EA040469B1 - 1,2,3',5'-TETRAHYDRO-2'H-SPIRO[INDOL-3,1'-PYRROLO[3,4-C]PYRRROLE]-2,3'-DIONE COMPOUNDS AS THERAPEUTIC AGENTS THAT ACTIVATE TP53 - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to novel 1,2,3',5'-tetrahydro-2H-spiro[indole-3,1'-pyrrolo[3,4c]pyrrole]-2,3'-dione compounds and these compounds for use in as a medicine, especially for the treatment of diseases in which p53-Mdm2 protein-protein interactions are impaired and/or which are susceptible to inhibition of p53-Mdm2 interactions, including proliferative diseases such as cancer. In addition, the present invention provides pharmaceutical compositions containing the above compounds.

Background Art p53 is a transcription factor that responds to cellular stress by regulating the transcription of numerous genes that determine cell fate. Under stressful conditions, p53 can induce cell cycle arrest and DNA repair processes or cell death programs such as apoptosis or senescence. The choice between these reactions depends on the type and intensity of stress signals. In human cells, p53 activity is tightly controlled by its negative regulator, a protein called Mdm2. Mdm2 forms a tight complex with the trans-activation domain of p53, blocking its ability to regulate target genes and exert antiproliferative effects. In addition, Mdm2 promotes nuclear export and rapid degradation of p53 via the ubiquitin-proteasome system.

Playing an important role in the cellular response to stress, p53 is the main obstacle to oncogenesis. Patients with Li-Fraumeni syndrome who inherit mutated p53 are highly susceptible to cancer. Mice with a damaged p53 gene appear normal, but by 6 months of age are prone to spontaneous development of various neoplasms. This significant role of p53 in tumor suppression results in its function being turned off in virtually all human cancers, either through mutation of the p53 gene or through aberrant expression of proteins that act as its negative regulators, such as Mdm2.

Mdm2 gene amplification has been reported in more than 10% of 8000 different human cancers, including sarcomas, lung and gastric tumors, in which the p53 gene is intact. Several other tumors acquire a single nucleotide polymorphism in the Mdm2 promoter, resulting in a 2-3-fold increase in Mdm2 expression, which correlates with accelerated tumor formation. These changes are perceived as the main mechanisms of inhibition of p53 function in cancers that retain wild-type p53.

Functional genetic studies in mice have shown that recovery of inactivated p53 is sufficient to induce rapid regression of several different types of tumors. Using this approach, targeting the p53-Mdm2 small molecule interaction to release and reactivate p53 has emerged as a promising therapeutic strategy for the treatment of human cancers that are wild-type p53. In recent years, several groups of low molecular weight non-peptide inhibitors of the p53-Mdm2 interaction have been registered, including nutlins, piperazine-4-phenyl derivatives, chalcones, sulfonamides, benzodiazepindiones, and spirooxindoles. MDM2 inhibitors produce both general and distinct cellular responses in normal and tumor cells, consistent with previous results from genetic studies. In normal cells, activation of p53 by MDM2 inhibitors causes cell cycle arrest but not cell death. In tumor cells, p53 activation by inhibitors causes not only cell cycle arrest, but also cell death. This profile provides the promise of high selectivity and low toxicity potential therapy. However, none of these Mdm2 antagonists have been proven effective in human clinical trials. Thus, there is still a need for new compounds with improved activity, favorable pharmacokinetics and toxicity profile.

The previous application of the inventors WO 2015/189799 discloses compounds containing 1,1',2,5'-tetrahydrospiro[indole-3,2'-pyrrole]-2,5'-dione system, which exhibit high and specific antitumor activity in in vitro studies. However, further studies have shown that their in vivo efficacy is moderate, and the most potent compounds exhibit unacceptably high clearance in human microsomes, precluding their clinical efficacy.

Therefore, there is still a need for compounds with excellent in vitro activity, having improved pharmacokinetics and thus exhibiting significant anti-cancer efficacy both in mouse in vivo models and in future clinical trials.

The present invention solves the problem by providing new compounds containing 1,2,3',5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'- dione system.

The essence of the invention

In a first aspect, the present invention provides a compound having the following structure

- 1 040469

wherein

R ¹ is meta-halophenyl which is optionally further substituted with one or two substituents independently selected from the group consisting of halo, -OH, -NH2, -NO2, -CN, -C ₁ -C ₆ -alkyl, -O-(C ₁ -C ₆ -alkyl), -S-(C ₁ -C ₆ -alkyl), -C(O)O-(C ₁ -C ₆ -alkyl), -NH(C ₁ -C ₆ -alkyl) and -N(C ₁ -C ₆ alkyl) ₂ ;

R ² and R ³ are independently H or halogen;

R ⁴ represents -C ₁ -C ₆ -alkyl;

R ⁷ is -OCH ₃ ;

R ⁵ , R ⁶ , R ⁸ , R ⁹ are independently H, halogen, -OCH ₃ , -NH(CH ₃ ) or -N(CH ₃ )2.

Z is CR ⁸ or N, Y is CR ⁹ or N, provided that Z is not CR ⁸ and Y is not CR ⁹ at the same time.

Preferably, in formula (I), R ¹ is meta-halophenyl which is optionally further substituted with one to two substituents independently selected from the group consisting of halo, -C1-C6-alkyl, -O-(C1-C6-alkyl), -NH(C1-C6-alkyl) and -N(C1-C6-alkyl)2. More preferably, in the definition of R ¹ substituent in formula (I), C 1 -C ₆ alkyl is C ₁ -C ₃ alkyl.

Even more preferably, in formula (I), R ¹ is meta-halophenyl which is optionally further substituted with one to two substituents independently selected from the group consisting of halogen, -CH3, -OCH3, -NH(CH3) and -N(CH3 )2. Even more preferably, R ¹ is meta-halophenyl which is optionally further substituted with one or two substituents independently selected from the group consisting of halo, -CH3 and _-OCH3 . Most preferably, R ¹ is meta-halophenyl which is optionally further substituted with one to two substituents independently selected from the group consisting of halo.

Preferably in formula (I), R ² is H and R ³ is Cl.

Preferably in formula (I), R ⁴ is isopropyl or isobutyl.

Preferably, in formula (I), Z and Y are both N. More preferably, in such an embodiment, R ⁵ and R ⁶ are both —OCH ₃ .

Preferably, in formula (I), Z is CR ⁸ and Y is N. More preferably, in such an embodiment, R ⁸ is H and at least one of R ⁵ and R ⁶ is -OCH3 and the second is selected from H, -N(CH3)2 and -OCH3.

As a specific compound of the invention, one of the following groups can be mentioned:

(1) (38)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)-1,2, 3',5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (2) (38)-6-chloro-2' -(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'- (propan-2-yl)-1,2,3',5'-tetrahydro-2 'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (3) (38)-6-chloro-2'-(5-chloro-2-methylphenyl )-6'-(propan-2-yl)-5'-(2,4,6- 2 040469 trimethoxypyrimidin-5-yl)-1,2,3',5'-tetrahydro-2'H-spiro[ indole-3,1'-pyrrolo[3,4c]pyrrole]-2,3'-dione (4) (38)-6-chloro-2'-(3-chloro-4-fluorophenyl)-5'-( 2,4-dimethoxypyrimidin-5-yl)-6'(propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indol-3,G-pyrrolo[3,4 -s]pyrrole]-2,3'-dione (5) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-6'-(propan-2-yl)-5'- (2,4,6|trimethoxypyrimidin-5-yl)-1,2,3\5<tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4c]pyrrole]-2,3'- dione (5) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-[6-(dimethylamino)-4-methoxypyridin-3yl]-6'-(propan-2- yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'dione (6) ( 38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan2-yl)-1,2,3',5 '-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (7) (38)-6-chloro-2'-(3, 4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3 ,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione (8) (38)-6-chloro-2%5-chloro-2-fluorophenyl)-5'-(4,6 -dimethoxypyridin-3-yl)-6'-(propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indol-3,G-pyrrolo[3,4-c ]pyrrole]-2,3'-dione (9) (38)-6-chloro-2'-(5-chloro-2,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl) -6'(propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3' -dione (10) (38)-6'-(butan-2-yl)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl )-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4c]pyrrole]-2,3'-dione

As a more specific compound of the invention, one of the following groups can be mentioned:

(1) (3X)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-propan-2yl)-1,2,3', 5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (2) (3X)-6-chloro-2'-(5 -chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'- (propan-2-yl)-1,2,3',5'-tetrahydro-2'R- spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (3) (3X)-6-chloro-2'-(5-chloro-2-methylphenyl)-6 '-(propan-2-yl)-5'-(2,4,6-trimethoxypyrimidin-5-yl)-1,2,3',5'-tetrahydro-2R-spiro[indole-3,r-pyrrolo [3,4с]pyrrole]-2,3'-dione (4) (3X)-6-chloro-2'-(3-chloro-4-fluorophenyl)-5'-(2,4-dimethoxypyrimidine-5- yl)-6'- (propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]- 2,3'-dione (5) (3X)-6-chloro-2'-(5-chloro-2-fluorophenyl)-6'-(propan-2-yl)-5'-(2,4,6 - trimethoxypyrimidin-5-yl)-1,2,3',5'-tetrahydro-2H-spiro[indole-3,G-pyrrolo[3,4c]pyrrole]-2,3'-dione (7) (30 )-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan2-yl)-1,2,3',5' -tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (10) (3X)-6-chloro-2'-(5-chloro -2,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'(propan-2-yl)-1,2,3',5'-tetrahydro-2'R-spiro [indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (11) (3X)-6'-(butan-2-yl)-6-chloro-2'-( 5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-1,2,3',5'-tetrahydro-2H-spiro[indole-3,1'-pyrrolo[3 ,4s]pyrrole]-2,3'-dione

Alternatively, as a more specific compound of the invention, one of the following groups can be mentioned:

- 3 040469 (6) (36)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-[6-(dimethylamino)-4-methoxypyridin-3yl]-6'-(propane- 2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'dione (8) (36 )-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2yl)-1,2,3',5' -tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione (9) (35)-6-chloro-2'-(5-chloro -2-fluorophenyl)-5'-(4,6-dimethoxypyr ide in-3-yl)-6'(propan-2-yl)-1,2,3',5'-tetrahydro-2'R-spiro [indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione

The most preferred compound of the invention is the compound represented by the following structure

which is (3S)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'(propan-2-yl)-1,2 ,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The second most preferred compound of the invention is the compound represented by the following structure

which is (3S)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'(propan-2-yl)-1,2,3 ,5'-tetrahydro-2'H-spiro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Another aspect of the invention relates to a compound of formula (I) for use as a drug.

Preferably, the drug is suitable for the prevention and/or treatment of diseases selected from the group consisting of cancer, immune diseases, inflammatory conditions, allergic skin diseases associated with excessive proliferation, blinding disease and viral infections.

Another aspect of the invention relates to a pharmaceutical composition containing, as an active ingredient, a compound of formula (I) in combination with at least one pharmaceutically acceptable excipient.

The last aspect of the invention relates to a method for the treatment and/or prevention of diseases selected from the group consisting of cancer, immune diseases, inflammatory conditions, allergic diseases of the skin associated with excessive proliferation, blinding disease and viral infections, comprising administering a therapeutically effective amount of a compound of the formula ( I) or a pharmaceutical composition as defined above.

Brief description of the drawings

In FIG. 1 shows the in vivo efficacy of compound 107 from WO 2015/189799 as a reference compound in a mouse model of human osteosarcoma (SJSA-1). SJSA-1 cells were inoculated subcutaneously (sc) at 3x10 ⁶ /mouse; the test compound was administered orally (po) according to the scheme q1dx 14; 7 mice per group.

In FIG. 2 shows the in vivo efficacy of compound 7, 8 and 11 of the present invention in a mouse model of human osteosarcoma (SJSA-1). SJSA-1 cells were inoculated subcutaneously (sc) at 3x10 ⁶ /mouse; the studied compounds were administered orally (p/o) according to the q1dx 14 scheme; 8 mice per group.

Detailed description of the invention

Where the compounds of the invention may exist in one or more tautomeric forms, all such forms, although not expressly indicated in the above formula, are within the scope of the present invention. Accordingly, the compounds may be present as a mixture of tautomers or as a single tautomer.

The terms used in the present invention have the following meanings. Other terms

- 4 040469 not defined below, have meanings as understood by a person skilled in the art.

The term C1- _C6 alkyl is a straight or branched saturated hydrocarbon containing from 1 to 6 carbon atoms. Examples of C ₁ -C ₆ alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, n-pentyl and n-hexyl. More preferably C ₁ C ₆ alkyl is C ₁ -C ₄ alkyl, C ₁ -C ₃ alkyl or C 1 -C 2 alkyl. Designation C1-C ₄ -alkyl, CrQ alkyl, C ₁ -C ₂ -alkyl represents a saturated hydrocarbon with a straight or branched chain containing from 1 to 4, 3 or 2 carbon atoms, respectively. Most preferably C ₁ -C ₆ alkyl is C ₁ alkyl which is a methyl group (abbreviated as Me).

The term halogen is selected from F, Cl, Br and I. Preferably halogen is selected from F and Cl.

The term m-halophenyl, which is present in the definition of the R ¹ group, means a phenyl group which is substituted by halogen as defined above in the meta position relative to the point of attachment of the phenyl group to the nitrogen atom of the pyrrolo[3,4-c]pyrrole ring system.

The expression Z is CR ⁸ or N, Y is CR ⁹ or N, provided that Z is not CR ⁸ and Y is not CR ⁹ simultaneously means that in the compound of the invention Z is CR ⁸ and Y is N , or Z is N and Y is CR ⁹ , or Z is N and Y is N.

Since the compounds of the invention may be acidic or basic, they may form suitable acid addition salts with base or acid, respectively.

A pharmaceutically acceptable acid addition salt refers to those salts which retain the biological effectiveness of the free bases and which are not biologically undesirable. Acid addition salts can be formed with inorganic (mineral) acids or organic acids. As examples of acids, mention may be made of hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric, carbonic, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamic, aspartic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic, such as 2-naphthalenesulfonic, pamic, xinafoic, hexanoic acid.

The acid addition salt can be prepared in a simple manner by reacting a compound of formula (I) with a suitable inorganic or organic acid in an amount substantially equimolar to a compound of formula (I), optionally in a suitable solvent such as an organic solvent to form a salt which is usually isolated , for example, by crystallization and filtration. For example, the free bases of the compounds can be converted to the corresponding hydrochloride salts by treating a solution of the compound, eg in methanol, with a stoichiometric amount of hydrochloric acid or hydrogen chloride in methanol, ethanol or diethyl ether, followed by evaporation of the solvents.

Similarly, pharmaceutically acceptable base addition salts include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Salts derived from pharmaceutically acceptable, non-toxic organic bases include salts of primary, secondary, and tertiary amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine, and triethanolamine .

The compounds of formula (I) can be obtained using the following methods.

Compounds based on 1',2,5'-tetrahydrospiro[indole-3,2'-pyrrolo]-2,5'-dione fused to a pyrrole ring (compounds of formula (I)) can be prepared according to the following scheme reactions 1.

- 5 040469

Reaction scheme 1.

First, methyl ketone C-1 was treated with isatin C-2 in the presence of a base, usually diethylamine (DEA) or lithium bis(trimethylsilyl)amide (LiHMDS).

The resulting C-3 aldol was subsequently dehydrated under acidic conditions, usually using conc. hydrochloric acid (12 M), providing unsaturated compound C-4.

In parallel, amide C-6 was prepared by coupling amine C-5 with prop-2-ic acid, preferably using coupling reagents, usually carbodiimide reagents such as dicyclohexylcarbodiimide (DCC) or (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC- HCl).

Hydroamination of alkyne С-6 with amine С-7 and subsequent interaction with enone С-4 leads to substituted pyrrole С-8. This one-pot reaction can usually be carried out in acetic acid under microwave irradiation.

Intermediate C-8 can be oxidized to the 3-hydroxy-2-oxindole derivative in the presence of an excess of a base, usually sodium tert-butoxide, a suitable trialkyl phosphite, usually trimethyl or triethyl phosphite, and atmospheric oxygen.

Such resulting C-9 compounds were cyclized in an acidic medium, typically in trifluoroacetic acid, to provide the desired racemic fused spirocyclic oxindoles.

Finally, the desired S-enantiomer was separated using chiral HPLC conditions.

More detailed information on the preparation of the 1,1',2,5'-tetrahydrospiro[indolo-3,2'-pyrrolo]-2,5'dione core has been described in our previous patent application WO 2015/189799 A1.

The corresponding C-1 ketones, C-2 isatins and amines are commercially available or can be prepared using the following methods.

Scheme 2 illustrates one typical method for the preparation of 5-amino-2,4-dimethoxypyrimidine (C7A) by nucleophilic substitution of the chlorine at positions 2 and 4 in 2,4-dichloro-5-nitropyrimidine (C-7A1) with a methoxy group and subsequent reduction of the nitro group at 2 ,4-dimethoxy-5nitropyrimidine (C-7A2).

- 6 040469

Reaction scheme 2.

Scheme 3 illustrates a typical method for the preparation of 3-amino-4,6-dimethoxypyridine (C-7B) by nucleophilic substitution of chlorine at positions 2 and 4 in 2,4-dichloro-5-nitropyridine (C-7B1) with a methoxy group followed by reduction of the nitro group at 2,4-dimethoxy-5-nitropyridine (C-7B2).

Reaction scheme 3.

Scheme 4 illustrates a typical method for the preparation of 5-amino-4-methoxy-2-(dimethylamino)pyridine (C-7C) by nucleophilic substitution of the chlorine at position 2 in 2-chloro-4-methoxy-5nitropyridine (C-7C1) with a methoxy group and subsequent reduction of the nitro group in 4-methoxy-2(dimethylamino)-5-nitropyridine (C-7 C2).

Reaction scheme 4.

NO

Me ₂ MN (aq.)

MeOH/THF

CI

NO

MeOH/THF

N.

H ₂ , Pd/C

NH-

N.

S-7S1

S-7S2

S-7S

Scheme 5 illustrates a typical preparation of 5-amino-2,4,6-trimethoxypyrimidine (C-7D) via nitration of 2-chloro-4,6-dimethoxypyrimidine (C-7D1) at position 5 after nucleophilic substitution of chlorine at position 2 to 2 -chloro-4,6-dimethoxy-5-nitropyrimidine (C-7D2) methoxy group. The last step was the reduction of the nitro group in 2,4,6-trimethoxy-5-nitropyrimidine (C-7D3).

Reaction scheme 5.

As mentioned above, the compounds of the invention are intended for use as a medicament which is useful in the prevention and/or treatment of diseases selected from the group consisting of cancer, immune diseases, inflammatory conditions, allergic skin diseases associated with excessive proliferation, blinding disease and viral infections.

In particular, the compounds according to the invention are suitable for the prevention and/or treatment of diseases associated with cell cycle dysregulation and apoptosis, i.e. immune diseases such as, for example, autoimmune diseases and conditions associated with tissue/organ transplant rejection, such as rheumatoid arthritis, graft versus host disease, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, polymyositis; chronic inflammatory conditions are asthma, osteoarthritis, atherosclerosis, Crohn's disease; inflammatory or allergic skin conditions are psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforme, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullous

- 7 040469 pemphigoid, pemphigus, epidermolysis bullosa acquired; the hyperproliferative disorder is Li-Fraumeni syndrome; cancer or neoplastic diseases are benign or malignant tumors, sarcomas such as rhabdomyosarcoma, bone cancer such as osteosarcomas, brain carcinoma such as soft tissue brain tumor, kidney, liver, adrenal, bladder, breast, stomach, gastric tumors, ovarian, colon, rectum, prostate, pancreas, lung, vagina or thyroid gland, glioblastoma, multiple myeloma, cancer of the gastrointestinal tract, in particular colon carcinoma or colorectal adenoma, tumor of the neck and head, melanoma, prostatic hyperplasia, neoplasia, epithelial neoplasia, breast carcinoma, leukemia such as B- or T-cell lymphomas, polycythemia vera, thrombocythemia, adrenocortical carcinoma, including metastasis to other organs, respectively; proliferative vitreoretinopathy, viral infections are herpes, papilloma, HIV, hepatitis.

In the treatment of the aforementioned diseases, the compounds of the invention may be administered in the form of a chemical compound, but they are usually used in the form of pharmaceutical compositions containing a compound according to the invention or a pharmaceutically acceptable salt thereof, as defined above, as an active ingredient in combination with pharmaceutically acceptable carriers and excipients. .

In the treatment of the aforementioned diseases, the pharmaceutical compositions of the invention may be administered by any route, preferably orally or parenterally, and will be in the form of a preparation intended for use in medicine, depending on the intended route of administration.

Solid preparations may take the form of, for example, tablets or capsules prepared by standard methods with pharmaceutically acceptable inactive ingredients such as binders (eg, pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (eg lactose, sucrose, carboxymethyl cellulose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (eg magnesium stearate, talc or silicon dioxide); disintegrants (eg crospovidone, potato starch or sodium starch glycolate); wetting agents (eg sodium lauryl sulfate). Tablets may be coated according to methods well known in the art, standard coatings, sustained/controlled release coatings, or enteric coatings. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by standard methods with pharmaceutically acceptable inactive ingredients such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl p- or propylhydroxybenzoate or sorbic acid). The formulations may also contain suitable buffers, flavors, colors and sweeteners.

Formulations for oral administration may be suitably formulated by methods known to those skilled in the art to obtain controlled release of the active compound.

Parenteral administration includes administration by intramuscular and intravenous injection and infusion (infusion) intravenously. Formulations for parenteral administration may be in unit dosage form, for example in ampoules or in multi-dose containers, with an added preservative. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, the active ingredient may be in the form of a powder for reconstitution with a suitable vehicle, eg sterile, pyrogen-free water.

A method of treatment using the compounds of this invention will comprise administering a therapeutically effective amount of a compound of the invention, preferably in the form of a pharmaceutical composition, to a subject in need of such treatment.

A suggested dosage of the compounds of the present invention is from about 0.1 to about 1000 mg per day, in single or divided doses. The skilled artisan will appreciate that the selection of the dose required to achieve the desired biological effect will depend on a number of factors, such as the particular compound, application, route of administration, age and condition of the patient, and the exact dosage will ultimately be at the discretion of the attending physician.

Examples

The following examples are not intended to limit the invention, but simply serve to illustrate the present invention.

- 8 040469

Abbreviations. AcOEt ethyl acetate ASON acetic acid br.s wide singlet CaCl ₂ calcium chloride SNS1 ₃ chloroform d doublet dd dd doublet of doublets doublet of doublets of doublets DC doublet of quartets DEA Α,Α'-diethylamine DCC Α,Α'-dicyclohexylcarbodiimide dhm dichloromethane EDCHC1 N-(3-dimethylaminopropyl)-A-ethylcarbodiimide hydrochloride EtOH ethanol equiv. equivalents ESI electrospray ionization h watch) HC1 hydrogen chloride HPLC high performance liquid chromatography l liter LiHMDS lithium bis(trimethylsilyl)amide m multiplet Meon methanol _MgSO4 magnesium sulfate ml milliliter(s) MSOH methanesulfonic acid MW microwave _NaHCO3 bicarbonate of soda NaOH sodium hydroxide _{Na2SO4 _} sodium sulfate NMR nuclear magnetic resonance NF HPLC normal-phase high-efficiency liquid chromatography -OMe methoxy group -OEt ethoxy group analytical grade clean for analysis ptsa-h _2o p-toluenesulfonic acid monohydrate sq. quartet RP-HPLC reverse phase high efficiency liquid chromatography With singlet sep septet

-9040469

SFH supercritical fluid chromatography SQDMS mass spectrometer with a single quadrupole detector T triplet TFA trifluoroacetic acid TFUA trifluoroacetic anhydride THF tetrahydrofuran those thin layer chromatography UEZHHMS ultra performance liquid chromatography with spectrometry MKL microliter

mass TLC was performed with silica gel 60 F254 on aluminum foil (Sigma-Aldrich, Merck) using appropriate solvent systems. Visualization was usually carried out using UV radiation (254 nm).

UVLC-MS method.

UPLCMS analyzes were performed on a UVLC liquid chromatograph equipped with an FDM detector and an SQD MS detector operating in ESI(+) or ESI(-) mode using a C18, 2.1 mm x 100 mm, 1.7 µm column (AQUITY UPLC VEN or equivalent) . HPLC or LC/MS purity methanol, HPLC purity water, HPLC or LC/MS purity formic acid, analytical grade purity 25% ammonia solution and their mixture was used as the mobile phase. Operating conditions were as follows: mobile phase flow 0.45 ml/min, wavelength 210-400 nm, injection volume 1 μl, column temperature 60°C, autosampler temperature 5°C. The analysis was carried out 5.5 min + 1.5 min to delay the next injection.

Linear Gradient Elution:

Time [min] % A % IN gradient curve θ,ο 80.0 20.0 - 4.0 0.1 99.9 linear (6) 5.5 80.0 20.0 straight (11)

Solutions were prepared as follows.

Preparation of mobile phase A1 - basic gradient: 25 μl of formic acid and 250 μl of 25% ammonia solution were added to 250 ml of water. Ultrasonic bath with degassing for 10 min.

Preparation of mobile phase A2 - acid gradient: 50 μl of formic acid was added to 250 ml of water. Ultrasonic bath with degassing for 10 min.

Mobile phase B: methanol supergradient.

Methods of synthesis.

Intermediate C-4A: 6-chloro-3-(3-methyl-2-oxobutylidene)-1 H-indol-2-one.

A 5 L reaction vessel was charged with C-3A aldol (1015 g, 3.79 mol, 1 eq) and EtOH (2.85 L) was added. The suspension was heated to 55° C. and 12 M HCl (202 ml, 2.42 mol, 0.64 eq.) was added in one portion. Then heating was continued until the boiling point of ethanol. It was found that more EtOH (500 ml) was required due to the rapid precipitation of the C-4A product. After 1 h, UELCMS analysis showed 98% of the peak area of the product. The heating was stopped and the cooling of the reaction mixture started. When the temperature of the reaction mixture reached 50°C, the entire mixture was transferred to a beaker and cooled to 0-5°C. The solid was filtered off and washed with 1.3 L of cold EtOH. The solid product was dried in a laboratory dryer (40° C.) for 3 h and then air dried overnight. As a result, compound C-4A was obtained as an orange solid (692 g, 73% yield, 98.1% purity according to UELCMS analysis).

Intermediate C-3A: 6-chloro-3-hydroxy-3-(3-methyl-2-oxobutyl)-2,3-dihydro-1H-indol-2-one.

- 10 040469

A 15 L reaction vessel was charged with 6-chloroisatin (1000 g, 5.5 mol, 1 eq) and EtOH (7 L) and then 3-methylbutanone (2.94 L, 27.5 mol, 5 eq) was added in one portion. ). The reaction mixture was heated to 40° C. and DEA (250 ml, 2.41 mol, 0.44 eq.) was added in one portion. Then heating was continued until the boiling point of ethanol. After 1 hour, UELCMS showed 71% product in the reaction mixture. Heating was continued for an additional 1 hour, and after this time the analysis UPLCMS showed 93% of the product. The reaction mixture was cooled to 50°C, then the entire mixture was transferred to a round bottom flask and all liquid ingredients were removed. The residue was suspended in DCM (3.4 L) and heated under reflux for 1 hour. After this time, heating was stopped and n-hexane (2 L) was added in one portion. The contents of the flask were cooled to approximately 5°C and stirred for 1.5 h at this temperature. The resulting mixture was filtered, and this resulting solid was washed with 500 ml of a DCM/n-hexane (1:1) mixture, followed by air drying overnight. As a result, the expected aldol product C-3A was obtained as a gray solid (965 g, 98.5% purity according to UELCMS analysis). The solvent mixture after filtration was reduced under vacuum to approximately 1.5 L, n-hexane (700 ml) was added and the resulting suspension was stirred for 0.5 h at room temperature. Another filtration followed by double washing with DCM/n-hexane (150 ml, 1:1 for each wash) and air drying provided a second portion of C-3A aldol product (50 g, 99.1% pure). The overall yield of C-3A aldol was 69% (1015 g, 98.5% purity according to UELCMS analysis).

Intermediate C-4B: 6-chloro-3-(3-methyl-2-oxopentylidene)-2,3-dihydro-1 H-indol-2-one.

H

A 250 ml reaction flask was charged with C-3B aldol (11 g, 39 mmol, 1 eq.) and EtOH (32 ml) was added in one portion. The suspension was heated to 55° C. and 12 M HCl (1.63 ml, 19.5 mmol, 0.5 eq.) was added in one portion. Then heating was continued until the boiling point of ethanol. After 1 hour, UELCMS showed 98% product. The reaction mixture was cooled to 0-5°C and stirred for 0.5 h. The solid residue was filtered off and washed with a small portion of cold EtOH. The solid product was air dried overnight. As a result, compound C-4B was obtained as an orange solid (5 g, 49% yield, 99% purity according to UELCMS analysis).

Intermediate C-3B: 6-chloro-3-hydroxy-3-(3-methyl-2-oxopentyl)-2,3-dihydro-1 H-indol-2-one.

ABOUT

In a round bottom flask (250 ml) 6-chloroisatin (15 g, 83 mmol, 1 eq.) was suspended in EtOH (70 ml) and then 3-methylpent-2-one (51.3 ml, 415 mmol, 5 equiv.). The reaction mixture was heated to 40° C. and DEA (4.34 ml, 42 mol, 0.5 eq.) was added in one portion. Then heating was continued until the boiling point of ethanol. After 1 hour, UELCMS showed 60% product. Heating was continued for an additional 2 hours, and after this time analysis UPLCMS showed 99% of the product. After cooling to room temperature, all liquid ingredients were removed under vacuum. The residue was suspended in AcOEt (50 ml) and stirred at room temperature for 1 hour. After this time, the contents of the flask were cooled to approximately 5°C and stirred for 1.5 hours at this temperature. The resulting mixture was filtered, and this resulting solid was washed with a small amount of cold EtOH, followed by air drying overnight. As a result, the expected aldol product C-3B was obtained as a light brown solid (5.1 g, 98.5% purity by UELCMS analysis). After filtration, the residue was pre-adsorbed on silica gel and purified by flash chromatography (30 to 60% AcOEt in n-hexane). After chromatography, the second part of the product C-3B was obtained as a light brown solid (5.9 g) with a purity of 97% (according to the analysis of UPLCMS). The overall yield of C-3B aldol was 47% (11 g, 98% purity according to UELCMS analysis).

Intermediate С-6А: N-(3-chlorophenyl)prop-2-inamide.

- 11 040469

In a 2 L two-necked round bottom flask equipped with a mechanical stirrer, 3-chloroaniline (75 g, 590 mmol, 1 eq.) was dissolved in 500 ml DCM (HPLC grade). Prop-2-ic acid (53.5 g, 760 mmol, 1.3 eq.) dissolved in 100 ml DCM was then added dropwise (amine salt appeared). In the next step, EDC-HCl (145 g, 760 mmol, 1.3 eq.) was added in several portions (the reaction flask was cooled in an ice bath during the addition to avoid refluxing with DCM). After complete addition of EDC-HCl, the reaction mixture was stirred for 2 h at room temperature and then the mixture was slowly transferred into a beaker containing 500 ml of water and 250 g of ice. Stirring was continued at 0-5° C. for approximately 15 minutes and then the white precipitate was filtered off, washed with 100 ml of cold water and air dried. As a result, the expected amide C-6A was obtained as a white solid (102 g, 96.6% yield, 96.3% purity according to UELCMS analysis).

Intermediate C-6B: N-(5-chloro-2-fluorophenyl)prop-2-inamide.

CI ά to

F

Prop-2-ic acid (15.16 g, 216.4 mmol, 1.05 eq.) was added in one portion to a stirred solution of 5-chloro-2-fluoroaniline (30 g, 206.1 mmol, 1 eq.) in toluene (400 ml) which was cooled in an ice/water bath. The mixture was stirred for 15 minutes, then DCC (44.65 g, 216.4 mmol, 1.05 eq.) was added portionwise while maintaining the temperature below 10°C. Stirring was continued at 5°C for 2 h, then the solid DCU was collected by filtration and washed with toluene (150 ml) and finally with AcOEt/n-hexane (150 ml, 1:9). The filtrate was concentrated to a volume of approximately 100 ml and stirred for 15 minutes at room temperature. The resulting precipitate was collected by filtration, washed with toluene/n-hexane (20 ml, 1:1), n-hexane (20 ml) and air-dried for 16 hours to give 15.29 g of the title product as a white solid. The filtrate was placed in a refrigerator for 3 hours and the resulting precipitate was collected by filtration, washed with toluene/nhexane (20 ml, 1:1), n-hexane (20 ml) and air-dried for 16 hours to give 13.29 g of amide C -6V. The filtrate was concentrated to a volume of approximately 20 ml and then hexane (400 ml) was added slowly with stirring. The mixture was heated at reflux for 30 min and the hot solution was filtered, concentrated to approximately 100 ml and placed in a refrigerator for 18 h. The resulting precipitate was collected by filtration, washed with n-hexane (2x20 ml) and air dried for 16 h to obtain an additional 7.19 g of C-6B amide. The overall yield of product C-B6 was 88% (35.77 g).

C-6C intermediate: N-(5-chloro-2-methylphenyl)prop-2-inamide.

CI rs?

In a 2 L two-necked round bottom flask equipped with a mechanical stirrer, 5-chloro-2-methylaniline (75 g, 530 mmol, 1 eq.) was dissolved in 1000 ml DCM (HPLC grade). Prop-2-ic acid (49 g, 690 mmol, 1.3 eq.) dissolved in 100 ml DCM (HPLC grade) was then added dropwise (amine salt appeared). In the next step, EDC-HCl was added in several portions (during the addition, the reaction flask was cooled in an ice bath to avoid refluxing with DCM). After complete addition of EDC-HCl, the reaction mixture was stirred for 1 h at room temperature. The entire mixture was transferred to a beaker containing 750 ml of water and 250 g of ice. Stirring was continued at 0-5° C. for approximately 15 minutes and then the white precipitate was filtered off, washed with 100 ml of cold water and air dried. As a result, the expected C-6C amide was obtained as a white solid (81 g, 80% yield, 99.1% purity by UELCMS analysis).

Intermediate C-6D: N-(3-chloro-4-fluorophenyl)prop-2-inamide.

- 12 040469

To a 1 L two-necked round bottom flask equipped with a magnetic stirrer and thermometer was added 3-chloro-4-fluoroaniline (14.8 g, 100 mmol, 1 eq.) followed by DCM (200 ml, HPLC grade) and prop-2 -ic acid (9.1 g, 130 mmol, 1.3 eq.). The reaction mixture was cooled to 0° C. and EDC-HCl (25.2 g, 130 mmol, 1.3 eq.) was added in several portions. The reaction is exothermic and exceeding +5°C during the addition should be avoided. After complete addition of EDC-HCl, the mixture was stirred for 1 h at 5°C. After this time, the ice bath was removed and 200 ml of cold water was added to the reaction mixture. The reaction mixture was stirred for about 0.5 h, the resulting precipitate was filtered off and washed with 100 ml of cold water. The solid thus obtained was redissolved in chloroform and the solution was washed with water, dried over Na ₂ SO ₄ and the solvent was evaporated to dryness. The resulting product was dried under vacuum. As a result, the expected amide C-6D was obtained as a light yellow solid (19.2 g, 98% yield, 98% purity according to UPLCMS analysis).

Intermediate C-6E: N-(3,4-difluorophenyl)prop-2-iminamide.

3,4-difluoroaniline (12.9 g, 9.9 ml, 100 mmol, 1 eq.) was added to a 1 L two-necked round bottom flask equipped with a magnetic stirrer and thermometer, followed by DCM (300 ml, HPLC grade) and prop-2-ic acid (9.1 g, 130 mmol, 1.3 eq.). The reaction mixture was cooled to 0° C. and EDC-HCl (24.9 g, 130 mmol, 1.3 eq.) was added in several portions. The reaction is exothermic and exceeding +5°C during the addition should be avoided. After complete addition of EDC-HCl, the mixture was stirred for 1 h at 5°C. After this time, the ice bath was removed and 300 ml of cold water was added to the reaction mixture. The reaction mixture was stirred for about 0.5 h, the resulting precipitate was filtered off and washed with 100 ml of cold water. The solid thus obtained was redissolved in AcOEt and the solution was washed with water, dried over Na ₂ SO ₄ and the solvent was evaporated to dryness. The product was then washed with 10 ml of cold DCM and dried under vacuum. As a result, the expected amide C-6E was obtained as a white with a grayish or yellowish tinge of a solid (17.3 g, yield 96%, purity 100% according to UPLCMS analysis).

Intermediate C-6F: N-(5-chloro-2,4-difluorophenyl)prop-2-ynamide.

In a 500 ml two-neck round bottom flask equipped with a magnetic stirrer, 5-chloro-2,4-difluoroaniline (10 g, 61 mmol, 1 eq.) was dissolved in DCM (150 ml, HPLC grade). Prop-2-ic acid (5.5 g, 78 mmol, 1.3 eq.) was then added dropwise (amine salt appeared). Then several portions of EDC-HCl (14 g, 78 mmol, 1.3 eq.) were added. (During the addition, the reaction flask was cooled in an ice bath to maintain room temperature.) After complete addition of EDC-HCl, the reaction mixture was stirred for another 1 h at room temperature. After this time, 150 ml of water was added and the mixture was transferred to a separating funnel. The phases were separated. The aqueous phase was extracted twice with DCM (2x100 ml). The organic fractions were combined, washed with brine, dried over MgSO ₄ and concentrated to approximately 50 ml under vacuum to obtain a thick suspension. The solids were filtered off and dried to give 8 g of creamy crystals. The filtrate was concentrated and then purified by flash chromatography (nhexane/AcOEt; 8:1->5:1) to give an additional 4.4 g of amide C-6F. As a result, the expected amide C-6F was obtained as an off-white solid (12.4 g, 94% yield, 100% purity according to UELCMS analysis).

Intermediate C-7A: 5-amino-2,4-dimethoxypyrimidine.

To a QianCap glass reactor (1850 ml) equipped with a magnetic stirrer was added C-7A2 (100 g, 540 mmol) followed by THF (800 ml). Then 10% palladium on carbon (2 g) was added in one portion and the reactor was connected to a source of hydrogen. The hydrogen pressure was set at the level

- 13 040469 bar and the reaction mixture was thoroughly stirred for 16 h with a continuous flow of hydrogen. After this time, UELCMS analysis showed complete conversion of the starting material. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo to about 150-200 ml. Then n-hexane (500 ml) was added dropwise and the suspension was stirred for 2 hours at room temperature. The precipitate was filtered off, washed twice with n-hexane (2x50 ml) and dried under vacuum. This gave amine C-7A as a yellow/green solid (77.94 g, 93% yield, 99% purity by UPLCMS analysis).

Intermediate C-7A2: 2,4-dimethoxy-5-nitropyrimidine.

NO ₂ fl I N^N

To a 2 L three-necked round-bottom flask containing a thermometer, a water condenser, a connection to an inert gas (argon) source and a mechanical stirrer, was added methanol (1 L, HPLC grade) and small pieces of sodium (65 g, 2.83 mol) under argon. , 2.2 eq.) was added slowly over about 1 hour with good stirring (the reaction is exothermic, but the reaction mixture was not cooled; another piece of sodium was usually added when the first one was used up). After dissolving the entire portion of sodium, the reaction mixture was cooled to -5°C and a freshly prepared suspension of C-7A1 (250 g, 1.29 mol, 1 eq.) in methanol (500 ml, HPLC grade) was carefully added (approximately 40 min.) portions with thorough mixing. (Caution: the reaction is very exothermic), avoid exceeding +10°C during the addition. In addition, a lot of solid product was formed during the reaction. After all of the substrate slurry had been added, the reaction mixture was held at 0-5° C. for about 0.5 h and then allowed to warm to room temperature (usually this took about 2 h). After this time, UELCMS analysis showed complete consumption of the substrate. Then the reaction mixture was cooled to approximately 5°C, the solid product was filtered off and washed with a small amount (100 ml) of cold methanol. The crude product was placed in a beaker with water (1 L) and thoroughly suspended with a mechanical stirrer (approximately 10 minutes of thorough mixing). The suspension was then filtered and the resulting solid was washed with water (500 ml), nhexane (200 ml) and air dried overnight. This yielded 211.5 g of C-7A2 as a light yellow solid (yield 88%, purity 99% according to UELCMS analysis).

Intermediate C-7B: 5-amino-2,4-dimethoxypyridine.

NH _? % _x o

To a QianCap glass reactor (1850 mL) equipped with a magnetic stirrer was added C-7B2 (40 g, 217 mmol) followed by THF (500 mL). Then 10% palladium on carbon (1.5 g) was added in one portion and the reactor was connected to a source of hydrogen. The hydrogen pressure was set at 2 bar and the reaction mixture was thoroughly stirred for 7 hours with a continuous flow of hydrogen. After this time, UELCMS analysis showed complete conversion of the starting material. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo. This gave the amine C-7B as a brown solid (33 g, 99% yield).

Intermediate С-7В2: 2,4-dimethoxy-5-nitropyridine.

NO ₂ %

To a 2 L three-necked round-bottom flask containing a thermometer, a water condenser, a connection to an inert gas (argon) source, and a mechanical stirrer, was added methanol (900 ml, HPLC grade) and, under argon, small pieces of sodium (26.7 g, 1. 16 mol, 2.1 eq.) was slowly added over about 1 hour with thorough stirring (note: the reaction is exothermic, but the reaction mixture was not cooled; another piece of sodium was usually added when the first one was used up). After dissolving the entire portion of sodium, the reaction mixture was cooled to -5°C and a freshly prepared suspension of compound C-7B1 (106.25 g, 0.55 mol, 1 eq.) in methanol (100 ml, HPLC grade purity) in small portions at

- 14 040469 thorough mixing (note: the reaction is highly exothermic, avoid exceeding +10°C during the addition). After all of the C7B1 substrate slurry had been added, the reaction mixture was kept at 0-5°C for approximately 40 minutes, then warmed to room temperature and stirred at 40°C for 3.5 hours. After this time, UPLCMS analysis indicated complete consumption of the substrate. Then the reaction mixture was cooled to a temperature below 10°C, the solid product was filtered off, washed with a small amount (50 ml) of cold methanol, water (100 ml), n-hexane (100 ml) and dried in air overnight. This yielded 99.3 g of C-7B2 as a light yellow solid (yield 98%, purity 97% according to UELCMS analysis).

Intermediate С-7С: 5-amino-4-methoxy-2-(dimethylamino)pyridine.

NH _?

A'

To a QianCap glass reactor (500 ml) equipped with a magnetic stirrer was added C-7C2 (4.5 g, 22.8 mol) followed by THF (70 ml) and methanol (70 ml). Then 10% palladium on carbon (0.48 g) was added in one portion, the reactor was connected to a source of hydrogen. The hydrogen pressure was set at 2 bar and the reaction mixture was thoroughly stirred for 17 hours with a continuous flow of hydrogen at room temperature. After this time, UELCMS analysis showed complete conversion of the starting material. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated to dryness in vacuo. This gave the C-7C amine as a dark solid (3.9 g; 96% yield; 95% purity by UELCMS analysis).

Intermediate С-7С2: 4-methoxy-2-(dimethylamino)-5-nitropyridine.

NO ₂

A'

A 0.5 L three-necked round bottom flask containing a thermometer, a water condenser and an addition funnel was charged with C-7Cl (10 g, 50.4 mmol, 1 eq.), THF (50 ml) and methanol (200 ml). The whole mixture was stirred at 22° C. for 10 minutes and then dimethylamine (13.7 ml, 60% solution in water) was added dropwise. After 1 hour a pale yellow solid began to precipitate. A further portion of dimethylamine (2 ml, 60% solution in water) was added and the reaction was continued for another 24 hours. After this time, the reaction mixture was concentrated under vacuum and the crude material was suspended in methanol (70 ml) and water (140 ml). After vigorous stirring for 1 hour at 0° C., the pale yellow solid was filtered off, washed with a small amount of methanol-water solution and dried under vacuum. As a result, compound C-7C2 was obtained as a pale yellow solid (10 g; 100% yield; 99% purity according to UELCMS analysis).

Intermediate C-7D: 5-amino-2,4,6-trimethoxypyrimidine.

। NH ₂ t t

NiyN

To a QianCap glass reactor (1850 ml) equipped with a magnetic stirrer was added C-7D3 (46 g, 231 mmol) followed by MeOH (750 ml). Then 10% palladium on carbon (2.5 g) was added in one portion and the reactor was connected to a source of hydrogen. The hydrogen pressure was set at 2 bar and the reaction mixture was thoroughly stirred for 24 hours with a continuous flow of hydrogen. After this time, TLC analysis showed complete conversion of the starting material. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated to dryness in vacuo. As a result, amine C-7D was obtained as a beige solid (38.3 g, 97% yield, 99% purity according to UELCMS analysis).

Intermediate C-7D3: 2,4,6-trimethoxy-5-nitropyrimidine.

I V°2

ABOUT

T it N^N

- 15 040469

Methanol (1000 ml) was added to a 2 L round bottom flask containing a thermometer and a magnetic stirrer, and the whole was cooled to 0° C. in a water/ice bath. Sodium hydride (13 g, 327 mmol, 1.2 eq., 60% in mineral oil) was then added in small portions over 30 minutes (caution: the reaction is exothermic). After 30 minutes of stirring, substrate C-7D2 (60 g, 272 mmol, 1 eq.) was added in several portions. The reaction is highly exothermic and the reaction flask was cooled in a water/ice bath during the addition. During the reaction, the formation of a solid yellow product was observed. The reaction mixture was allowed to warm to room temperature (1 h) and after this time TLC analysis (DCM as eluent) showed complete conversion of the starting material. 1 L of water was added to the reaction mixture and the methanol was evaporated. The precipitate was filtered off, washed with 200 ml of n-hexane, and dried in air. As a result, compound C-7D3 was obtained as a yellow solid (46.5 g, 79% yield, 100% purity according to UELCMS analysis).

Intermediate C-7D2: 2-chloro-4,6-dimethoxy-5-nitropyrimidine.

I?°2

IO O.

g ¥ N^N Cl

Compound C-7D1 (50 g, 286 mmol, 1 eq.) was added to a 0.5 L two-necked round-bottom flask containing a thermometer and equipped with a magnetic stirrer followed by TFAA (80 ml, 572 mmol, 2 eq.)/DCM mixture (1:1). The mixture was then cooled in a brine-ice bath to -5°C. Concentrated fuming nitric acid (14.3 ml, 343 mmol, 1.2 eq.) was added dropwise to the thoroughly stirred mixture (note: the reaction is highly exothermic) at such a temperature not to exceed +40°C. During the reaction, a lot of solid product was formed. After adding the entire amount of nitric acid, the reaction mixture was left to reach room temperature. After this time, TLC analysis showed complete consumption of the substrate (an aliquot which was quenched with water and extracted with DCM; the plate was eluted with DCM). The resulting thick white precipitate was poured onto ice and stirring was continued for 10 minutes. The aqueous solution was extracted with DCM (3x100 ml).

The combined organic layers were then washed with saturated aqueous NaHCO ₃ until the washings remained at pH 7. The solution was dried over MgSO ₄ and the solvent was removed under vacuum to give a yellow-white crystalline compound C-7D2 (61 g, 97% yield , purity 99% according to UELCMS analysis).

Compound (1), C1: (3S)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2yl)-1, 2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

CI

H

The title compound was obtained after resolution by preparative chiral SFC (method A) or chiral RP-HPLC (method R) of racemic compound C-10.C1; >99% e.i.; tr: 7.77 min (method R'); ¹ H NMR (500 MHz, DMSO^) δ 11.18 (s, 1H), 8.55-8.42 (m, 1H), 7.37 (s, 1H), 7.36-7.27 ( m, 2H), 7.14 (s, 1H), 7.08 (dd, J=8.1, 1.9 Hz, 1H), 6.98 (d, J= 7.8 Hz, 1H), 6.93 (d, J= 1.9 Hz, 1H), 3.98 (s, 3H), 3.94 (br.s, 3H), 2.43 (sep, J= 7.0 Hz, 1H ), 0.86 (d, J= 7.0 Hz, 3H), 0.43 (d, J= 7.0 Hz, 3H); ¹³ C NMR (126 MHz, DMSO-d6) δ 175.42, 166.58, 164.87, 164.85, 157.89, 144.17, 138.80, 135.26, 134.00, 133, 38, 130.76, 127.52, 127.42, 127.30, 126.96, 126.02, 123.70, 123.04, 120.65, 118.41, 116.45, 111.18, 70.20, 55.52, 54.85, 25.47, 21.41, 21.25.

Compound C-10.C1: 6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)1,2,3 ',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Cl

ml of TFA was placed in a 100 ml flask equipped with a magnetic stirrer. Then intermediate С-9.С1 (17.8 g, 30.6 mmol) was added in several portions and the reaction mixture was vigorously stirred.

- 16 040469 were sewn for 3 hours at 40°C. After this time UPLCMS analysis showed 89% of the product peak with some minor peaks from impurities. Most of the acid was then evaporated and the residue was dissolved in DCM (100 ml). Added 100 ml of water and the mixture was treated with 3 M NaOH to pH 8. The phases were separated and the aqueous phase was extracted with DCM (2x100 ml). The combined organic phases were washed with brine (30 ml), dried over MgSO ₄ and evaporated to dryness to give 18 g of a beige solid compound C-10.C1 with a purity of 90%. The crude product C-10.C1 was dissolved in 50 ml of methanol (HPLC grade). Then, 25% sodium methoxide solution (10 ml) was added dropwise over 10 minutes and the mixture was stirred at room temperature. After 24 hours, UELCMS showed 97% of the expected product C-10.C1. The mixture was concentrated to half volume, slowly transferred to a stirred mixture of ice (100 g) and then acidified with 3 M HCl to neutral pH. The precipitate was filtered off, washed with 50 ml of water and dried under vacuum to give compound C-10.C1 as a beige-orange solid (14.5 g, 84% yield, 98% purity according to UELCMS analysis).

Alternative procedure for the preparation of C-10.C1: 12.0 g (19 mmol) of intermediate C-9.C1 was dissolved in 50 ml of ice-cold AcOH. MsOH (1 eq) was added and the mixture was stirred at 40°C. After 16 hours, the mixture was transferred to a beaker containing 100 g of ice and 100 ml of 25% ammonia. The solid product was filtered off, washed with 50 ml of water and dried in air. Purification by flash chromatography (DCM/MeOH, 100:0^98:2) gave 9.6 g of a beige-orange solid with a purity of 94% (according to UELCMS analysis). Yield 78%.

Compound С-9.С1: 4-(6-Chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3-chlorophenyl)-1(2,4 -dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1 H-pyrrol-3-carboxamide.

BUT

ABOUT

Compound C-8.C1 (15 g, 26.6 mmol, 1 eq.) and THF (200 ml) were added to a 500 ml flask equipped with a magnetic stirrer, followed by triethyl phosphite (6.81 ml, 39.8 mmol, 1.5 eq.). Sodium tert-butoxide (5.11 g, 53.2 mmol, 2 eq.) was then added in several portions. The reaction mixture was stirred for 3 hours at room temperature under air (the flask was fitted with a CaCl ₂ tube). After this time, UELCMS analysis showed 82% of the desired product and 10% of the main impurity. The reaction mixture was slowly transferred into a chilled (0-5°C) mixture of water (150 ml) and 12 M HCl (5 ml). After addition of AcOEt (100 ml), the entire mixture was transferred to a separating funnel. The layers were separated and the aqueous phase was re-extracted with AcOEt (100 ml). The combined organic phases were washed with saline, dried over MgSO ₄ and the solvent was removed under vacuum. The crude products were purified by flash chromatography (CHCl ₃ /MeOH 100:0^98:2). This gave 5.86 g of C-9.C1 with a purity of 93.7%. Yield 38%.

Compound С-8.С1: 4-(6-Chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3-chlorophenyl)-1-(2,4-dimethoxypyrimidine- 5-yl)-5-(propan-2-yl)-III-pyrrole-3-carboxamide.

In a 500 ml round bottom flask equipped with a magnetic stirrer, compounds C-4A (21 g, 117 mmol, 1 eq), C-6A (29.25 g, 117 mmol, 1 eq) and C-7A (20 g, 130 mmol , 1.1 eq) was suspended in 90% aqueous AcOH and the flask was tightly closed with a plastic stopper. The mixture was heated to 70°C and stirred at this temperature for 24 h. After this time, UPLCMS showed 60% of the expected product. The reaction mixture was cooled to room temperature and evaporated to dryness. The reaction was repeated and the remainder of the two batches were combined and purified according to the following procedure.

The solid residue was pre-adsorbed on silica gel and purified by flash chromatography (30% to 50% AcOEt in n-hexane). All fractions that contained product were concentrated to 500 ml and left at room temperature. After 24 hours, the pink solid from 17 040469 was filtered, washed with 50 ml of n-hexane and air dried (40.8 g, 96.5% purity according to UPLCMS analysis). The filtrate was evaporated to dryness to give an additional 6.6 g of product C-8

C1 with a purity of 50%. Yield 33%.

Compound (2), C2: (3S)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'(propan-2- yl)-1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral SFC (method B) or chiral RP-HPLC (method N) of racemic compound C-10 C2, >99%, ae, t _r 9.31 min (method N '), 1H NMR (500 MHz, DMSO-de) δ 11.23 (br s, 1H), 8.50 (br s, 1H), 7.45 (ddd, J= 8.9, 4.1, 2.7 Hz, 1H), 7.39 (s, 1H), 7.32 (t, J= 9.1 Hz, 1H), 7.21-7.13 (m, 1H), 7.08 ( dd, J= 8.1, 1.9 Hz, 1H), 7.05 (dd, J= 6.3, 2.7 Hz, 1H), 6.90 (d, J= 1.9 Hz, 1H ), 3.98 (s, 3H), 3.94 (br s, 3H), 2.48-2.39 (m, 1H), 0.85 (d, J= 7.0 Hz, 3H), 0.44 (d J= 7.0 Hz, 3H), ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 175.51, 166.44, 164.70, 164.24, 158.75, 157, 96, 156.74, 144.03, 135.39, 134.14, 130.82, 130.75, 129.58, 128.33, 127.30, 125.99, 125.66, 125.54, 123.64, 123.04, 119.63, 118.75, 118.68, 118.57, 116.25, 111.12, 70.04, 55.67, 55.01, 49.04, 25, 34, 22.00, 21.50-20.80.

Compound C-10.C2: 6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2yl)-1, 2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Compound C-9.C2 (2.86 g, 4.76 mmol) was added to a 100 ml flask equipped with a magnetic stirrer and the flask was cooled in an ice bath. TFA (25 ml) was then added (approximately 2 ml/min). The cooling bath was removed and the reaction mixture was stirred for 2 hours at room temperature. After this time, UELCMS analysis showed 72% of the peak area of the product. The mixture was then poured into ice (approximately 100 g) and diluted with DCM (50 ml). The phases were separated and the aqueous phase was extracted three times with DCM. The combined organic phases were washed with water and brine and the solvent was removed in vacuo. The crude mixture was pre-adsorbed on silica gel and purified by flash chromatography (AcOEt/hexane 40%-60%). The brown solid obtained after chromatography (1.51 g) was stirred in 60% AcOEt in n-hexane (10 ml) for 0.5 h and then filtered, washed with 60% AcOEt in n-hexane and air dried. As a result, the compound C-10.C2 was obtained as a light brown solid (1.35 g, 46% yield) with a purity of 96% according to the analysis of UPLCMS.

Compound C-9.C2: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indole-3ul)-1-( 2,4-dimethoxypyrimidin-5-yl)-5-(propan-2-ul)-1H-pyrrole-3-carboxamide.

CI

Compound C-8.C2 (5 g, 8.6 mmol, 1 eq.) and THF (80 ml) were added to a 500 ml flask equipped with a magnetic stirrer, followed by the addition of trimethyl phosphite (2 ml, 17.2 mmol, 2 eq. .). The reaction mixture was then cooled in an ice bath and sodium tert-pentoxide (3.79 g, 34.4 mmol, 4 eq.) was added in one portion. The cooling bath was removed and the reaction mixture was stirred for 1 h at room temperature (the flask was equipped with a CaCl ₂ tube). After this time, UELCMS analysis showed 91% of the peak area of the product. Approximately 90% of the solvent was removed and this resulting mixture was diluted with 100 ml of water. The resulting suspension was acidified with 3 M HCl to pH~5 and diluted with 100 ml DCM. The phases were separated and the aqueous phase was extracted three times with DCM. The combined organic phases were washed with water, brine, dried over Na ₂ SO ₄ and the solvent was removed under vacuum. The crude product C-9.C2 was obtained as a red-brown solid/foam (purity

- 18 040469

72% according to UELCMS analysis) and used in the next step without any further purification.

Compound C-8.C2: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-1-(2, 4-dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1H-pyrrol-3-carboxamide.

Compounds C-4A (12.48 g, 50 mmol, 1 eq.), C-7A (7.76 g, 50 mmol, 1 eq.) and C-6B (9 .88 g, 50 mmol, 1 eq.) followed by the addition of ice-cold AcOH (125 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 90° C. (heating bath temperature) and stirred at this temperature for 16 hours. After this time, UPLCMS analysis showed almost complete consumption of the starting materials (corresponding to 40% of the peak area of the product). The reaction mixture was cooled to room temperature and AcOH was evaporated to dryness. The residue was pre-adsorbed on silica gel and purified by flash chromatography (30 to 60% AcOEt in n-hexane). After removal of the solvents, the product C-8.C2 was obtained as a dark red solid/foam (8.7 g, 30% yield) with a purity of 84% according to UPLCMS analysis.

Compound (3), C3: (3S)-6-chloro-2'-(5-chloro-2-methylphenyl)-6'-(propan-2-yl)-5'-(trimethoxypyrimidin5-yl)-1, 2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral SFC (method C) or chiral RP-HPLC (method T) of racemic compound C-10.C3; >99% e.i.; t _r : 16.7 min (method T'). 1H NMR (600 MHz, DMSO-d ₆ ) rotamer mixture: δ 11.31 (br.s, 1H), 10.90 (br.s, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.33-7.18 (m, 4H), 7.02 (dd, J= 8.1, 2.0 Hz, 1H), 6.91 (d, J= 1.9 Hz, 1H ), 6.86 (d, J= 1.9 Hz, 1H), 6.44 (d, J= 2.2 Hz, 1H), 3.97 (d, J= 1.4 Hz, 3H), 3.93 (d, J= 4.3 Hz, 3H), 3.90 (d, J= 2.4 Hz, 3H), 2.38-2.32 (m, 1H), 2.24 (s , 1H), 2.07 (s, 2H), 0.80 (dd, J= 13.5, 7.0 Hz, 3H), 0.41 (dd, J= 10.0, 7.0 Hz, 3H); ¹³ C NMR (151 MHz, DMSO^) rotamer mixture: δ 176.16, 174.19, 167.19, 167.05, 166.99, 166.89, 163.67, 163.37, 162.67, 143.81, 137.44, 136.75, 136.68, 136.17, 134.82, 134.74, 133.24, 133.19, 132.37, 132.31, 129.95, 129, 22, 128.11, 127.96, 127.92, 127.45, 127.11, 127.05, 125.63, 122.64, 122.44, 122.19, 119.53, 119.28, 117.89, 110.67, 110.59, 99.16, 70.13, 69.70, 55.04, 54.78, 54.74, 54.71, 40.41, 40.03, 39, 89, 24.99, 24.97, 21.84, 21.06, 21.02, 20.85, 20.78, 17.97, 17.82.

Compound C-10.C3: 6-chloro-2'-(5-chloro-2-methylphenyl)-6'-(propan-2-yl)-5'-(trimethoxypyrimidin5-yl)-1,2,3' ,5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Compound C-9.C3 (6.7 g, 1.19 mmol) and AcOH (10 ml) were added to a 25 ml flask equipped with a magnetic stirrer at room temperature. Then MsOH (0.077 ml, 1.19 mmol) was added and the reaction mixture was stirred for 3 h at 80°C. After 1 hour, UELCMS analysis showed 93% of the peak area of the product. AcOH was evaporated and the residue was dissolved in AcOEt, washed with saturated NaHCO ₃ , brine and dried over MgSO ₄ . The mixture was concentrated to approximately 5 ml AcOEt and the suspension was filtered. The collected solid was washed with 5 ml of AcOEt and dried under vacuum. Target product C-10.C3 was obtained as a white solid (0.31 g, 43% yield, purity

98% according to UELCMS analysis).

Compound С-9.С3: N-(5-chloro-2-methylphenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indole- 19 040469

3-yl)-5-(propan-2-yl)-1-(trimethoxypyrimidin-5-yl)-1 H-pyrrol-3-carboxamide.

Compound C-8.C3 (0.8 g, 1.3 mmol, eq.) and THF (15 ml) were added to a 50 ml flask equipped with a magnetic stirrer. The reaction mixture was then cooled in an ice bath to 0° C. and triethyl phosphite (0.45 ml, 2.6 mmol, 2 eq.) was added. After 10 min at 0° C., sodium tertpentoxide (0.6 g, 5.2 mmol, 4 eq.) was added in several portions. The reaction mixture was stirred at room temperature (the flask was fitted with a CaCl ₂ tube) and monitored by TLC (5% MeOH in CHCl ₃ ). After 24 h the mixture was poured onto ice and the reaction mixture was acidified with 1 M HCl to pH 5. After the addition of AcOEt (50 ml) the mixture was transferred to a separating funnel. The layers were separated and the aqueous phase was re-extracted with AcOEt (25 ml). The combined organic phases were washed with saline, dried over Na ₂ SO ₄ and the solvent was removed under vacuum. The residue was purified by column chromatography using 1% MeOH in CHCl ₃ as eluent. Target product C-9.C3 was obtained as a pink solid (0.8 g, 90% yield, 93% purity by UELCMS analysis).

Compound С-8.С3: N-(5-chloro-2-methylphenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-5-(propan2- yl)-1-(trimethoxypyrimidin-5-yl)-1 H-pyrrole-3-carboxamide.

n

To a 25 ml round bottom flask equipped with a magnetic stirrer were added compounds C-4A (0.67 g, 2.7 mmol, 1 eq.), C-7D (0.5 g, 2.7 mmol, 1 eq.) and C-6C (0.52 g, 2.7 mmol, 1 eq.) followed by the addition of ice-cold AcOH (10 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 90°C and stirred at this temperature overnight. After this time, UELCMS analysis showed almost complete consumption of the starting materials (which is 45% of the peak area of the product). AcOH was then evaporated under vacuum. The residue was purified by flash chromatography (n-hexane/AcOEt, 4:1^1:1) to give the expected product C-8.C3 as a red solid (0.8 g, 40% yield, 82% purity in according to the analysis of UELCMS).

Compound (4), C4: (3S)-6-chloro-2'-(3-chloro-4-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan2-yl )-1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral SFC (method D) or chiral RP-HPLC (method K) of racemic compound C-10.C4; >99% e.i.; tr: 3.79 min (method K'). ¹ H NMR (500 MHz, CDCl3) δ 8.47 (br. s, 1H), 8.23 (s, 1H), 7.24-7.19 (m, 1H), 7.18-7.12 (m, 1H), 7.12-7.06 (m, 1H) 7.05-6.96 (m, 2H), 6.96-6.86 (m, 2H), 4.07 (s, 3H), 3.99 (s, 3H), 2.49-2.38 (m, 1H), 0.90 (d, J= 7.0 Hz, 3H), 0.52 (d, J= 7 .0 Hz, 3H); ¹³ C NMR (125 MHz, CDCl3) δ 175.2, 166.5, 165.6, 164.9, 158.5, 157.0, 156.5, 142.0, 136.4, 134.3, 132.6, 132.5, 131.0, 128.6, 128.5, 126.5, 126.2, 123.8, 122.8, 121.4, 121.3 120.7, 117.8 , 117.0, 116.8, 116.2, 111.6, 70.1, 55.6, 54.7, 25.6, 21.2, 1.9.

Compound C-10.C4: 6-chloro-2'-(3-chloro-4-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2ul)-1, 2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

- 20 040469

Compound C-9.C4 (10.34 mmol) and TFA (70 ml) were added to a 250 ml flask equipped with a magnetic stirrer at room temperature (TFA was added at approximately 5 ml/min). The reaction mixture was then stirred for 3 hours at 40° C. and monitored by TLC. After this time, UELCMS analysis showed 71% of the peak area of the product. The mixture was cooled to room temperature and evaporated to dryness. The crude product was purified by column chromatography (10% 50% AcOEt/n-hexane). After removal of the solvents, the product C-10.C4 was obtained as a light brown solid/foam (5.44 g, 80% yield after two steps based on C-8.C4) with a purity of 88% according to UPLCMS analysis.

Compound С-9.С4: 4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3-chloro-4-fluorophenyl)1- (2,4-dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1H-pyrrol-3-carboxamide.

Compound C-8.C4 (6.04 g, 10.34 mmol, 1 eq.) and THF (140 ml) were added to a 500 ml flask equipped with a magnetic stirrer. The reaction mixture was then cooled in an ice bath to 0°C and trimethyl phosphite (1.83 ml, 15.51 mmol, 1.5 eq.), sodium tert-peptoxide (2.28 g, 20.68 mmol, 2 equiv.). The reaction mixture was stirred for 4 h at this temperature (the flask was equipped with a tube with CaCl ₂ ) and monitored by TLC. After this time, UELCMS analysis showed 87% of the peak area of the desired product. Then cold water (50 ml) was added to the mixture and the reaction mixture was acidified with 5% HCl to pH~5. The reaction mixture was stirred for approximately 0.5 h at room temperature and the resulting precipitate was filtered off, washed with 100 ml of cold water. The solid thus obtained was redissolved in AcOEt, washed with water, dried over Na ₂ SO ₄ and the solvent was evaporated. The crude product C-9.C4 was obtained as a brown solid (purity 85% according to UPLCMS analysis) and was used in the next step without any further purification.

Compound C-8.C4: 4-(6-chloro-2-oxo-2,3-dihydro-1 H-indol-3 -yl)-N-(3-chloro-4-fluorophenyl)-1-(2 ,4dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1H-pyrrole-3-carboxamide.

Compounds C-4A (12.48 g, 50 mmol, 1 eq), C-7A (8.5 g, 55 mmol, 1.1 eq) and C-6D ( 9.9 g, 50 mmol, 1 eq.) followed by the addition of ice-cold AcOH and the MB reactor was tightly capped. The mixture was heated to 90°C (300 W) and stirred for 5 h at this temperature. After this time, analysis by UPLCMS showed almost complete consumption starting materials (representing 40% of the peak area of the product) The reaction mixture was cooled to room temperature and AcOH evaporated to dryness The residue was pre-adsorbed on silica gel and purified using column chromatography (10% acetone/DCM) After removal of solvents, the product C-8 C4 was obtained as dark brown solid/foam (6.96 g, 20.7% yield) 87% pure by UPLCMS analysis.

Compound (5), C5 (3S)-6-chloro-2'-(5-chloro-2-fluorophenyl)-6'-(propan-2-yl)-5'-(trimethoxypyrimidin-5yl)-1,2 ,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

- 21 040469

The title compound was obtained after resolution by preparative chiral SFC (method E) or chiral RP-HPLC (method L) of racemic compound C-10 C5, >99% ee, t _r 7.18 min (method L' ), ¹ H NMR (600 MHz, DMSOL) δ 11.20 (s, 1H), 7.44 (ddd, J= 8.9, 4.1, 2.7 Hz, 1H), 7.31 (t , J= 9.1 Hz, 1H), 7.25 (s, 1H), 7.15 (dd, J= 8.1, 1.7 Hz, 1H), 7.08 (dd, J= 8, 1, 1.9 Hz, 1H), 7.05 (dd, J= 6.3, 2.7 Hz, 1H), 6.89 (d, J= 1.9 Hz, 1H), 3.97 ( s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 2.40-2.31 (m, 1H), 0.82 (d, J = 7.1 Hz, 3H ), 0.41 (d, J= 7.0 Hz, 3H), ¹³ C NMR (151 MHz, DMSO^) δ 175.64, 167.54, 167.40, 164.33, 163.18, 158 .6, 156.93, 144.05, 135.29, 133.95, 130.69, 129.61, 128.30, 127.19, 126.20, 125.74, 125.65, 123.01 , 119.35, 118.69, 118.55, 111.07, 99.55, 70.08, 55.50, 55.20, 49.02, 25.42, 21.46, 21.26.

Compound С-10.С5 6-chloro-2'-(5-chloro-2-fluorophenyl)-6'-(propan-2-yl)-5'-(trimethoxypyrimidin-5yl)-1,2,3', 5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Compound C-9 C5 (6.7 g, 10.6 mmol) and TFA (15 ml) were added to a 100 ml flask equipped with a magnetic stirrer at room temperature. The reaction mixture was stirred for 3 hours at room temperature. showed 85% of the peak area of the product. The reaction mixture was poured onto ice and extracted twice with DCM (2x30 ml). The combined organic phases were washed with water (30 ml), dried over Na ₂ SO ₄ and evaporated to dryness. AcOEt (50 ml) was added to the residue and a pinkish precipitate formed. The solid was filtered off, washed with several portions of cold AcOEt and dried in air. Target product C-10.C5 was obtained as a pale pink solid (5.17 g, 80% yield, 99% purity by UPLCMS analysis).

Compound C-9.C5: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3yl)-5-( propan-2-yl)-1-(trimethoxypyrimidin-5-yl)-1 H-pyrrol-3-carboxamide.

H

Compound C-8.C5 (5 g, 8.14 mmol, 1 eq.) and THF (150 ml) were added to a 500 ml flask equipped with a magnetic stirrer. The reaction mixture was then cooled in an ice bath to 0° C. and trimethyl phosphite (1.92 ml, 16.3 mmol, 2 eq.) was added. After 10 minutes at 0° C., sodium tertpeptoxide (3.6 g, 32.6 mmol, 4 eq.) was added in several portions. The reaction mixture was stirred at room temperature (the flask was fitted with a CaCl ₂ tube) and monitored by TLC (40% AcOEt in n-hexane). The mixture was then poured onto ice and the reaction mixture was acidified with 0.5 M aqueous HCl to pH 5. After addition of DCM (100 ml), the mixture was transferred to a separating funnel. The layers were separated and the aqueous phase was re-extracted with DCM (100 ml). The combined organic phases were washed with saline, dried over Na ₂ SO ₄ and the solvent was removed under vacuum. The residue was purified by column chromatography (30%-50% AcOEt/n-hexane). Target product C-9.C5 was obtained as a brownish solid (2.41 g, 47% yield, 96% purity by UELCMS analysis).

Compound C-8.C5: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1 H-indol-3-yl)-5-(propane2 -yl)-1-(trimethoxypyrimidin-5 -yl)-1 H-pyrrol-3-carboxamide.

- 22 040469

Compounds C-4A (6.3 g, 25.3 mmol, 1 eq.), C-6B (4.7 g, 130 mmol, 1 eq.) and C- 7D (5 g, 117 mmol, 1 eq.) followed by the addition of AcOH (40 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 80°C and stirred at this temperature overnight. After this time, UELCMS analysis showed almost complete consumption of starting materials (which is 66% of the peak area of the product). The reaction mixture was cooled to room temperature and the red solid product C-8 C5 was filtered off, washed with AcOH and air dried (10 g, 64% yield, 82% purity according to UPLCMS analysis).

Compound (6), С6 (3S)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-[6-(dimethylamino)-4-methoxypyridin-3yl]-6'-(propane- 2-yl)-1,2,3',5'-tetrahydro-2Ή-spiro[indol-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral NP-HPLC (method F) or chiral RP-HPLC (method J) of racemic compound C-10 C6, >99% ee, tr 14.66 min (method J '), ¹ H NMR (500 MHz, DMSO-de) δ 11.20 (br s, 1H), 7.91 (d, J= 22.0 Hz, 1H) 7.43 (ddd, J= 8, 9, 4.1, 2.7 Hz, 1H), 7.30 (t, J= 9.1 Hz, 1H), 7.23 (s, 1H), 7.16 (dd, J= 8.0 , 2.1 Hz, 1H), 7.107.03 (m, 2H), 6.89 (d, J= 1.9 Hz, 1H), 6.25 (d, J= 5.6 Hz, 1H), 3.82 (d, J= 20.8 Hz, 3H), 3.09 (s, 6H), 2.47-2.37 (m, 1H), 0.83 (dd, J= 12.2, 7.0 Hz, 3H), 0.41 (t, J= 7.0 Hz, 3H), ¹³ C NMR (126 MHz, DMSO^) δ 175.77, 175.72, 164.50, 162.08 161.95 161.08 158.79 156.79 147.23 144.06 135.26 134.43 134.39 130.67 130.60 129.61 128 .30, 127.36, 127.20, 126.29, 126.23, 125.85, 125.75, 123.20, 122.99, 122.94, 118.84, 118.71, 118.64 , 118.53, 115.45, 111.06, 88.48, 70.09, 56.06, 56.03, 38.38, 25.38, 22.01,21.63,21,27.21 .04.

Compound С-10.С6 6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-[6-(dimethylamino)-4-methoxypyridin-3yl]-6'-(propan-2-yl) -1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Compound C-9 C6 (2.96 g, 4.8 mmol) and TFA (30 ml) were added to a 100 ml flask equipped with a magnetic stirrer. The reaction mixture was stirred for 1 hour at 40°C. After this time, UELCMS analysis showed 97% of the desired C-10 C6 product. The reaction mixture was evaporated to dryness. To the residue was added 20 ml of methanol and saturated NaHCO ₃ (200 ml). The suspension was heated under reflux for 2 h and then cooled to room temperature. The precipitate was filtered off and soaked in methanol (20 ml). The solid was collected by filtration, washed with a small amount of methanol and dried under vacuum to give the desired product C-10 C6 as a white to yellowish or grayish solid (2.3 g, 83% yield, >99% purity according to UPLCMS analysis ).

Compound C-9.C6: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3yl)-1 -[ 6-(dimethylamino)-4-methoxypyridin-3-yl]-5-(propan-2-yl)-1 H-pyrrol-3-carboxamide.

- 23 040469

Compound C-8.C6 (4 g, 6.7 mmol, 1 eq.) and THF (30 ml) were added to a 100 ml flask equipped with a magnetic stirrer, followed by triethyl phosphite (1.72 ml, 10.1 mmol, 1.5 eq.). The mixture was then cooled to 3° C. and sodium tert-pentoxide (1.48 g, 13.4 mmol, 2 eq.) was added in several portions. After warming to room temperature, the reaction mixture was stirred for 22 h (the flask was equipped with a CaCl ₂ tube). After this time, UELCMS analysis showed 94% of the desired product C9.C6. Water (200 ml) and 1 M HCl were added to the mixture until pH 8 was reached. The aqueous phase was extracted with AcOEt (3x150 ml). The organic phases were combined and dried over MgSO ₄ . After concentration, the crude material was further purified by chromatography (10-30% iso-PrOH/n-hexane) to give compound C-9.C6 (3 g, 72% yield, 94% purity according to UELCMS analysis).

Compound C-8.C6: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3 -yl)-1-[6( dimethylamino)-4-methoxypyridin-3-yl]-5-(propan-2-yl)-1H-pyrrole-3-carboxamide.

H

In a pressurized round-bottom flask (250 ml) equipped with a magnetic stirrer, compounds C-4A (5.37 g, 21.5 mmol, 1 eq.), C-7C (3.6 g, 21.5 mmol, 1 eq. .), C-6B (4.25 g, 21.5 mmol, 1 eq.) and PTSA-H ₂ O (4.09 g, 21.5 mmol, 1 eq.) were suspended in water (9 ml) and methanol (36 ml). The flask was tightly capped, then the mixture was heated to 60° C. and stirred at this temperature for 23 hours. After this time, UPLCMS analysis showed 17% of the expected product C-8.C6. Then an additional portion of compound C-6B (1.06 g, 5.4 mmol) was added, the mixture was heated to 75°C and stirred at this temperature for 42 hours. .C6. The reaction mixture was evaporated to dryness. DCM (200 ml), 5% NaHCO ₃ solution (150 ml) was added to the residue and the mixture was stirred for 20 minutes. The aqueous phase was extracted with DCM (4x200 ml). The organic phases were combined and dried over MgSO4. After concentration, the crude material was purified by column chromatography (20-50% AcOEt/n-hexane) to give compound C-8.C6 (4.1 g, 32% yield, 90% purity according to UELCMS analysis).

Compound (7), C7: (3S)-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(p-propan2-yl)- 1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral SFC (method G) or chiral RP-HPLC (method O) of racemic compound C-10.C7; >99% e.i.; tr: 5.61 min (method O'). ¹ H NMR (500 MHz, CDCl3) δ 8.76 (br. s, 1H), 8.23 (s, 1H), 7.23-7.18 (m, 1H), 7.11-7.06 (m, 1H), 7.06-6.98 (m, 2H) 6.98-6.90 (m, 1H), 6.90-6.85 (m, 1H), 6.83-6, 76 (m, 1H), 4.06 (s, 3H), 3.99 (s, 3H), 2.49-2.39 (m, 1H) 0.90 (d, J = 7.0 Hz, 3H), 0.51 (d, J=7.0 Hz, 3H); ¹³ C NMR (125 MHz, CDCl3) δ 175.3, 166.5, 165.7, 164.9, 157.0, 150.9, 149.0, 148.9, 148.8, 142.2, 136.4, 134.3, 132.3, 126.5, 126.2, 124.9, 123.7, 122.9, 120.6, 118.1, 118.0, 117.8, 117, 5, 117.4, 116.1, 111.7, 77.3, 77.0, 76.7, 70.2, 55.6, 54.7.25.6.21.3.

Compound C-10.C7: 6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2yl)-1,2, 3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

- 24 040469

Compound C-9.C7 (7.34 g, 15.57 mmol) and TFA (80 ml) were added to a 250 ml flask equipped with a magnetic stirrer at room temperature (TFA was added approximately 5 ml/min). Then the reaction mixture was stirred for 3 h at 40°C. After this time, UELCMS analysis showed 84% of the peak area of the product. The mixture was cooled to room temperature and evaporated to dryness. The crude product was purified by flash column chromatography (20%-50% AcOEt/n-hexane). After removal of the solvents, product C-10.C7 was obtained as a light brown solid/foam (4.45 g, 63% yield after two-step synthesis) with a purity of 93% according to UPLCMS analysis.

Compound С-9.С7: 4-(6-Chloro-3-hydroxu-2-oxo-2,3-dahydro-1H-undol-3-yl)-N-(3,4-difluorophenyl)-1(2 ,4-dimethoxypyrimidin-5-yl)-5-(propan-2-ul)-1H-pyrrole-3-carboxyamide.

Compound C-8.C7 (7.14 g, 12.57 mmol, 1 eq.) and THF (140 ml) were added to a 500 ml flask equipped with a magnetic stirrer. The reaction mixture was then cooled in an ice bath to 0°C and trimethyl phosphite (2.25 mL, 18.86 mmol, 1.5 eq.) was added in one portion followed by sodium tert pentoxide (2.77 g, 25.14 mmol, 2 equiv.). The reaction mixture was stirred for 1.5 h at 0°C (the flask was equipped with a tube with CaCl ₂ ). After this time, UELCMS analysis showed 88% of the peak area of the product. Cold water (50 ml) was added and the reaction mixture was acidified with 5% HCl to pH~5. The reaction mixture was stirred for approximately 0.5 h at room temperature and the resulting precipitate was filtered off and washed with 100 ml of cold water. The solid thus obtained was redissolved in AcOEt, washed with water, dried over Na ₂ SO ₄ and the solvent was evaporated. The crude product C-9.C7 was obtained as a brown solid (purity 83% according to UPLCMS analysis) and was used in the next step without any further purification.

Compound С-8.С7: 4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3,4-difluorophenyl)-1-(2,4dimethoxypyrimidine- 5-yl)-5-(propan-2-yl)-1 H-pyrrole-3-carboxamide.

Compounds C-4A (12.48 g, 50 mmol, 1 eq.), C-7A (7.76 g, 50 mmol, 1 eq.) and C-6E ( 9.06 g, 50 mmol, 1 eq.) followed by the addition of AcOH (100 ml) and the tube was tightly closed with a plastic stopper. The mixture was heated to 80° C. (heating bath temperature) and stirred at this temperature overnight. After this time, UELCMS analysis showed almost complete consumption of starting materials (which is 40% of the peak area of the product). The reaction mixture was cooled to room temperature and AcOH was evaporated to dryness. The residue was pre-adsorbed on silica gel and purified by chromatography (50% AcOEt/n-hexane). After removal of the solvents, product C-8.C7 was obtained as a dark brown solid/foam (7.63 g, 24.9% yield, 84% purity by UPLCMS analysis).

Compound (8), C8: (3S)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2ul)- 1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

- 25 040469

The title compound was obtained after resolution by preparative chiral SFC (method H) or chiral RP-HPLC (method N) of racemic compound C-10.C8; >99% e.i.; tr: 6.2 min (method N'); ¹ H NMR (500 MHz, DMSO-d6) δ 11.15 (br. s, 1H), 8.07 (d, J = 34.9 Hz, 1H), 7.467.37 (m, 1H), 7, 37-7.27 (m, 2H), 7.15 (m, 1H), 7.08 (dd, J= 8.0, 1.9 Hz, 1H), 6.92 (m, J= 2, 2 Hz, 1H), 6.85 (m, 1H), 6.64 (d, J= 4.0 Hz, 1H), 3.90 (s, 3H), 3.83 (d, J= 23, 6 Hz, 3H), 2.37 (m, 1H), 0.85 (dd, J= 13.0, 7.0 Hz, 3H), 0.41 (dd, J= 9.4, 6.9 Hz, 3H); ¹³ C NMR (125 MHz, DMSO-d6) δ 175.18, 175.05, 165.31, 164.56, 162.80, 162.74, 149.75, 149.64, 147.79, 147, 68, 147.33, 147.24, 145.92, 145.79, 143.48, 143.43, 134.62, 134.59, 133.42, 127.08, 126.18, 126.10, 122.71, 122.67, 122.58, 120.23, 119.41, 117.79, 117.70, 117.55, 117.23, 117.09, 110.62, 93.37, 93, 29, 69.61, 56.21, 53.64, 24.90, 21.39, 21.08, 20.64, 20.50.

Compound C-10.C8: 6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2yl)-1,2, 3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

Compound C-9.C8 (2 g, 3.5 mmol) was added to a 100 ml flask equipped with a magnetic stirrer and the flask was cooled in an ice bath. TFA (20 ml) was then slowly added (approximately 2 ml/min). The cooling bath was removed and the reaction mixture was stirred for 1 h at room temperature. After this time, UELCMS analysis showed 75% of the peak area of the product. The reaction mixture was stirred for an additional 1 hour, the mixture was poured onto ice (ca. 50 g) and diluted with DCM (50 ml). The phases were separated and the aqueous phase was extracted three times with DCM (3x50 ml). The combined organic phases were washed with water, brine and the solvent was removed in vacuo. The residue was pre-adsorbed on silica gel and purified by flash chromatography (50% AcOEt in n-hexane). After removal of the solvents, the product C-1O.C8 was obtained as a red-brown solid/foam (815 mg, 41% yield) with a purity of 98% according to UELCMS analysis. The above reaction was repeated three more times, all obtained samples (5.76 g) of compound C-10.C8 were combined and stirred in 25 ml of AcOEt/n-hexane (1:5). The mixture was heated to reflux and AcOEt was added until all remaining solid had dissolved. Then 75 ml of n-hexane was added dropwise and the mixture was stirred for 16 hours at room temperature. The solid was filtered off, washed with AcOEt/n-hexane (1:10, 25 ml) and dried under high vacuum. As a result, the final product C-10.C8 was obtained as a light red solid (4.77 g, 98% purity according to UELCMS analysis).

Compound С-9.С8: 4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3,4-difluorophenyl)-1(4 ,6-dimethoxypyridin-3-yl)-5-(p-propan-2-yl)-1H-pyrrole-3-carboxamide.

Compound C-8.C8 (2 g, 3.5 mmol, 1 eq.) and THF (35 ml) were added to a 100 ml flask equipped with a magnetic stirrer, followed by the addition of trimethyl phosphite (0.83 ml, 7 mmol, 2 eq. .). The reaction mixture was then cooled in an ice bath and sodium tert-peptoxide (1.54 g, 14 mmol, 4 eq.) was added in one portion. The cooling bath was removed and the reaction mixture was stirred for 1 h at room temperature (the flask was equipped with a CaCl ₂ tube). After this time, UELCMS analysis showed

- 26 040469

80% of the peak area of the product. After another hour of stirring, approximately 90% of the solvent was removed and this resulting mixture was diluted with 50 g of ice. The resulting suspension was acidified with 1 M HCl to pH~5 and further diluted with 50 ml DCM. The phases were separated and the aqueous phase was extracted with DCM (2x30 ml). The combined organic phases were washed with water, brine, dried over Na ₂ SO ₄ and the solvent was removed under vacuum. The crude product C-9.C8 was obtained as a red black solid/foam (purity 70% according to UPLCMS analysis) and was used in the next step without any further purification (yield exceeded 100%).

Compound C-8.C8: 4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-(3,4-difluorophenyl)-1-(4,6dimethoxypyridine -3-yl)-5-(propan-2-yl)-1H-pyrrole-3-carboxamide.

Compounds C-4A (8.1 g, 32.4 mmol, 1 eq.), C-7B (5 g, 32.4 mmol, 1 eq.) and C-6E were added to a 500 ml flask equipped with a magnetic stirrer. (5.9 g, 32.4 mmol, 1 eq.) followed by the addition of AcOH (80 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 85° C. (heating bath temperature) and stirred at this temperature for 16 hours. After this time, UPLCMS analysis showed almost complete consumption of the starting materials (corresponding to 55% of the peak area of the product). The reaction mixture was cooled to room temperature and AcOH was evaporated to dryness. The residue was pre-adsorbed on silica gel and purified by flash chromatography (30% to 60% AcOEt in n-hexane). After removal of the solvents, the product C-8.C8 was obtained as a light red solid (8 g, 44% yield) with a purity of 87% according to UPLCMS analysis.

Compound (9), C9: (3S)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan2-yl )-1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after resolution by preparative chiral SFC (method I) or chiral RP-HPLC (method S) of racemic compound C-10.C9; >99% e.i.; t _r : 8.85 min (method S'). ¹ H NMR (500 MHz, DMSO-d6) δ 11.23 (br. s, 1H), 8.10 (d, J= 26.0 Hz, 1H), 7.44 (ddd, J= 8.9 , 4.1, 2.7 Hz, 1H), 7.35-7.27 (m, 2H), 7.17 (dd, J= 8.1, 1.8 Hz, 1H), 7.10- 7.02 (m, 2H), 6.90 (d, J= 1.9 Hz, 1H), 6.64 (d, J= 2.1 Hz, 1H), 3.90 (s, 3H), 3.83 (d, J= 20.7 Hz, 3H), 2.45-2.32 (m, 1H), 0.84 (dd, J= 9.4, 7.0 Hz, 3H), 0 .42 (dd, J= 7.1, 4.0 Hz, 3H); ¹³ C NMR (126 MHz, DMSO-d6) δ 175.68, 165.85, 164.38, 163.36, 163.24, 158.77, 156.77, 146.44, 144.17, 135, 31, 134.21, 130.70, 129.60, 128.33, 127.33, 126.18, 125.78, 125.66, 123.46, 122.92, 120.71, 119.27, 118.72, 118.54, 111.11, 93.90, 70.08, 56.78, 54.18, 25.42, 22.00, 21.64, 21.24, 21.04.

Compound C-10.C9: 6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2yl)-1, 2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

In a 100 ml flask equipped with a magnetic stirrer, compound C-9.C9 (5.8 g, 9.68 mmol) was dissolved in 30 ml anhydrous DCM and 10 ml TFA was added in one portion. The reaction mixture was stirred for 3 h at room temperature. After this time, UELCMS analysis showed 54% of the peak area of the product. The reaction mixture was evaporated to dryness. The residue was stirred in 30 ml AcOEt for 1 hour and a pale gray solid precipitated from the mixture. The solid was separated by filtration and dried in air. This gave the product C-10.C9 (2.2 g, 34.1% yield after 2 steps, based on C-8.C9,

- 27 040469 purity 94% according to UELCMS analysis).

Compound C-9.C9: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3yl)-1 -( 4,6-dimethoxypyridin-3-yl)-5-(propan-2-yl)-1 H-pyrrol-3-carboxamide.

Compound C-8.C9 (6.08 g, 10.5 mmol, 1 eq.) and THF (62 ml) were added to a 100 ml flask equipped with a magnetic stirrer, followed by the addition of trimethyl phosphite (2.47 ml, 21 mmol, 2 eq.). The reaction mixture was then cooled in an ice bath and sodium tert-pentoxide (3.47 g, 31.5 mmol, 3 eq.) was added in one portion. The cooling bath was removed and the reaction mixture was stirred for 1 h at room temperature (the flask was equipped with a CaCl ₂ tube). After this time, UELCMS analysis showed 94% of the peak area of the product. The reaction mixture was cooled to approximately -10°C, then poured onto ice and the reaction mixture was acidified with 0.5 M HCl to pH 5. The aqueous phase was extracted with AcOEt (3x100 ml), dried over Na ₂ SO ₄ and the solvent was evaporated under vacuum. The crude product C-9.C9 was obtained as a red-black solid/foam (purity 68% according to UPLCMS analysis) and was used in the next step without any further purification (yield exceeded 100%).

Compound C-8.C9: N-(5-chloro-2-fluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1H-indol-3-yl)-1-(4, 6dimethoxypyridin-3-yl)-5-(propan-2-yl)-1H-pyrrol-3-carboxamide.

Compounds C-4A (7.79 g, 30 mmol, 1 eq.), C-7B (4.68 g, 30 mmol, 1 eq.) and C-6B (6 g, 30 mmol, 1 equiv.) followed by the addition of AcOH (60 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 85° C. (heating bath temperature) and stirred at this temperature for 16 hours. After this time, UPLCMS analysis showed almost complete consumption of the starting materials (corresponding to 40% of the peak area of the product). The reaction mixture was cooled to approximately 15° C. and an abundant red solid precipitate was filtered off from the reaction mixture. The filtrate was evaporated to dryness. The residue was pre-adsorbed on silica gel and purified by flash chromatography (10% to 60% AcOEt in nhexane). After removal of the solvents, the product C-8.C9 was obtained as a light red solid (5.58 g, 31.5% yield) with a purity of 93% according to UPLCMS analysis.

Compound (10), C10: (3S)-6-chloro-2'-(5-chloro-2,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)6'-(propane -2-yl)-1,2,3',5'-tetrahydro-2Ή-spiro[indol-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

The title compound was obtained after preparative chiral RP-HPLC resolution of racemic C-10.C10 using method P; >99% e.i.; t _r : 9.96 min (method P'); ¹ H NMR (500 MHz, DMSO^) δ 11.22 (br. s, 1H), 8.50 (br. s, 1H), 7.64 (t, J = 9.4 Hz, 1H), 7 .40 (s, 1H), 7.20 (t, J= 7.5 Hz, 2H), 7.10 (dd, J= 8.0, 2.0 Hz, 1H), 6.91 (d, J= 1.9 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 2.47-2.41 (m, 1H), 0.85 (d, J= 7 .0 Hz, 3H), 0.44 (d, J= 6.9 Hz, 3H); ¹³ C NMR (126 MHz, DMSO^) δ 175.36, 170.76, 166.43, 164.71, 164.35, 159.05, 158.96, 158.36, 158.26, 157.95 157.03 156.93 156.36 156.26 144.01 135.46 134.20 131.13 127.41 125.84 123.55 123.09 121 .72, 121.63, 119.46, 118.77, 116.23, 115.59, 115.44, 111.19, 107.31, 107.10, 106.90, 70.05, 60.19 .55.66.55.01.25.34, 22.31-20.83.

- 28 040469

Compound C-10.C10: 6-chloro-2'-(5-chloro-2,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'(propan-2-yl) -1,2,3',5'-tetrahydro-2H-spiro[indole-3,Γ-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

AcOH (25 ml) was placed in a 100 ml flask equipped with a magnetic stirrer. C-9.C10 (2.3 g, 4 mmol, 1 eq.) was then added in one portion. MsOH (0.25 ml, 4.85 mmol, 1.2 eq) was added dropwise and the mixture was stirred for 2 h at 45°C. Most of the AcOH was evaporated and the residue was dissolved in DCM (100 ml), then 100 ml of water was added and the mixture was treated with 3 M NaOH until a pH~8 was reached. The phases were separated and the aqueous phase was extracted with DCM (2x100 ml). The combined organic phases were washed with brine (50 ml), dried over MgSO ₄ and concentrated. The residue was purified by flash chromatography (CHCl ₃ :MeOH; 100:0^98:2) to give 1.7 g of product C-10.C10 with a purity of 91% according to UELCMS analysis.

Compound С-9.С10: N-(5-chloro-2,4-difluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro-1Hindol-3-yl)-1 -(2,4-dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1H-pyrrole-3-carboxamide.

To a 100 ml flask equipped with a magnetic stirrer was added compound C-8.C10 (8.9 g, 14.8 mmol, 1 eq.) and THF (50 ml) followed by the addition of triethyl phosphite (3.8 ml, 22.2 mmol, 1.5 eq.). The solution was cooled to 0° C. and then sodium tert-butoxide (2.85 g, 29.6 mmol, 2 eq.) was added in several portions. The reaction mixture was stirred for 3 h at room temperature (the flask was equipped with a tube with CaCl ₂ ). After this time, UELCMS analysis showed 82% of the desired product. The reaction mixture was slowly poured into a chilled (0-5°C) mixture of water (150 ml) and 36% HCl (5 ml). After adding ethyl acetate (100 ml) the mixture was transferred to a separating funnel. The layers were separated and the aqueous phase was re-extracted with AcOEt (100 ml). The combined organic phases were washed with brine, dried over MgSO4 and concentrated in vacuo. The oily residue was pre-adsorbed on silica gel and purified by flash chromatography (DCM/MeOH 100:0-98:2). Two fractions of the product C-9.C10 were isolated: 3 g with a purity of 85% and 3 g with a purity of 41% according to the analysis of UELCMS. Yield: 50% (including both fractions).

Compound C-8.C10: N-(5-chloro-2,4-difluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1 H-indol-3 -yl)-1 ( 2,4-dimethoxypyrimidin-5-yl)-5-(propan-2-yl)-1H-pyrrole-3-carboxamide.

In a 250 ml round bottom flask equipped with a magnetic stirrer, compounds C-4A (11.6 g, 47 mmol, 1 eq.), C-7A (8.7 g, 56 mmol, 1.2 eq.) and C-6F (10 g, 47 mmol, 1 eq) was suspended in 75 ml AcOH and the flask was tightly closed with a plastic stopper. The mixture was heated to 70° C. and stirred at this temperature for 24 hours. After this time, UELCMS analysis showed 47% of the expected product. The reaction mixture was evaporated to dryness. The solid residue was pre-adsorbed on silica gel and purified by flash chromatography (20% to 50% AcOEt in n-hexane). All fractions that contained product were evaporated to dryness to give 6.6 g of the desired product C-8.C10 with a purity of 83% according to UPLCMS analysis. Yield: 32%.

Compound (11), C11: (3S)-6'-(butan-2-yl)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidine5- yl)-1,2,3',5'-tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

- 29 040469

The title compound (diastereomeric mixture) was obtained after preparative chiral HPLC-RP separation of racemic C-1O.C11 using method M; >99% e.i.; 0 9.96 min (method M'); 1H NMR (500 MHz, DMSO-di) δ 11.24 (s, 1H), 8.50 (br. s, 1H), 7.44 (ddd, J= 8.9, 4.2, 2.7 Hz, 1H), 7.38 (s, 1H), 7.31 (t, J = 9.1 Hz, 1H), 7.23-7.14 (m, 1H), 7.03 (dd, J = 6.4, 2.7 Hz, 1H), 6.84 (ddd, J= 10.5, 8.4, 2.4 Hz, 1H), 6.72 (dd, J= 8.9, 2 .4 Hz, 1H), 3.98 (s, 3H), 3.95 (br.s, 3H), 3.09 (kv, J= 7.3 Hz, 2H), 2.48-2.39 (m, 1H), 1.18 (t, J= 7.2 Hz, 3H), 0.85 (d, J= 7.0 Hz, 2H), 0.44 (d, J= 7.0 Hz , 3Н); ¹³ C

NMR (126 MHz, DMSO-d ₆ ) δ 175.83, 166.45, 164.83, 164.70, 164.26, 162.87, 158.76, 157.95, 156.76, 144.36 , 144.26, 134.08, 130.72, 130.66, 129.55, 128.30, 128.28, 127.61, 127.53, 125.73 88, 119.71, 118.70, 118.59, 118.52, 116.26, 109.68, 109.50, 99.43, 99.21, 70.02, 55.66, 55.01, 46.14, 25.33, 21.51, 11.45, 9.08.

Compound C-10.C11: 6'-(butan-2-yl)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin5-yl)-1, 2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione.

To a 250 ml flask equipped with a magnetic stirrer was added compound C-9.C11 (2.8 g, 4.56 mmol) along with DCM (60 ml). TFA (30 ml) was then added (approximately 2 ml/min). The reaction mixture was stirred for 2 hours at room temperature. After this time, UELCMS analysis showed 65% of the peak area of the expected product. The mixture was then poured onto ice (approximately 200 ml) and diluted with DCM (100 ml). The phases were separated and the aqueous phase was extracted three times with DCM (3x50 ml). The combined organic phases were washed with water, brine, dried over MgSO ₄ and concentrated in vacuo. The crude product C-10.C11 was purified by column chromatography (10 to 40% acetone in n-hexane). This gave 1.5 g of a brown solid C-10.C11. This solid was stirred in AcOEt (5 ml), separated by filtration, washed with 20 ml AcOEt and air dried. As a result, the product C-10.C11 was obtained as white solids (1.4 g, 98% purity according to UELCMS analysis).

Compound С-9.С11: 5-(bugan-2-yl)-Ni(5-chloro-2-fluorophenyl)-4-(6-chloro-3-hydroxy-2-oxo-2,3-dihydro1H-indole -3-yl)-1-(2,4-dimethoxypyrimidin-5-yl)-1H-pyrrol-3-carboxamide.

H

To a 250 ml flask equipped with a magnetic stirrer was added compound C-8.C11 (3 g, 5 mmol, equiv.) and THF (80 ml) followed by the addition of trimethyl phosphite (1.21 ml, 10 mmol, 2 equiv.). The reaction mixture was then cooled in an ice bath and sodium tert-pentoxide (2.2 g, 20 mmol, 4 eq.) was added in small portions. The cooling bath was removed and the reaction mixture was stirred for 5 hours at room temperature (the flask was fitted with a CaCl ₂ tube). After this time, the analysis

UELCMS showed 71% of the two (diastereomeric) peaks of the C-9.C11 product. The reaction mixture was diluted with 200 ml of ice water. The resulting suspension was acidified with 3 M HCl to pH 5 and diluted with 100 ml AcOEt. The phases were separated and the aqueous phase was extracted three times with AcOEt (3x50 ml). The combined organic phases were washed with water, brine, dried over Na ₂ SO ₄ and the solvent was removed under vacuum. The crude product C-9.C11 was obtained as a dark red oil (purity 72% according to UPLCMS analysis) and was used in the next step without any further purification.

Compound С-8.С11: 5-(butan-2-yl)-N-(5-chloro-2-fluorophenyl)-4-(6-chloro-2-oxo-2,3-dihydro-1H-indole- 30 040469

3-yl)-1-(2,4-dimethoxypyrimidin-5-yl)-1H-pyrrol-3-carboxamide. Η

Compounds C-4B (4.44 g, 16.8 mmol, 1 eq.), C-7A (2.87 g, 18.5 mmol, 1.1 eq.) were added to a 250 ml flask equipped with a magnetic stirrer. and C-6F (3.32 g, 16.8 mmol, 1 eq.) followed by the addition of AcOH (30 ml) and the flask was tightly closed with a plastic stopper. The mixture was heated to 90° C. (heating bath temperature) and stirred at this temperature for 16 hours. After this time, UPLCMS analysis showed almost complete consumption of the starting materials (corresponding to 50% of the peak area of the product). The reaction mixture was cooled to room temperature and evaporated to dryness. The residue was pre-adsorbed on silica gel and purified by flash chromatography (30% to 60% AcOEt in n-hexane). After removal of the solvents, the product C-8.C11 was obtained as a dark red solid/foam (3.5 g, 35% yield) with a purity of 83% according to UPLCMS analysis.

Preparation and analysis of enantiomers

All enantiomers were separated on preparative SFC or HPLC with chiral columns.

Conditions for chiral purification - SFC.

Method A: column: Lux Amylose-1 (21.2 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 25:75, detection: UV 210 nm.

Method B: column: Lux Cellulose-1 (21.2 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 25:75, detection: UV 210 nm.

Method C: column: Lux Cellulose-4 (21.2 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 45:55, detection: UV 215 nm.

Method D: column: Chiralpak IC (20 mm x 250 mm, 5 μm), flow 21 ml/min, isocratic elution EHN:CO ₂ , 45:55, detection: UV 210 nm.

Method E: column: Lux Cellulose-4 (21.2 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 40:60, detection: UV 210 nm.

Method F: column: Chiralpak AS-H (20 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 25:75, detection: UV 210 nm.

Method G: column: Chiralpak IC (20 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 45:55, detection: UV 210 nm.

Method H: column: Lux Cellulose-4 (30 mm x 250 mm, 5 μm), flow 50 ml/min, isocratic elution with MeOH:CO ₂ , 40:60, detection: UV 210 nm.

Conditions for chiral purification - NF-HPLC.

Method I: column: Chiralpak IC (20 mm x 250 mm, 5 μm), flow 21 ml/min, isocratic elution with MeOH, detection: UV 220 nm.

Conditions for chiral purification - RP-HPLC.

Method J: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:MeOH:H ₂ O, 50:20:30, detection: UV 254 nm.

Method K: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 80:20, detection: UV 254 nm.

Method L: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 65:35, detection: UV 254 nm.

Method M: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O+HCO ₂ NH ₄ (mobile phase A1), 90:10, detection: UV 254 nm.

Method N: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 70:30, detection: UV 254 nm.

Method O: column: Lux Cellulose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, gradient elution, A=ACN, B=H ₂ O, detection: UV 254 nm.

Time [min] % A %IN gradient curve 0.0 60 40 - 1.0 60 40 linear (6) 5.0 90 10 linear (6) 10.0 60 40 straight (11)

- 31 040469

Method P: column: Lux Amylose-2 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 50:50, detection: UV 254 nm.

Method R: column: Lux Amylose-2 (21 mm x 250 mm, 5 µm), flow: 30 ml/min, gradient elution; A=ACN, B=H ₂ O, detection: UV, 254 nm. ___________________________________

Time [min] % A %IN gradient curve 0.0 60 40 - 1.0 60 40 linear (6) 7.0 90 10 linear (6) 12.0 60 40 straight (I)

Method S: column: Lux Amylose-2 (21 mm x 250 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 60:40, detection: UV 254 nm.

Method T: column: Lux Cellulose-4 (21 mm x 150 mm, 5 μm), flow: 30 ml/min, isocratic elution, ACN:H ₂ O, 60:40, detection: UV 254 nm.

Conditions for the analysis of chiral purity - SFC.

Method A': column: Lux Amylose-1 (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 25:75, detection: UV 211 nm and 254 nm.

Method B': column: Lux Cellulose-1 (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 25:75, detection: UV 211 and 254 nm.

Method C: column: Lux Cellulose-4 (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 50:50, detection: UV 210 -400 nm.

Method D': column: Chiralpak IC (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, EtOH:CO ₂ , 45:55, detection: UV 210- 400 nm.

Method E': column: Lux Cellulose-4 (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 40:60, detection: UV 210-400 nm.

Method F': column: AMS (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 30:70, detection: UV 211 and 254 nm.

Method G': column: Chiralpak IC (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 40:60, detection: UV 210- 400 nm.

Method H': column: Lux Cellulose-4 (4.6 mm x 250 mm, 5 μm), column temperature: 40°C, flow: 4 ml/min, isocratic elution, MeOH:CO ₂ , 40:60, detection: UV 211 and 254 nm.

Analysis of chiral purity - NF-HPLC.

Method I': column: Lux Cellulose-5 (4.6 mm x 150 mm, 5 µm), column temperature: ambient, flow: 1 ml/min, isocratic elution, EtOH, detection: UV 254 nm

Conditions for analysis of chiral purity - RP-HPLC.

Method J': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 µm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:MeOH:H ₂ O, 50:20 :30, detection: UV 254 nm.

Method K': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 µm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 80:20, detection : UV 254 nm.

Method L': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 µm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 65:35, detection : UV 254 nm.

Method M': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 μm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O+HCO ₂ NH ₄ ( mobile phase A1), 90:10 detection: UV 254 nm.

Method N': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 μm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 70:30, detection : UV 254 nm.

Method O': column: Lux Cellulose-2 (4.6 mm x 150 mm, 5 μm), column temperature: ambient, flow: 1.23 ml/min, gradient elution, A=ACN, B=H ₂ O, detection : UV 254 nm.

Time [min] % A %IN gradient curve oh oh 60 40 - 1.0 60 40 linear (6) 5.0 90 10 linear (6) 10.0 60 40 straight (I)

Method P': column: Lux Amylose-2 (4.6 mm x 150 mm, 5 µm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 50:50, detection : UV 254 nm.

Method R': column: Lux Amylose-2 (4.6 mm x 250 mm, 5 µm), column temperature: ambient

- 32 040469 medium, flow: 1.23 ml/min, gradient elution; , A=ACN, B=H ₂ O, detection: UV 254 nm.

Time [min] % A %IN gradient curve 0.0 60 40 - 1.0 60 40 linear (6) 7.0 90 10 linear (6) 12.0 60 40 straight (11)

Method S': column: Lux Amylose-2 (4.6 mm x 250 mm, 5 µm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 60:40, detection : UV 254 nm.

Method T': column: Lux Cellulose-4 (4.6 mm x 150 mm, 5 μm), column temperature: ambient, flow: 1.23 ml/min, isocratic elution, ACN:H ₂ O, 60:40, detection : UV 254 nm.

The following examples were synthesized in accordance with the methods described in this application or known literary methods using appropriate starting materials and methods known to a person skilled in the art.

Table 1

Comm. no Structure Chiral purity analysis method Name Analytical data 1 CI Zz n R' (35)-6-chloro-2'-(3chlorophenyl)-5'-(2,4dimethoxypyrimidin-5yl)-6'-(propan-2-yl)1,2,3',5'-tetrahydro-2' Jaspiro[indole-3, Gpyrrolo[3,4-c]pyrrole]-2,3'- DION acid gradient analysis: 98.08%, 564 [M+H] ⁺ , retention time: 3.52 minutes; main gradient analysis: 98.13%, 564 [M+H] ⁺ , retention time: 3.52 minutes; chiral analysis: 99.71%, retention time: 7.77 min. 2 o XX x o ^x n N' (3X)-6-chloro-2'-(5-chloro-2-ph orphenyl)-5'-(2,4-dimethoxy pyrimidin-5-yl)-6'-(propa n-2-yl)- 1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrr olo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 99.43%, 582 [M+H] ⁺ , retention time: 3.48 minutes; main gradient analysis: 99.02%, 582 [M+H] ⁺ , retention time: 3.48 min; chiral analysis: 99.44%, retention time: 9.31 min.

- 33 040469

3 £Y ° / ii θ' H\ Τ' (35)-6-chloro-2'-(5-chloro-2-methylphenyl)-6'-(propan-2-yl)-5'-(2,4,6-trimethoxypyrim id in-5-yl )-1,2,3',5'-tetragy dro-2'H-spiro[indole-3, Mr. irrolo[3,4-c]pyrrole]-2,3'-d ion acid gradient analysis: 97.4%, 608 [M+H] ⁺ , retention time: 3.71 minutes; main gradient analysis: 97.4%, 608 [M+H] ⁺ , retention time: 3.71 minutes; chiral analysis: 99.7%, retention time: 16.70 min. 4 ABOUT TO' (35)-6-chloro-2'-(3-chloro-4-ph orphenyl)-5'-(2,4-dimethoxy pyrimidin-5-yl)-6'-(propa n-2-yl)- 1,2,3',5'-tetrahydro-2H-spiro[indole-3,1'-pyrr olo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 98.96%, 582 [M+H] ⁺ , retention time: 3.58 minutes; main gradient analysis: 99.08%, 582 [M+H] ⁺ , retention time: 3.57 minutes; chiral analysis: 99.97%, retention time: 3.79 min. 5 GG o _z ciAA ji _o ^z H °\ L' (35)-6-chloro-2'-(5-chloro-2-ph-orphenyl)-6'-(propan-2-yl)5'-(2,4,6-trimethoxypyrimide in-5-yl) -1,2,3',5'-tetragy dro-2'H-spiro[indole-3, Mr. irrolo[3,4-c]pyrrole]-2,3'-d ion acid gradient analysis: 99.89%, 612 [M+H] ⁺ , retention time: 3.65 minutes; main gradient analysis: 99.95%, 612 [M+H] ⁺ , retention time: 3.65 min; chiral analysis: 99.74%, time retention: 7.18 min. 6 H J' (35)-6-chloro-2'-(5-chloro-2-ph-orphenyl)-5'-[6-(dimethylamino)-4-methoxypyridin-3yl]-6'-(propan-2- yl)-1,2,3',5'-tetrahydro-2H-spiro[indol-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 99.88%, 594 [M+H] ⁺ , retention time: 3.94 minutes; main gradient analysis: 99.89%, 594 [M+H] ⁺ , retention time: 3.58 min; chiral analysis: 100%, retention time: 14.66 min. 7 O- x rA oAJ °=\ Ml ^{z 1} o 0' (35)-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyr imidin-5-yl)-6'-(propan-2yl)-1,2,3 ',5'-tetrahydro-2H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 98.89%, 566 [M+H] ⁺ , retention time: 3.48 minutes; main gradient analysis: 98.79%, 566 [M+H] ⁺ , retention time: 3.47 minutes; chiral analysis: 100%, retention time: 5.61 min. 8 O^ Πγΰ w o N' (35)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2-yl)-1,2 ,3',5'-tetrahydro-2'H-sp iro[indole-3,G-pyrrolo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 99.75%, 565 [M+H] ⁺ , retention time: 3.57 min. main gradient analysis: 99.77%, 565 [M+H] ⁺ , retention time: 3.57 min.

- 34 040469

chiral analysis: 100%, retention time: 6.20 min. 9 O , A n S' (3X)-6-chloro-2'-(5-chloro-2-ph-orphenyl)-5'-(4,6-dimethoxy-pyridin-3-yl)-6'-(propan-2-yl)-1 ,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrole o[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 99.68%, 581 [M+H] ⁺ , retention time: 3.60 min. main gradient analysis: 99.63%, 581 [M+H] ⁺ , retention time: 3.59 min. chiral analysis: 100%, retention time: 8.85 min. 10 Yr about Η P' (3X)-6-chloro-2'-(5-chloro-2,4-d ifluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(p-propan-2- yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione acid gradient analysis: 97.97%, 600 [M+H] ⁺ , retention time: 3.59 min. main gradient analysis: 97.47%, 600 [M+H] ⁺ , retention time: 3.59 min. chiral analysis: 100%, retention time: 9.96 min. AND Q /l whether II Ί o ₄ M' (3X)-6'-(butan-2-yl)-6-chloro2'-(5-chloro-2-fluorophenyl)-5'(2,4-dimethoxypyrimidin5-yl)-1,2,3',5 '-tetrahydro2'H-spiro[indole-3, G- acid gradient analysis: 97.73%, 596 [M+H] ⁺ , retention time: 3.62 min. main gradient analysis: 99.54%, 596 [M+H] ⁺ , retention time: 3.62 pyrrolo[3,4-c]pyrrole]-2,3'- DION min. chiral analysis: 100%, retention time: 9.96 min.

biological examples.

Biological example 1. Fluorescent polarization analysis.

Inhibition of the p53-Mdm2 interaction was measured using a fluorescent polarization (FP) binding assay. FP measures the rotational motion of molecules in a homogeneous suspension. For this assay, the N-terminal domain of the Mdm2 protein (amino acids 1-111) is combined with a fluorescein-labeled (FAM) peptide derived from the p53 transactivation domain (sequence: 5FAM-TSFAEYWNLLSP). When a fluorescent ligand is excited by linearly polarized light, the peptide emits perpendicularly polarized light. If the peptide is bound to Mdm2, the rotation will slow down and the perpendicular component will proportionally decrease. In contrast, disruption of the peptide-Mdm2 complex due to inhibitor binding to the p53 Mdm2 binding site results in the release of the peptide and a decrease in the polarization of the emitted light.

Fluorescent polarization experiments were read on a Biotek Cytation 5 reader with 470 nm excitation and 520 nm emission filters for fluorescein. Fluorescent polarization was measured in black 96-well plates (Corning, CLS3991) at room temperature. The purity of Mdm2 was controlled at >95%. The reaction buffer was optimized by adding 5 mM DTT and 0.1% zwitterionic detergent CHAPS to reduce the effect of non-specific interactions.

The assay was performed by combining serial dilutions of compounds diluted in dimethyl sulfoxide (DMSO, 5% final concentration) with 75 nM Mdm2 in reaction buffer (PBS, 0.1% CHAPS, 5 mM DTT (dithiothreitol)). After 15 minutes of incubation at room temperature, 10 nM FAM-labeled peptide was added. The final reading was performed after 90 minutes of incubation. Dose-dependent binding curves and IC50 values were calculated using GraphPad Prism5 and then converted to Ki values using the Kenakin equation (Table 2).

- 35 040469

table 2

Compound Ki (nM) p53-Mdm2 1 1.8 2 1.7 3 2.4 4 1.7 5 2.2 6 1.7 7 1.9 8 1.9 9 2.0 10 2.0 AND 2.5

Examination of measured Ki values indicates that all of the disclosed compounds are effective (Ki in the range of 1.7-2.5 nM) inhibitors of Mdm2-p53 interactions.

Biological example 2 Cell viability assay.

The effect of the invented p53-Mdm2 inhibitors on cell viability was evaluated using the MTT assay. This is a colorimetric assay that measures the conversion of the tetrazolium ring of a soluble yellow dye (MTT) to insoluble purple formazan. This process is catalyzed exclusively in mitochondrial dehydrogenases of living cells. Dead cells do not cause this change. To measure the specific cytotoxicity of Mdm2p53 inhibitors, an MTT assay was performed with the SJSA-1 osteosarcoma cell line, which demonstrates amplification of the wild-type MDM2 and p53 gene.

Cells were seeded in 96-well plates and then treated with serial dilutions of test compounds. After 72 hours of incubation, MTT was added to a final concentration of 0.5 mg/ml. The cells were additionally incubated for a further 4 hours. The solution was then decanted and the remaining formazan crystals were dissolved in 100 μl of DMSO. Absorbance readings were taken at 570 nm showing relative cell viability between cells treated with compounds of interest and DMSO control. All MTT experiments were independently repeated 2-5 times. Dose-dependent binding curves and IC50 values were calculated using GraphPad Prism 5. The IC50 values presented are the average of all experiments performed (Table 3).

Table 3

Compound IC50 (µM) SJSA-1 1 0.18 2 0.16 3 0.07 4 0.32 5 0.18 6 0.03 7 0.22 8 0.15 9 0.12 10 0.40 AND 0.05

Biological example 3 Measurement of intrinsic clearance using microsomes in vitro.

The metabolic stability of the compounds of the invention was evaluated by measuring intrinsic clearance in mouse and human microsomes in vitro.

mM stock solutions of markers and test compounds were prepared in DMSO. They were diluted 100-fold in MeCEEDMS0 91:9 to give a 100 μM assay stock solution. 10 mM NADPH

-36040469 was prepared in 0.1 M phosphate buffer (pH 7.4). The microsomes were thawed in a water bath at 37° C. and diluted to a final assay concentration of 0.5 mg/mL.

100 μM assay stock solutions were added to give a final concentration of 1 μM in incubation tubes containing buffer and NADPH (final assay concentration is 1 mM). Incubation tubes and microsomes were preheated at 37°C for 3 min. The microsomes were then added to incubation tubes, which were stored at 37° C. and shaken using an orbital shaker throughout the assay. Samples were taken at 6 predetermined time points up to 1 hour and transferred to prepared quenched tubes containing the appropriate solvent with an internal standard.

The quenched samples are mixed thoroughly and the protein is precipitated at -20°C for a minimum of 12 hours. The samples are then centrifuged at 4°C. The supernatants were transferred to a fresh 96-well autosampler compatible plate. The plate was sealed with a pre-cut silicone mat and analyzed by LC-MS/MS.

Compound mouse MC clear, own. [µl/min/mg] human MC clear, own. [µl/min/mg] WO2015/189799-107 9.5 49.7 1 8.8 <3.0 2 <4.0 <3.0 3 <3.0 <3.0 4 <3.0 <3.0 5 <3.0 <3.0 6 33.6 19.8 7 6.6 <3 8 12.8 <3 9 n.a. n.a. 10 <3 <3.2 AND n.a. n.a.

Compared to the compounds described in WO 2015/189799, all currently disclosed compounds exhibit low intrinsic clearance in both human and mouse microsomes. Compound 6 is the only exception. However, the slightly lower stability of this compound is offset by a marked in vitro potency (SJSA-1 IC50=0.03 μM).

Biological Example 4 In vivo efficacy in the SJSA-1 xenograft mouse model.

The experiment was carried out on female mice from the strain Crl: CD-1-Foxnlnu. Mice were inoculated subcutaneously in the right flank with the SJSA-1 cancer cell line at 3x106 cells suspended in 100 μl of HBSS:Matrigel matrix at a ratio of 3:1 per mouse. On the 17th day after inoculation, mice were divided into groups so that in each group the average tumor volume was the same and averaged approximately 200 mm ³ . Experimental groups were selected, each of which consisted of 8 mice: control NaCl 0.9% and compounds. Compounds 1-11 were dissolved in 56.60% PEG 400, 9.43% Cremophor RH40, 9.43% ETOH, 18.87% Labrafil M1944CS, 5.67% DMSO. Compound 107 of WO 2015/189799 used a group of 7 mice and was dissolved in 15% PEG400, 10% Cremophor EL, 75% H2O.

Mice used in the experiment were administered orally (po) compounds or 0.9% NaCl qldx14 (14 doses daily). During the experiment, mice were weighed before each administration twice/thrice a week. Animal welfare was monitored daily. No significant difference in body weight or well-being was observed between the experimental groups during and at the end of the study.

The change in tumor volume was monitored twice/thrice a week, starting from the first day of administration. Tumor volume was calculated based on its length and width measured using electronic calipers:

V [mm ³ ]=d ² xD/2 in which d is the width, D is the length.

The tumor volume in the groups was measured up to 101 days after inoculation (72 days after the last injection). Experimental results were expressed as mean tumor growth inhibition (TGI)±SEM values (Table 4). Tumor growth inhibition was calculated using the following formula:

TGI [%]=[100-(T/Cx100)] where C is the average tumor size in the control group, T is the average tumor size in the treated group. All calculations and plots were performed using GraphPad Prism 5 software.

-

Claims (14)

Table 4 Compound TGI po 12.5 mg/kg (7 doses) [%] ± SEM TGI po 12.5 mg/kg (12 doses) [%] ± SEM Standard: Compound 107 from WO2015/189799 49.8 17.3 60.1 13.4 1 94.97 1.09 99.53 0.30 2 94.36 1.23 98.91 0.44 3 94.22 1.25 99.57 0.29 4 94.89 1.57 99.47 0.34 5 85.78 5.59 95.99 2.29 6 92.03 1.88 99.09 0.46 7 95.00 1.29 99.97 0.01 8 96.39 1.57 99.71 0.27 9 89.46 2.72 97.73 0.92 10 93.48 1.44 98.79 0.50 AND 93.74 1.35 99.69 0.28 The results of this experiment are summarized in table. 4 and in FIG. 1 and 2. As can be seen, the improved metabolic stability of the currently disclosed compounds results in exceptional in vivo efficacy. The tumor growth inhibition values observed for compounds 111 are significantly higher than for the best compound disclosed in WO 2015/189799 after both 7 (difference 36.0-46.6%) and 12 (difference 35.9-39 .9%) doses of the substance. Moreover, as can be seen in FIG. 1, treatment of SJSA-1 xenograft with 14 doses of 12.5 mg/kg p/o WO 2015/189799 only slows tumor growth. The same treatment protocol but with compounds 7, 8 and 11 resulted in complete tumor resection. Moreover, none of the lesions showed regrowth during follow-up until day 101. CLAIM 1. A compound having the following structure wherein R ¹ is meta-halophenyl which is optionally further substituted with one or two substituents independently selected from the group consisting of halo and -C1- _C6 alkyl, R ² and R ³ are independently H or halogen; R ⁴ represents -C1-C ₆ -alkyl; R ⁷ is -OCH ₃ ; Z is CR ⁸ or N, Y is CR ⁹ or N, provided that Z is not CR ⁸ and Y is not CR ⁹ at the same time, R ⁵ , R ⁶ are independently H, -OCH ₃ or -N(CH ₃ ) ₂ , R ⁸ , R ⁹ are independently H. 2. The compound of claim 1 wherein R ¹ is meta-halophenyl which is optionally further substituted with one to two substituents selected from halo. 3. The compound of claim 1 wherein R ¹ is meta-halophenyl which is optionally further substituted with one substituent selected from -CH ₃ . 4. A compound according to any one of claims 1 to 3, wherein R ² is H and R ³ is Cl. 5. A compound according to any one of claims 1 to 4, wherein R ⁴ is isopropyl or isobutyl. 6. A compound according to any one of claims 1 to 5, wherein Z and Y are both N. 7. A compound according to any one of claims 1-5, wherein Z is CR ⁸ and Y is N. 8. A compound according to claim 6 wherein R ⁵ and R ⁶ are both OMe. 9. A compound according to claim 7, wherein R ⁸ is H, at least one of R ⁵ and R ⁶ is OMe, and the second is selected from H, -N(Me) ₂ and OMe. 10. The compound according to claim 2 selected from (1) (3S)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propane -2-yl)-1,2,3',5'-teIrahygro2'H-spiro[indol-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, - 38 040469 (2) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2- yl)-1,2,3',5'tetrahydro-2H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (3) (38)- 6-chloro-2'-(5-chloro-2-methylphenyl)-6'-(propan-2-yl)-5'-(2,4,6-trimethoxypyrimidin-5-yl)-1,2,3 ',5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (4) (38)-6-chloro-2' -(3-chloro-4-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)-1,2,3',5'tetrahydro-2' H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (5) (38)-6-chloro-2'-(5-chloro-2- fluorophenyl)-6'-(propan-2-yl)-5'-(2,4,6-trimethoxypyrimidin-5-yl)-1,2,3',5'tetrahydro-2'H-spiro[indol- 3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (6) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'- [6-(dimethylamino)-4-methoxypyridin-3-yl]-6'-(propan2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indol-3,1'- pyrrolo[3,4-c]pyrrole]-2,3'-dione, (7) (38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidine- 5-yl)-6'-(propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4 -c]pyrrole]-2,3'-dione, (8) (38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)- 6'-(propan-2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2.3 '-dione, (9) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2 -yl)-1,2,3',5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (10) ( 38)-6-chloro-2'-(5-chloro-2,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)- 1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (11) (38)- 6'-(butan-2-yl)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-1,2,3', 5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione. 11. The compound according to claim 6 selected from (1) (38)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-propan- 2-yl)-1,2,3',5'-tetrahydro2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (2) ( 38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)-1,2, 3',5'tetrahydro-2Ή-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (3) (38)-6-chloro-2'- (5-chloro-2-methylphenyl)-6'-(propan-2-yl)-5'-(2,4,6-trimethoxypyrimidin-5-yl)-1,2,3',5'tetrahydro-2 'H-spiro [indole-3,1'-pyrrolo[3,4-c]pyrrole] -2,3'-dione, (4) (38)-6-chloro-2'-(3-chloro-4 -fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)-1,2,3',5'teΊrahydro-2Ή-spiro[indole-3,1 '-pyrrolo[3,4-c]pyrrole] -2,3'-dione, (5) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-6'-(propane- 2-yl)-5'-(2,4,6-trimethoxypyrimidin-5-yl)-1,2,3',5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo [3, 4-c]pyrrole]-2,3'-dione, (7) (38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl) -6'-(propan-2-yl)-1,2,3',5'tetrahydro-2'H-sp iro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (10) (38)-6-chloro-2'-(5-chloro-2,4- difluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'-(propan-2-yl)- 1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (11) (38)- 6'-(butan-2-yl)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-1,2,3', 5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione. 12. The compound according to claim 7 selected from (6) (38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-[6-(dimethylamino)-4-methoxypyridine-3- yl]-6'-(propan2-yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2, 3'-dione, (8) (38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2- yl)-1,2,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione, (9) ( 38)-6-chloro-2'-(5-chloro-2-fluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'-(propan-2-yl)-1,2, 3',5'tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione. 13. Compound Formula which is (38)-6-chloro-2'-(3,4-difluorophenyl)-5'-(4,6-dimethoxypyridin-3-yl)-6'(propan-2-yl)-1,2 ,3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione. 14. Compound of formula o n which is (38)-6-chloro-2'-(3-chlorophenyl)-5'-(2,4-dimethoxypyrimidin-5-yl)-6'(propan-2-yl)-1,2, 3',5'-tetrahydro-2'H-spiro[indole-3,1'-pyrrolo[3,4-c]pyrrole]-2,3'-dione. -