EA045742B1 - CD73 INHIBITOR CRYSTAL FORMS - Google Patents

Description

The present invention relates to crystalline forms of 5-[5-[2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione and pharmaceutical compositions containing them, which inhibit CD73 activity and are useful for the treatment of cancer.

CD73, also known as 5'-nucleotidase or ecto-5'-nucleotidase (EC 3.1.3.5), is an enzyme that converts 5'-mononucleotides into nucleosides. CD73 is expressed in many tissues and is activated in cancerous tissues, and the CD73 pathway promotes tumor growth by limiting antitumor T-cell immunity through adenosine receptor signaling (Zhang B., Cancer Research, 2010, 70:6407-6411; Antonioli L. et al. al., Drug Discovery Today, 2017, 22:1686-1696). CD73-deficient mice have enhanced antitumor immunity and are resistant to experimental metastasis (Stagg J. et al., Cancer Research, 2011, 71:2892-2900). Extracellular adenosine produced by tumor CD73 accumulates in the tumor microenvironment, disrupts antitumor T-cell immunity (Zhang B., Antonioli L.) and is associated with tumor immune evasion, proliferation, migration, neovascularization, metastasis and chemoresistance of tumor cells (Inoue Y et al., Oncotarget, 2017, 8:8738-8751). It has also been reported that increased CD73 expression is associated with increased immunosuppression (Jin D. et al., Cancer Res., 70:2245-2255 (2010)).

Thus, the CD73 pathway has immunosuppressive effects, and it has been suggested that blocking the CD73 pathway may be useful for treating cancer (Allard D. et al., Immunotherapy, 2016, 8:145-163). CD73 inhibitors are being tested in clinical trials for cancer treatment (Hay C.M. et al., Oncoimmunol., 2016, 5(8):e1208875, Allard D).

US Application No. 16/481146, entitled CD73 Inhibitors, is the US national phase of International Application No. PCT/US2019/019074 and discloses certain CD73 inhibitor molecules, including the compound

N,? /\

N—N = N Η (formula I, which is 5-[5-[2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]1H-pyrimidin-2,4-dione).

Accordingly, the present invention provides crystalline forms of the compound

Н 7° / \

Ν—У Ν = Ν

H (5-[5-[2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione).

In a preferred embodiment, the crystalline form is the compound form

(5-[5-[(1 S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1 H-pyrimidin-2,4-dione.

The present invention also provides a pharmaceutical composition containing a crystalline form of the compound 5-[5-[2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidine2,4-dione, and one or more pharmaceutically acceptable carriers, diluents or excipients . In a preferred embodiment, the pharmaceutical composition contains a crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione and one or more pharmaceutically acceptable carriers, diluents or fillers.

Crystal form 1.

In one preferred embodiment, the present invention provides a composition containing at least 500 g of an anhydrous crystalline form of a compound of the formula

(5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione).

In another preferred embodiment, the anhydrous crystalline form in the composition is characterized by at least one of

- 1 045742 (a) a peak in the X-ray diffraction pattern at a 2-theta diffraction angle of 4.7° in combination with one or more peaks at 11.7, 15.8, 16.0, 26.8 and 27.2°; with a tolerance for diffraction angles of 0.2°; and (b) a solid-phase ¹³ C NMR spectrum, which includes peaks assigned to the high-field resonance of adamantane (δ=29.5 ppm) at 25.4, 28.9, 43.1, 109.3, 124 .4, 128.2 and 160.7 ppm. (±0.2 ppm respectively).

In another preferred embodiment, the anhydrous crystalline form is further characterized by a solid phase ¹³ C NMR spectrum that includes one or more peaks assigned to the high field resonance of adamantane (δ=29.5 ppm) at 16.6, 18.3, 20.7, 22.1, 23.9, 26.6, 27.6, 28.1, 30.2, 31.2, 32.2, 38.6, 39.7, 41.2, 111, 0, 112.0, 130.7, 145.1, 146.3, 150.1, 151.0, 153.4, 156.7, 157.6, 158.6, 159.3, 160.7, 163.4, 165.9, 169.4 and 170.2 ppm. (±0.2 ppm respectively).

In another preferred embodiment, the anhydrous crystalline form is characterized by a solid phase ¹³ C NMR spectrum that includes peaks assigned to the adamantane high field resonance (δ=29.5 ppm) at 16.6, 18.3, 20.7, 22.1, 23.9, 25.4, 26.6, 27.6, 28.1, 28.9, 30.2, 31.2, 32.2, 38.6, 39.7, 41, 2, 43.1, 109.3, 111.0, 112.0, 124.4, 128.2, 130.7, 145.1, 146.3, 150.1, 151.0, 153.4, 156.7, 157.6, 158.6, 159.3, 160.7, 163.4, 165.9, 167.0, 169.4 and 170.2 ppm. (±0.2 ppm respectively).

In another preferred embodiment, the anhydrous crystalline form in the composition is the compound 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione.

In another preferred embodiment, the composition is at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% free from impurities. In another preferred embodiment, the composition is at least 95% free from impurities. In another preferred embodiment, the composition is at least 96% free from impurities. In another preferred embodiment, the composition is at least 97% free from impurities. In another preferred embodiment, the composition is at least 98% free from impurities. In another preferred embodiment, the composition is at least 99% free from impurities.

In another preferred embodiment, the composition is a granular composition. In another preferred embodiment, the granular composition is a wet granular composition. In another preferred embodiment, the granular composition is a dry granular composition.

In another preferred embodiment, the present invention provides a production container containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 kg of composition. In another preferred embodiment, the present invention provides a production container containing at least 1 kg of the composition. In another preferred embodiment, the present invention provides a production container containing at least 5 kg of the composition.

In another preferred embodiment, the present invention provides a pharmaceutical composition comprising the composition and one or more pharmaceutically acceptable carriers, diluents or excipients.

Crystal form 2.

In another preferred embodiment, the crystalline form is the desolvated (anhydrous) crystalline form of the compound of the formula (5-[5-[(1S,2R)-2-isonropylcyclonropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin- 2,4-dione) which is characterized by at least one of (a) an X-ray diffraction peak at a 2-theta diffraction angle of 4.3-5.5° in combination with one or more peaks at 6.4, 9.6, 11.0 and 11.6°; with a tolerance for diffraction angles of 0.2°; and (b) a solid-phase ¹³ C NMR spectrum, which includes peaks assigned to the high-field resonance of adamantane (δ=29.5 ppm) at 17.2, 28.7, 122.0, 126.1, 143 .6 and 155.5 ppm (±0.2 ppm respectively).

In another preferred embodiment, the peak in the x-ray diffraction pattern at a 2-theta diffraction angle of 4.3-5.5° is at a 2-theta diffraction angle of 4.3°.

In another preferred embodiment, the desolvated (anhydrous) crystalline form is further characterized by a solid phase ¹³ C NMR spectrum that includes one or more peaks assigned to the high field resonance of adamantane (δ=29.5 ppm) at 18.9, 21 ,1, 22.5, 24.4, 26.7, 27.8, 29.7, 39.7, 41.3, 45.5, 50.1, 110.7, 111.7, 112.4 , 125.0, 129.1, 144.8, 146.2, 147.2, 149.9, 151.1, 153.0, 155.5, 157.2, 159.0, 160.6, 161 .4, 162.7, 164.8, 167.0, 168.3 and 170.4 ppm. (±0.2 ppm respectively).

- 2 045742

In another preferred embodiment, the desolvated (anhydrous) crystalline form is characterized by a solid phase ¹³ C NMR spectrum that includes peaks assigned to the adamantane upfield resonance (δ=29.5 ppm) at 17.2, 18.9, 21 ,1, 22.5, 24.4, 26.7, 27.8, 28.7, 29.7, 39.7, 41.3, 45.5, 50.1, 110.7, 111.7 , 112.4, 122.0, 125.0, 126.1, 129.1, 143.6, 144.8, 146.2, 147.2, 149.9, 151.1, 153.0, 155 .5, 157.2, 159.0, 160.6, 161.4, 162.7, 164.8, 167.0, 168.3 and 170.4 ppm. (±0.2 ppm respectively).

In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free from impurities. In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 95% free of impurities. In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 96% free from impurities. In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 97% free from impurities. In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 98% free of impurities. In another preferred embodiment, the desolvated (anhydrous) crystalline form is at least 99% free of impurities.

In another preferred embodiment, the present invention provides a composition containing at least 100, 200, 300, 400, 500 or 1000 g of desolvated (anhydrous) crystalline form. In another preferred embodiment, the present invention provides a composition containing at least 100 g of a desolvated (anhydrous) crystalline form. In another preferred embodiment, the composition is a granular composition. In another preferred embodiment, the granular composition is a wet granular composition. In another preferred embodiment, the granular composition is a dry granular composition.

In another preferred embodiment, the present invention provides a production container containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 kg of desolvated (anhydrous) crystalline form. In another preferred embodiment, the present invention provides a production container containing at least 1 kg of the desolvated (anhydrous) crystalline form. In another preferred embodiment, the present invention provides a pharmaceutical composition comprising a desolvated (anhydrous) crystalline form and one or more pharmaceutically acceptable carriers, diluents or excipients.

Crystal form 3.

In another preferred embodiment, the present application provides a hemihydrate crystalline form of the compound of the formula (5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4- dione).

In another preferred embodiment, the hemihydrate crystalline form is characterized by at least one of (a) an X-ray diffraction peak at a 2-theta diffraction angle of 5.0° in combination with one or more peaks at 16.4, 17.8 and 26.6° ; with a tolerance for diffraction angles of 0.2°; and (b) a solid phase ¹³ C NMR spectrum, which includes peaks assigned to the high field resonance of adamantane (δ=29.5 ppm) at 23.5, 34.6, 110.2, 124.1, 126 .8 and 166.2 ppm (±0.2 ppm respectively).

In another preferred embodiment, the hemihydrate crystalline form is further characterized by a ¹³ C solid phase NMR spectrum that includes one or more peaks assigned to the high field resonance of adamantane (δ=29.5 ppm) at 16.5, 18.9, 19.6, 20.4, 22.6, 24.4, 26.0, 27.4, 28.3, 30.0, 30.9, 38.4, 40.5, 110.8, 130, 4, 131.2, 146.0, 147.1, 150.4, 151.2, 153.4, 157.1, 157.7, 158.3, 159.6, 163.1 and 169.7 m .d. (±0.2 ppm respectively).

In another preferred embodiment, the hemihydrate crystalline form is characterized by a ¹³ C solid phase NMR spectrum that includes peaks assigned to the adamantane upfield resonance (δ=29.5 ppm) at 16.5, 18.9, 19.6, 20.4, 22.6, 23.5, 24.4, 26.0, 27.4, 28.3, 30.0, 30.9, 34.6, 38.4, 40.5, 110, 2, 110.8, 124.1, 126.8, 130.4, 131.2, 146.0, 147.1, 150.4, 151.2, 153.4, 157.1, 157.7, 158.3, 159.6, 163.1, 166.2 and 169.7 ppm. (±0.2 ppm respectively).

In another preferred embodiment, the hemihydrate crystalline form is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free from impurities. In another preferred embodiment, the hemihydrate crystalline form is at least 95% free

- 3 045742 from impurities. In another preferred embodiment, the hemihydrate crystalline form is at least 96% free from impurities. In another preferred embodiment, the hemihydrate crystalline form is at least 97% free from impurities. In another preferred embodiment, the hemihydrate crystalline form is at least 98% free from impurities. In another preferred embodiment, the hemihydrate crystalline form is at least 99% free from impurities.

In another preferred embodiment, the present invention provides a composition containing at least 100, 200, 300, 400, 500 or 1000 g of the hemihydrate crystalline form. In another preferred embodiment, the present invention provides a composition containing at least 100 g of the hemihydrate crystalline form.

In another preferred embodiment, the composition is a granular composition. In another preferred embodiment, the granular composition is a wet granular composition. In another preferred embodiment, the granular composition is a dry granular composition.

In another preferred embodiment, the present invention provides a production container containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 kg hemihydrate crystalline form. In another preferred embodiment, the present invention provides a production container containing at least 1 kg of the hemihydrate crystalline form.

In another preferred embodiment, the present invention provides a pharmaceutical composition comprising a hemihydrate crystalline form and one or more pharmaceutically acceptable carriers, diluents or excipients.

Treatment methods.

The present invention provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of a crystalline form of the compound 5-[5-[2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidine-2,4-dione.

In another preferred embodiment, the cancer is bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, colon cancer, stomach cancer, gallbladder cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, lymphoma, medulloblastoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or kidney cancer. In one embodiment, the breast cancer is triple negative breast cancer. In another embodiment, the lung cancer is non-small cell lung cancer.

The present invention also provides a crystalline form of the compound 5-[5-[2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione for use in therapy.

The present invention also provides a crystalline form of the compound 5-[5-[2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione for use in the treatment of cancer. In a preferred embodiment, the crystalline form is that of the compound 5-[5-[(1S,2S)-2-ethylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione.

The present invention also provides a crystalline form of the compound 5-[5-[2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione in the production of drugs for the treatment of cancer.

Subjects for whom CD73 inhibitor treatment is effective include subjects with tumors resistant or refractory to anti-PD1/PDL-1 inhibitors, such as non-small cell lung cancer, bladder cancer and melanoma; subjects with EGFR/BRAF/Kras mutation cancers such as non-small cell lung cancer, bladder cancer, melanoma, colon and pancreatic cancer; subjects with estrogen receptor negative (-) cancer, such as triple negative breast cancer; subjects with high levels of CD73 expression, such as pancreatic cancer and colorectal cancer. If necessary, such a subject may be selected for therapy with a compound described herein, or a pharmaceutically acceptable salt thereof, based on the presence of high levels of CD73 expression in their tumors, as determined by immunohistochemical (IHC) analysis; or based on the presence of EGFR and BRAF mutations in their tumors detected in RT-PCR analysis; or based on loss of estrogen receptor in their tumors, as determined by IHC or RT-PCR analysis; or based on the presence of high levels of adenosine and AMP in their tumors or plasma, as determined by LC-MS analysis. For pharmacodynamic evaluation, the ex vivo LC-MS assay described herein can be used to measure the effect of a CD73 inhibitor on the conversion of AMP to adenosine in the blood.

In a preferred embodiment, the patient is a patient whose serum CD73 activity has been determined. In one preferred embodiment, determining CD73 activity means determining whether CD73 activity is present. Methods for determining the level of CD73 expression or CD73 activity are known to those skilled in the art, for example, see S Morello et al., J. Trans. Med., 2017, 15:244. In another preferred embodiment, determining CD73 activity means quantifying the degree of conversion of AMP to adenosine by CD73, and

- 4 045742 This document proposes an LC-MS based assay to facilitate the quantification of CD73 activity levels.

In another preferred embodiment, the patient is a patient whose tissue expression of CD73 is determined. In another embodiment, the tissue is tumor tissue. Methods for determining the level of CD73 expression in tissue are known to those skilled in the art, for example, using Western blotting or immunohistochemistry (X-RWu et al., J. Surg. Oncol., 2012, 106:130-137).

In one embodiment, the present invention provides a method of treating cancer comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or sequential combination with one or more antineoplastic agents. Non-limiting examples of antineoplastic agents include ramucirumab, necitumumab, olaratumab, gemcitabine, pemetrexed, galunisertib, abemaciclib, gefitinib, vemurafenib, dabrafenib, trametinib, cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxora bicine, navelbine, eribulin, paclitaxel, bound particles with paclitaxel protein for injection suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (leucovorin, fluorouracil and irinotecan), cetuximab and EGFR inhibitor, Raf inhibitor, B-Raf inhibitor, ERK inhibitor, CDK4/6 inhibitor, indoleamine 2,3-dioxygenase inhibitor, TGFe inhibitor and TGFe receptor inhibitor.

In another embodiment, the present invention provides a method of treating cancer comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or sequential combination with one or more immuno-oncology agents. In one preferred embodiment, the immuno-oncology agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD137 agonist antibody, or an anti-CTLA4 antibody. Non-limiting examples of immuno-oncology agents include nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, durvalumab and the anti-PD-L1 antibody LY3300054 (the heavy and light chain sequences of which are described in WO 2017/034916 and US 2017/005803 3 as SEQ ID NO: 10 and 11 respectively). In one preferred embodiment, the immuno-oncology agent is an anti-PD-1 antibody. In another preferred embodiment, the immuno-oncology agent is an anti-PD-L1 antibody. In another preferred embodiment, the compound of the invention is 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione and the immuno-oncology agent is LY3300054.

In one embodiment, the present invention provides a method of treating non-small cell lung cancer, comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate, or sequential combination with another agent. In one preferred embodiment, the other agent is osimertinib, cetuximab, or abemaciclib. In another preferred embodiment, the other agent is osimertinib. In another preferred embodiment, the other agent is cetuximab. In another preferred embodiment, the other agent is abemaciclib.

In another preferred embodiment, the present invention provides a method of treating melanoma, comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or sequential combination with another agent. In one preferred embodiment, the other agent is a BRAF inhibitor, an anti-PD-1 antibody, or an anti-PD-L1 antibody. In another preferred embodiment, the other agent is a BRAF inhibitor. In another preferred embodiment, the other agent is an anti-PD-1 antibody. In another preferred embodiment, the other agent is an anti-PD-L1 antibody. In another preferred embodiment, the anti-PD-L1 antibody is LY3300054 (the heavy and light chain sequences of which are provided in WO 2017/034916 and US 2017/0058033 as SEQ ID NO: 10 and 11, respectively).

In another preferred embodiment, the present invention provides a method of treating colorectal cancer comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or sequential combination with another agent. In one preferred embodiment, the other agent is abemaciclib. In another preferred embodiment, the compound of the invention is 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]N-pyrimidin-2,4-dione and the other agent is abemaciclib .

In another preferred embodiment, the present invention provides a method of treating pancreatic cancer comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or sequential combination with another agent. In one preferred embodiment, the other agent is abemaciclib. In another preferred embodiment, the compound of the invention is 5-[5-[(1S,2R)-2-isonropylcyclopropyl]-6-methylpyridazin3-yl]-N-pyrimidin-2,4-dione and the other agent is abemaciclib.

In another preferred embodiment, the present invention provides a method of treating triple-negative breast cancer, comprising administering an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, in simultaneous, separate, or sequential combination with another agent. In one preferred embodiment, the compound of the invention is 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione.

It should be understood that the following terms mentioned above and throughout the specification of this invention, unless otherwise specified, have the following meanings.

A pharmaceutically acceptable carrier, diluent or excipient is a medium conventional in the art for delivering biologically active agents to mammals, such as humans.

Terms treatment, treat, treating, etc. include slowing or reversing the progression of the disorder. These terms also include alleviation, amelioration, reduction, elimination or attenuation of one or more symptoms of a disorder or condition, even if the disorder or condition is not actually eliminated and even if the progression of the disorder or condition is not itself slowed or reversed.

An effective amount means that amount of a compound or a pharmaceutically acceptable salt thereof of this invention or a pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof of this invention that produces a biological or medical response or desired therapeutic effect in a patient as observed by a treating clinician. In one embodiment, the present compound or a pharmaceutically acceptable salt thereof inhibits the conversion of AMP to adenosine in an in vitro or ex vivo CD73 enzyme assay. In another embodiment, the present compound or a pharmaceutically acceptable salt thereof inhibits the conversion of AMP to adenosine in whole blood of mice obtained from animals administered various doses of the compound.

In this context, the term patient refers to a person.

An effective amount can be readily determined by a physician skilled in the art using known techniques and by observing results obtained under similar circumstances. When determining the amount effective for a patient, the observing diagnostician considers many factors, including, but not limited to: the patient's biological species; its size, age and general health; the specific disease or disorder involved; degree of involvement or severity of disease or disorder; individual patient response; the specific compound administered; method of administration; characteristics of the bioavailability of the administered drug; chosen treatment regimen; use of concomitant medications; and other relevant circumstances.

The compounds of this invention are preferably formulated into pharmaceutical compositions that are administered by any route that provides bioavailability of the compound, including oral, intravenous and transdermal routes. Most preferably, said compositions are for oral administration. Such pharmaceutical compositions and methods for their preparation are well known in the art. (See, for example, Remington: The Science and Practice of Pharmacy (D.B. Troy, ed., 21st edition, Lippincott, Williams & Wilkins, 2006)).

Those skilled in the art will appreciate that the compounds of this invention may form salts. The compounds of this invention contain basic heterocycles and accordingly react with any of a variety of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Such pharmaceutically acceptable acid addition salts and general procedures for their preparation are known in the art. See, for example, P. Stahl et al., Handbook of pharmaceutical salts: properties, selection and use (VCHA/Wiley-VCH, 2008).

As used herein, the term pharmaceutically acceptable salts or pharmaceutically acceptable salt refers to the relatively non-toxic inorganic and organic salts and salts of the compounds of this invention (S. M. Berge et al., Pharmaceutical Salts, Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January, 1977).

As used herein, the term granular composition refers to a composition in granular form that, in the pharmaceutical manufacturing process, is a precursor composition to a pharmaceutical composition.

As used herein, the term production container refers to containers that are used in the production of pharmaceutical containers, but not in a medicinal chemistry laboratory. Examples of production containers include, but are not limited to, hopper, pallet, drying tray,

- 6 045742 tray for granulator, drying tray, ladle for granulator and mixing bowl.

The compounds of formula I can be prepared by synthetic methods well known and recognized in the art. Suitable reaction conditions for the various steps of these reactions are known in the art, and appropriate substitutions of solvents and co-reagents are well known in the art. It will also be appreciated by those skilled in the art that synthetic intermediates can be isolated and/or purified by various known techniques as needed or desired, and that the various intermediates can often be used directly in subsequent synthetic steps with little or no purification. In addition, those skilled in the art will understand that in some cases the order in which the fragments are introduced is not critical. The specific order of steps required to prepare the compounds of this invention depends on the particular compound being synthesized, the starting compound, and the relative mobility of the substituted moieties, as is well known to skilled chemists. All substituents, unless otherwise indicated, are as described above, and all reagents are well known and generally accepted in the art.

As used herein, the following terms have the following specified meanings: ACN refers to acetonitrile; DAST refers to diethylaminosulfur trifluoride; DCM refers to dichloromethane; DMAP refers to 4-dimethylaminopyridine; DMSO or DMSO refers to dimethyl sulfoxide; e.i. refers to enantiomeric excess; ER/MS refers to electrospray ionization mass spectrometry; EtOAc refers to ethyl acetate; Et2O refers to diethyl ether; FBS refers to fetal bovine serum; GC-MS refers to gas chromatography-mass spectrometry; HBSS refers to Hanks' balanced salt solution; IC50 refers to the half-maximal inhibitory concentration; LAH refers to lithium aluminum hydride; LC-ES/MS refers to liquid chromatography-mass spectrometry with electrospray ionization; MS refers to mass spectrometry; MeOH refers to methanol; MTBE refers to methyl tert-butyl ether; nBuLi refers to n-butyllithium; nm refers to nanometer or nanometers; NMR refers to nuclear magnetic resonance; OAc refers to acetate; psi inch refers to pounds per square inch; room t-ra refers to room temperature or ambient temperature; MSD refers to strong cation exchange; SLC refers to supercritical liquid chromatography; SNAr refers to nucleophilic aromatic substitution; TEA refers to triethylamine; THF refers to tetrahydrofuran; t _R refers to retention time; and w/w refers to the mass/mass ratios in solution.

The compounds of formula I can be synthesized as shown in the following schemes.

Scheme 1

Scheme 1 shows the preparation of compounds of formula I. Using well known Suzuki reaction conditions, commercially available (2,4-dimethoxypyrimidin-5-yl)boronic acid 1 can be condensed with 4,6-dichloro-3-methylpyridazine. The Suzuki coupling of the chlorine moiety of compound 3 with an appropriately substituted trans-cyclopropylboronate can be performed to give compound 4. Those skilled in the art will appreciate that the enantiomers of compound 4 can be separated using chiral resolution techniques known in the art. Compounds of formula I can be prepared by deprotecting the methoxy groups in compound 4 under a number of demethylation conditions described in detail in the prior art.

6 7

Formula II

Scheme 2

- 7 045742

Scheme 2 shows the essential cyclopropylboronic esters required to prepare the compounds of formula II. Those skilled in the art will appreciate that hydroboration of an appropriately substituted alkyne 5 can be carried out under a number of conditions, particularly in the presence of a transition metal catalyst such as Cu or Zr, to yield alkenyl boronate 6. The alkenyl boronate can be cyclopropanated under the preparation conditions stabilized carbenoids known in the art, for example, the Simmons-Smith cyclopropanation, the Corey-Tchaikovsky reaction and the cyclopropanation method with diazomethane (or diazo compounds), both with and without transition metal catalysts (for example, Cu, Pd or Ni) them (e.g., thermally or photochemically), to obtain the appropriately substituted trans-cyclopropylboronate 7. Those skilled in the art will understand that the thermodynamically favorable cyclopropanation product is a mixture of trans-enantiomers in compound 7.

Scheme 3

Scheme 3 shows the chiral synthesis of compounds of formula III. Diazotization of the appropriately substituted amino acid 8 under modified Sandmeyer conditions (radical SNAr), resulting in the formation of compound 9, is known in the art. Subsequent reduction to alcohol 10 can be accomplished using a number of reducing agents known in the art, including aluminum hydrides and diborane as reducing agents. Cyclization to chiral epoxide 11 under alkaline conditions is described in detail in the prior art. The stereoselective Horner-Wadsworth-Emmons reaction can be used by treating the chiral epoxide 11 with a suitable phosphonate ester (e.g. ethyl 2-diethoxyphosphorylacetate or triethylphosphonoacetate) and a suitable base (e.g. alkyllithium, metal alkoxides or metal hydrides) to obtain the trans-cyclopropane derivative 12 (See, for example, L. Delhaye, A. Merschaert, P. Delbeke, W. Brione, Org. Proc. Res. & Dev., 2007, 11, 689-692). Hydrolysis of compound 12 to the corresponding acid 13 can be accomplished under a wide range of basic conditions, which are described in detail in the art. Subsequent coupling of acid 13 with an appropriately substituted N-hydroxyphthalimide is described in detail in the art using a suitable acid activating agent, for example carbonyldiimidazole, in the presence of a weak non-nucleophilic base, to obtain compound 14. Decarboxylation borylation of N-hydroxyphthalimide ester 14 can be carried out in various conditions known in the art, including in the presence of a transition metal catalyst (for example, the Suzuki-Miyaura reaction), under photolytic conditions, or through single electron transfer reactions, including, for example, the formation of an N-hydroxyphthalimide complex ester with diboron by a radical condensation reaction accelerated by a pyridine-boron radical to give trans-cyclopropaneboronate 15 (see, for example, W.-M. Cheng, S. Rui, B. Zhao, W.-L. Xing, Y Fu., Org. Lett., 2017, 19, 4291-4294). Coupling of boronate 15 with aryl chloride 4 and subsequent demethylation can be carried out in the same manner as described in Scheme 1 to produce the types of chiral compounds of formula III.

Methods of obtaining and examples

The following preparation methods and examples further illustrate the present invention and represent typical methods for synthesizing the compounds of this invention, but should in no way be construed as limiting the scope of the present invention. Reagents and starting materials without

- 8 045742 works are available or can be easily synthesized by specialists in the art. It should be understood that the following preparation methods and examples are presented for purposes of illustration and not limitation, and that various modifications may be made by those skilled in the art.

LC-ES/MS was performed on an Agilent HP1100 liquid chromatography system. Electrospray mass spectrometry measurements (recorded in positive and/or negative mode) were performed on a quadrupole mass spectrometer with a mass selective detector connected to a PH 1100 HPLC system.

LC-MS conditions (low pH): column: PHENOMENEX® GEMINI® NX C-18 2.1x50 mm, 3.0 µm; gradient: 5-100% B over 3 min, then 100% B over 0.75 min, column temperature: 50±10°C; flow rate: 1.2 ml/min; solvent A: deionized water with 0.1% HCOOH, solvent B: ACN with 0.1% formic acid; wavelength 214 nm. Alternative LC-MS conditions (high pH): Column: WATERSTM XTERRA® MS C-18 columns, 2.1x50 mm, 3.5 µm; gradient: 5% solvent A in 0.25 min, gradient from 5 to 100% solvent B in 3 min and 100% solvent B in 0.5 min, or from 10 to 100% solvent B in 3 min and at 100% solvent B for 0.75 min; column temperature: 50±10°C; flow rate: 1.2 ml/min; solvent A: 10 mM NH4HCO3 pH 9; solvent B: ACN; wavelength: 214 nm.

Preparative reverse phase chromatography was performed on an Agilent 1200 LC-ER/MS system equipped with a mass spectrometer with a mass selective detector and a Leap autosampler. High pH methods were performed on a 75x30 mm PHENOMENEX® GEMINI®NX 5 µm particle size column with 10x20 mm guard. Flow rate 85 ml/min. The eluent used was 10 mM ammonium bicarbonate (pH 10) in ACN.

NMR spectra were recorded on a Bruker AVIII HD NMR spectrometer at 400 MHz, samples were prepared as solutions in CDCl ₃ or (CD ₃ ) ₂ SO, the results were recorded in ppm using the residual solvent [CDCl ₃ , 7.26 m. d.; (CD ₃ ) ₂ SO, 2.50 ppm] as the reference standard. When recording multiplicity of peaks, the following abbreviations can be used: s (singlet), d (doublet), t (triplet), k (quartet), m (multiplet), shs (wide singlet), dd (doublet of doublets), dt (doublet triplets). Spin-spin coupling constants (J), when indicated, are written in hertz (Hz).

Preparation method 1. 4,4,5,5-Tetramethyl-2-[(E)-3-methylbut-1-enyl]-1,3,2-dioxaborolane.

Pinacolborane (9.5 mL, 64 mmol) was added dropwise to ice-cold 3-methylbut-1yne (4.91 g, 72.0 mmol) over 5 min. Cap the pressure vessel, warm to room temperature, and stir for 18 hours. Add bis(cyclopentadienyl)zirconium(IV) chloride hydride (2.0 g, 7.4 mmol) and TEA (1.1 mL, 7.9 mmol). The pressure vessel was sealed, placed in an oil bath at 60°C and stirred for 10 minutes. Cool the resulting red solution to room temperature over 2.5 hours. The reaction mixture was diluted with DCM (200 ml), washed with saturated aqueous NaHCO ₃ (100 ml), saturated aqueous NaCl (50 ml), dried over MgSO ₄ , filtered through layer of silica gel (150 ml), washed silica gel with DCM (700 ml) and concentrated in vacuo to give the title compound (11.5 g, 82%).

ER/MS (m/z): 196 (M+H).

^1H NMR ( _CDCl3 ) δ 1.03 (d, J= 6.7 Hz, 6H), 1.29 (s, 12H), 2.37 (m, 1H), 5.40 (dd, 1H) , 6.64 (dd, 1H).

Preparation method 2. Rac-trans-2-[2-isopropylcyclopropyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane.

'v/o

N-nitroso-N-methylurea was added piecemeal to an ice-cold two-phase mixture of Et ₂ O (70 ml) and aqueous KOH (30.5 g, 435 mmol, 70 ml H ₂ O). Stir until solid dissolves (<5 min). The resulting diazomethane solution was pipetted into a rapidly stirred ice-cold suspension of Pd(OAc) ₂ (237 mg, 1.05 mmol) and 4,4,5,5-tetramethyl-2-[(E)-3-methylbut-1-enyl] -1,3,2-dioxaborolane (4.00 g, 20.4 mmol) in Et ₂ O (70 ml). After addition was complete, warm the reaction mixture to room temperature, filter through diatomaceous earth, and concentrate the filtrate in vacuo. Dissolve the resulting residue in DCM, filter through a pad of silica gel (25 g) and concentrate in vacuo to give the title compound (4.32 g, >99%).

ER/MS (m/z): 210 (M+H).

^1H NMR ( _CDCl3 ) δ -0.35 (m, 1H), 0.45 (m, 1H), 0.65 (m, 1H), 0.78 (m, 1H), 0.98 (m , 7H), 1.23 (s, 12H).

Method of preparation 3. 4-Chloro-6-(2,4-dimethoxypyrimidin-5-yl)-3-methylpyridazine.

- 9 045742\

О Cl N=<

/ N—N=N

Mix 2,4-dimethoxy-5-pyrimidinylboronic acid (6.75 g, 36.7 mmol), 4,6-dichloro-3-methylpyridazine (5.98 g, 36.7 mmol) in a pressure flask. [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (0.55 g, 0.73 mmol), Cs ₂ CO ₃ (29.9 g, 91.8 mmol) in 4:1 mixture 1, 4-dioxane/H ₂ O (151 ml). The air was pumped out and N2 was pumped in. Cap the container and heat at 70°C for 3 hours. Filter the residue through diatomaceous earth and wash with EtOAc. The organic mixture was washed with water, then with a saturated aqueous NaCl solution, dried over MgSO ₄ and evaporated to dryness. Purify the resulting black residue by chromatography on silica gel using a 330 g REDISEP® column with a 0-30% DCM/(33% MeOH in DCM) gradient over 15 min at a flow rate of 200 ml/min. to obtain the title compound (6.2 g, 63%) after evaporation of the chromatographic fractions.

ER/MS (m/z) ( ³⁵ Cl/ ³⁷ Cl) 267/269 [M+1]+.

H NMR (d ₆ -DMSO δ 2.74 (s, 3H), 4.00 (s, 3H), 4.03 (s, 3H), 8.20 (s, 1H), 8.87 (s, 1H).

Alternative procedure for production method 3.

A nitrogen stream was passed through a mixture of (2,4-dimethoxypyrimidin-5-yl)boronic acid (85 g, 439 mmol), 4,6-dichloro-3-methylpyridazine (75 g, 437 mmol) and Cs ₂ CO ₃ (358 g , 1099 mmol) in 1,4-dioxane (1175 ml) and H2O (340 ml) for 5 min. Add [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (6.6 g, 8.7 mmol) and stir the resulting mixture at 75°C for 16 hours. Cool the reaction mixture to room temperature, filter the mixture through diatomaceous earth and washed the precipitate on a filter with EtOAc. The resulting layers were separated and the organic phase was washed twice with a saturated aqueous NaCl solution, dried over MgSO4, filtered and concentrated in vacuo. Water (500 ml) was added to the resulting residue, stirred for 16 hours at room temperature, and the resulting solid was filtered. Wash the collected solid with H ₂ O and dry under vacuum for 16 hours to obtain the desired compound (70 g, 54%) as a brown solid.

ER/MS (m/z) (35Cl/37Cl) 267/269 [M+1] ⁺ .

Preparation method 4. trans-5-[2-Isopropylcyclopropyl]-6-methylpyridazin-3-yl]-2,4-dimethoxypyrimidine.

/ N—N=N

Combine 4-chloro-6-(2,4-dimethoxypyrimidin-5-yl)-3-methylpyridazine (1.97 g, 7.39 mmol), ractrans-2-[2-isopropylcyclopropyl]-4,4,5, 5-tetramethyl-1,3,2-dioxaborolane (3.32 g, 15.8 mmol), bis(ditert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium (II) (1.35 g, 1.85 mmol) , 1,4-dioxane (37 ml) and 1 M aqueous Na2CO3 solution (18 ml, 18 mmol). Purge the reaction vessel with N2 and heat to 90°C for 18 hours. Cool to room temperature, dilute with EtOAc (150 ml) and separate the layers. The organic layer was washed successively with a 1 M aqueous solution of Na ₂ CO ₃ , a saturated aqueous solution of NaCl, dried over MgSO ₄ , filtered, and the filtrate was concentrated in vacuo. The resulting residue was purified by chromatography on silica, eluting with a gradient of 60-100% EtOAc/DCM, to obtain the title compound as an essentially racemic mixture of isomers (1.48 g, 64%) after evaporation of the chromatographic fractions.

The isomers were purified by SLC (column: PHENOMENEX® LUX® Cellulose-4, 4.6x150 mm; isocratic elution 40% MeOH/CO ₂ ; flow rate: 5 ml/min, UV 250 nm) to obtain 647 mg of isomer 1: t _R =2.56 min and 647 mg of isomer 2: t _R =3.75 min.

ER/MS (m/z): 315 (M+H).

Preparation method 5. (2S)-2-Chloro-3-methylbutanoic acid.

I ⁰ ci

Cool a solution of L-valine (286 g, 2.44 mol) and 5 M aqueous HCl solution (3.25 L, 16.3 mol) at 0°C. 4 M aqueous NaNO2 solution (1 L, 4 mol) was added dropwise over 2 h, maintaining the internal temperature below 5°C. The reaction mixture was stirred for 2 hours, warming to room temperature, and stirred for another 16 hours at room temperature. Na ₂ CO ₃ (242 g, 2.28 mol) was added piecemeal over 30 minutes. The resulting solution was extracted with MTBE (3x1000 ml), the combined organic extracts were washed with saturated aqueous NaCl (500 ml), the organic extracts were dried over MgSO ₄ and concentrated in vacuo. The resulting residue was purified by vacuum distillation (15 mbar (1.5 kPa)/140°C) to give the title compound (248 g, 68%) as an oil

- 10 045742 total substance.

¹ H NMR (400 MHz, CDCl·,) δ 1.1 (dt, J=6.6, 2.6 Hz, 6H), 2.34-2.42 (m, 1H), 4.19-4 .23 (m, 1H), 10.0-12.0 (shs, 1H).

Preparation method 6. (2S)-2-Chloro-3-methylbutan-1-ol.

Cool a solution of (2S)-2-chloro-3-methylbutanoic acid (137 g, 1 mol) in 2-methyltetrahydrofuran (500 ml) to 0°C. A 2.3 M solution of LAH in methyltetrahydrofuran (480 mL, 1.1 mol) was added dropwise over 2.5 h while maintaining the internal temperature below 10°C. The mixture was heated to room temperature and stirred for 1 hour at room temperature and 1 hour at 50°C. Cool the reaction mixture to 0°C and sequentially and slowly add H ₂ O (1.48 ml), 15% aqueous NaOH (1.48 ml) and H2O (4.46 ml). Allow the mixture to warm to room temperature, filter through a pad of diatomaceous earth and evaporate the solvent in vacuo to give the title compound (110 g, 81%) as a colorless oil.

1H NMR (400 MHz, CDCl3) δ 0.97-1.1 (m, 6H), 1.94-2.18 (m, 1H), 2.60-3.09 (brs, 1H), 3, 71-3.78 (m, 1H), 3.80-3.85 (m, 1H), 3.90-3.96 (m, 1H).

Preparation method 7. (2R)-2-Isopropyloxirane.

Cool a solution of KOH (195 g, 3.48 mol) in H2O (195 ml) to 0°C and add undiluted (2S)-2-chloro-3-methylbutan-1-ol (110 g, 801 mmol) over 20 min ), maintaining the internal temperature below 5°C. The reaction mixture was allowed to warm to room temperature. The reaction mixture was purified by vacuum distillation at 100 mbar (10 kPa), heating from 23 to 50°C, to obtain the title compound (47 g, 64%) as a colorless oil.

¹ H NMR (400 MHz, CDCl3) δ 0.98 (d, J=6.9 Hz, 3H), 1.05 (d, J=6.9 Hz, 3H), 1.51 (o, J= 6.9 Hz, 1H), 2.52-2.54 (m, 1H), 2.70-2.75 (m, 2H).

Preparation method 8. Ethyl (1S,2R)-2-isopropylcyclopropanecarboxylate.

A 2.5 M solution of nBuLi in hexanes (310 ml, 780 mmol) was added dropwise over 25 min to a solution of ethyl 2-diethoxyphosphorylacetate (153 ml, 772 mmol) in 1,4-dioxane (870 ml), cooled in ice sauna (internal temperature: 8°C). Heated to room temperature and stirred for 40 minutes. The solution was transferred via cannula into a 3 L pressure flask and (2R)-2-isopropyloxirane (70 g, 772 mmol) in 1,4-dioxane (180 ml) was added. The reaction mixture was stirred at 150°C at 50 psi. inch (345 kPa) for 14 hours. Cool the reaction mixture to room temperature and add H ₂ O (700 ml). The layers were separated and the aqueous phase was re-extracted with MTBE (2x500 ml). Combine the organic phases, wash with saturated aqueous NaCl (2x350 ml), dry over MgSO ₄ and concentrate in vacuo to give the crude title compound (107.7 g, >99%) as a yellow oil, suitable for further use without additional cleaning.

GC-MS (m/z): 156 (M+).

Preparation method 9. (1S,2R)-2-Isopropylcyclopropanecarboxylic acid.

I HE

Stir a mixture of ethyl (1S,2R)-2-isopropylcyclopropanecarboxylate (107.7 g, 482.6 mmol) in 1,4-dioxane (800 ml) containing 25% aqueous NaOH (800 ml) at 100°C for 7 hours. Cool the mixture to room temperature, add water (300 ml), extract with MTBE (2x500 ml) and remove the organic phase. The aqueous phase was acidified with 37% aqueous HCl solution (approximately 500 ml) to pH ~ 1-2.

The acidified aqueous mixture was extracted with MTBE (2x600 ml), the organic layer was washed with saturated aqueous NaCl, dried over MgSO4 and concentrated in vacuo to give the title compound (54.2 g, 75%) as an amber oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 0.81-0.86 (m, 1H), 1.01 (dd, J=5.9, 3.7 Hz, 6H), 1.04-1, 13 (m, 1H), 1.20-1.24 (m, 1H), 1.28-1.37 (m, 1H), 1.39-1.44 (m, 1H).

Preparation method 10. (1,3-Dioxoisoindolin-2-yl)-(1S,2R)-2-isopropylcyclopropanecarboxylate.

- 11 045742

A suspension of (1S,2R)-2-isopropylcyclopropanecarboxylic acid (54.2 g, 359 mmol), 2-hydroxyisoindoline-1,3-dione (59.8 g, 359 mmol) and DMAP (4.44 g, 35 mmol) was stirred. 9 mmol) in DCM (690 ml) at 0°C. N,N'-diisopropylcarbodiimide (50.4 g, 395 mmol) was added dropwise, the mixture was warmed to room temperature, and the resulting reaction mixture was stirred for 2 hours at room temperature. H ₂ O (600 ml) was added and the phases were separated. The aqueous phase was extracted with DCM (2x300 ml), the organic phases were combined, dried over MgSO ₄ , filtered and evaporated. The resulting solid was purified by chromatography on silica gel, eluting with 100% DCM, to give the title compound (96 g, 88%) as a pale yellow solid after evaporation of the chromatography fractions.

ER/MS (m/z): 274 (M+1).

Preparation method 11. 2-[(1S,2S)-2-Isopropylcyclopropyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. y+y.

Pass a stream of nitrogen through a solution of (1,3-dioxoisoindolin-2-yl)-(1S,2S)-2isopropylcyclopropanecarboxylate (96 g, 316 mmol) and bis(pinacolato)diboron (160 g, 630 mmol) in EtOAc (480 ml) within 15 min. Stir the mixture at 85°C and add ethyl isonicotinate (24.38 g, 157 mmol) dropwise over 10 minutes. The resulting mixture was stirred at 85°C for 16 hours. The resulting suspension was cooled, filtered and the solid was removed, and the brown filtrate was evaporated under reduced pressure. The crude residue was filtered through a pad of silica gel, eluting with 2% EtOAc/hexanes. The solvent was removed from the filtrate and the resulting residue was purified again by chromatography on silica gel, eluting with 3% EtOAc/hexanes, to give the title compound (25.3 g, 38%) as a colorless oil after removal of the solvent from the chromatography fractions.

1H NMR (400 MHz, CDCl ₃ ) δ -0.33-(-0.38) (m, 1H), 0.42-0.47 (m, 1H), 0.63-0.67 (m, 1H), 0.75-0.82 (m, 1H), 0.89-1.02 (m, 1H), 0.98 (m, 6H), 1.24 (s, 12H).

Preparation method 12. 5-[5-[(1S,2R)-2-Isopropylcyclopropyl]-6-methylpyridazin-3-yl]-2,4-dimethoxypyrimidine.

Mixed 4-chloro-6-(2,4-dimethoxypyrimidin-5-yl)-3-methylpyridazine (19.1 g, 70.6 mmol), K3PO4 (45.9 g, 212 mmol), 1,4-dioxane (300 ml) and H ₂ O (75 ml) and degassed the mixture with N ₂ for 10 minutes. [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (7.98 g, 10.6 mmol) was added. Nitrogen gas was passed through for another 2 min and 2-[(1S,2S)-2-isopropylcyclopropyl]4,4,5,5-tetramethyl-1,3,2-dioxaborolane (22.2 g, 106 mmol) was added in one portion. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was cooled to room temperature, H2O was added and extracted with EtOAc. The organic layer was separated, dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The resulting residue was purified by chromatography on silica, eluting with 85% hexanes/EtOAc to isolate the title compound (21.5 g, 92%) as an amber oil upon removal of the solvent from the chromatography fraction. The oily substance solidified upon standing at room temperature to an off-white solid.

ER/MC (m/z): 315 (M+1).

Example. 5-[5-[(1S,2R)-2-Isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione.

Trans-5-[5-[2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-2,4-dimethoxypyrimidine isomer 1 (628 mg, 2.00 mmol) was dissolved in MeOH (3 ml). Add a 1 M aqueous solution of HCl (5 ml) and heat to 70°C for 3 hours. Cool to room temperature, load the reaction mixture onto an SKO column washed with MeOH (20 g, Silicycle SILIABOND-p-toluenesulfonic acid), wash with SKO MeOH column (140 ml) and eluted the desired product using 2 M NH ₃ /MeOH (140 ml).

- 12 045742

Concentrate the NH3/MeOH fractions to give the title compound as a cream solid (546 mg, 95%).

ER/MS (m/z): 287 (M+H).

1H NMR ( _d6 -DMSO) δ 0.99 (m, 9H), 1.24 (m, 1H), 1.81 (m, 1H), 2.72 (s, 3H), 7.67 (s , 1H), 8.23 (s, 1H), 11.43 (shs, 2H).

X-ray powder diffraction of crystalline forms.

The free base compound was dissolved in methanol, stirred at 50°C for 1 hour and allowed to cool to room temperature while it crystallized from solution. The solid was then isolated by vacuum filtration and briefly dried under vacuum at 70°C.

RPD diagrams of crystalline substances were recorded on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKa radiation source, λ=1.54060 A) and a Vantec sensor operating at 35 kV and 50 mA. The sample was scanned from 4 to 40° 2Θ with a step of 0.008° 2Θ and a scanning speed of 0.5 s/step and with a divergence of 0.6 mm, a fixed anti-scatter slit of 5.28 mm and a detector slit of 9.5 mm. The dry powder was placed on a quartz sample holder and a glass slide was used to ensure a smooth surface. X-ray diffraction patterns of the crystalline form were recorded at ambient temperature and relative humidity. It is well known in the field of crystallography that for any given crystal form, the relative intensities of the diffraction peaks can vary due to preferred orientation caused by factors such as morphology and crystal structure. In the presence of a preferred orientation effect, the peak intensities are variable, but the positions of the polymorph's characteristic peaks do not change. See, for example, USP No. 23, National Formulary No. 18, p. 1843-1844, 1995. Additionally, it is also well known in the field of crystallography that for any given crystal shape the angular positions of the peaks may vary slightly. For example, peak positions shift due to changes in the temperature or humidity at which the sample is analyzed, sample drift, or the presence or absence of an internal standard. In this case, the peak position variability of ±0.2 2Θ accounts for these potential changes without interfering with the unambiguous identification of the indicated crystalline form. Confirmation of the crystal form can be made based on any unique combination of distinctive peaks (in units of °2Θ), typically the more prominent peaks. Crystal form diffraction patterns recorded at ambient temperature and relative humidity were corrected to NIST 675 standard peaks at 8.853 and 26.774° 2Θ.

Solid phase ¹³ C NMR ( ¹³ C ssNMR).

¹³ C Cross-polarization/magic angle spinning NMR spectra (solid-state NMR or ssNMR) obtained using a Bruker Avance II 400 MHz NMR spectrometer operating at a carbon frequency of 100.622 MHz and a proton frequency of 400.131 MHz and equipped with a 4 mm dual resonant probe Bruker. TOSS sideband suppression is used in conjunction with cross polarization using SPINAL64 decoupling and an H-core CP pulse in the form of RAMP 100. Data acquisition parameters are as follows: 90° proton RF pulse width 2.6 μs, contact time 2.0 ms, pulse repetition time 3 s, MAS frequency 10 kHz, spectral width 30 kHz, acquisition time 34 ms and number of scans 18661. Chemical shifts were correlated with adamantane (δ=29.5 ppm) in a separate experiment.

Method of obtaining 13.

Crystalline Form 1: 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione (anhydrous).

A suspension of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-2,4dimethoxypyrimidine (21.5 g, 65.6 mmol) and 1 M aqueous HCl (165 ml, 135 mmol) at 45°C for 16 hours. Cool to room temperature and extract with MTBE. The organic phase was removed and a 2M aqueous solution of K ₂ HPO ₄ was added to the aqueous phase to pH~6 (approximately 150 ml). The resulting mixture was stirred at room temperature for 16 hours. The resulting solid was filtered and collected, washed with water and dried in a vacuum oven at 45°C for 16 hours to obtain the title compound (14.3 g, 76%) as white solid.

ER/MS (m/z): 287 (M+1).

The obtained sample of the anhydrous crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 1) is characterized by an X-ray diffraction pattern using CuKa radiation, having diffraction peaks (2-theta values) described in table. 1 below. Specifically, the pattern contained a peak at 4.7° in combination with one or more peaks selected from the group consisting of 11.7, 15.8 and 16.0° with a tolerance for diffraction angles of 0.2° .

Table 1

X-ray powder diffraction peaks of the anhydrous crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 1)

Peak Angle (2-theta°) +/- 0.2° Relative intensity (% of the most intense peak) 1 4.7 100% 2 I,7 6% 3 15.8 6% 4 16.0 10% 5 26.8 5% 6 27.2 4%

Representative ^13C ssNMR resonances for the anhydrous crystalline form of 5-[5-[(1S,2R)-2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 1) include 16.6 , 18.3, 20.7, 22.1, 23.9, 25.4, 26.6, 27.6, 28.1, 28.9, 30.2, 31.2, 32.2, 38 ,6, 39.7, 41.2, 43.1, 109.3, 111.0, 112.0, 124.4, 128.2, 130.7, 145.1, 146.3, 150.1 , 151.0, 153.4, 156.7, 157.6, 158.6, 159.3, 160.7, 163.4, 165.9, 167.0, 169.4 and 170.2 m. d. (±0.2 ppm respectively).

Method of obtaining 14.

Crystalline Form 2: 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione (desolvated/anhydrous).

Crystalline form of 1,5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin3-yl]-1H-pyrimidin-2,4-dione (15.4 kg, 53.8 mol) was added. to water (770 L) in a 3500 L Hastelloy reactor at room temperature. The resulting suspension was stirred at room temperature for 19 hours. The resulting solid was filtered and collected, washed with water and dried in a vacuum oven at 40°C for 3 days to obtain the title compound (13.8 kg, 89%) as white solid. Purity by HPLC: 99.8%.

A family of related crystalline forms obtained from a sample of the desolvated crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione (Form 2) was observed. One member of the family is characterized by an X-ray diffraction pattern using CuKa radiation having diffraction peaks (2-theta values) described in Table. 2 below.

table 2

X-ray powder diffraction peaks of the desolvated crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 2)

Peak Corner (2-theta°) +/- 0.2° Relative intensity (% of the most intense peak) 1 4.3 100 2 6.4 56 h 9.6 13 4 and about AND 5 I,6 36

Representative ^13C ssNMR resonances for the desolvated (anhydrous) crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione ( form 2) include 17.2, 18.9, 21.1, 22.5, 24.4, 26.7, 27.8, 28.7, 29.7, 39.7, 41.3, 45, 5, 50.1, 110.7, 111.7, 112.4, 122.0, 125.0, 126.1, 129.1, 143.6, 144.8, 146.2, 147.2, 149.9, 151.1, 153.0, 155.5, 157.2, 159.0, 160.6, 161.4, 162.7, 164.8, 167.0, 168.3 and 170, 4 ppm (± 0.2 ppm respectively).

Other members of the family are characterized by an X-ray diffraction pattern using CuKa radiation having diffraction peaks (2-theta values) at greater than 4.3 and up to 5.5° in combination with one or more peaks at 4.7, 6.4, 9.3, 11.0, 11.6, 18.4 and 27.9° with a diffraction angle tolerance of 0.2°.

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Method of obtaining 15.

Crystalline Form 3: 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione (hemihydrate).

Add 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-2,4-dimethoxypyrimidine (316 g, 0.92 mol) to a 1 M aqueous solution of HCl (2, 6 L, 2.6 mol) in a 5 L flask at room temperature. The resulting suspension was heated to 45°C and stirred at this temperature for 16 hours. Cooled to room temperature and a 2 M aqueous solution of K ₂ HPO ₄ was added to pH~6. The resulting mixture was stirred at room temperature for 1 hour. The resulting solid was filtered and collected, dried in a vacuum oven at 40°C for 3 days to obtain the title compound (250 g, 92%) as a white solid. Purity by HPLC: 99%.

The resulting sample of the hemihydrate crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 3) is characterized by an X-ray diffraction pattern using CuKa radiation , having diffraction peaks (2-theta values) described in table. 3 below. Specifically, the pattern contained a peak at 5.0° in combination with one or more peaks selected from the group consisting of 16.4, 17.8 and 26.6° with a tolerance for diffraction angles of 0.2°.

Table 3

X-ray powder diffraction peaks of the hemihydrate crystalline form of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 3)

Peak Corner (2-theta°) +/- 0.2° Relative intensity (% of the most intense peak) 1 5.0 100 2 16.4 15 3 17.8 10 4 26.6 23 5 28.8 8

Representative ^13C ssNMR resonances for the hemihydrate crystalline form of 5-[5-[(1S,2R)2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione (Form 3) include 16. 5, 18.9, 19.6, 20.4, 22.6, 23.5, 24.4, 26.0, 27.4, 28.3, 30.0, 30.9, 34.6, 38.4, 40.5, 110.2, 110.8, 124.1, 126.8, 130.4, 131.2, 146.0, 147.1, 150.4, 151.2, 153, 4, 157.1, 157.7, 158.3, 159.6, 163.1, 166.2 and 169.7, etc. (±0.2 ppm respectively).

Method of obtaining 16.

Conversion of crystal form 2 to crystal form 1.

Add crystalline form of 2,5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin3-yl]-1H-pyrimidin-2,4-dione (5.0 kg, 17.4 mol) to a mixture of THF (100 l) and water (10 l). The resulting solution was concentrated to 10-15 L under reduced pressure below 50°C. THF (5 L) and water (60 L) were loaded, heated to 50-60°C and stirred for 2-4 hours. Cooled to 20-25°C and stirred for 1-2 hours. The resulting solid was filtered and collected. the material was washed with water (10 L) and dried to obtain the title compound (4.2 kg, 84%) as an off-white solid. HPLC purity: 99.4%.

Mass spectroscopy for determination of adenosine and purification of adenosine.

An Agilent 300 RapidFire automated extraction system (Agilent, Santa Clara, CA) with three quaternary HPLC pumps coupled to a Sciex 6500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA) with a surface electrospray ionization (ESI) source was used. . The RapidFire mass spectrometer system was equipped with a reusable RapidFire HILIC solid phase extraction (SPE) cartridge (G9203-80109) (H1).

Solvent A used to load and wash samples was 50 mM ammonium formate pH 4.0 containing 5% (v/v) ACN. Solvent B used to elute the samples was 0.3% formic acid + 2% ammonium hydroxide in 70% ACN/30% MeOH. Samples were analyzed sequentially by withdrawing 10 μL per sampling loop under vacuum directly from the multiwell plates. Load 10 μL of sample onto the HILIC cartridge and wash with quaternary pump 1 using solvent A at a flow rate of 1.25 mL/min for 3000 ms. The retained analyte was eluted into the mass spectrometer using quaternary pump 3 using solvent B at a flow rate of 1.25 mL/min. for 3000 ms. The system was re-equilibrated using quaternary pump 1 using solvent A at a flow rate of 1.25 mL/min for

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3000 ms.

A triple quadrupole mass spectrometer was equipped with an electrospray ionization (ESI) source and the analytes were studied using selective reaction monitoring (SRM) in positive mode (M+H)+. Adenosine was recorded at m/z 268.05/136.0 and adenosine monophosphate at m/z 348.1/136.0. Area ratio values for adenosine and adenosine monophosphate were calculated using 13C5-adenosine and 15Ν5-ΑΜΦ as internal standards, respectively.

Biochemical analysis of human CD73.

The purpose of this assay is to identify and characterize the activity of 5-[5-[(1S,2R)-2isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione against the CD73 enzyme. Reaction mixtures (20 μl) containing 2 μM adenosine monophosphate (Sigma, no. 01930), 10 mM Tris pH 7.5, 100 mM NaCl, 0.01% BSA, 0.2 mM octyl glucoside and 50 pM CD73 protein were added to 384 -well plate (Nunc, No. 264573). After 30 min of incubation at room temperature, the reaction was stopped by adding 20 μl of a stop solution containing 2% formic acid and 10 μM ¹³ C5-adenosine (ribose labeled ¹³ C5) (Cambridge Isotope Laboratories, No. CLM-3678-0), then 40 µl dH2O was added. Levels of adenosine and adenosine ribose- ^13C5 (internal standard) were determined using mass spectrometry as described above. The signal ratio (adenosine peak integration/adenosine internal standard peak integration) was used to quantify each reaction. Percent inhibition was calculated using the equation % inhibition=100χ[1-(XMIN)/(MAX-MIN)]}, where X is equal to the ratio of signals in the well, MAX is equal to the average ratio of signals of the DMSO control sample, and MIN is equal to the ratio of enzyme activity signals in presence of >10X IC50 of a known competitive inhibitor. For screening, each compound was tested at a concentration of 50 μM in 1% DMSO. The IC ₅₀ for 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione was determined by testing each compound at 10 concentrations from 0.0025 to 50 µM (using a 1:3 dilution scheme).

The IC ₅₀ for 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione was 0.028 µM.

Mechanistic analysis of mouse CD73.

The purpose of this assay is to evaluate 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3yl]-1H-pyrimidine-2,4-dione for inhibition of murine CD73 enzyme activity. The assay was performed as described above for the human CD73 biochemical assay, except that 3 μM AMP and 50 pM mouse CD73 enzyme were used.

The IC ₅₀ for 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione was 0.175 μM.

Analysis of human Calu6 cells.

The purpose of this assay is to test 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin3-yl]-1H-pyrimidine-2,4-dione against CD73 in a cell-based assay. Calu6 cells (1500 cells/well) were grown in a 96-well poly-O-lysine-coated plate (BD, no. 356640) containing 100 μl MEM (Gibco, no. 11095-072) + 1% sodium pyruvate (Gibco, #11360-070)+1% NEAA (Gibco, #11140-050)+10% FBS (Hyclone, #SH30071) Plates were incubated at room temperature for 30 min, then incubated overnight at 37°C/5% CO2 Cells were washed twice with assay buffer (10 mM Tris-HCl pH 7.2, 10 mM D-glucose, 1 mM KCl, 125 mM NaCl, 2 mM MgCl ₂ ) (90 μl/well). Then 90 μl of assay buffer, then 10 μl per well of AMP and compound premix (50 μM AMP, various concentrations of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H- pyrimidine-2,4-dione in 1% DMSO) The plates were incubated at room temperature for 60 min. Then 10 μl of the supernatant solution was removed from each well and placed in a new plate, then 20 μl of stop solution (2% formic acid) was added , 1.2 µM adenosine ribose- ¹³ C5 (Cambridge Isotope Laboratories, No. CLM-3678-0) and 90 µl ddH ₂ O for mass spectroscopic analysis. Levels of adenosine and adenosine ribose- ^13C5 (internal standard) were determined using mass spectrometry (Agilent RapidFire) as described above for biochemical analysis of human CD73. Percent inhibition was also calculated as described above.

IC ₅₀ for 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione was 0.0073 μM (compound of Example 2) .

Ex vivo target inhibition assay.

The purpose of this assay is to test 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin3-yl]-1H-pyrimidine-2,4-dione against murine CD73 in mouse blood in an ex vivo assay. Animals (6 per group) were orally dosed with each compound formulated in 20% HPBCD (2-hydroxypropyl-b-cyclodextrin), pH 2, when the tumor volume reached approximately 400 mm ³ . After treatment, blood was collected in heparin tubes and used for ex vivo analysis of the conversion of ^{13 C10-15} N5-AMP to labeled adenosine, inosine and hypoxanthine as described for the ex vivo analysis, using whole blood collected from animals treated by oral administration. - 16 045742 dose of the compound.

5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione inhibited the conversion of AMP to adenosine, inosine and hypoxanthine in mouse whole blood collected in animals that were administered various doses of the specified compound, as shown in table. 4.

Table 4

Inhibition of the conversion of AMP to adenosine

The IC ₅₀ for 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione was 0.0073 µM.

In vivo analysis of human CD73 inhibition in Calu6 tumor.

The purpose of this assay is to test 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6methylpyridazin-3-yl]-1H-pyrimidin-2,4-dione against human CD73 in xenograft tumors derived from Calu6 cancer cells human in vivo target inhibition assay. Calu6 cells (ATCC) were grown in HBSS supplemented with 10% fetal bovine serum. Subconfluent cells were collected using trypsin and washed twice with serum-free culture medium. Subcutaneous tumor growth was initiated by injecting 5 x 10 ⁶ cells in a 1:1 mixture of HBSS and MATRIGEL® (BD Biosciences, Franklin Lakes, NJ) into the posterior flank of nude mice (The Harlon Laboratory). Once the average tumor volume reached approximately 400-500 mm ³ , animals were randomly assigned to tumor size and body weight and assigned to their respective designated experimental groups. After treatment, tumor samples (50-80 mg each) were collected and processed in 1 ml of ice-cold extraction buffer containing internal standards as described below.

A strip of foil and liquid _N2 were added to the mortar for pre-cooling. A drop of tumor tissue was placed on a strip of foil and liquid _N2 was added. Another strip of foil was placed on top of the tumor tissue and beaten with a pestle until the tumor was completely crushed. 50-100 mg of tumor tissue was placed in tubes (Fishers Scientific, cat. no. 02-681-302) and placed on dry ice. One metal bead (Qiagen, cat. no. 69989) and 1 ml of 80% methanol containing internal standards ¹³ C5-adenosine, ¹³ C5-AMP, ¹⁵ Ν5-ΓΤΦ, ¹⁵ N4-inosine-5'-monophosphate and ¹³ were added to the tubes. C- ¹⁵ N-hypoxanthine (Cambridge Isotope Lab and Cayman Chemical), and stored samples at -80°C until used for LC/MS analysis.

Blood was also collected in heparin tubes and used for ex vivo analysis of the conversion of ¹³ C5- ¹⁵ N5-AMP to labeled adenosine, inosine and hypoxanthine as described for the ex vivo analysis, using whole blood collected from animals exposed to oral dosing. connections.

As shown in table. 5, 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1Hpyrimidin-2,4-dione inhibited the conversion of AMP to adenosine in Calu6 tumors treated with different doses of the compound.

Table 5

Inhibition of the conversion of AMP to adenosine

Compound treatment group (mg/kg) Inhibition (%) p-value 0.0 0 1,000 1.0 39.1 0.0057 * 2.5 68.5 <0.0001* 6.4 64.3 <0.0001* 16.0 77.5 <0.0001* 40.0 82.1 <0.0001* 100.0 89.3 <0.0001*

* Statistical significance.

LC/MS Assay to Measure CD73 Activity in Human Serum Fresh blood from a healthy subject was centrifuged at 1500 g for 15 min at room temperature and the upper fraction containing serum was collected. The collected serum (25 μl/well) was transferred into a 96-well deep well plate (DWP, Analytical Sales & Services Inc., cat. no. 968820) containing various concentrations of 5-[5-[(1S,2R)-2- isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidine2,4-dione and a fixed concentration of levamisole (1500 μM). After incubation on ice for 60 min, ^{13 C5-15} N5-AMP (50 μM) was added to each well on the plate and the plate was incubated at room temperature for 15 min. The plate was then placed on dry ice with 200 μl per well of 17.3 TCA added, followed by shaking at 26 rps for 3 min on a plate shaker (Qiagen). The plate was then centrifuged at 2940 g for 20 min at 4°C. After centrifugation, transfer 100 µl per well of the supernatant solution from each solution to a new 96-well deep well plate and mix with 18.4 µl per well of 2.5 M Na ₂ CO ₃ on ice, then add 200 µl of the extraction solution containing the internal standard (internal st.: ¹³ C5-AMP, ¹³ C5-adenosine, ¹³ C5-hypoxanthine and ¹⁵ N4-uho3uh). After further centrifugation at 2940 g for 20 min at 4°C, 200 µl per well of the supernatant was used to analyze ¹³ C10- ¹⁵ N5-adenosine, ¹³ C10- ¹⁵ N5-inosineα and ¹⁵ X5-hypoxanthine by LC/MS as described above. To calculate EC50, serum concentrations of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3yl]-1H-pyrimidin-2,4-dione were corrected for the unbound fraction (%) obtained in computer models or experimentally.

In table Figure 6 presents data on the inhibition of CD73 activity in human serum under the influence of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-ul]-1H-pyrimidine-2,4-dione.

Table 6

5-[5-[(18.2K)-2-Isopropylcyclopropyl]-6methylpyridazin-3-yl]-1 H-pyrimidin-2,4-dione (µM) Inhibition (%) 4.6800 76.9 1.5600 71.1 0.5200 56.8 0.1733 39.6 0.0578 25.4 0.0193 15.4 0.0064 8.4 0.0021 3.7 0.0007 0.7 0 0.0

In vivo models.

If necessary, preclinical modeling of the inhibitory effect of 5-[5-[(1S,2R)-2-isopropylcyclopropyl]-6-methylpyridazin-3-yl]-1H-pyrimidin-2,4-dioneα or its pharmaceutically acceptable salt on CD73, for example, in accordance with methods described in the art, for example in the publication Rongvaux A. et al., Annual Rev. Immunology, 2013, 31:635-74, and in the literature cited therein; Sanmamed M.F. et al., Annals of Oncology, 2016, 27:1190-1198, and in the literature cited therein.

Claims (15)

1. Crystalline form of the compound formula which is characterized by at least one of (a) a peak in the X-ray diffraction pattern at a 2-theta diffraction angle of 4.3° in combination with one or more peaks at 6.4, 9.6, 11.0 and 11.6°; with a tolerance for diffraction angles of 0.2°; and (b) a solid phase ¹³ C NMR spectrum which contains peaks assigned to the high field resonance of adamantane (δ=29.5 ppm) at 17.2, 28.7, 122.0, 126.1, 143 .6 and 155.5 ppm (±0.2 ppm respectively). 2. A precursor composition to a pharmaceutical composition containing a crystalline form according to claim 1. 3. A precursor composition to a pharmaceutical composition according to claim 2, characterized in that the composition is a granular composition. 4. A pharmaceutical composition containing the crystalline form of claim 1 and one or more pharmaceutically acceptable carriers, diluents or excipients. - 18 045742 5. The crystalline form according to claim 1, which is at least 95% free from impurities. 6. Hemihydrate crystalline form of the compound of formula N N 7. The hemihydrate crystalline form of claim 6, which is characterized by at least one of (a) a peak in an x-ray diffraction pattern at a 2-theta diffraction angle of 5.0° in combination with one or more peaks at 16.4, 17.8, and 26.6°; with a tolerance for diffraction angles of 0.2°; and (b) solid-phase ¹³ C NMR spectrum, which contains peaks assigned to the high-field resonance of adamantane (5 = 29.5 ppm) at 23.5, 34.6, 110.2, 124.1, 126 .8 and 166.2 ppm (±0.2 ppm respectively). 8. A pharmaceutical precursor composition containing the hemihydrate crystalline form according to claim 6 or 7. 9. A precursor composition to a pharmaceutical composition according to claim 8, characterized in that the composition is a granular composition. 10. A pharmaceutical composition comprising the hemihydrate crystalline form of claim 6 or 7 and one or more pharmaceutically acceptable carriers, diluents or excipients. 11. The hemihydrate crystalline form according to claim 6 or 7, characterized in that the crystalline form is at least 95% free from impurities. 12. A pharmaceutical precursor composition containing 500 g of an anhydrous crystalline form of a compound of the formula 13. The composition of claim 12, wherein the anhydrous crystalline form is characterized by at least one of (a) an X-ray diffraction peak at a 2-theta diffraction angle of 4.7° in combination with one or more peaks at 11.7, 15.8, 16.0, 26.8 and 27.2°; with a tolerance for diffraction angles of 0.2°; and (b) solid-phase ¹³ C NMR spectrum, which contains peaks assigned to the high-field resonance of adamantane (5 = 29.5 ppm) at 25.4, 28.9, 43.1, 109.3, 124 .4, 128.2 and 160.7 ppm. (±0.2 ppm respectively). 14. The composition according to claim 12 or 13, characterized in that the composition is a granular composition. 15. A pharmaceutical composition comprising a composition according to claim 12 or 13 and one or more pharmaceutically acceptable carriers, diluents or excipients.