy. Adjuvant therapy is a therapy for treating cancer in addition to the primary therapy or initial therapy (referred to herein as "other therapy") that maximizes its therapeutic effect.

The other therapy b) may be immunotherapy, chemotherapy and/or radiotherapy. Preferably, the other therapy b) is immunotherapy and/or chemotherapy.

"immunotherapy" refers to a therapy capable of inducing, enhancing or suppressing an immune response. The immunotherapy is preferably selected from: cytokines, chemokines, growth factors, growth inhibitors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, monospecific antibodies, bispecific antibodies or multispecific antibodies, monomeric (monobody), multimeric (polybody), vaccination, or adoptive specific immunotherapy.

Preferably, the immunotherapy is selected from monoclonal or polyclonal antibodies, monospecific antibodies, bispecific antibodies or multispecific antibodies, monomers, multimers, such as anti-angiogenic agents like bevacizumab (mAb, VEGF-A inhibiting, Genentech), IMC-1121B (mAb, VEGFR-2 inhibiting, Imclone Systems), CDP-791(Pegylated DiFab, VEGFR-2, Celltech), 2C3(mAb, VEGF-A, Peregrine Pharmaceuticals), VEGF-trap (soluble hybrid receptor VEGF-A, PIGF (placental growth factor) Aventis/Regeneron).

Preferably, the immunotherapy is a monoclonal antibody, preferably an anti-checkpoint antibody.

Anti-checkpoint antibodies comprise antibodies directed against an immune checkpoint which may be selected from PD1, PDL1, PDL2, CTLA4, BTLA, CD27, CD40, OX40, GITR (also referred to as "tumor necrosis factor receptor superfamily member 18" or TNFRSF18), CD137 (also referred to as 4-1BB or TNFRS9), CD28, ICOS, IDO (indoleamine 2, 3-dioxygenase), B7H3 (also referred to as CD276), KIR2DL2 (also referred to as killer immunoglobulin-like receptor 2DL2), NKG2 (C-type lectin receptor family), LAG3 (also referred to as lymphocyte activator gene 3) and CD 70. Preferably, the anti-checkpoint antibody is an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-PDL 2 antibody or an anti-CTLA 4 antibody. anti-PD 1 antibodies include nivolumab (nivolumab) and pembrolizumab (pembrolizumab). anti-CTLA 4 antibodies include Epipilimumab (ipilimumab) and tremelimumab (tremelimumab).

Preferably, the invention also relates to a product comprising:

a) at least one compound selected from the group consisting of compounds of formula (I), preferably compounds of formula (I '), (II) or (II'), enantiomers thereof and pharmaceutically acceptable salts thereof, and

b) at least one further therapy selected from an anti-checkpoint antibody, preferably selected from an anti-PD 1 antibody, more preferably selected from nivolumab and pembrolizumab, as a combination product for simultaneous, separate or sequential use in the treatment and/or prevention of cancer.

"chemotherapy" or "chemotherapeutic agent" refers to a compound used to treat cancer and having functional properties that inhibit the development or progression of tumors, particularly malignant (cancerous) lesions, in humans.

Chemotherapeutic agents have different effects, for example, by affecting DNA or RNA and interfering with cell cycle replication.

Examples of chemotherapeutic agents that act at the DNA level or RNA level are:

-antimetabolites, such as azathioprine, cytarabine, fludarabine phosphate, fludarabine, gemcitabine, cytarabine, cladribine, capecitabine 6-mercaptopurine, 6-thioguanine, methotrexate, 5-fluorouracil and hydroxyurea;

alkylating agents, such as melphalan, busulfan, cisplatin, carboplatin, cyclophosphamide, ifosfamide, dacarbazine (dacarbazine), Fotemustine (Fotemustine), procarbazine, chlorambucil, thiotepa, lomustine, temozolomide;

antimitotic agents, such as vinorelbine, vincristine, vinblastine, docetaxel, paclitaxel;

-topoisomerase inhibitors, such as doxorubicin, amsacrine, irinotecan, daunorubicin, epirubicin, mitomycin, mitoxantrone, idarubicin, teniposide, etoposide, topotecan;

antibiotics, such as actinomycin and bleomycin;

-an asparaginase enzyme;

anthracyclines or taxanes.

Other chemotherapeutic agents are Tyrosine Kinase Inhibitors (TKIs). Many TKIs are under late and early development for the treatment of various types of cancer. Exemplary TKIs include, but are not limited to: BAY 43-9006(Sorafenib,) And SU11248(Sunitinib,)、Imatinib mesylate(Novartis)；Gefitinib(AstraZeneca)；Erlotinib hydrochloride(Genentech)；Vandetanib(AstraZeneca)、Tipifarnib(Janssen-Cilag)；Dasatinib(Bristol Myers Squibb)；Lonafarnib(Schering Plough)；Vatalanib succinate(Novartis,Schering AG)；Lapatinib(GlaxoSmithKline); nilotinib (novartis); lestaurtinib (Cephalon); pazopanib hydrochloride (GlaxoSmithKline); axitinib (pfizer); canertinib dihydrochloride (Pfizer); pelitinib (National Cancer Institute, Wyeth); tandutinib (millennium); bosutinib (wyeth); semaxanib (Sugen, Taiho); AZD-2171 (AstraZeneca); VX-680(Merck, Vertex); EXEL-0999 (Exelixis); ARRY-142886(Array BioPharma, AstraZeneca); PD-0325901 (Pfizer); AMG-706 (Amgen); BIBF-1120(Boehringer Ingelheim); SU-6668 (Taiho); CP-547632 (OSI); (AEE-788 (Novartis); BMS-582664(Bristol-Myers Squibb); JNK-401 (Celgene); R-788 (Rigel); AZD-1152HQPA (AstraZeneca); NM-3(Genzyme Oncolog); CP-868596 (Pfizer); BMS-599626(Bristol-Myers Squibb); PTC-299(PTC Therapeutics); ABT-869 (Abbott); EXEL-2880 (Exelixis); AG-024322 (Pfizer); XL-820 (Exelixis); OSI-930 (OSI); ABT-184 (Exelix); KRN-951(Kirin Brewery); CP-724714(OSI E-7080 (Eisisein I-ai) (Wsanth 272); CHIR-258 (Bayer-258; Boryeyne-WO-7692); Biotin AG-5952 (Adelyne); AGE-598 (AViserin), midostaurin (PKC412, Novartis); perifosine (AEterna Zentaris, Keryx, National Cancer Institute); AG-024322 (Pfizer); AZD-1152 (AstraZeneca); ON-01910Na (Onconova); and AZD-0530 (AstraZeneca).

The inflammatory disease is preferably a chronic inflammatory disease and may be selected from rheumatoid arthritis, crohn's disease, Inflammatory Bowel Disease (IBD), osteoarthritis, osteoporosis, dermatitis, psoriasis, asthma, respiratory distress syndrome and Chronic Obstructive Pulmonary Disease (COPD).

The cancer is for example selected from colon cancer, colorectal cancer, melanoma, breast cancer, thyroid cancer, prostate cancer, ovarian cancer, lung cancer, pancreatic cancer, glioma, cervical cancer, endometrial cancer, head and neck cancer, liver cancer, kidney cancer, skin cancer, stomach cancer, testicular cancer, urothelial cancer or adrenocortical cancer, and non-solid cancers such as lymphoma.

The cancer may or may not be metastatic cancer. The cancer may or may not be susceptible to immunotherapy. Preferably, the cancer is lung cancer, preferably Lewis lung cancer.

Also described herein is a method of preventing and/or treating cancer comprising administering to a subject in need thereof an effective amount of at least one compound of formula (I) as defined above (preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof.

The term "subject" refers to any subject and generally refers to a patient, particularly a subject undergoing a cancer treatment (such as immunotherapy, chemotherapy and/or radiotherapy), or a subject at risk of developing cancer or suspected of developing cancer.

Also described herein is a method of preventing and/or treating an inflammatory disease comprising administering to a subject in need thereof an effective amount of at least one compound of formula (I) as defined above (preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof.

The term "subject" refers to any subject and generally refers to a patient, particularly a subject undergoing treatment for, or at risk of developing, an inflammatory disease.

In any case, the subject is preferably a mammal, even more preferably a human, e.g. a human suffering from cancer or from an inflammatory disease.

The compounds of the present invention are preferably administered in a therapeutically effective amount or dose. As used herein, "therapeutically effective amount or dose" refers to an amount of a compound of the invention that prevents, eliminates, slows cancer or an inflammatory disease in a subject (preferably a human), or reduces or delays one or more symptoms or conditions caused by or associated with the disease. The effective amount of the compounds of the present invention and pharmaceutical compositions thereof, and more generally the dosage regimen, can be determined and adjusted by those skilled in the art. The effective dose can be determined by using conventional techniques and by observing the results obtained under similar circumstances. The therapeutically effective dose of the compounds of the present invention will vary depending on the disease to be treated or prevented, its severity, the route of administration, any combination therapy involved, the age, weight, general medical condition, medical history of the patient, and the like.

Generally, for human patients, the amount of compound administered may be from about 0.01 to 500mg/kg body weight. In a specific embodiment, the pharmaceutical composition of the invention comprises 0.01mg/kg to 300mg/kg, preferably 0.01mg/kg to 3mg/kg, for example 25 to 300mg/kg of a compound of the invention.

In particular aspects, the compounds of the invention can be administered to a subject by parenteral route, topical route, oral route, or intravenous injection. The compounds or nanoparticles of the invention may be administered to a subject daily (e.g., 1,2, 3,4, 5, 6, or 7 times per day) over a period of several consecutive days, e.g., 2 to 10 consecutive days (preferably 3 to 6 consecutive days). The treatment may be repeated over a period of 1,2, 3,4, 5, 6 or 7 weeks, or every two or three weeks or months. Alternatively, multiple treatment cycles may be performed, optionally with an interruption time of, for example, 1,2, 3,4, or 5 weeks between two treatment cycles. The compounds or nanoparticles of the invention may be administered, for example, in a single dose once a week, once every two weeks, or once a month. The treatment may be repeated one or more times per year.

The doses are administered at appropriate intervals as can be determined by the skilled person. The choice of amount will depend on a number of factors including: the route of administration, the duration of administration, the number of administrations, the elimination rate of the selected compound of formula (I) or of the various products used in combination with said compound, the age, weight and physical condition of the patient and his/her medical history, as well as any other information known medically.

The route of administration may be oral, topical or parenteral, and is typically rectal, sublingual, intranasal, Intraperitoneal (IP), Intravenous (IV), Intraarterial (IA), Intramuscular (IM), intracerebral, intrathecal, intratumoral and/or intradermal. The pharmaceutical compositions are suitable for one or more of the above routes. The pharmaceutical compositions are preferably administered by injection or intravenous infusion of a suitable sterile solution, or in the form of a liquid or solid dose through the digestive tract.

Another object of the present invention is a composition comprising at least one compound of formula (I), preferably at least one compound of formula (I '), (II) or (II'), an enantiomer thereof or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical compositions may be formulated as solutions in pharmaceutically compatible solvents, or as gels, oils, emulsions, suspensions or dispersions in suitable pharmaceutical solvents or carriers, or as pills, tablets, capsules, powders, suppositories and the like containing solid carriers in a manner known in the art, possibly by providing sustained and/or delayed release dosage forms or devices. For this type of formulation, agents such as cellulose, lipids, carbonates or starches are advantageously used.

The agents or carriers that can be used in the formulations (liquid and/or injectable and/or solid) are excipients or inert carriers, i.e. pharmaceutically inactive and non-toxic carriers.

Mention may be made, for example, of saline, physiological, isotonic and/or buffer solutions compatible with pharmaceutical use and known to the person skilled in the art. The composition may comprise one or more agents or carriers selected from dispersants, solubilizers, stabilizers, preservatives, and the like.

Specific examples are methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, cyclodextrin, polysorbate 80, mannitol, gelatin, lactose, liposomes, vegetable or animal oils, gum arabic, and the like. Preferably, vegetable oils are used.

Formulations of the invention suitable for oral administration may be in the form of discrete units such as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient, in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid or in the form of an oil-in-water or water-in-oil emulsion.

Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Each such formulation may also contain other pharmaceutically compatible and non-toxic adjuvants such as stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring agents.

Drawings

The drawings used in this application are as follows and are for illustration purposes only.

FIG. 1: HEI2314 and HEI2328 increased macropore opening in the presence of ATP in a heterologous cell line expressing mouse P2RX 7.

Calcium channel activity (Fluo4AM probe) and the macropore opening (Topro3 probe) were analyzed by FACS on mP2RX7 HEK cells. Cells were unstimulated (NS), or treated with BzATP (500 μ M), either alone or in the presence of HEI2314, HEI2328, HEI2333, or HEI2336(50 μ M). Each panel shows a representative dot plot, and the red numbers indicate the percentage of positive cells in each sub-panel.

FIG. 2: mP2RX7 and ATP mediated macropores are required to respond to HEI 2328.

Calcium channel activity (Fluo4AM probe) and the macropore opening (Topro3 probe) were analyzed by FACS on mock and mP2RX7 HEK cells. Cells were unstimulated (NS), or treated with BzATP (500 μ M), either alone or in the presence of 50 μ M HEI 2328. Each panel shows a representative dot plot, and the red numbers indicate the percentage of positive cells in each sub-panel. The histogram shows the results of 9 independent experiments. P <0.001 (mann-whitney test).

FIG. 3: HEI2328 significantly ameliorated DSS-induced colitis in vivo.

C57BL/6J treated with 3% DSS over 5 days (J1 to J5) followed by 10 days with normal tap water (A) weight loss and (B) clinical score. The treatment group received HEI2328(ip, 2.3mg/kg in PBS, 10% DMSO). Data are representative of 2 independent experiments (n-20). Mean ± SEM are shown. P < 0.05; p <0.01 (mann-whitney test).

FIG. 4: HEI2328 significantly reduced inflammatory lesions in the colon of DSS-treated mice.

The inflammation index is scored in longitudinal (a) and transverse (B) sections of the colon. Data are representative of 2 independent experiments (n-20). Mean ± SEM are shown. P < 0.05; p <0.01 (mann-whitney test).

FIG. 5: HEI2328 reduced the rate of inflammatory monocytes in the spleen.

(A-D) phenotypic analysis of spleen immune cells from 20 mice treated or untreated with HEI 2328. Data are representative of 2 independent experiments (n-20). Mean ± SEM are shown. P < 0.05; p <0.01 (mann-whitney test).

FIG. 6: HEI2328 increased macropore opening in the B16F10 melanoma cell line in the presence of ATP.

Calcium channel activity (Fluo4AM probe) and the large pore opening (Topro3 probe) were analyzed by FACS. Cells were analyzed unstimulated (NS), or treated with HEI2328 (50. mu.M) or BzATP (500. mu.M) alone. (A) Representative dot plots. The numbers highlight the percentage of positive cells. (B) The histogram shows the results of 3 independent experiments. P <0.05 (mann-whitney test).

FIG. 7: HEI2328 reduced tumor growth in vivo.

Will be 5X 10⁵The B16F10 cells were injected subcutaneously into the right flank of C57BL/6 mice. Mice received either HEI2328 (n-8, ip, 2.3mg/kg in 10% DMSO/PBS) or placebo (n-8, 10% DMSO/PBS) daily. A. Incidence of tumor appearance b. tumor size (mm2) and c. tumor weight at day of sacrifice (J12). P <0.05 (mann-whitney test).

FIG. 8: HEI3090 increased the macropore opening in the presence of BzATP.

A.From control or p2rx7^{exon2 KO}Splenocytes from mice were isolated and treated with HEI3090(50 μ M) in the presence or absence of BzATP (500 μ M). B. Macropore opening induced by increasing BzATP concentration in human P2RX7 expressing cells in the presence or absence of HEI 3090.

FIG. 9: HEI3090 induced B16F10 cell death in the presence of BzATP.

A. The large hole is open. Dot plots of representative experiments. The numbers highlight the percentage of positive cells. The histogram shows the results of 3 independent experiments. P <0.05 man-whitney test. Representative curve of xtt absorbance over time. The histogram shows the percentage of dead cells. Mean ± SEM are shown. P < 0.001; p <0.0001 mann-whitney test.

FIG. 10: HEI3090 is a potent mouse tumor inhibitor.

Will be 5X 10⁵LLC or 5 x 10⁵The B16F10 cells were injected subcutaneously into the right flank of C57BL/6 mice. Mice received HEI3090(ip, 2.5mg/kg in 10% DMSO/PBS) or placebo (10% DMSO/PBS) daily. A, c. incidence of tumor appearance B, d. tumor size (mm2) and tumor weight on day of sacrifice (J12). Hei treated tumors were more immunogenic. Dot plots show LLC (black) and B16F10 (grey) tumor vs CMH1 (H2K)^b/D^b) And expression of PD-L1. Number highlighting the pair H2K^b/D^bPercentage of tumor cells positive for and PD-L1. The histogram shows mean ± SEM. Cd-45 positive infiltration was increased in LLC and B16F10 tumors. A, P<0.05; p <0.001 mann-whitney test.

FIG. 11: the HEI3090 modified tumors infiltrated the environment with less immunosuppression.

Will be 5X 10⁵LLC or 5 x 10⁵The B16F10 cells of (a) were injected subcutaneously into the right flank of C57BL/6 mice. Mice received HEI3090(ip, 2.5mg/kg in 10% DMSO/PBS) or placebo (10% DMSO/PBS) daily. A. The number of immunosuppressive cells is reduced. Dot plots of representative experiments. The numbers highlight the percentages of M-MDSC and PMN-MDSC. The histogram shows the results for 8 mice. B. The number of effector T cells (T γ δ, NK, CD4+) was increased.The histogram shows the results for 8 mice. A, P<0.05; p <0.01 mann-whitney test.

FIG. 12: t CD4+ cells are part of the essential material mediating the anti-tumor effect of HEI 3090.

Will be 5X 10⁵The LLC cells were injected subcutaneously into the right flank of C57BL/6 mice. Mice received HEI3090(ip, 2.5mg/kg in 10% DMSO/PBS) or placebo (10% DMSO/PBS) either alone or in the presence of anti-CD 4 antibody daily. A. Tumor growth was followed daily in 3 groups. B. Tumor weight on sacrifice day (J12). A, p<0.05; p <0.0001 (mann-whitney test).

FIG. 13: ADME (absorption, distribution, metabolism and excretion) studies.

HEI-2328 and-3090 are stable molecules. Mouse liver microparticles from BD Gentest (ref 5345001) were incubated with 1. mu.M HEI-2314, -2328, -2333 and-3090 for 0, 5, 10, 20, 30 and 40 minutes. The stability of the compounds was analyzed by mass spectrometry.

B. HEI3090 was not found in plasma until 18h after intraperitoneal administration. Intraperitoneal administration of HEI-3090 (0.166 mg/mL in 10% DMSO) was performed and sera were collected at 0, 10, 20, 30, 0, 60, 120, 240, 360, 1080 and 1440 minutes. Plasma concentrations were determined by LC-MS/MS and reached a maximum of 2.5. mu.M.

FIG. 14: HEI-3090 is a positive allosteric modulator of mouse P2RX 7.

Calcium influx (Fluo-AM positive cells) and large cation infiltration (Topro3 positive cells) were determined by FACS 15 minutes after stimulation of HEK cells transfected with mouse P2RX7 treated with increasing doses of BzATP.

In the presence of HEI3090, EC50 was reduced by 22%.

FIG. 15: HEI-3090 is a positive allosteric modulator of human P2RX 7.

Calcium influx (Fluo-AM positive cells) and macrocation infiltration (Topro3 positive cells) were determined by FACS 15 minutes after stimulation of HEK cells transfected with increasing doses of HEI3090 treated human P2RX7 in the presence of 7.8 μ M BzATP. In the presence of HEI3090, both Ca and bulk cation influx were increased by 3-fold, indicating that this molecule is a positive allosteric modulator.

FIG. 16: HEI3090 enhances its anti-tumor effect by stimulating P2RX 7-expressing immune cells rather than Immunogenic Cell Death (ICD).

In vivo assays for evaluating HEI3090 as an inducer of immunogenic cell death.

To induce B16-F10 death, cells were exposed to 3mM ATP and 50 μ M of HEI3090 and 1.105 dying cells or PBS were injected subcutaneously into the right flank (n-4) as controls. 7 days later, 0.5.105 live B16-F10 cells (a) were injected subcutaneously in the contralateral flank. The curves show the mean value of tumor growth at the contralateral site (b).

(c-f) HEI3090 vs. p2rx7 deficient mice (p2rx 7)^-/-) The effect of tumor growth. 5.105B16-F10(c) or 5.105LLC (d) was injected subcutaneously into p2rx7^-/-Hypochondriac regions of mice. Mice received intraperitoneal treatment with vehicle (PBS, 10% DMSO) or HEI3090 (1.5 mg/kg in PBS, 10% DMSO) from day 1 to day 12. (e, f) p2rx7^-/-Mice were adoptively transferred with 5.106 splenocytes from WT C57BL/6J mice and injected subcutaneously into the flank with 5.105 LLC. The mice then received intraperitoneal treatment with vehicle (PBS, 10% DMSO) or HEI3090 (1.5 mg/kg in PBS, 10% DMSO) from day 1 to day 12.

FIG. 17: HEI3090 stimulated potent function of NK cells and CD4+ T cells (supplementary results of fig. 12).

a. Phenotypic flow cytometric analysis of tumor infiltrating cells. LLC tumors were collected on day 12. Ratio of PMN-MDSC in CD45+ within LLC tumors from mice treated with vehicle or HEI3090 (a). Ratio of NK, CD4+ or CD8+ T cells on PMN-MDSC (b).

c. d.5.105LLC was injected subcutaneously into the flank of C57BL6/J mice. The mice were then treated intraperitoneally with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO) or with HEI3090 and depleted of antibodies to NK1.1, CD4+ or CD8+ T cells. Antibodies were injected intraperitoneally every 3 days (200. mu.g/injection). Day 12 summary (Spaghetti plot) and tumor weight (d). Each curve represents one mouse.

e-g functional flow cytometry analysis of tumor infiltrating immune cells or spleen cells. Tumors were stimulated with PMA/ionomycin and simultaneously analyzed for IFN-. gamma.and IL-10 production in CD45+, NK, CD4+, or CD8+ T cells. The figure shows the ratio of IFN-. gamma.producing cells in CD45+ cells or the geometric mean of fluorescence (e). Representative dot plots of IFN-. gamma.and IL-10 stained into TIL (f). The ratio of IFN- γ on IL-10 positive cells per TIL. Degranulation assay of splenocytes from LLC tumor-bearing mice treated with vehicle or HEI3090 (g) splenocytes were re-stimulated in vitro with LLC and displayed CD107a positive cells among NK, CD4+ and CD8+ T cells.

FIG. 18: HEI3090 induces IL-18 production in vivo.

Flow cytometry analysis of P2RX7 expression in conventional dendritic cells CD4+ or CD8+ within llc tumors.

b. The protein levels of IL-1. beta. and IL-18 in the serum of LLC tumor-bearing mice treated with vector or HEI3090 were determined by ELISA.

c.5.105LLC was injected subcutaneously into the flank of C57BL6/J mice. The mice then received intraperitoneal injections from day 1 to day 12 with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO) or with HEI3090 and neutralized antibodies to IL-1 β or IL-18. Antibodies were injected intraperitoneally every 3 days (200. mu.g/injection).

d. Flow cytometric analysis of IFN-. gamma.and IL-10 production by intratumoral NK, CD4+ T or CD8+ T cells. The figure presents the ratio of IFN- γ on IL10 positive cells.

HEI3090 in IL-18 deficient mice (IL-18)^-/-) Effects of tumor growth. 5.105LLC injection into IL-18^-/-Hypochondriac regions of mice. Mice received intraperitoneal treatment with vehicle (PBS, 10% DMSO) or HEI3090 (1.5 mg/kg in PBS, 10% DMSO) from day 1 to day 12.

f. IL-18IHC staining in 4 LLC tumors of C57BL6/J mice treated with vehicle and IL-18IHC staining in 4 LLC tumors of C57BL6/J mice treated with HEI 3090.

FIG. 19: the combination of HEI3090 and anti-PD-1 immune checkpoint inhibitors cured LLC tumor-bearing mice (supplementary results of fig. 10).

a. Flow cytometry analysis of MHC-1 and PD-L1 expression on CD45 negative cells in LLC tumors from mice treated with vector or HEI 3090.

Effect of b-g HEI3090 and anti-PD-1 checkpoint inhibitors on LLC tumor growth. 5.105LLC was injected subcutaneously into the flank of C57BL/6J mice. Mice received intraperitoneal treatment with vehicle (PBS, 10% DMSO) or HEI3090 (1.5 mg/kg in PBS, 10% DMSO) from day 1 to day 18. Each mouse received 200. mu.g of anti-PD-1 intraperitoneal injection on days 4, 7, 10, 13 and 16. Tumor size was measured from day 1 to day 90. Mice were sacrificed when tumors reached 100mm 2. For contralateral re-challenge, cured mice or young matched C57BL/6J mice were injected subcutaneously with 2.105LLC at the opposite flank. On day 150, the re-challenged mice or C57BL/6J young mice were sacrificed to sort CD8+ T cells. 2.105CD8+ T cells were adoptively transferred to other C57BL/6J young mice. The mice were then injected subcutaneously with 2.105llc (b). Schematic of LLC tumor growth of vehicle and anti-PD-1 treated mice or HEI3090 and anti-PD-1 treated mice (c). Each curve represents one mouse. Survival curves of vehicle or HEI3090 and isotype control or vehicle or HEI3090 and anti-PD-1 treated mice (d). Tumor growth and survival curves after re-challenge of cured HEI3090 and anti-PD-1 treated mice or age-matched C57BL/6J mice (e, f). Tumor growth (g) after intravenous injection of CD8+ T cells in young mice or re-challenged treated mice.

Detailed Description

Examples

Example 1: synthesis of compounds HEI2314, HEI2328, HEI2333, HEI2336 and HEI3090

General procedure for the synthesis of compounds of the invention:

a mixture of N- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-2-carboxamide (1 equivalent) and the corresponding isocyanate (1 equivalent) in toluene was stirred under nitrogen at reflux for 1-24 h. The resulting mixture was purified by direct crystallization in MeOH to afford the pure compound.

The reaction scheme is as follows:

a)synthesis of compound HEI 2347:

(S)-N²- (2, 4-dichlorophenyl) -5-oxo-N¹-phenylpyrrolidine-1, 2-dicarboxamide (HEI _ 2347). The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (0.90g, 3.3mmol) and phenyl isocyanate (0.39g, 3.3 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2347(1.05g,2.7mmol) in 81% yield. mp 214 + 217 deg.C (MeOH); TLC Rf (CH)₂Cl₂/MeOH:98/2)0.9；¹H NMR(DMSO-d₆,400MHz)δppm 1.98-2.10(m,1H,CH₂CH₂CH),2.38-2.48(m,1H,CH₂CH₂CH),2.60-2.81(m,2H,CH₂CH₂CH),5.08(dd,J＝5.6,2.4Hz,1H,CH₂CH₂CH),7.10(t,J＝7.2Hz,1H,ArH),7.34(t,J＝7.2Hz,2H,ArH),7.42(dd,J＝8.8,2.4Hz,1H,ArH),7.51(d,J＝7.2Hz,2H,ArH),7.68(d,J＝2.4Hz,1H,ArH),7.73(d,J＝8.8Hz,1H,ArH),10.08(br s,1H,NH),10.51(br s,1H,NH)；¹³C NMR(DMSO-d₆,100MHz)δppm 21.4(CH₂),31.7(CH₂),58.8(CH),119.6(2CH),123.9(CH),127.3(CH),127.5(CH),127.6(CH),129.0(C),129.1(2CH),129.7(C),133.6(C),137.2(C),149.3(C),170.2(C),177.8(C)；LC-MS(APCI⁺)m/z:392.1(MH⁺),tr4.38min；IRν(cm^-1):3275,2922,1723,1693,1662,1559,1473,1219,1100,819；C₁₈H₁₅Cl₂N₃O₃C, 55.12; h, 3.85; n, 10.71. Actually measuring as follows: c, 54.91; h, 3.89; n,10.75 percent.

b)Synthesis of compound HEI 2346:

(S)-N²- (cyclohexylmethyl) -5-oxo-N¹-phenylpyrrolidine-1, 2-dicarboxamide (HEI _ 2346). The general procedure was followed using (S) -N-cyclohexylmethyl-5-oxo-pyrrolidine-2-carboxamide (0.40g, 1.8mmol) and phenyl isocyanate (0.21g, 1.8 mmol). The desired product was purified on a silica gel column (EtOAc/n-heptane 4/6 to 5/5) to give pure HEI _2346(1.05g, 8.8mmol) in 88% yield as a white solid. mp 122-127 deg.C (EtOAc/n-heptane); TLC Rf (EtOAc/n-heptane: 1/1) 0.3;¹H NMR(CDCl₃400MHz) delta ppm 0.85-0.97(m,2H, cyclohexane-H), 1.08-1.28(m,3H, cyclohexane-H), 1.45-1.46(m,1H, cyclohexane-H), 1.60-1.74(m,5H, cyclohexane-H), 2.15-2.25(m,1H, CH)₂CH₂CH),2.30-2.40(m,1H,CH₂CH₂CH),2.55-2.62(m,1H,CH₂CH₂CH),3.00-3.20(m,3H,NHCH₂ and CH₂CH₂CH),4.79(dd,J＝8.4,1.6Hz,1H,CH₂CH₂CH),6.59(s,1H,NHCH₂),7.11(t,J＝7.6Hz,1H,ArH),7.32(t,J＝7.6Hz,2H,ArH),7.47(d,J＝7.6Hz,2H,ArH),10.56(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.6(CH₂),25.9(2CH₂),26.4(CH₂),30.8(2CH₂),32.8(CH₂),38.0(CH₂),46.0(CH),59.4(CH),120.5(2CH),124.6(CH),129.1(2CH),137.1(C),150.6(C),170.3(C),177.8(C)；LC-MS(APCI⁺)m/z:344.2(MH⁺),tr4.16min；IRν(cm^-1):3289,2922,2851,1718,1654,1552,1446,1217,747；C₁₉H₂₅N₃O₃C, 66.45; h, 7.34; and N, 12.24. Actually measuring as follows: c, 66.71; h, 7.42; n,11.68 percent.

c)Synthesis of compound HEI 2269:

(S)-N²-benzyl-5-oxo-N¹-phenylpyrrolidine-1, 2-dicarboxamide (HEI — 2269). The general procedure was followed using (S) -N-benzyl-5-oxo-pyrrolidine-2-carboxamide (0.35g, 1.4mmol) and phenyl isocyanate (0.15g, 1.4 mmol). The desired product is in silica gelColumn (CH)₂Cl₂MeOH 1/0 to 98/2) to give pure HEI _2269(0.33g, 1.0mmol) as a 70% yield white solid. mp 164-₂Cl₂/MeOH)；TLC Rf(CH₂Cl₂/MeOH:97/3)0.2；¹H NMR(DMSO-d₆,400MHz)δppm 1.90-2.10(m,2H,CH₂CH₂CH),2.22-2.40-2.42(m,2H,CH₂CH₂CH),4.27(m,3H,CH₂CH₂CH and NHCH₂),7.25(m,3H,ArH),7.28-7.41(m,5H,ArH),7.47(t,J＝7.6Hz,2H,ArH),8.39(t,J＝5.2Hz,1H,NHCH₂),8.51(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm 27.4(CH₂),30.1(CH₂),42.1(CH₂),55.7(CH),126.7(CH),126.8(2CH),127.2(2CH),127.7(CH),128.3(2CH),128.7(2CH),132.2(C),139.5(C),155.6(C),171.0(C),173.1(C)；LC-MS(APCI⁺)m/z:338.2(MH⁺),tr 3.07min；IRν(cm^-1):3367,3183,1705,1645,1416,1175,701；C₁₉H₁₉N₃O₃Analytically calculated as C, 67.64; h, 5.68; n, 12.45. Actually measuring as follows: c, 67.72; h, 5.82; and N, 12.13%.

d)Synthesis of compound HEI 2298:

(S)-N¹adamantyl-N²- (2-chlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2298). The general procedure was followed using (S) -N- (2-chlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.43g, 5.6mmol) and adamantane isocyanate (1.00g, 5.6 mmol). The desired product was purified on a silica gel column (EtOAc/n-heptane 4/6 to 6/4) to give pure HEI _2298(1.50g, 3.5mmol) in 62% yield as a white solid. mp 79-80 deg.C (EtOAc/n-heptane); TLC Rf (CH)₂Cl₂/MeOH:98/2)0.6；¹H NMR(CDCl₃,400MHz)δppm 1.68(s,6H,ADM-H),1.98(s,6H,ADM-H),2.09(s,3H,ADM-H),2.10-2.18(m,1H,CH₂CH₂CH),2.38-2.45(m,1H,CH₂CH₂CH),2.46-2.52(m,1H,CH₂CH₂CH),2.89-2.97(m,1H,CH₂CH₂CH),4.51(dd,J＝14.2,5.0Hz,1H,NHCH₂),4.55(dd,J＝14.2,5.0Hz,1H,NHCH₂),4.76(dd,J＝8.8,1.6Hz,1H,CH₂CH₂CH),7.14(t, J ═ 5.0Hz,1H, ArH),7.21(m,2H, ArH),7.35(m,2H, ArH and NHCH)₂),8.39(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.1(CH₂),29.5(2CH₂),32.9(CH₂),36.4(2CH₂),41.6(CH₂),41.7(2CH₂),51.9(CH),59.0(CH),127.1(2CH),128.9(2CH),129.6(2CH),129.7(C),133.6(C),135.4(C),151.4(C),170.5(C),177.3(C)；LC-MS(APCI⁺)m/z:430.1(MH⁺),tr 4.60min；IRν(cm^-1):3289,2906,2849,1715,1663,1538,1223,1033,749；C₂₃H₂₈ClN₃O₃C,64.25 by analysis; h, 6.56; and N, 9.77. Actually measuring as follows: c, 64.32; h, 6.89; and N, 9.58%.

e)Synthesis of compound HEI 2535:

(S)-N¹-butyl-N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2535).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.97g, 6.9mmol) and N-butyl isocyanate (0.75g, 7.6 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2535(1.89g,4.9mmol) in 71% yield. mp 162-; TLC Rf (CH)₂Cl₂/MeOH:99/1)0.4；¹H NMR(CDCl₃,400MHz)δppm 0.92(t,J＝7.4Hz,3H,CH₃CH₂),1.35(m,2H,CH₃CH₂) 1.51 (quintuple, J ═ 7.8Hz,2H, CH)₂CH₂CH₂),2.12(td,J＝18.0,11.3,8.7Hz,1H,CH₂CH₂CH),2.39(m,1H,CH₂CH₂CH),2.49(td,J＝17.7,9.4,2.0Hz,1H,CH₂CH₂CH),2.92(td,J＝17.5,11.3,9.4Hz,1H,CH₂CH₂CH),3.27(dq,J＝7.1,1.6Hz,2H,CH₂NHCO),4.47(dd,J＝6.0,2.8Hz,2H,NHCH₂Ar),4.75(dd,J＝9.0,1.6Hz,1H,CH₂CH₂CH),7.08(br t,J＝5.7Hz,1H,NHCH₂Ar),7.20(dd,J＝8.2,2.3Hz,1H,ArH),7.29(d,J＝8.2Hz,1H,ArH),7.36(d,J＝2.0Hz,1H,ArH),8.43(br t,J＝5.9Hz,1H,NHCH₂CH₂)；¹³C NMR(CDCl₃,100MHz)δppm 13.7(CH₃),20.0(CH₂),21.1(CH₂),31.6(CH₂),32.5(CH₂),39.7(CH₂),41.1(CH₂),59.0(CH),127.3(CH),129.3(CH),130.6(CH),133.9(C),134.0(C),134.1(C),153.1(C),170.3(C),177.2(C)；IRν(cm^-1):3270,1712,1683,1649,1465,1236,1101,810,653；C₁₇H₂₁Cl₂N₃O₃Analytically calculated as C, 52.86; h, 5.48; n, 10.88. Actually measuring as follows: c, 52.50; h, 5.88; n, 11.11%.

f)Synthesis of compound HEI 2541:

(S)-N²- (2, 4-dichlorobenzyl) -5-oxo-N¹-pentylpyrrolidine-1, 2-dicarboxamide (HEI _ 2541).

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.73g, 6.0mmol) and N-pentyl isocyanate (0.75g, 6.6 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2541(1.67g,4.2mmol) in 70% yield. mp 159 and 160 deg.C (MeOH); TLC Rf (CH)₂Cl₂/MeOH:99/1)0.7；¹H NMR(CDCl₃,400MHz)δppm 0.96(t,J＝6.9Hz,3H,CH₃CH₂),1.39(m,4H,CH₂(CH₂)₂CH₂) 1.59 (quintuple, J ═ 7.3Hz,2H, CH)₃CH₂),2.19(m,1H,CH₂CH₂CH),2.41(m,1H,CH₂CH₂CH),2.56(td,J＝17.6,9.4,1.9Hz,1H,CH₂CH₂CH),2.99(td,J＝17.6,11.3,9.3Hz,1H,CH₂CH₂CH),3.32(qd,J＝7.4,2.8Hz,2H,CH₂(CH₂)₃),4.54(dd,J＝7.5,6.3Hz,2H,NHCH₂Ar),4.82(dd,J＝9.1,1.6Hz,1H,CH₂CH₂CH),7.18(br t,J＝6.0Hz,1H,NHCH₂Ar),7.26(dd,J＝8.2,2.4Hz,1H,ArH),7.36(d,J＝8.2Hz,1H,ArH),7.43(d,J＝2.3Hz,1H,ArH),8.51(br t,J＝5.7Hz,1H,NH(CH₂)₂)；¹³C NMR(CDCl₃,100MHz)δppm 13.9(CH₃),21.2(CH₂),22.3(CH₂),29.0(CH₂),29.2(CH₂),32.5(CH₂),40.0(CH₂),41.1(CH₂),59.0(CH),127.3(CH),129.3(CH),130.5(CH),133.9(C),134.1(C),153.1(C),170.4(C),177.2(2C)；IRν(cm^-1):3318,1710,1664,1561,1530,1358,1259,830,630；C₁₈H₂₃Cl₂N₃O₃Analytically calculated as C, 54.01; h, 5.79; n, 10.50. Actually measuring as follows: c, 53.70; h, 5.95; n,10.59 percent.

g)Synthesis of compound HEI 2534:

(S)-N²- (2, 4-dichlorobenzyl) -N¹-hexyl-5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2534)

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.54g, 5.4mmol) and N-hexyl isocyanate (0.75g, 5.9 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2534(1.47g,3.5mmol) in 66% yield. mp 141-; TLC Rf (CH)₂Cl₂/MeOH:99/1)0.4；¹H NMR(CDCl₃,400MHz)δ0.90(t,J＝6.9Hz,3H,CH₃CH₂),1.32(m,6H,CH₂(CH₂)₃CH₂),1.54(q,J＝6.7Hz,2H,CH₃CH₂),2.14(m,1H,CH₂CH₂CH),2.41(m,1H,CH₂CH₂CH),2.52(td,J＝17.5,9.3,2.0Hz,1H,CH₂CH₂CH),2.94(td,J＝17.9,11.4,9.6Hz,1H,CH₂CH₂CH),3.82(dq,J＝7.3,1.7Hz,2H,CH₂(CH₂)₄),4.50(dd,J＝5.8,4.5Hz,2H,NHCH₂Ar),4.77(dd,J＝8.6,1.4Hz,1H,CH₂CH₂CH),7.11(br t,J＝5.5Hz,1H,NHCH₂Ar),7.22(dd,J＝8.2,2.0Hz,1H,ArH),7.31(d,J＝8.2Hz,1H,ArH),7.39(d,J＝2.1Hz,1H,ArH),8.46(br t,J＝5.7Hz,1H,NH(CH₂)₂)；¹³C NMR(CDCl₃,100MHz)δppm 14.0(CH₃),21.1(CH₂),22.5(CH₂),26.5(CH₂),29.5(CH₂),31.4(CH₂),32.5(CH₂),40.1(CH₂),41.1(CH₂),59.0(CH),127.3(CH),129.3(CH),130.6(CH),133.9(C),134.0(C),134.1(C),153.1(C),170.3(C),177.2(C)；IRν(cm^-1):3313,2926,2865,1708,1664,1355,1253,1153,829,654；C₁₉H₂₅Cl₂N₃O₃Analytically calculated as C, 55.08; h, 6.08; n, 10.14. Actually measuring as follows: c, 55.02; h, 6.15; n, 10.27%.

h)Synthesis of compound HEI 2542:

(S)-N²- (2, 4-dichlorobenzyl) -N¹Dodecyl-5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2542)

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (0.93g, 3.2mmol) and N-dodecyl isocyanate (0.75g, 3.5 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2542(0.78g,1.6mmol) in 49% yield. mp 124-; TLC Rf (CH)₂Cl₂/MeOH:99/1)0.8；¹H NMR(CDCl₃,400MHz)δppm 0.88(t,J＝6.6Hz,3H,CH₃CH₂),1.26(m,18H,CH₂(CH₂)₉CH₂) 1.53 (quintuple, J ═ 6.7Hz,2H, CH)₃CH₂),2.13(m,1H,CH₂CH₂CH),2.40(m,1H,CH₂CH₂CH),2.51(td,J＝17.6,9.3,1.8Hz,1H,CH₂CH₂CH),2.93(td,J＝17.7,11.3,9.5Hz,1H,CH₂CH₂CH),3.29(q,J＝7.2Hz,2H,CH₂(CH₂)₁₀),4.48(dd,J＝6.2Hz,2H,NHCH₂Ar),4.76(dd,J＝8.9,1.2Hz,1H,CH₂CH₂CH),7.11(br t,J＝5.9Hz,1H,NHCH₂Ar),7.21(dd,J＝8.3,2.2Hz,1H,ArH),7.30(d,J＝8.2Hz,1H,ArH),7.37(d,J＝2.1Hz,1H,ArH),8.45(br t,J＝6.0Hz,1H,NH(CH₂)₂)；¹³C NMR(CDCl₃,100MHz)δppm 14.1(CH₃),21.1(CH₂),22.7(CH₂),26.9(CH₂),29.3(CH₂),29.3(CH₂),29.5(CH₂),29.5(CH₂),29.6(CH₂),29.6(CH₂),29.6(CH₂),31.9(CH₂),32.5(CH₂),40.1(CH₂),41.1(CH₂),59.0(CH),127.3(CH),129.3(CH),130.5(CH),133.9(C),133.9(C),134.1(C),153.1(C),170.3(C),177.2(C)；IRν(cm^-1):3302,2918,2850,1718,1658,1541,1349,1251,832,648；C₂₅H₃₇Cl₂N₃O₃C, 60.2; h, 7.48; n, 8.43. Actually measuring as follows: c, 59.8; h, 7.5; and N, 8.5%.

i)Synthesis of compound HEI 2537:

(S)-N²- (2, 4-dichlorobenzyl) -5-oxo-N¹- (propan-2-yl) pyrrolidine-1, 2-dicarboxamide (HEI _ 2537).

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (2.30g, 8.0mmol) and propan-2-isocyanate (0.75g, 8.8 mmol). The desired product was purified on a silica gel column (EtOAc/n-heptane 1/9 to 1/0) to give pure HEI _2537(1.65g, 4.4mmol) in 55% yield as a yellow solid. mp 111-; TLC Rf (CH)₂Cl₂/MeOH:99/1)0.4；¹H NMR(CDCl₃,400MHz)δppm 1.20(dd,J＝6.5,2.7Hz,6H,(CH₃)₂CH),2.12(td,J＝18.1,11.4,9.0Hz,1H,CH₂CH₂CH),2.38(m,1H,CH₂CH₂CH),2.50(td,J＝17.5,9.3,1.8Hz,1H,CH₂CH₂CH),2.86(td,J＝17.6,11.4,9.4Hz,1H,CH₂CH₂CH),3.95(m,1H,(CH₃)₂CH),4.48(d,J＝6.2Hz,2H,NHCH₂),4.76(dd,J＝8.6,1.4Hz,1H,CH₂CH₂CH),7.18(br t,J＝6.2Hz,1H,NHCH₂),7.20(dd,J＝8.3,2.2Hz,1H,ArH),7.30(d,J＝8.0Hz,1H,ArH),7.37(d,J＝1.8Hz,1H,ArH),8.33(br t,J＝5.7Hz,1H,NHCH(CH₃)₂)；¹³C NMR(CDCl₃,100MHz)δppm 21.1(CH₂),22.6(CH₃),22.7(CH₃),32.6(CH₂),41.1(CH₂),42.4(CH),59.0(CH),127.3(CH),129.3(CH),130.5(CH),133.9(C),134.1(C),152.3(C),170.4(C),177.2(2C)；IRν(cm^-1):3317,1703,1643,1523,1226,817,590；C₁₆H₁₉Cl₂N₃O₃Analytically calculated as C, 51.63; h, 5.14; n, 11.29. Actually measuring as follows: c, 51.46; h, 5.55; and N, 11.70%.

j)Synthesis of compound HEI 2538:

(S)-N¹-tert-butyl-N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2538)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.97g, 6.9mmol) and tert-butyl isocyanate (0.75g, 7.6 mmol). The desired product was purified on a silica gel column (EtOAc/n-heptane 3/7 to 1/0) to give pure HEI _2538(0.95g, 2.5mmol) in 36% yield as a white solid. mp 99-101 deg.C (EtOAc/n-heptane); TLC Rf (CH)₂Cl₂/MeOH:99/1)0.7；¹H NMR(CDCl₃,400MHz)δppm 1.35(s,9H,C(CH₃)₃),2.05-2.16(m,1H,CH₂CH₂CH),2.36(dd,J＝11.9,2.0Hz,1H,CH₂CH₂CH),2.49(ddd,J＝17.6,9.4,2.0Hz,1H,CH₂CH₂CH),2.93(ddd,J＝17.7,11.4,9.4Hz,1H,CH₂CH₂CH),4.42(dd,J＝15.2,5.9Hz,1H,NHCH₂),4.53(dd,J＝15.7,6.7Hz,1H,NHCH₂),4.76(dd,J＝8.6,1.6Hz,1H,CH₂CH₂CH),7.18(dd,J＝8.2,2.1Hz,1H,ArH),7.20(br s,1H,NHCH₂),7.29(d,J＝8.2Hz,1H,ArH),7.36(d,J＝2.3Hz,1H,ArH),8.48(br s,1H,NHC(CH₃)₃)；¹³C NMR(CDCl₃,100MHz)δppm 20.9(CH₂),28.7(3CH₃),32.7(CH₂),41.0(CH₂),51.1(C),58.8(CH),127.2(CH),129.2(CH),130.3(CH),133.8(C),133.9(C),134.0(C),151.7(C),170.6(C),177.1(C)；IRν(cm^-1):3280,1717,1689,1656,1547,1267,1191,824,657；C₁₇H₂₁Cl₂N₃O₃Analytically calculated as C, 52.86; h, 5.48; n, 10.88. Actually measuring as follows: c, 52.63; h, 6.03; n,10.91 percent.

k)Synthesis of compound HEI 2315:

(S)-N¹-cyclohexyl-N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2315).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (2.00g, 7.0mmol) and cyclohexyl isocyanate (0.87g, 7.0 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2315(2.35g,5.7mmol) in 82% yield. mp 137-; TLC Rf (CH)₂Cl₂/MeOH:98/2)0.5；¹H NMR(CDCl₃400MHz) delta ppm 1.15-1.45(m,5H, cyclohexane-H), 1.59(m,1H, cyclohexane-H), 1.71(m,2H, cyclohexane-H), 1.88(m,2H, cyclohexane-H), 2.05-2.20(m,1H, cyclohexane-H), 2.25-2.40(m,1H, CH)₂CH₂CH),2.42-2.56(m,1H,CH₂CH₂CH),2.82-2.96(m,1H,CH₂CH₂CH),3.62-3.66(m,1H,CH₂CH₂CH),4.44(dd,J＝15.2,5.6Hz,1H,NHCH₂),4.51(dd,J＝15.2,5.6Hz,1H,NHCH₂),4.73-4.79(m,1H,CH₂CH₂CH),7.19(dd,J＝8.4,2.0Hz,1H,ArH),7.29(d,J＝8.4Hz,1H,ArH),7.30(br s,1H,NHCH₂),7.37(d,J＝2.0Hz,1H,ArH),8.42(d,J＝7.6Hz,1H,NHCH)；¹³C NMR(CDCl₃,100MHz)δppm 21.2(CH₂),24.5(CH₂),25.4(CH₂),32.5(CH₂),32.7(CH₂),32.8(CH₂),41.0(CH₂),49.0(CH₂),58.9(2CH),127.3(CH),129.3(CH),130.5(CH),133.9(C),134.0(C),134.1(C),152.2(C),170.5(C),177.2(C)；LC-MS(APCI⁺)m/z:412.0(MH⁺),tr 4.45min；IRν(cm^-1):3303,2928,2853,1703,1656,1534,1224；C₁₉H₂₃Cl₂N₃O₃Analytically calculated as C, 55.35; h, 5.62; n, 10.19. Actually measuring as follows: c, 55.36; h, 6.14; n,10.39 percent.

l)Synthesis of compound HEI 2329:

(S)-N¹adamantyl-N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI-2329).

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.50g, 5.2mmol) and adamantane isocyanate (0.93g, 5.2 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI 2329(2.34g,5.0mmol) in 97% yield. mp 85-87 ℃ (MeOH); TLC Rf (CH)₂Cl₂/MeOH:98/2)0.5；¹H NMR(CDCl₃400MHz) delta ppm 1.64-1.70(m,6H, ADM-H),1.95-1.99(m,6H, ADM-H),2.00-2.16(m,4H, ADM-H and CH₂CH₂CH),2.30-2.40(m,1H,CH₂CH₂CH),2.42-2.54(m,1H,CH₂CH₂CH),2.86-2.98(m,1H,CH₂CH₂CH),4.45(dd,J＝15.2,6.0Hz,1H,NHCH₂),4.51(dd,J＝15.2,6.0Hz,1H,NHCH₂),4.76(dd,J＝8.8,1.6Hz,1H,CH₂CH₂CH),7.17(dd,J＝8.0,2.0Hz,1H,ArH),7.26(t,J＝6.0Hz,1H,NHCH₂),7.28(d,J＝8.0Hz,1H,ArH),7.36(d,J＝2.0Hz,1H,ArH),8.39(br s,1H,NHCH)；¹³C NMR(CDCl₃,100MHz)δppm 21.1(CH₂),29.5(2CH₂),32.9(CH₂),36.4(2CH₂),41.1(CH₂),41.7(2CH₂),51.9(CH),59.0(CH),127.4(CH),129.4(2CH),130.5(2CH),134.0(C),134.1(2C),134.2(C),151.4(C),170.7(C),177.3(C)；LC-MS(APCI⁺)m/z:464.2(MH⁺),tr 4.80min；IRν(cm^-1):3289,2906,2848,1715,1661,1538,1222,1048,829；C₂₃H₂₇Cl₂N₃O₃Analytically calculated as C, 59.49; h, 5.86; and N, 9.05. Actually measuring as follows: c, 59.59; h, 6.26; n,8.71 percent.

m)Synthesis of compound HEI 2339:

(S)-N²- (2, 4-dichlorobenzyl) -5-oxo-N¹-phenylpyrrolidine-1, 2-dicarboxamide (HEI _ 2339).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (0.40g, 1.4mmol) and phenyl isocyanate (0.17g, 1.4 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2339(0.43g,1.1mmol) in 76% yield. mp 216-219 ℃ (MeOH); TLC Rf (CH)₂Cl₂/MeOH:96/4)0.8；¹H NMR(DMSO-d₆,400MHz)δppm 1.85-1.95(m,1H,CH₂CH₂CH),2.30-2.40(m,1H,CH₂CH₂CH),2.58-2.79(m,2H,CH₂CH₂CH),4.36(m,2H,NHCH₂),4.80(dd,J＝9.2,2.4Hz,1H,CH₂CH₂CH),7.11(t,J＝7.2Hz,1H,ArH),7.35(t,J＝7.2Hz,2H,ArH),7.43-7.44(m,2H,ArH),7.51(d,J＝7.2Hz,2H,ArH),7.61-7.62(m,1H,ArH),8.85(t,J＝5.6Hz,1H,NHCH₂),10.51(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm 21.4(CH₂),31.8(CH₂),40.0(CH₂),58.7(CH),119.5(2CH),123.8(CH),127.3(CH),128.6(CH),129.1(2CH),130.1(CH),132.3(C),132.9(C),135.2(C),137.3(C),149.4(C),171.2(C),177.8(C)；LC-MS(APCI⁺)m/z:408.1(MH⁺),tr 4.37min；IRν(cm^-1):3276,1718,1644,1543,1217,1054,748；C₁₉H₁₇Cl₂N₃O₃Analytically calculated as C, 56.17; h, 4.22; n, 10.34. Actually measuring as follows: c, 56.12; h, 3.96; n, 10.24%.

n)Synthesis of compound HEI 2761:

(S)-N¹- (biphenyl-4-yl) -N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2761).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and biphenyl-4-isocyanate (0.75g, 3.8 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2761(1.09g,2.3mmol) in 65% yield. mp 191-; TLC Rf (CH)₂Cl₂/MeOH:98/2)＝0.9；¹H NMR(CDCl₃,400MHz)δppm 2.16-2.30(m,1H,CH₂CH₂CH),2.34-2.43(m,1H,CH₂CH₂CH),2.57-2.67(m,1H,CH₂CH₂CH),2.99-3.12(m,1H,CH₂CH₂CH),4.52(dd,J＝13.4,6.7Hz,2H,NCH₂Ar),4.84(d,J＝8.8Hz,1H,CH₂CH₂CH),6.93(s,1H,NH),7.21(dd,J＝8.2,1.8Hz,1H,ArH),7.30-7.38(m,3H,ArH),7.44(t,J＝7.3Hz,2H,ArH),7.52-7.61(m,6H,ArH),10.60(s,1H,NH)；¹³C NMR(CDCl₃,100MHz)δppm 21.2(CH₂),29.7(CH₂),32.6(CH₂),41.2(CH₂),59.2(CH),126.8(2CH),126.9(CH),127.4(CH),127.7(2CH),128.8(2CH),129.4(2CH),130.8(CH),133.8(C),134.1(C),134.2(C),136.2(C),137.5(C),140.5(C),150.5(C),170.2(C),177.5(C)；IRν(cm^-1):3311,3083,1715,1542,1225,764,693；C₂₅H₂₁Cl₂N₃O₃Analysis ofCalculated as C, 62.25; h, 4.39; n, 8.71. Actually measuring as follows: c, 62.06; h, 4.39; n, 8.73%.

o)Synthesis of compound HEI 2314:

(S)-N¹- (2-chlorophenyl) -N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2314).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (2.00g, 7.0mmol) and 2-chlorophenyl isocyanate (1.07g, 7.0 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2314(2.75g,6.2mmol) in 90% yield. mp 213-219 deg.C (MeOH); TLC Rf (CH)₂Cl₂/MeOH:98/2)0.5；¹H NMR(CDCl₃,400MHz)δppm 2.18-2.36(m,2H,CH₂CH₂CH),2.58-2.68(m,1H,CH₂CH₂CH),2.95-3.06(m,1H,CH₂CH₂CH),4.47(dd,J＝15.6,5.6Hz,1H,NHCH₂),4.54(dd,J＝15.6,5.6Hz,1H,NHCH₂),4.80(dd,J＝8.4,2.4Hz,1H,CH₂CH₂CH),7.06(t,J＝7.6Hz,1H,NHCH₂),7.21(dd,J＝8.0,2.0Hz,1H,ArH),7.26(t,J＝7.6Hz,1H,ArH),7.34(d,J＝8.4Hz,1H,ArH),7.37(d,J＝2.0Hz,1H,ArH),7.40(m,2H,ArH),8.15(d,J＝8.0Hz,1H,ArH),11.04(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.7(CH₂),32.3(CH₂),41.1(CH₂),59.2(CH),121.8(CH),124.0(C),125.0(CH),127.4(CH),127.5(CH),129.4(CH),129.5(CH),130.6(CH),133.9(C),134.0(C),134.1(C),134.4(C),150.3(C),171.0(C),177.6(C)；LC-MS(APCI⁺)m/z:442.0(MH⁺),tr 4.45min；IRν(cm^-1):3271,1713,1643,1597,1545,1222,1054,749；C₁₉H₁₆Cl₃N₃O₃Analytically calculated as C, 51.78; h, 3.66; and N, 9.53. Actually measuring as follows: c, 51.80; h, 3.73; and N,9.59 percent.

p)Synthesis of compound HEI 2760:

(S)-N¹- (2-bromophenyl) -N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2760).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 2-bromophenyl isocyanate (0.76g, 3.8 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2760(0.95g,2.0mmol) in 56% yield. mp 217-; TLC Rf (CH)₂Cl₂/MeOH:95/5)0.7；¹H NMR(CDCl₃,400MHz)δppm 2.18-2.43(m,2H,CH₂CH₂CH),2.58-2.69(m,1H,CH₂CH₂CH),2.98-3.11(m,1H,CH₂CH₂CH),4.52(dd,J＝9.1,5.7Hz,2H,NHCH₂),4.81(dd,J＝9.2,2.0Hz,1H,CH₂CH₂CH),6.81(br s,1H,NHCH₂),7.00(ddd,J＝7.9,7.9,1.6Hz,1H,ArH),7.20(dd,J＝8.6,2.4Hz,1H,ArH),7.30-7.35(m,2H,ArH),7.37(d,J＝2Hz,1H,ArH),7.58(dd,J＝8.1,1.8Hz,1H,ArH),8.13(dd,J＝8.4,2.1Hz,1H,ArH),10.94(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.3(CH₂),33.4(CH₂),41.3(CH₂),59.2(CH),111.3(C),122.3(CH),125.5(CH),127.4(CH),128.0(CH),129.4(CH),130.8(CH),132.7(CH),133.7(C),134.1(C),134.2(C),135.7(C),150.5(C),170.1(C),177.3(C)；IRν(cm^-1):3268,3202,3063,1714,1644,1541,1218,748；C₁₉H₁₆BrCl₂N₃O₃Analytically calculated as C, 47.04; h, 3.32; and N, 8.66. Actually measuring as follows: c, 47.03; h, 3.30; n,8.66 percent.

q)Synthesis of compound HEI 2759:

(S)-N²- (2, 4-dichlorobenzyl) -N^1-(2-iodophenyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2759)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 2-iodophenyl isocyanate (0.94g, 3.8 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2759(1.07g,2.0mmol) in 58% yield. mp 203-; TLC Rf (CH)₂Cl₂/MeOH:95/5)0.7；¹H NMR(CDCl₃,400MHz)δppm 2.18-2.31(m,1H,CH₂CH₂CH),2.36-2.45(m,1H,CH₂CH₂CH),2.59-2.68(m,1H,CH₂CH₂CH),3.00-3.12(m,1H,CH₂CH₂CH),4.52(t,J＝5.8Hz,2H,NHCH₂),4.82(dd,J＝8.7,1.7Hz,1H,CH₂CH₂CH),6.82(s,1H,NHCH₂),6.88(td,J＝7.6,1.6Hz,1H,ArH),7.21(dd,J＝8.1,2.3Hz,1H,ArH),7.30-7.39(m,3H,ArH),7.84(dd,J＝8.1,1.5Hz,1H,ArH),7.96(dd,J＝8.3,1.5Hz,1H,ArH),10.68(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.3(CH₂),32.5(CH₂),41.3(CH₂),59.2(CH),90.4(C),123.0(CH),126.4(CH),127.4(CH),128.8(CH),129.4(CH),130.8(C),133.8(C),134.1(C),134.2(C),138.5(C),139.5(CH),150.7(C),170.1(C),177.2(C)；IRν(cm^-1):3265,3207,3076,1714,1644,1533,1217,754；C₁₉H₁₆Cl₂IN₃O₃C, 42.88; h, 3.03; and N, 7.90. Actually measuring as follows: c, 42.82; h, 2.98; n, 8.04%.

r)Synthesis of compound HEI 2331:

(S)-N²- (2, 4-dichlorobenzyl) -N^1-(2, 4-dichlorophenyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2331)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 2, 4-dichlorophenyl isocyanate (0.66g, 3.5 mmol). The desired product was crystallized from MeOH as a white powder, which was filteredPure HEI _2331(1.45g,3.1mmol) was obtained in 87% yield. mp 203-; TLC Rf (CH)₂Cl₂/MeOH:98/2)0.6；¹H NMR(DMSO-d₆,400MHz)δppm 1.86-1.96(m,1H,CH₂CH₂CH),2.29-2.43(m,1H,CH₂CH₂CH),2.60-2.81(m,2H,CH₂CH₂CH),4.36(m,2H,CH₂CH₂CH and NHCH₂),4.81(dd,J＝9.6,2.8Hz,1H,NHCH₂),7.41(d,J＝8.0Hz,1H,ArH),7.43(d,J＝2.0Hz,1H,ArH),7.47(dd,J＝9.2,2.0Hz,1H,ArH),7.62(d,J＝2.0Hz,1H,ArH),7.74(d,J＝2.0Hz,1H,ArH),8.24(d,J＝9.2Hz,1H,ArH),8.88(t,J＝2.0Hz,1H,NHCH₂),11.11(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm 21.5(CH₂),31.7(CH₂),39.5(CH₂),58.7(CH),121.9(CH),123.0(CH),127.3(CH),127.7(C),128.1(CH),128.6(CH),128.8(CH),130.1(C),132.3(C),133.0(C),133.6(C),135.2(C),149.3(C),171.0(C),178.2(C)；LC-MS(APCI⁺)m/z:475.9(MH⁺),tr 4.78min；IRν(cm^-1):3263,1716,1656,1582,1537,1224,823；C₁₉H₁₅Cl₄N₃O₃C, 48.03; h, 3.18; n, 8.84. Actually measuring as follows: c, 48.02; h, 3.17; n,8.76 percent.

s)Synthesis of compound HEI 2333:

(S)-N²- (2, 4-dichlorobenzyl) -N^1-(2, 4-Dimethoxyphenyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2333)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 2, 4-dimethoxyphenyl isocyanate (0.62g, 3.5 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2333(1.35g,2.9mmol) in 83% yield. mp 203-; TLC Rf (CH)₂Cl₂/MeOH:98/2)0.6；¹H NMR(CDCl₃,400MHz)δppm 2.15-2.23(m,1H,CH₂CH₂CH),2.35-2.43(m,1H,CH₂CH₂CH),2.55-2.63(m,1H,CH₂CH₂CH),2.92-3.05(m,1H,CH₂CH₂CH),3.80(s,3H,OCH₃),3.88(s,3H,OCH₃),4.45(dd,J＝15.2,6.0Hz,1H,NHCH₂),4.54(dd,J＝15.2,6.0Hz,1H,NHCH₂),4.84(dd,J＝8.8,1.6Hz,1H,CH₂CH₂CH),6.44(dd,J＝8.8,2.8Hz,1H,ArH),6.50(d,J＝2.8Hz,1H,ArH),7.10(t,J＝6.0Hz,1H,NHCH₂),7.17(dd,J＝8.0,2.0Hz,1H,ArH),7.31(d,J＝8.0Hz,1H,ArH),7.34(d,J＝2.0Hz,1H,ArH),7.93(d,J＝8.8Hz,1H,ArH),10.71(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.4(CH₂),32.7(CH₂),41.3(CH₂),55.7(CH),56.0(CH₃),59.2(CH₃),98.9(CH),103.9(CH),120.3(CH),121.1(CH),127.5(CH),129.5(C),130.7(CH),134.0(C),134.1(C),134.2(C),150.4(C),150.5(C),157.0(C),170.5(C),177.2(C)；LC-MS(APCI⁺)m/z:466.0(MH⁺),tr4.33min；IRν(cm^-1):3294,1712,1657,1547,1299,1205,1033,818；C₂₁H₂₁Cl₂N₃O₅Analytically calculated as C, 54.09; h, 4.54; and N, 9.01. Actually measuring as follows: c, 53.91; h, 3.81; n,8.91 percent.

t)Synthesis of compound HEI 2338:

(S)-N²- (2, 4-dichlorobenzyl) -5-oxo-N¹- (2,4, 6-trimethylphenyl) pyrrolidine-1, 2-dicarboxamide (HEI _ 2338).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (0.70g, 2.4mmol) and 2,4, 6-trimethylphenyl isocyanate (0.39g, 2.4 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2338(0.60g,1.3mmol) in 55% yield. mp 156-; TLC Rf (CH)₂Cl₂/MeOH:95/5)0.8；¹H NMR(CDCl₃,400MHz)δppm 2.14(s,6H,CCH₃),2.18-2.24(m,1H,CH₂CH₂CH),2.27(s,3H,CCH₃),2.38-2.45(m,1H,CH₂CH₂CH),2.55-2.65(m,1H,CH₂CH₂CH),2.98-3.10(m,1H,CH₂CH₂CH),4.42(dd,J＝6.0,2.0Hz,2H,NHCH₂),4.83(dd,J＝8.4,1.6Hz,1H,CH₂CH₂CH),6.89(s,2H,ArH),7.09-7.15(m,2H,NHCH₂ and ArH),7.23(d,J＝8.8Hz,1H,ArH),7.34(d,J＝2.4Hz,1H,ArH),9.74(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 18.4(2CH₃),21.1(CH₃),21.3(CH₂),32.7(CH₂),41.1(CH₂),59.2(CH),127.4(CH),129.1(2CH),129.4(C),130.4(C),130.5(CH),134.0(CH),134.1(C),134.2(C),135.1(2C),137.3(C),151.7(C),170.5(C),177.8(C)；LC-MS(APCI⁺)m/z:448.1(MH⁺),tr 4.57min；IRν(cm^-1):3275,1714,1658,1514,1358,1242,1219,1033,826；C₂₂H₂₃Cl₂N₃O₃Analytically calculated as C, 58.94; h, 5.17; n, 9.37. Actually measuring as follows: c, 58.80; h, 5.26; and N, 9.25%.

u)Synthesis of compound HEI 2337:

(S)-N²- (2, 4-dichlorobenzyl) -5-oxo-N¹- [ (3-trifluoromethyl) phenyl]Pyrrolidine-1, 2-dicarboxamide (HEI _ 2337).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 3-trifluoromethylphenyl isocyanate (0.65g, 3.5 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2337(1.20g,2.5mmol) in 73% yield. mp 171-; TLC Rf (CH)₂Cl₂/MeOH:98/2)0.6；¹H NMR(DMSO-d₆,400MHz)δppm 1.90-1.98(m,1H,CH₂CH₂CH),2.30-2.42(m,1H,CH₂CH₂CH),2.60-2.80(m,2H,CH₂CH₂CH),4.35-4.39(m,2H,CH₂CH₂CH and NHCH₂),4.81(dd,J＝9.6,2.8Hz,1H,NHCH₂),7.38(dd,J＝8.8,2.4Hz,1H,ArH),7.45(d,J＝8.4Hz,1H,ArH),7.47(d,J＝8.4Hz,1H,ArH),7.59(t,J＝8.4Hz,1H,ArH),7.61(d,J＝2.4Hz,1H,ArH),7.72(d,J＝8.8Hz,1H,ArH),8.07(s,1H,ArH),8.88(t,J＝6.0Hz,1H,NHCH₂),10.68(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm 21.8(CH₂),32.2(CH₂),40.6(CH₂),59.2(CH),116.3(q,J＝3.8Hz,CH),127.0(m,CH),124.0(CH),124.3(q,J＝272.0Hz,CF₃),127.6(CH),129.0(CH),130.0(q,J＝32Hz,C),130.2(CH),130.6(CH),132.8(C),133.4(C),135.7(C),138.6(C),150.1(C),171.5(C),178.2(C)；¹⁹F NMR(CDCl₃,376MHz)δppm-61.4(CF₃)；LC-MS(APCI⁺)m/z:474.0(MH⁺),tr 4.58min；IRν(cm^-1):3298,1727,1658,1556,1450,1333,1251,1112,831；C₂₀H₁₆Cl₂F₃N₃O₃The analysis calculation is that C is 50.65; h, 3.40; and N, 8.86. Actually measuring as follows: c, 50.34; h, 3.30; n,8.74 percent.

v)Synthesis of compound HEI 2328:

(S)-N²- (2, 4-dichlorobenzyl) -N^1-(3, 4-dichlorophenyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2328)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.50g, 5.2mmol) and 3, 4-dichlorophenyl isocyanate (0.98g, 5.2 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI2328 (2.00g,4.2mmol) in 81% yield. mp 220 + 224 deg.C (MeOH); TLC Rf (CH)₂Cl₂/MeOH:98/2)0.7；¹H NMR(DMSO-d₆,400MHz)δppm 1.84-1.97(m,1H,CH₂CH₂CH),2.25-2.40(m,1H,CH₂CH₂CH),2.60-2.80(m,2H,CH₂CH₂CH),4.36(s,2H,NHCH₂),4.79(d,J＝8.5Hz,1H,CH₂CH₂CH),7.38-7.65(m,5H,NHCH₂ and ArH),7.94(s,1H,ArH),8.86(s,1H,ArH),10.59(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm21.5(CH₂),31.7(CH₂),39.5(CH₂),58.7(CH),120.0(CH),121.1(CH),125.4(C),127.3(CH),128.6(CH),130.2(CH),130.8(CH),131.2(C),132.3(C),133.0(C),135.2(C),137.5(C),149.4(C),171.0(C),177.7(C)；LC-MS(APCI⁺)m/z:474.0(MH⁺),tr 4.67min；IRν(cm^-1):3270,1718,1644,1598,1547,1476,1218,1134,818；C₁₉H₁₅Cl₄N₃O₃C, 48.03; h, 3.18; n, 8.84. Actually measuring as follows: c, 47.75; h, 3.03; n,8.71 percent.

w)Synthesis of compound HEI 2336:

(S)-N¹- [3, 5-bis (trifluoromethyl) phenyl group]-N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2336).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 3, 5-bis (trifluoromethyl) phenyl isocyanate (0.89g, 3.5 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI 2337(1.20g,2.2mmol) in 64% yield. TLC Rf (CH)₂Cl₂/MeOH:98/2)0.6；mp 223–225℃(MeOH)；¹H NMR(CDCl₃,400MHz)δppm 2.23-2.36(m,2H,CH₂CH₂CH),2.60-2.70(m,1H,CH₂CH₂CH),2.98-3.12(m,1H,CH₂CH₂CH),4.49(dd,J＝15.2,5.6Hz,1H,NHCH₂),4.57(dd,J＝15.2,5.6Hz,1H,NHCH₂),4.78(dd,J＝7.2,2.8Hz,1H,CH₂CH₂CH),6.66(t,J＝5.6Hz,1H,NHCH₂),7.20(dd,J＝8.4,2.4Hz,1H,ArH),7.34(d,J＝8.4Hz,1H,ArH),7.36(d,J＝2.4Hz,1H,ArH),7.62(s,1H,ArH),7.99(s,2H,ArH),10.91(br s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm21.4(CH₂),32.4(CH₂),41.4(CH₂),59.2(CH),117.7(q,J＝3.9Hz,CH),119.8(m,2CH),123.0(q,J＝272.0Hz,2CF₃),127.5(CH),129.5(CH),130.9(CH),132.4(q,J＝32.0Hz,2C),133.5(C),134.2(C),134.4(C),138.6(C),150.2(C),170.0(C),177.8(C)；¹⁹F NMR(CDCl₃,100MHz)δppm-63.1(CF₃)；LC-MS(APCI⁺)m/z:543.8(MH⁺),tr 5.05min；IRν(cm^-1):3290,3089,1725,1656,1561,1284,1172,1128,1042,890；C₂₁H₁₅Cl₂F₆N₃O₃Analytically calculated as C, 46.51; h, 2.79; and N, 7.75. Actually measuring as follows: c, 46.49; h, 2.61; n,7.69 percent.

x)Synthesis of compound HEI 2278:

(S)-N¹- (4-chlorophenyl) -N²- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2278).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 4-chlorophenyl isocyanate (0.54g, 3.5 mmol). The desired product is purified on a silica gel Column (CH)₂Cl₂MeOH 1/0 to 99/1) to give pure HEI _2278(1.05g, 2.4mmol) as a 68% yield white solid. mp 188-₂Cl₂MeOH); TLC Rf (EtOAc/n-heptane: 4/6) 0.3;¹H NMR(DMSO-d₆,400MHz)δppm 1.85-1.95(m,1H,CH₂CH₂CH),2.30-2.40(m,1H,CH₂CH₂CH),2.58-2.78(m,2H,CH₂CH₂CH),4.35(m,2H,CH₂CH₂CH and NHCH₂),4.79(dd,J＝9.2,6.0Hz,1H,NHCH₂),7.40(d,J＝8.8Hz,2H,ArH),7.42(m,2H,ArH),7.57(d,J＝8.8Hz,2H,ArH),7.62(d,J＝1.5Hz,1H,ArH),8.85(t,J＝6.0Hz,1H,NHCH₂),10.54(br s,1H,NHAr)；¹³C NMR(DMSO-d₆,100MHz)δppm 21.4(CH₂),31.8(CH₂),38.3(CH₂),58.7(CH),121.2(2CH),127.3(CH),127.5(C),128.6(CH),128.9(2CH),130.1(CH),132.3(C),132.9(C),135.2(C),136.3(C),149.4(C),171.1(C),177.8(C)；LC-MS(APCI⁺)m/z:439.9(MH⁺),tr 4.50min；IRν(cm^-1):3283,2922,1712,1649,1600,1545,1388,1224,1051,828；C₁₉H₁₆Cl₃N₃O₃Analytically calculated as C, 51.78; h, 3.66; and N, 9.53. Actually measuring as follows: c, 52.02; h, 3.92; and N,9.38 percent.

y)Synthesis of compound HEI 2817:

(S)-N¹,N²-bis (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2817).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 2, 4-dichlorobenzyl isocyanate (0.77g, 3.8 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2817(1.31g,2.7mmol) in 78% yield. TLC Rf (CH)₂Cl₂/MeOH:95/5)0.7；mp 224–226℃(MeOH)；¹H NMR(DMSO-d₆,400MHz)δppm 1.81-1.91(m,1H,CH₂CH₂CH),2.24-2.37(m,1H,CH₂CH₂CH),2.43-2.72(m,2H,CH₂CH₂CH),4.31(d,J＝5.7Hz,2H,NCH₂),4.44(d,J＝5.7Hz,2H,NCH₂),4.69(dd,J＝9.2,2.6Hz,1H,CH₂CH₂CH),7.24-7.43(m,4H,ArH),7.61(dd,J＝7.4,2.0Hz,2H,ArH),8.77(t,J＝6.3Hz,1H,NH),8.86(t,J＝6.3Hz,1H,NH)；¹³C NMR(DMSO-d₆,100MHz)δppm 22.0(CH₂),32.1(CH₂),40.6(CH₂),40.9(CH₂),59.0(CH),127.6(CH),127.7(CH),128.9(CH),129.0(CH),130.4(CH),130.6(CH),132.6(CH),132.8(CH),133.2(C),133.3(C),135.7(C),135.8(C),152.5(C),171.8(C),177.6(C)；IRν(cm^-1):3251,3089,1709,1653,1538,1239；C₂₀H₁₇Cl₄N₃O₃Analytically calculated as C, 49.11; h, 3.50; and N, 8.59. Actually measuring as follows: c, 49.12; h,3.50；N,8.60％。

z)Synthesis of compound HEI 2820:

(S)-N²- (2, 4-dichlorobenzyl) -N^1-(3, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 2-dicarboxamide (HEI _2820)

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.5mmol) and 3, 4-dichlorobenzyl isocyanate (0.67g, 3.3 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2820(0.54g,1.1mmol) in 32% yield. mp 201-; TLC Rf (CH)₂Cl₂/MeOH:95/5)0.7；¹H NMR(CDCl₃,400MHz)δppm 2.12-2.25(m,1H,CH₂CH₂CH),2.31-2.41(m,1H,CH₂CH₂CH),2.49-2.59(m,1H,CH₂CH₂CH),2.89-3.01(m,1H,CH₂CH₂CH),4.33-4.57(m,4H,NCONHCH₂,CHCONHCH₂,CH₂CH₂CH),4.74(d,J＝9.3Hz,1H,CHCONHCH₂),6.86(s,1H,CHCONHCH₂),7.11(d,J＝9.3Hz,1H,ArH),7.16-7.22(m,1H,ArH),7.27-7.34(m,1H,ArH),7.36-7.42(m,3H,ArH),8.88(s,1H,NCONHCH₂)；¹³C NMR(CDCl₃,100MHz)δppm 21.3(CH₂),32.4(CH₂),41.2(CH₂),42.8(CH₂),59.1(CH),126.8(CH),127.4(CH),129.3(CH),129.4(CH),130.6(CH),130.7(CH),132.8(C),133.7(C),134.1(C),134.1(C),138.2(C),153.3(C),170.2(C),177.4(C),184.0(C)；IRν(cm^-1):3320,3274,3058,2943,1707,1650,1542,1243,816；C₂₀H₁₇Cl₄N₃O₃Analytically calculated as C, 49.11; h, 3.50; and N, 8.59. Actually measuring as follows: c, 49.14; h, 3.58; n,8.53 percent.

a') Synthesis of Compound HEI 2548:

N²- (2, 4-dichlorobenzyl) -5-oxo-N¹- (thien-2-yl) pyrrolidine-1, 2-dicarboxamide (HEI _ 2548).

The general procedure followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (0.83g, 2.9mmol) and thiophen-2-isocyanate (0.40g, 3.2 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2548(0.31g,0.8mmol) in 26% yield. mp 141 and 143 deg.C (MeOH); TLC Rf (CH)₂Cl₂/MeOH:99/1)0.5；¹H NMR(CDCl₃,400MHz)δppm 2.20-2.31(m,1H,CH₂CH₂CH),2.35-2.41(m,1H,CH₂CH₂CH),2.62(td,J＝18.1,9.4Hz,1H,CH₂CH₂CH),3.06(td,J＝17.6,9.4Hz,1H,CH₂CH₂CH),4.50-4.55(m,2H,NHCH₂),4.85(dd,J＝9.0,1.6Hz,1H,CH₂CH₂CH),6.73(dd,J＝3.9,1.5Hz,1H,ArH),6.89(dd,J＝5.5,3.5Hz,1H,ArH),6.92(dd,J＝5.5,1.5Hz,1H,ArH),6.95(br t,J＝4.6Hz,1H,NHCH₂),7.21(dd,J＝8.2,2.0Hz,1H,ArH),7.34(d,J＝8.3Hz,1H,ArH),7.37(d,J＝1.9Hz,1H,ArH),11.00(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.6(CH₂),32.2(CH₂),41.2(CH₂),59.1(CH),113.4(CH),118.4(CH),124.5(CH),127.3(CH),129.3(CH),130.6(CH),133.6(C),134.0(C),134.1(C),137.9(C),149.6(C),170.2(C),177.5(C)；IRν(cm^-1):3291,1712,1660,1556,1510,1343,1245,1218,815,689；C₁₇H₁₅Cl₂N₃O₃S is analyzed and calculated to be C, 49.52; h, 3.67; n, 10.19; and S, 7.78. Actually measuring as follows: c, 49.23; h, 3.80; n, 10.00; s,7.03 percent.

b') Synthesis of Compound HEI 3090:

(S)-N¹- (6-chloropyridin-3-yl) -N²- (2, 4-dichlorobenzyl) -5-Oxopyrrolidine-1, 2-dicarboxamide (HEI-3090).

The general procedure was followed using (S) -N- (2, 4-dichlorobenzyl) -5-oxo-pyrrolidine-2-carboxamide (1.86g, 6.5mmol) and 6-chloropyridin-3-isocyanate (1.00g, 6.5 mmol). The desired product is purified on a silica gel Column (CH)₂Cl₂MeOH 1/0 to 9/1) to give pure HEI — 3090(0.30g, 0.7mmol) as an 11% yield white solid. mp 187-₂Cl₂/MeOH)；TLC Rf(CH₂Cl₂/MeOH:95/5)0.8；¹H NMR(CDCl₃,400MHz)δppm 2.12-2.37(m,2H,CH₂CH₂CH),2.59-2.68(m,1H,CH₂CH₂CH),2.99-3.10(m,1H,CH₂CH₂CH),4.49(dd,J＝15.2,6.2Hz,1H,NHCH₂),4.55(dd,J＝15.2,6.2Hz,1H,NHCH₂),4.77(dd,J＝8.7,2.1Hz,1H,CH₂CH₂CH),6.65(br t,J＝6.2Hz,1H,NHCH₂),7.22(dd,J＝8.1,2.0Hz,1H,ArH),7.29(d,J＝8.6Hz,1H,ArH),7.33(d,J＝8.1Hz,1H,ArH),7.38(d,J＝2.0Hz,1H,ArH),7.92(dd,J＝8.6,2.5Hz,1H,ArH),8.49(d,J＝2.5Hz,1H,ArH),10.66(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.4(CH₂),32.4(CH₂),41.4(CH₂),59.2(CH),124.3(CH),127.5(CH),129.5(CH),130.2(CH),130.9(CH),133.1(C),133.6(2C),134.2(C),141.3(CH),146.2(C),150.3(C),170.0(C),177.7(C)；IRν(cm^-1):3271,3095,2935,1721,1655,1594,1542,1464,1217,1104。

N, N' -bis (6-chloropyridin-3-yl) urea (HEI _3091)

It was isolated from the previous experiment during column chromatography to give pure HEI — 3091(0.50g, 1.8mmol) as a 54% yield white solid. Mp>260℃；TLC Rf(CH₂Cl₂/MeOH:95/5)0.4；¹H NMR(CDCl₃,400MHz)δppm 7.45(d,J＝8.7Hz,2H,ArH),7.99(dd,J＝8.7,3.1Hz,2H,ArH),8.49(d,J＝3.1Hz,2H,ArH),9.21(s,2H,NH)；¹³C NMR(CDCl₃,100MHz)δppm 124.0(2CH),129.2(2CH),135.6(2C),139.8(2CH),142.6(2C),152.3(C)；IRν(cm^-1):3267,3180,3095,3052,1711,1531,1465,1285,1208,1107,833。

c') synthesis of compound HEI 2773:

(S)-N¹- (3, 4-dichlorophenyl) -N^2-[2- (2, 4-dichlorophenyl) ethyl group]-5-oxopyrrolidine-1, 2-dicarboxamide (HEI _ 2773).

The general procedure follows from the use of (S) -N- (2, 4-dichlorophenyl) ethyl]-5-oxo-pyrrolidine-2-carboxamide (1.00g, 3.3mmol) and 3, 4-dichlorophenyl isocyanate (0.69g, 3.7 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _2773(0.80g,1.6mmol) in 51% yield. mp 213-; TLC Rf (CH)₂Cl₂/MeOH:95/5)0.7；¹H NMR(DMSO-d₆,400MHz)δppm 1.70-1.85(m,1H,CH₂CH₂CH),2.19-2.36(m,1H,CH₂CH₂CH),2.57-2.71(m,2H,CH₂CH₂CH),2.78-2.92(m,2H,CH₂CH₂Ar),3.25-3.46(m,2H,CH₂CH₂Ar),4.63(dd,J＝9.5,2.9Hz,1H,CH₂CH₂CH),7.30-7.38(m,2H,ArH),7.49(dd,J＝8.6,2.4Hz,1H,ArH),7.54-7.65(m,2H,ArH),7.94(d,J＝2.5Hz,1H,ArH),8.36(s,1H,NH),10.60(s,1H,NH)；¹³C NMR(DMSO-d₆,100MHz)δppm 22.1(CH₂),32.1(CH₂),32.6(CH₂),38.5(CH₂),59.1(CH),120.3(CH),121.4(CH),125.8(C),127.6(CH),129.0(CH),131.2(CH),131.6(C),132.2(C),133.0(CH),134.5(C),136.2(C),137.9(C),149.7(C),171.0(C),178.2(C)；IRν(cm^-1):3267,3105,1716,1652,1592,1542,1220,807；C₂₀H₁₇Cl₄N₃O₃Analytically calculated as C, 49.11; h, 3.50; and N, 8.59. Actually measuring as follows: c, 48.99; h, 3.37; n,8.50 percent.

d') synthesis of compound HEI 3127:

N¹- (6-chloropyridin-3-yl) -N³- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-1, 3-dicarboxamide (HEI _ 3127).

The general procedure was followed using N- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-3-carboxamide (0.83g, 2.9mmol) and 4-chloropyridin-3-yl isocyanate (0.40g, 3.2 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _3127(0.31g,0.8mmol) in 26% yield. TLC Rf (CH)₂Cl₂/MeOH:99/1)＝0.5；mp(EtOH)＝141–143℃；¹H NMR(CDCl₃,400MHz)δppm 2.20-2.31(m,1H,CH₂CH₂CH),2.35-2.41(m,1H,CH₂CH₂CH),2.62(td,J＝18.1,9.4Hz,1H,CH₂CH₂CH),3.06(td,J＝17.6,9.4Hz,1H,CH₂CH₂CH),4.50-4.55(m,2H,NHCH₂),4.85(dd,J＝9.0,1.6Hz,1H,CH₂CH₂CH),6.73(dd,J＝3.9,1.5Hz,1H,ArH),6.89(dd,J＝5.5,3.5Hz,1H,ArH),6.92(dd,J＝5.5,1.5Hz,1H,ArH),6.95(br t,J＝4.6Hz,1H,NHCH₂),7.21(dd,J＝8.2,2.0Hz,1H,ArH),7.34(d,J＝8.3Hz,1H,ArH),7.37(d,J＝1.9Hz,1H,ArH),11.00(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.6(CH₂),32.2(CH₂),41.2(CH₂),59.1(CH),113.4(CH),118.4(CH),124.5(CH),127.3(CH),129.3(CH),130.6(CH),133.6(C),134.0(C),134.1(C),137.9(C),149.6(C),170.2(C),177.5(C)；IRν(cm^-1):3291,1712,1660,1556,1510,1343,1245,1218,815,689；C₁₇H₁₅Cl₂N₃O₃S is analyzed and calculated to be C, 49.52; h, 3.67; n, 10.19; and S, 7.78. Actually measuring as follows: c, 49.23; h, 3.80; n, 10.00; s,7.03 percent.

e') synthesis of compound HEI 3204:

N³- (2, 4-dichlorobenzyl) -N^1-(3, 4-dichlorophenyl) -5-oxopyrrolidine-1, 3-dicarboxamide (HEI _3204)

The general procedure was followed using N- (2, 4-dichlorobenzyl) -5-oxopyrrolidine-3-carboxamide (0.83g, 2.9mmol) and 3, 4-dichlorophenyl isocyanate (0.40g, 3.2 mmol). The desired product crystallized in MeOH as a white powder which was filtered to give pure HEI _3204(0.31g,0.8mmol) in 26% yield. TLC Rf (CH)₂Cl₂/MeOH:99/1)＝0.5；mp(EtOH)＝141–143℃；¹H NMR(CDCl₃,400MHz)δppm 2.20-2.31(m,1H,CH₂CH₂CH),2.35-2.41(m,1H,CH₂CH₂CH),2.62(td,J＝18.1,9.4Hz,1H,CH₂CH₂CH),3.06(td,J＝17.6,9.4Hz,1H,CH₂CH₂CH),4.50-4.55(m,2H,NHCH₂),4.85(dd,J＝9.0,1.6Hz,1H,CH₂CH₂CH),6.73(dd,J＝3.9,1.5Hz,1H,ArH),6.89(dd,J＝5.5,3.5Hz,1H,ArH),6.92(dd,J＝5.5,1.5Hz,1H,ArH),6.95(br t,J＝4.6Hz,1H,NHCH₂),7.21(dd,J＝8.2,2.0Hz,1H,ArH),7.34(d,J＝8.3Hz,1H,ArH),7.37(d,J＝1.9Hz,1H,ArH),11.00(s,1H,NHAr)；¹³C NMR(CDCl₃,100MHz)δppm 21.6(CH₂),32.2(CH₂),41.2(CH₂),59.1(CH),113.4(CH),118.4(CH),124.5(CH),127.3(CH),129.3(CH),130.6(CH),133.6(C),134.0(C),134.1(C),137.9(C),149.6(C),170.2(C),177.5(C)；IRν(cm^-1):3291,1712,1660,1556,1510,1343,1245,1218,815,689；C₁₇H₁₅Cl₂N₃O₃S is analyzed and calculated to be C, 49.52; h, 3.67; n, 10.19; and S, 7.78. Actually measuring as follows: c, 49.23; h, 3.80; n, 10.00; s,7.03 percent.

Example 2: biological applications of the Compounds of the invention

Statistical analysis

Results are expressed as mean ± SEM. We performed statistical analysis using Prism 6 software (GraphPad). The Mann-Whitney test was used to assess the significance of the differences between the two groups (, p < 0.05;, p < 0.01;, p < 0.001;, p < 0.0001). The Mantel-Cox (log rank) test was used to analyze the incidence curves.

1) Study 1-toxicity:

toxicity of HEI2333 and HEI2336 have been tested on NCI panels of 60 tumor cell lines (NCI 60 cell panel).

All test molecules (10 μ M) did not show any toxicity (i.e., significant inhibition of proliferation) in the absence of extracellular ATP (data not shown).

2) Study 2-biological activity of compounds of the invention on a heterologous cell line expressing mouse P2RX7(mP2RX 7):

2.1. HEK cells that do not express any P2X receptor have been transfected with an expression vector encoding the mP2rx7 gene (mock). Cation channel activity was measured with the Fluo4am (Invitrogen) probe, while the opening of the macropores was measured with Topro3 (Invitrogen). In each experiment, 2X 10 will be used⁶The cells were incubated with 50nM Fluo4-AM for 30 min. After washing, the cells were treated with 500. mu.M BzATP (3' -O- (4-benzoyl) benzoylATP) for 1 hour, or untreated, in the presence of 50. mu.M of a compound of the invention. When indicated, the Topro3 probe (8.5nM) was added over 1h of stimulation time. Fluorescence was measured by flow cytometry.

Each compound was tested in the absence (data not shown) or presence of BzATP. Figure 1 shows that compounds HEI2314 and HEI2328 only increase Topro3 entry in the presence of BzATP.

HEI2328 was the most active and was used by the inventors for further characterization. When used alone in mock and mP2RX7 cells, HEI2328 did not induce either channel activity or macropore opening. As expected, BzATP was inactive in mock cells. In contrast, in cells expressing mP2RX7, it increased 15-fold Ca2+ entry and 4-fold macropore opening in the presence of BzATP: HEI2328 enhanced the large open porosity of 61% of Topro 3-positive cells when used in combination with BzATP, and 24.7% of Topro 3-positive cells when cells were stimulated with BzATP alone. Interestingly, the inventors note here that: HEI2328 did not affect channel activity under this stimulation condition (fig. 2).

In summary, HEI2328 enhances the macropore opening in the presence of ATP, a mechanism that leads to cell death. Since no toxic effect was seen in the absence of ATP, the inventors considered: HEI2328 is active only in the inflammatory microenvironment (100 μ M ATP), thus reducing the likelihood of off-target effects.

ADME studies were also performed and show that: both HEI2328 and HEI3090 are stable molecules.

The stability studies were directed to mouse liver microsomal systems (and humans not shown) (fig. 13A) and directly injected in vivo HEI3090 molecules (fig. 13B). These experiments highlight that: in the plasma of mice injected with HEI3090(ip, 1.5mg/kg in 10% DMSO), the maximum concentration of HEI3090 was found to be 2.5. mu.M.

A complementary assay was also performed to demonstrate the biological activity of HEI3090 (a more soluble version of HEI 2328) on two HEK cells expressing mouse P2RX7(mP2RX7) (fig. 14) or human P2RX7(hP2RX7) (fig. 15). BzATP or ATP stimulates channel activity, an event that occurs during the first minutes of P2RX7 stimulation. 1h stimulation triggered only greater cation penetration (Topro3 influx, FIG. 2), while 15min stimulation demonstrated that EC50 for BzATP moved to the left in the presence of low dose HEI3090(250 nM). The same positive effect was observed on cells expressing human P2RX7 (fig. 15).

These results are the first evidence that HEI3090 acts as a positive allosteric modulator of P2RX 7.

3) Study 3-colitis mouse model:

clinical score

P2RX7 has been shown in the past to modulate the inflammatory response of the colon in patients with Crohn's disease, a chronic inflammatory disease of the colon (Cesaro A et al, activation of the Amplification loop of the inflammatory process induced by P2X7R in intestinal epithelial cells in response to transepithelial migration of neutrophils (Amplification loop of the inflammatory process induced by P2X7R activation in intestinal epithelial cells in response to a neutral inflammatory shift), Am J physical gastrointestinal tract physiology, 2010.299(1): p.G32-42), the inventors have tested the effect of HEI2328 on a mouse model of DSS-induced colitis.

All mice received dextran sodium sulfate (3% DSS, TbS challenge) ad libitum from day 1 to day 5 and returned to normal tap water for the next 10 days. Control mice (n-20) were injected intraperitoneally daily with placebo (PBS/10% DMSO), while treated mice received 2.3mg/kg HEI2328 in PBS/10% DMSO. The inventors used the following semi-quantitative clinical scores:

body weight loss was scored as follows: 0, no loss; 1, loss < 5%; 2, loss < 10%; 3, loss < 20%; and 4, loss > 20%.

The stool consistency score was measured as: 0, normal; 1, loose stool; 2, watery diarrhea; or 3, severe watery diarrhea.

Rectal bleeding was scored as: 0, no blood; 1, presence of petechiae; 2, the excrement has a silk bloodstain; or 3, bleeding.

Clinical scores were stable for the first 5 days of treatment, as expected for DSS-induced colitis (figure 3). From day 6 to day 9, the body weight loss of the treated mice was statistically lower than that of the control group. More importantly, HEI 2328-treated mice recovered their original body weight after 15 days. Clinical scores confirmed that: HEI 2328-treated mice were healthier and recovered more rapidly than control mice (fig. 3B).

In summary, the inventors show here: compound HEI2328 reduced clinical signs of colitis. Interestingly, the inventors noted that: compound HEI2328 was more effective than a classical P2RX7 antagonist in reducing clinical signs of Mouse DSS-induced Colitis (Hofman, P. et al, Genetic and Pharmacological Inactivation of Purinergic P2RX7 receptors inhibits Inflammation but Increases Tumor Incidence in a Mouse Model of Colitis-Associated Cancer (Genetic and Pharmacological Inactivation of the Purinergic P2RX7Receptor or Dampens Inflammation bucket incorporation a patient Model of Colitis-Associated Cancer), Cancer Res, 2015; Marques, c.c. et al, a Prophylactic systemic P2X 7Receptor blocker can prevent experimental Colitis (therapeutic system P2X 36 Receptor expression), biophysical experiment, 539, biophysical 2.78).

Histological scoring

On day 15, the mice were euthanized, a segment of the colon from the rectum to the cecum was excised, measured, and a histological score was established on a cross section reaching the lower colon 1/3 and on a longitudinal section from the lower 1/3 to the rectum. Inflammation scoring is performed by a trained pathologist. Briefly, the severity of inflammation (none, mild, moderate, severe), the extent of inflammation (none, mucosal and submucosal, transmural), crypt damage (none, basal to 1/3, basal to 2/3, crypt loss, crypt and epithelial cell loss), and the percentage of tissue affected by inflammation (0%, 25%, 50%, 75% and 100%) were scored.

Fig. 4A shows: the inflammatory score of HEI 2328-treated mice demonstrated a statistically significant very low inflammatory index compared to the control group.

In conclusion, compound HEI2328 is a very potent colitis inhibitor.

Immunization Pattern

The observation that HEI2328 effectively inhibited mouse DSS-induced colitis was counter-intuitive. Indeed, by enhancing BzATP to increase the macropore opening, one would expect increased production of IL1B, leading to worsening colitis. Since P2RX7 was expressed by immune cells, the inventors also characterized the ratio of each immune cell population in the spleen of treated versus untreated mice. As shown in fig. 5A, the inventors observed that: a significant reduction in myeloid infiltration (28% versus 17%) favoured lymphoid population in HEI 2328-treated mice (67% versus 76%). In lymphoid populations, both Treg and T γ δ lymphocytes were elevated (fig. 5B). Within myeloid infiltration, HEI2328 statistically reduced the rate of inflammatory monocytes (CD11 b)⁺CD11c^-Ly6c⁺Ly6g^-CD3^-NK1.1^-B220^-). In contrast, treatment with HEI2328 did not alter the ratio of conventional dc (cdc) and plasma cell-like dc (pdc) (fig. 5C-D).

In summary, compound HEI2328 reduced the rate of inflammatory monocytes. This may explain how HEI2328 inhibits DSS-induced colonic inflammation.

Thus, in a mouse model of colitis, the inventors have shown that: compound HEI2328 can reduce DSS-induced colonic inflammation. This result opens up a very interesting view for the treatment of inflammatory diseases.

4) Study 4-syngeneic tumor mouse model:

4.1. HEI2328 has been shown to affect the ratio of M-MDSC (myeloid derived suppressor cells) in the spleen of treated mice, and the inventors hypothesized that: HEI2328 reduces immunosuppression and thus may enhance anti-tumor responses. To test this hypothesis, immunocompetent C57B16J (Envigo, Gannat France) mice were injected with the melanoma cell line B16-F10. First, the inventors demonstrated in vitro that B16F10 cells were sensitive to HEI2328 treatment. As shown in fig. 6, the addition of HEI2328 on BzATP-treated cells increased the opening of the macropores by more than 6 times; this is illustrated by the increased number of Topro3 positive cells. Interestingly, the inventors noted that HEI2328 alone had no toxic effects in the absence of BzATP.

These results indicate that HEI2328 has a cytotoxic effect on B16F10 cells in the presence of ATP. Thus, the inventors tested the compound on a classical tumor mouse model. Right flank subcutaneous injections of 5X 10 of C57B16 mice receiving intraperitoneal injections of 2.3mg/kg HEI2328 (in PBS/10% DMSO) or placebo (PBS/10% DMSO) daily for 12 days⁵B16F10 cells (in 200. mu.l PBS). As shown in fig. 7A, tumor incidence was delayed and tumor growth decreased by 20% in the treated group (fig. 7B, C). However, this encouraging result was not statistically significant.

Due to the poor solubility of HEI2328, one chlorine was directly replaced with one azo compound in the aromatic cycle to synthesize a new molecule, giving compound HEI 3090. This novel compound HEI3090 enhanced the macropore opening in the presence of BzATP and required functional P2RX7 to mediate its effect, since the incorporation of Topro3 was lost in splenocytes isolated from transgenic P2RX7 KO mice (fig. 8A). By using a heterologous cell line expressing human P2RX7, the inventors demonstrated that: HEI3090 enhanced the macropore opening in the presence of BzATP (fig. 8B). Indeed, at the same BzATP concentration, more cells stained positive for Topro 3.

Interestingly, the enhancement of the macropore openings correlated with increased B16F10 cell death, as shown in fig. 9. BzATP and HEI3090 treated cells for 1h incorporated 3-fold more Topro3 compared to BzATP-only treated cells (fig. 9A). Prolonged incubation increased cell death, as up to 80% dead cells were observed after 3h treatment (fig. 9B).

4.2. By killing tumor cells, HEI3090 may represent a new anti-tumor therapy. To test this hypothesis, immunocompetent C57B16J mice were injected subcutaneously with 5X 10⁵B16F10 or 5X 10⁵LLC (Lewis lung carcinoma) cells and treated daily with HEI3090(ip, 2.5mg/kg in 10% DMSO/PBS) or placebo (10% DMSO/PBS). As shown in fig. 10, HEI3090 was effective in inhibiting tumor growth. First, the appearance of tumors was delayed by 1 to 4 days in the group receiving HEI3090 treatment (fig. 10A, C), and second, the tumor volume and tumor weight were greatly reduced (fig. 10B, D). In both tumor mouse models, the tumor weight of the treated group was reduced 10-fold in the B16F10 model (8mg versus 81mg) and 4-fold in the LLC model (80mg versus 295 mg). Interestingly, tumors from the HEI3090 treated group were more immunogenic than tumors from the placebo group. Indeed, in the presence of HEI3090, the tumor expressed 2-fold more major complex histocompatibility 1(MCH1) and PD-L1 (fig. 10E), and the tumor was enriched for CD45 positive cells (fig. 10F).

The inventors performed a dedicated experiment to test the hypothesis that HEI3090 might induce Immunogenic Cell Death (ICD). They clearly demonstrate that: in fig. 16, HEI3090 did not stimulate ICD, but rather promoted its anti-tumor effect by stimulating P2RX7 expressing immune cells.

In addition, figure 10 shows that HEI3090 treatment increased the immunogenicity of tumors. Thus, the inventors combined HEI3090 with anti-PD-1 treatment and showed that: these combination treatments not only cured tumor bearing mice, but also established memory T cell responses (fig. 19).

Since P2RX7 is highly expressed by immune cells, the inventors believe that the effect of HEI3090 on the immune compartment was determined and characterized the composition of tumor infiltrating immune cells. As shown in FIG. 11A, tumor infiltrates from the HEI3090 treated group contained fewer immunosuppressive cells (fewer M-MDSCs in B16F10 tumors and fewer PMN-MDSCs in LLC tumors). In addition, HEI3090 treatment may enhance the effector response, since the ratio between effector cells (T γ δ, NK, CD4+) and total MDSCs favors effector cells (fig. 11B).

HEI3090 has been shown to affect anti-tumor immune responses, and the inventors aimed to determine which immune cells are key players. First, the inventors consumed T CD4+ lymphocytes. As shown in fig. 12, the tumors in the group treated with HEI3090 and anti-T CD4+ antibody were larger than in the HEI3090 group and smaller than in the placebo group. This intermediate phenotype suggests that other immune cells are involved in the HEI-induced anti-tumor response. As shown in fig. 17, both NK and CD4+ cells were required to deliver HEI3090 anti-tumor effects, whereas T CD8+ cells were not. The inventors have also shown that: these two immune cell populations were activated and expressed more IFN- γ in tumors of mice treated with HEI3090 (fig. 17E-G).

Since IFN- γ secreting cells were activated by IL18, the inventors tested whether the anti-tumor effect of HEI3090 was dependent on IL18 (fig. 18). First, they show: HEI3090 increased P2RX7 expression on dendritic cells (well known as IL18 producing cells). Secondly, they show: both in plasma and intratumorally, IL18 was overexpressed in mice receiving HEI 3090. Third, they demonstrated that by using two different approaches (blocking IL18 antibody and IL 18-/-mice): in the absence of IL18, HEI3090 no longer blocked tumor growth. These results highlight IL18 as a target for HEI 3090.

In summary, it appears that:

-HEI3090 is a first molecule that potentiates P2RX7 in an ATP-rich microenvironment;

-HEI3090 is non-toxic in the absence of endogenous P2RX7 ligand (ATP), which is only found in inflammatory and/or tumor microenvironments. Tests were performed on hP2RX7 or mP2RX7 stably transfected HEK cell lines and on NCI panels of 60 human tumor cell lines;

-HEI3090 is specific for P2RX7, as in the case of P2RX7^-/-No effect was demonstrated when splenocytes prepared from mice were tested;

-HEI3090 is an orthosteric modulator of P2RX7, which reduces EC of ATP and 3' -O- (4-benzyl) benzoyl ATP (BzATP)₅₀And increasing ATP-induced death of P2RX 7-expressing cells;

HEI3090 blocked the growth of lung cancer (Lewis lung cancer) and melanoma (B16-F10) tumors in syngeneic mouse tumor models, as evidenced by an increase in overall survival of the mice from 22 days to 50 days in treated mice;

-the anti-tumor effect of HEI3090 is mediated in part by T CD4+ lymphocytes;

HEI3090 triggers IL18 and NK cell mediated anti-tumor responses, as in IL18 depletion (blocking antibody and Il 18)^-/-Mice) and NK cells (anti-NK 1.1 antibody) in mice;

-HEI3090 increased the immunogenicity of the tumor as demonstrated by an increased number of MHC-1 and PD-L1 positive tumor cells;

when used in combination with anti-PD-1 treatment, HEI3090 cured LLC tumor-bearing mice;

-HEI3090 induced memory T cell responses.

Interestingly, the inventors were unable to detect any modification of immune cells in the spleens of treated mice. In combination with these in vitro results demonstrating that HEI3090 is active only in the presence of ATP, it is believed that: in vivo HEI3090 is only active in inflammatory and tumor microenvironments known to contain high levels of ATP. Taken together, these data indicate that: there was no or little off-target effect with the HEI3090 compound.