CARBOXAMIDE

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an isotopologue of Compound 1 substituted with one or more deuteriums. The isotopologues of this invention are selective CDK4/6 kinase inhibitors and possess unique biopharmaceutical and metabolic properties compared to Compound 1. They may also be used to accurately determine the concentration of Compound 1 in biological fluids and to determine metabolic patterns of Compound 1 and its isotopologues. The invention further provides compositions comprising these deuterated isotopologues and methods for treating pRb-positive tumor types including HR-positive and HER2-negative breast cancer in combination with additional agents (i.e. letrozole). See Rader J, el al. "Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma." Clin Cancer Res. 2013, 19(22), 6173-82.

BACKGROUND OF THE INVENTION

Compound 1 , chemically described as 7-cyclopentyl-N, N-dimethyl-2-((5-(pipera/in- 1 -yl)pyridin-2-yl)amino)-7H-py rrolo[2,3-d ]pyrimidine-6-carboxamide and its

pharmaceutically acceptable

Compound 1 salts, solvates, hydrates, and polymorphs thereof, are known as selective CDK4/6 inhibitors. This comrjound and oharmaceutical comDositions comDrisine it mav have utility in the treatment of pRb-positive tumor types including HR-positive and HER2- negative breast cancer in combination with additional agents {i.e. letro/.ole).

Definitions and descriptions of these conditions are known to the skilled practitioner and are further delineated, for instance, in the above patents and patent applications and references contained therein. See also: Harrison's Principles of Internal Medicine 16th Edition, Kasper D L et. al. Eds., 2004, McGraw-Hill Professional; and Robbins & Cotran Pathologic Basis of Disease, Kumar V et al. Eds., 2004, W.B. Saunders.

Compound 1 , also known as Ribociclib (LEE-01 1 ), selectively binds to cyclin- dependent kinases 4 and 6 (CDK4/6) and inhibits the activity of CDK4/6 thereby inhibiting retinoblastoma (Rb) protein phosphorylation. Inhibition of Rb phosphorylation prevents CDK-mediated G l -S phase transition, therefore arresting the cell cycle in the Gl phase, suppressing DNA synthesis and inhibiting cancer cell growth. Overexpression of CDK.4/6, as seen in certain types of cancer, causes cell cycle deregulation and uncontrolled cellular proliferation. The first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, often resulted in limited activity and poor safety profiles in the clinic. The emergence of a new generation of selective CDK.4/6 inhibitors has enabled tumor types in which CDK4/6 has a pivotal role in the Gi-to-S-phase cell- cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Ribociclib is one of this type of drugs currently in clinical development. See Shapiro GI. "Cyclin dependent kinase pathways as targets for cancer treatment." J Clin Oncol. 2006, 24, 1770- 1783. Lim S, Kalciis P "Cdks, cyclins and CKIs: roles beyond cell cycle regulation. " Development, 2013, 140, 3079 93.

In in vitro tests, Compound 1 showed good CDK4 and CDK6 inhibitory activities

|IC5() (CDK4) = 10 nMol/L, IC50 (CDK6) = 39 nmol/L] and excellent selectivity over other CDK family kinases | IC50(CDK 1 ) > 100 μmol/L, IC50 (CDK2) > 50 μmol/L]. See Asghar U, et al. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov 2015, 14, 130-46. Kim S A, et al. "Abstract PRO 2: LEE01 1 : an orally bioavailable, selective small molecule inhibitor of CDK4/6 reactivating Rb in cancer." Mot Cancer Ther 2013, 12, 1 1 S:PR02. Treatment with compound 1 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations

(mean IC50 = 307 ± 68 nmol/L in sensitive lines). Compound 1 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated Rb and FOXM 1 , respectively. In addition, it has demonstrated dose-dependent growth inhibition in tumor xenograft models. Rader J, et al. "Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma." Clin Cancer Res. 2013, 19(22), 6173-82.

Ribociclib has been investigated for the safety and efficacy in patients with HR- positive, HER2-negative advanced breast cancer. One of the Phase III studies

(MONALEESA-2) was stopped on May 18, 2016 due to positive efficacy results at interim analysis in HR+/HER2- advanced breast cancer. A pre-planned interim analysis showed the trial met the primary endpoint of clinically meaningful improvement in PFS (progression-free survival). MONALEESA-2 is a pivotal Phase III trial of ribociclib in combination with letro/ole, compared to lelro/ole alone in postmenopausal women who had received no prior therapy lor their HR+/HER2- advanced breast cancer. It has validated the belief that ribociclib in combination with letro/ole can be a beneficial treatment option for women diagnosed with HR+/HER2- advanced breast cancer. See Novartis announcement, https://www.novartis.com/news/media-releases/monaleesa-2- Irial-novartis-leeO 1 1 -ribociclib-stopped-due-positive-efficacy

Ribociclib is currently evaluated in combination with additional endocrine agents as part of the MONALEESA clinical trial program. The MONALEESA-3 trial is evaluating ribociclib in combination with fulvestrant compared to fulvestrant alone in men and postmenopausal women with HR+/HER2- advanced breast cancer who have received no or a maximum of one prior endocrine therapy. The MONALEESA-7 trial is investigating ribociclib in combination with endocrine therapy and goserelin compared to endocrine therapy and goserelin alone in pre-menopausai women with HR+/HER2- advanced breast cancer who have not previously received endocrine therapy.

Ribociclib has also been evaluated in a phase I monotherapy study and shown to have an acceptable safety profile with less prominent hematologic and gastrointestinal toxicity than the other two CDK4/6 inhibitors, palbociclib and abemaciclib. The recommended dose was 600 mg daily, 3 weeks on, i week off. See, Infante JR, Shapiro G, Witteveen P. "A phase I study of the single-agent CDK 4/6 inhibitor LEE01 1 in patients with advanced solid tumors and lvmDhomas." J Clin Oncol. 32:5s. 2014 tsuDol: abstr 2528V

There remains potential unmet clinical need for a method of administering higher doses of Compound 1 to a patient in a manner that eliminates or minimizes adverse events, such as less prominent hematologic and gastrointestinal toxicity and other potentially dangerous side effects that can occur with ribociclib therapy.

It is therefore desirable to create a compound displaying the beneficial activities of Compound 1 , but with a decreased metabolic liability, to further extend its

pharmacological effective life in increasing the concentration of drug in the blood, and improving the effective bioavailability, reducing the dosage therefore reducing the toxicity and other side effects.

SUMMARY OF THE INVENTION

Disclosed herein is a deuterated compound having the structure of Formula I:

or a pharmaceutically acceptable salt, or solvate thereof; or a hydrate or polymorph thereof; wherein:

Without being bound by any theory of operation, it is believed that compound 1 may be primarily metabolized by CYP3A and sulfotransferase in a similar manner to that of its structurally related analog, palbociclib. The primary metabolic pathways for compound 1 may involve oxidation and sulfonation.

Limiting the production of the oxidation metabolites has the potential to decrease the danger of the administration of such drugs and may even allow increased dosage and concomitant increased efficacy. All of these transformations can occur through polymorphically-expressed enzymes, thus exacerbating the interpatient variability.

Limiting the production of metabolites has the potential to allow increased dosage and concomitant increased efficacy. All of these transformations can occur through polymorphically-expressed enzymes, thus exacerbating the interpatient variability. For all of foregoing reasons, there is a strong likelihood that a longer half-life medicine will diminish these problems with greater efficacy and cost savings.

Various deuteration patterns can be used to a) reduce or eliminate unwanted metabolites, b) increase the half-life of the parent drug, c) decrease the number of doses needed to achieve a desired effect, d) decrease the amount of a dose needed to achieve a desired effect, e) increase the formation of active metabolites, if any are formed, and/or 0 decrease the production of deleterious metabolites in specific tissues and/or create a more effective drug and/or a safer drug for polypharmacy, whether the polypharmacy be intentional or not. The deuteration approach has strong potential to slow the metabolism via various oxidative and other modification mechanisms.

In particular, this would produce benefits in the use of combinations of medications, which is common in patients suffering from cancer. Moreover, it would be useful in patients taking Compound 1 , while being treated by different healthcare providers without disclosing all of their medications to each of them. It would also be beneficial in patients who are using drugs of abuse while taking Compound 1 without the knowledge of their Dhvsician.

The compounds and compositions of this invention are also useful as analytical reagents for determining the concentration of the Compound 1 in solution. "Compound 1 " as used herein refers to a compound wherein all hydrogen and all carbon atoms are present at their natural isotopic abundance percentages. It is recognized that some variation of natural isotopic abundance occurs depending upon the origin of chemical materials. The concentration of naturally abundant stable hydrogen and carbon isotopes, not withstanding this variation, is small and immaterial with respect to the degree of stable isotopic substitution of compounds of this invention. (See for instance Wada E and Hanba Y, Seikagaku 1994 66: 15; Ganes L Z et. al., Comp, Biochem. Physiol. A Mol. Integr.

Physiol. 1998 1 19: 725.)

The altered properties of the compounds of this invention will not obliterate their ability to bind to their protein target. This is because such binding is primarily dependent upon non-covalent binding between the protein and the inhibitor which may be impacted both positively and negatively by isotopic substitution, depending on the specific substitution involved, and any negative effects that a heavy atom of this invention may have on the highly optimized non-covalent binding between compounds of formula I and its target proteins will be relatively minor. Major factors contributing to the noncovalent recognition of small molecules by proteins and the binding strength between them include: Van der Waals forces, hydrogen bonds, ionic bonds, molecular reorganization, desolvation energy of the small molecule, hydrophobic interactions and, in certain instances, displacement energy for pre-existing bound ligands. See, for instance, Goodman & Gilman's The Pharmacological Basis of Therapeutics, Tenth Edition, Hardman J G and Limbird L E, eds. McGraw-Hill, 2001 and The Organic Chemistry of Drug Design and Drug Action, Silverman R B, 2004, Academic Press.

The compounds of this invention possess molecular topology that is very similar to Compound 1 , since exchange of deuterium for hydrogen does not alter molecular shape and exchange of ^U C for ^{l 2} C is conformatioinally neutral (Holtzer M E et. al., Biophys. J. 2001 80: 939). Deuterium replacement does cause a slight decrease in Van der Waals radius (Wade D, Chem. Biol. Interact. 1999 1 17: 191 ); but applicant believes that such decrease will not greatly reduce binding affinity between the molecule and its receptor. Furthermore, the slightly smaller size of the deuterated compounds of this invention Drevents their beine involved in new undesirable steric clashes with the bindine Drotein relative to the Compound 1.

Neither deuterium nor ¹³ C atoms in the compounds of this invention contribute significantly to hydrogen bonding or ionic interactions with the protein receptors. This is because the major hydrogen bond and ionic: interactions formed by Compound 1 with serotonin uptake proteins are mediated by the oxygens, nitrogens, and the amine-bound hydrogens within Compound 1. Any deuterium atoms attached to the amine nitrogen will be rapidly exchanged with bulk solvent protons under physiological conditions. Protein reorganization or side chain movement will be identical between a compound of this invention and Compound 1. Desolvation energy of a compound of this invention will be equivalent to or less than that of Compound 1, resulting in neutral or increased binding affinity for the receptor; Turowski M et. al., J. Am. Chem. Soc. 2003 125: 13836. The replacement of ⁿ C in place of ^{l 2} C in compounds of this invention will have no practical change in desolvation.

Thus, a compound of this invention advantageously retains substantial selective CDK4/6 inhibitory activity with reduced rate of metabolite generation.

The compounds and compositions of this invention are also useful as analytical reagents for determining the concentration of the Compound 1 (the active component) in solution.

The contents of the patents and publications cited herein and the contents of documents cited in these patents and publications are hereby incorporated herein by reference to the extent permitted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated compound of formula I:

the compounds of formula (I) are each deuterium;

In another embodiment, at least one of the positions represented as D independently has deuterium enrichment of no less than about 1 %, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about K()%, no less than about 90%, or no less than about 98%.

The deuterated compound as disclosed herein may also contain less prevalent isotopes for other elements, including, but not limited to, ^,3 C or ¹⁴ C for carbon, ¹⁵ N for nitrogen, and ¹⁷ 0 or ¹⁸ 0 for oxygen.

In one embodiment, the deuterated compounds disclosed herein maintain the beneficial aspects of the corresponding non-isotopically enriched molecules while substantially increasing the maximum tolerated dose, decreasing toxicity, increasing the half-life (ΊΥ ₂ ), lowering the maximum plasma concentration (C _max ) of the minimum efficacious dose (MED), lowering the efficacious dose and thus decreasing the non- mechanism-related toxicity, and/or lowering the probability of drug-drug interactions.

The term "compound" as used herein, is intended to include salts, prodrugs, and prodrug salts of a compound of formula I. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "prodrug," "prodrug salt," "solvate," "hydrate," or "polymorph" in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term "compound" is used without recitation of these other forms. A salt of a compound of this invention is formed between an acid and a basic group of the corrmound. such as an amino functional eroiiD. or a base and an acidic crouD of the compound, such as a carboxyl functional group. According to another preferred embodiment, the compound is a pharmaceutically acceptable acid addition salt.

As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds of any one of the formulae disclosed herein that comprise—NO,— NO.sub.2,— ONO, or— ONO.sub.2 moieties. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery ( 1995) 172- 178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransforrnation of Drugs".

As used herein and unless otherwise indicated, the terms "biohydrolyzable amide", "biohydrolyzable ester", "biohydrolyzable carbamate", "biohydrolyzable carbonate", "biohydrolyzable ureide" and "biohydrolyzable phosphate analogue" mean an amide, ester, carbamate, carbonate, ureide, or phosphate analogue, respectively, that either: 1 ) does not destroy the biological activity of the compound and confers upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is itself biologically inactive but is converted in vivo to a biologically active compound. Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, ' and ■ oolv ml ether amines.

A prodrug salt is a compound formed between an acid and a basic group of the prodrug, such as an amino functional group, or a base and an acidic group of the prodrug, such as a carboxyl functional group. In a preferred embodiment, the prodrug salt is a pharmaceutically acceptable salt. According to another preferred embodiment, the counterion to the saltable prodrug of the compound of formula I is pharmaceutically acceptable. Pharmaceutically acceptable counterions include, for instance, those acids and bases noted herein as being suitable to form pharmaceutically acceptable salts.

Particularly favored prodrugs and prodrug salts are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or central nervous system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. See, e.g., Alexander, J. et al. Journal of Medicinal Chemistry 1988, 31 , 3 18-322; Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985; pp 1 -92; Bundgaard, H.; Nielsen, N. M Journal of Medicinal Chemistry 1987, 30, 451-454; Bundgaard, H. A Textbook of Drug Design and Development; Harwood Academic Publ.: Switzerland, 1991 ; pp 1 13- 191 ; Digenis, G. A. et al. Handbook of Experimental Pharmacology 1975, 28, 86- 1 12; Friis, G. J.; Bundgaard, H. A Textbook of Drug Design and Development; 2 ed.; Overseas Publ.: Amsterdam, 1996; pp 351-385; Pitman, I. H. Medicinal Research Reviews 1981 , 1 , 189-214.

The term "pharmaceutically acceptable," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.

Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para- bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,

dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1 ,4- dioate, hexyne- l ,6-dioate, ben/oate, chlorobenzoate, methylben/oate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacelate, phenylpropionate, phenylbulyrate, citrate, lactate, .beta. -hydroxy butyrale, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate and the like salts. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as

hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

As used herein, the term "hydrate" means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent

intermolecular forces.

As used herein, the term "solvate" means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, elhanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.

As used herein, the term "polymorph" means solid crystalline forms of a compound or complex thereof which may be characterized by physical means such as, for instance, X- ray powder diffraction patterns or infrared spectroscopy. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat, light or moisture) '. ' comD l ressibilitv ml and densitv ml ( \im Άrjortant in formulation and 1 Droduct manufacturing), hygroscopicity, solubility, and dissolution rates and solubility (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing. For example, one polymorph might be more likely to form solvates or might be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution of particles of it.

Abbreviations

Ac acetyl

ACN acetonitrile

AcOH or HOAc acetic acid

aq. aqueous

anhyd. anhydrous

ATP adenosine triphosphate

Bn benzyl

Bu butyl

Boc tert-butoxycarbonyl

BOP Benzotriazole- 1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate

CD1 carbonyldiimidazole

°C degrees Centigrade

Cbz carbobenzyloxy

Cone. concentration

d days

DAST (diethylamino)sulfur trifluoride

DBU 1 ,8-Diazabicycloj 5.4.0 jundec-7-ene DCE 1 ,2-dichloroethane

DCM dichloromethane

DEA diethylamine

DIEA or DIPEA diisopropylethylamine

DMAP dimethylaminopyridine

DMA N,N-dimethylacetamide

DME 1 ,2-dimethoxyethane

DMF dimethylformamide

DMSO dimethylsulfoxide

DPPA diphenylphosphorylazide

dppf 1 , 1 '-B;is(diphenylphosphino)ferrocene

DTT dilhiothreitol

EDC or EDCI or ED AC l -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol letraacelic acid

eq. or Eq. or equiv. equivalents

EtOAc or EA Ethyl acetate

Et Ethyl

EtOH Ethanol

Ex example

GST glutathione S-transferase

H Hydrogen

HATU N,N,N,N-tetramethyl-0-(7-a/abenzotriazol- 1 - yl)uronium hexafluorophosphate

Hex hexanes

HIS histidine

h or hr Hours

i iso

IPA isopropanol

Hz hertz

MHz megahertz HPLC High pressure liquid chromatography

RP-HPLC Reverseohase Hieh Dressure liauid chromatoeraDhv

HOBT 1 -Hydroxybenzotria/ole hydrate

Lawesson's Reagent [2,4-bis(4-methoxyphenyl)- 1 ,3-dithia-2,4- diphosphetane-2-4-disufide

LC liquid chromatography

LCMS or LC/MS liquid chromatograph mass spectrometry

LDA lithium diisopropylamide

m-CPBA or MCPBA meta-chloroperbenzoic acid

Me methyl

MeOH methanol

min. minutes

M+ (M+H) ⁺

M " (M+H) '

MS mass spectrometry

MSA meth∑inesulfonic acid

MTBE methyl tert-butyl ether

ml/. mass to charge ratio

N Normal

NMP N-methy lpyrrol idinone

NMR nuclear magnetic resonance

PBMC peripheral blood mononuclear cells

PhCONCS ben/yolyisothiocyanale

Pd/C palladium on carbon

Ph phenyl

Pr propyl

PHA phytohemagglutinin

ppm parts per million

PS I or psi pounds per square inch

quant. quantitative

Ret Time or Rt retention time

rt or RT room temperature sat. or sat'd. saturated

sec seconds

S-Tol-BINAP (S)-(-)-2,2'-Bis(di-p-tolylphosphino)-l , r-binapthyl

SM or sm starting material

t tert

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMS-I or TMSI iodotrimethylsilane

p-TSA para-toluenesulfonic acid

Xantphos® (9,9-dimethyl-9H-xanthene-4,5- diyl)bisfdiphenylphosphine]

t triplet

m multiplet

s singlet

d doublet

br. s. broad singlet

dd doublet of doublets

tt triplet of triplets

ddd doublet of doublet of doublets

q quartet

quin. Quintet

W/V or w/v weight to volume

X-Phos dicycllohexyl(2 ^, ,4',6 ^, -triisopropylbiphenyl-2- yl)phosphine

The following examples are given as specific illustrations of the invention. It should be understood, however, that the invention is not limited to the specific details set forth in the examples. All parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified. Further, any range of numbers recited in the specification or paragraphs hereinafter describing or claiming various asDects of the invention, such as that reDresentine a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers or ranges subsumed within any range so recited. The term "about" when used as a modifier for, or in conjunction with, a variable, is intended to convey that the numbers and ranges disclosed herein are flexible and that practice of the present invention by those skilled in the art using temperatures, concentrations, amounts, contents, carbon numbers, and properties that are outside of the range or different from a single value, will achieve the desired result, namely, using the compounds of the present invention as selective CDK.4/6 inhibitors with the potential for treatment of pRb-positive tumor types including HR-positive and HER2-negative breast cancer in combination with additional agents (i.e. letro/ole). Example 1

Methods of Preparation and Analysis

The compounds of formula I may be prepared by the processes described herein m the following reaction scheme (Scheme A). Examples of suitable reagents and procedures for conducting these reactions appear hereinafter and in the working examples included therein. Protection and deprotection in the schemes herein may be carried out by procedures generally known in the art [See, for example, T.W. Greene & P.G. M.Wuts, P r otecting G roups in Or ganic S ynth esis,3r d Editio n , Wi ley , ( 1 999 ) \ .

SCHEME A

(1)

Compounds of formula I can be prepared from compounds of formula V and VI as depicted in Scheme A as shown in WO 2010/020675.

Reaction of the compound of formula II wherein X = halogen, such as Br, Ci, or I, with a compound of formula III wherein P is a N-protecting group, to obtain a compound of formula IV, can be achieved in a solvent, preferably an organic solvent or a mixture of an organic solvent with water, such as DMSO/water. Further, preferably an organic or inorganic alkaline compound, preferably TEA, can be added to the mixture. The reaction can be carried out at a temperature in the range of 40 to 90 °C, preferably at 65 °C.

Reduction of the compound of formula IV to the compound of formula V can be carried out in an organic solvent or in an organic solvent in mixture with water. Suitable organic solvents are for example alcohol and ethers, preferably methanol. Further, reaction can be carried out in the presence of a catalyst, such as 10% Pd/C or Raney Nickel and/or hydrogen. Preferably, the reaction is carried out in an autoclave reactor pressurized to 1 to 10 bar hydrogen. Particularly preferred conditions are 10% Pd/C in methanol under hydrogen at atmospheric pressure.

Reaction of the compound of formula V with a compound of formula VI to obtain a compound of formula VII can be achieved through the Buchwald coupling reaction. The reaction can be carried out in a suitable organic solvent, such as dioxane, DME, DMF or toluene, preferably toluene, in the presence of a palladium catalyst such as Pd2(dba); and a phosphorous compound such as BINAP. The reaction can be carried out with addition of an alkaline compound, such as sodium tert-butoxide. Further, the reaction is performed at temperature of 50 °C to 120 °C, preferably 100 °C. jV-deproiection of the compound of formula VII using procedures known in the art can afford the compound of formula I.

The compound of formula VI may be prepared from the compound of formula VIII by the processes similar to those described in the reference [Ref: WO 2010/02067S] (Scheme B).

Analytical LCMS Conditions:

Method A: HPLCSHIMAZU LC-2010 /MS: ThermoFisher LTQ XL. Column:

Hypersil Gold 2.1 x 50 mm 3 μτη; Mobile phase: 3% solvent B/A for 0.5 min, gradient 3- 95% B/A in 5 min with a 0.2-min hold at 95% B/A, gradient 95-3% B/A in 0.01 min with a 2.5-min hold at 3% B/A; Flow rate: 0.4 mL/min; Solvent A: 5/95 MeOH/water w/ 0.1 % formic acid; Solvent B: 5/95 MeOH/acetonitrile w/ 0.1% formic acid; Products detected at 220 or 254 nM wavelength w/ positive ionization mode

Analytical GCMS Conditions:

Method B: Shima/u GC-2010/GCMS-QP201 OS. Primary column SLB-5ms 30m x 0.25 mm, 0.25 μπι; GC oven temperature program total 15 min, 45 °C to 300 °C at 40 °C /min with 10 min-hold at 300 °C; Carrier gas He; Inlet pressure 50 kPa; Column flow rate 1.0 tnl/min. Product detected by electron ionization mode.

Example 2

7-Cyclopentyl-N, N-dimethyl-2-((5-(piperazin- 1 -yl)pyridin-2-yl-3,4,6-d ₃ )amino)-7H- pyrrolo[2,3-dJpyrimidine-6-carboxamide

Step 1: tert-Butyl 4-(6-nitropyridin-3-yl-2,4,5-d3)pipera/.ine-l -carboxylate

To a stirred solution of 5-bromo-2-nitropyridine-3,4,6-d3 (commercially available) (3.0 g, 14.6 mmol) and teri-bulyl pipera/ine- 1 -carboxylate (4.08 g, 21.9 mmol) in DMSO (35 ml) were added TEA (2.22 g, 21.9 mmol) and lithium chloride (0.63 g, 14.9 mmol). The reaction solution was healed at 63 °C for 12h. Water (35 ml) was slowly added. The product mixture was stirred at 65 °C for one hour and at room temperature for additional one hour. After filtration, the filter cake was washed with water, and dried under vacuum at 50-55 °C overnight to afford /cr/-butyl 4-(6-nitropyridin-3-yl-2,4,5- d.Opiperazine- l -carboxylate as a pale yellow crystal (3.3 g, 73 % yield).

Step 2: tert-butyl 4-(6-aminopyridin-3-yl-2,4,5-d;0piperazine-l -carboxylate

A mixture of tert-butyl 4-(6-nilropyridin-3-yl-2,4,5-d-,)piperazine- l -carboxylate (3.3 g, 10.6 mmol) and 10% Pd/C (230 mg) in methanol ( 100 ml) was stirred under hydrogen at atmospheric pressure for 30 min. The reaction was filtered through celite and concentrated under reduced pressure to afford tert-butyl 4-(6-aminopyridin-3-yl-2,4,5-d3)piperazine- l- carboxyiate (3.0 g. 73 % yield) as a light brown solid.

Step 3: ter/-Butyl 4-(6-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrroloL2,3- d |pyrimidin-2-yl)amino)pyridin-3-yl-2,4,5-di)piperazine- 1 -carboxylate

A mixture of /erf-butyl 4-(6-aminopyridin-3-yl-2,4,5-d3)piperazine-l-carboxylate (600 mg, 2.1 mmol) and 2-chloro-7<yclopentyl-N,NKlimethyl-7H-pyiTolof2,3^1pyrimi dine-6- carboxamide (630 mg, 2.2 mmol), Pd(dba) ₂ (62 mg, 0.1 1 mmol), BINAP (200 mg, 0.32 mmol) and CS2CO3 (1.4 g, 4.2S mmol) in dioxane (IS mL) is degassed and heated to 1 10 °C over a period of 24 hours. The reaction mixture is partitioned between

dichloromethane and saturated NaHCOj solution. After separation, the aqueous layer was further extracted with dichloromethane. The combined organic layers were washed with brine, dried (MgSO.0, filtered and concentrated. The crude product was purified by flash column chromatography (Combiflash RF+, 0 to 10% methanol / dichloromethane) to give the title product ( 1.1 g, 93% Yield).

Step 4: 7-Cyclopentyl-N,N-dimethyl-2-((5-(pipera/.in-l-yl)pyridin-2- yl-3,4,6- d3)amino)-7H-pyrrolo| 2,3-d |pyrimidine-6-carboxamide

A solution of /erz-butyl 4-(6-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolof2,3- d|pyrimidin-2-yl)amino)pyridin-3-yl-2A5-d3)piDeni/ine-l-carb oxy late ( 1.1 g, 2.0S mmol) in toluene (7 mL) was treated with HC1 (2.1 mL, 6 M solution) dropwise over a period of

30 min. After 16h, the reaction solution was evaporated, and the residue was taken into a mixture of water and dichloromethane. The mixture was adjusted to pH>8 by slow addition of a solution of NaOH. After separation, the aqueous layer was further extracted with dichloromethane. The combined organic layers were washed with brine, dried (MgS0 ₄ ), filtered and concentrated. The crude product was purified by flash column chromatography (Combiflash RF+, 0 to 10% methanol / dichloromethane) to give 7- cyclopenty 1-N, N-dimethyl-2-((5-(piperazin- 1 -yl)pyridin-2-yl-3,4,6-d.i)amino)-7H- pyrroIo[2,3-d]pyrimidine-6-carboxamide as a white solid (710 mg, 79 % yield). ¹ H NMR (300MHz, CDClj): δ 8.72 (s, 1 H), 8.15 (s, 1 H), 6.44 (s, 1 H), 4.79 (m, 1 H), 3.20 - 3.06 (m, 14H), 2.09 - 2.03 (m, 4H), 1.78 - 1.76 (m, 4H). LCMS (Method A): m/z 438.4 (|M+H| ⁴ ), HPLC Rt S.30 min (98.5 % purity).

Example 3

According to the procedures described for Example 2, Example 3 was prepared from 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pynolo[2,3-d|pyrimidine-6-carboxamide (800 mg, 2.74 mmol), and /erf-butyl 4-(6-aminopyridin-3-yl-2A5<h)piperazine-l- carboxylate-2,2,3,3,5,5,6,6-d8 (610 mg, 2.1 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. /er/-butyl 4-(6-aminopyridin-3-yl-2,4,5-dj)piperazine-l- carboxylate-2,2,3,3,5,5,6,6-d8 was prepared from 5-bromo-2-n itropyridine-3 ,4,6-d3 (commercially available) and tert-butyl pipera/ine-l-carboxylate-2,2,3,3,5,5,6,6-d8 (commercially available) following the procedures similar to Steps 1 and 2 in Example 2. 0.45 g (48 % Yield). ¹ H NMR (300MHz, CDC 13): 5 ;8.74 (s, 1H), 8.27 (s, 1H), 6.44 (s, 1 H), 4.80 (m, 1 H), 3.16 (s, 6H), 2.06 - 1.67 (m, 8H). LCMS (Method A): m/z 446.4 (I M+H f ), HPLC Rt 5.10 min (98 % purity). s

Examples 4

7-CyclopentyI-N, N-dimelhyl-2-((5-(pipera/in-l-yl-2,2,3,3,5,5,6,6-de)pyridin- 2- yl)amino)-7H-pyiTolo[2,3-d]pyrimidine-6-carboxamide

According to the procedures described Tor Example 2, Example 4 was prepared from 2-chloro-7-cyclopentyl-N, Nslimemyl-7H-pyrrolo|2,3-d|pyrimidine-6-carboxamide (350 mg, 1.19 mmol), and tert-butyl 4-(6-aminopyridin-3-yl)pipera/ine-l-carboxylale- 2,2,3,3,5,5,6,6-dg (610 mg, 1.05 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. /erf-butyl 4-(6-aniinopyridin-3-yl)piperazine-l-carboxylate-2,2,3,3,5,5 ,6,6- ds was prepared from 5-bromo-2-nitropyridine and /w/-butyl piperazine- 1 -carboxy late- 2,2,3,3,5,5,6,6-ds (commercially available) following the procedures similar to Steps 1 and 2 in Example 2. 0.26 g (56 % Yield). Ή NMR (300MHz, CDClj): δ ;8.72 (s, 1H), 8.36 (d, J = 9 Hz, 1H), 8.15 (s, 1H), 8.03 (d, J = 3 Hz, 1H), 7.32 (dd, J = 9.3 Hz, 3Hz, 1 H), 6.44 (s, 1 H), 4.79 (m, 1 H), 3.16 (s, 6H), 2.12 - 1.78 (m, 8H). LCMS (Method A): ml/. 443.4 (|M+H| ⁺ ), HPLC Rt 5.34 min (98.5 % purity).

SCHEME B

Examples 5

7-Cyclopentyl-N, N-dimethyl-2-((5-(piperazin- 1 -yl)pyridin-2-yl-3,4,6-d.0amino)-7H- pynOlo[2,3-d]pyrimidine-6-carboxamide

Step 1: tert-Bulyl 4-(pyridin-3-yl-d ₄ )piperazine-l-carboxylate

A toluene solution (10 ml) of 3-bromopyridine-2,4,5,6-d| (commercially available, 500 mg, 3.09 mmol), piperazine-l-carboxylic acid ten-butyl ester (690 mg, 3.70 mmol), PdC12(dppf) (1 13 mg, 0.15 mmol), and potassium tert butoxide (800 mg, 7.1 mmol) was degassed and stirred at 100°C for 14 hours under a nitrogen atmosphere. After the reaction mixture was cooled to room temperature, water (10 mL) was added, followed by extraction twice with dichloromethane (50 ml). The organic layer was dried overMgS04, followed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=30/ 1 ). A crude product of tert- Butyl 4-(pyridin-3-yl-d ₄ )pipera/ine-l-carboxylate was obtained as a liquid (350 mg, 43 % Yield).

Step 2: tert-butyl 4-(6-bromopyridin-3-yl-2,4,5-d ₃ )piperazine-l-carboxylate

A solution of tert-Butyl 4-(pyridin-3-yl-d ₄ )piperazine-l-carboxylate (200 mg, 0.75 mmol) in 20 mL of acetonitrile was cooled in an ice bath before a solution of N- bromosuccinimide ( 120 mg, 0.67 mmol) in 5 mL of acetonitrile was added over a period of 10 minutes. The solution was then stirred in the ice bath for 2 hours. Added silica gel and DCM, and the slurry was then concentrated under vacuum. Purified on CombiFlash column (dry loaded), eluting with a gradient of 5% ethyl acetate/hexane to 40% ethyl acetate/hexane, the fractions containing the product were combined and concentrated under vacuum. tert-Butyl 4-(6-bromopyridin-3-yl-2,4,5-d.0piperazine- 1 -carboxylate ( 180 mg, 78 % yield) was obtained as a solid. Step 3: tert-Butyl 4-(6-((7-cyclopentyI-6-(dimethylcarbainoyI)-7H-pyrrolof2,3- d]pyrimidin-2-y l)amino)pyridin-3-yl-2,4,5-d^iperazine- 1 -carboxylate

A mixture of tert-butyl 4-(6-bromopyridin-3-yl-2,4,5-dj)piperazine-l -carboxylate (150 mg, 0.43 mmol) and 2-amino-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d|pyrimidi ne-

6- carboxamide (120 mg, 0.44 mmol), Pd(dba) ₂ (12.6 mg, 0.022 mmol), BINAP (20 mg, 0.032 mmol) and CS2CO3 (420 mg, 1.29 mmol) in dioxane (3 mL) is degassed and heated to 1 10 °C over a period of 24 hours. The reaction mixture is partitioned between dichloromethane and saturated NaHCOj solution. After separation, the aqueous layer was further extracted with dichloromethane. The combined organic layers were washed with brine, dried (MgSO.0, filtered and concentrated. The crude product was purified by flash column chromatography (Combiflash RF+, 0 to 10% methanol / dichloromethane) to give the title product ( 108 mg, 47 % Yield).

Step 4: 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin- 1 -yl)pyridin-2-yl-3 ,4,6- d3)amino)-7H-pyrrolo| 2,3-d |pyrimidine-6-carboxamide

According to the procedure described for Step 4 in Example 2, Example S was prepared from /erf-butyl 4-(6-((7-cyclopentyl-6-(diniethylcarbamoyl)-7H-pyrrolo|2,3- d|pyrimidin-2-yl)amino)pyridin-3-yl-2,4,5-d3)pipera/ine-l -carboxylate and HC1 solution. ¹ H NMR (300MHz, CDCI3): δ 8.72 (s, 1H), 8.15 (s, 1H), 6.44 (s, 1 H), 4.79 (m, 1 H), 3.20 - 3.06 (m, 14H), 2.09 - 2.03 (m, 4H), 1.78 - 1.76 (m, 4H). LCMS (Method A): m/z 438.4 ([M+H] ⁺ ), HPLC Rt 5.30 min (98.5 % purity).

Examples 6

7- Cyclopentyl-N, N-dimethyl-2-((5-(piperazin- 1 -yl)pyridin-2-yl)amino)-7H-pyrrolo(2,3- d]pyrimidine-6-carboxamide-4,5-d2

According to the procedures described for Example S, Example 6 was prepared from 2-amino-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo[2,3-d|pyrimidine-6^arboxarnide- 4,5-d ₂ (commercially available, 100 mg, 0.36 mmol), and /erf-butyl 4-(6-bromopyridin-3- yl)piperazine-l-carboxylate (160 mg, 0.47 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. 120 mg (76 % Yield) product was obtained. LCMS (Method A): m/z 437.3 ([Μ+ΗΓ).

Examples 7

7<:yclopentyl-N,N-dimethyl-2K(5-(piperazin-l-yl-2,2,3, 3,5^,6,frde)pyridin-2- yl)amino)-7H-pyiTolo|2,3-d|pyrimidine-6-carboxamide-4,S-d2

According to the procedures described for Example S, Example 7 was prepared from 2-amino-7-cyclopentyl-N, N-dimemyl-7H-pyrrolo|2,3-d|pyrimidine-6-carboxarnide-4,5- d2 (commercially available, 100 mg, 0.36 mmol), and /erf-butyl 4-(6-bromopyridin-3- yl)pipera/ine-l-carboxylate-2,2,3 ,3,5,5 ,6,6-d8 (comercially available, 160 mg, 0.46 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. 1 10 mg (69 % Yield) product was obtained. LCMS (Method A): m/x 445.3 (|Μ+Η|').

Example 8

7-Cyclopentyl-N, N-bis(methyl-dj)-2-((5-(piperazin- 1 -yl)pyridin-2-yl)amino)-7H- pyrrolol 2,3-d]pyrimidine-6-carboxamide-4,5-d2

According to the procedures described for Example 5, Example 8 was prepared from 2-amino-7-cyclopentyl-N, N-bis(methyl-d3>-7H-pynrolo|2,3-d|pyrimidine-6- carboxamide-4,5 -di (commercially available, 100 mg, 0.35 mmol), and terf-butyl 4-(6- bromopyridin-3-yl)pipera/ine-l-carboxylate (160 mg, 0.47 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. 98 mg (63 % Yield) product was obtained. LCMS (Method A): m/z 443.3 ([M+H]').

Example 9

7-Cyclopentyl-N, N-bis(methyl-d3)-2-((5-(pipera/in- 1 -yl-2,2,3,3,5,5,6,6-dg)pyridin-2- yl)amino)-7H-pynOlo[2,3-d]pyrimidine-6-carboxamide

According to the procedures described for Example S, Example 9 was prepared from 2-amino-7-cyclopentyl-N, N-bis(methyl-d3)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (commercially available, 100 mg, 0.36 mmol), and /erf-butyl 4-(6-bromopyridin-3- yl)pipera/ine-l-carboxylale-2,2,3,3,5,5,6,6-d8 (comercially available, 160 mg, 0.46 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. 100 mg (62 % Yield) product was obtained. LCMS (Method A): m/z 443.3 (|M+H] ⁺ ).

Example 10

7-Cyclopentyl-N, N-bisimethyl-d3)-2-((5-(piperazin-l-yl)-yridin-2-ylOAe- cb)amino)-7H-pyrrolo| 2,3-d |pyrimidine-6-carboxamide

According to the procedures described for Example 5, Example 10 was prepared from 2-amino-7-cyclopentyl-N, N-bis(meuhyl-dj)-7H-py-TOlo[2,3-dJpyrimidine-6-carboxamide (commercially available, 100 mg, 0.36 mmol), and /er/-butyl 4-(6-bromopyridin-3-yl- 2,4,S-d3)pipera/jne-l-carboxylate (comercially available, ISO mg, 0.43 mmol) under the conditions simialr to Steps 3 and 4 in Example 2. 104 mg (65 % Yield) product was obtained. LCMS (Method A): m/z 444.3 ([M+H]*).

Example 11

Microsomal Assay: Certain in vitro liver metabolism studies have been described previously in the following references: Obach, R. S. Drug Metab Disp 1999, 27, p. 13S0 "Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes"; Houston, J. B. et al., Drug Metab Rev 1997, 29, p. 891 "Prediction of hepatic clearance from microsomes, hepatocytes, and liver slices"; Houston, J. B.

Biochem Pharmacol 1994, 47, p. 1469 "Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance"; Iwatsubo, T et al., Pharmacol Ther 1997, 73, p. 147 "Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data"; and Lave, T. et al., Pharm Res 1997, 14, p. 1S2 "The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans".

The objectives of the present study were to determine the metabolic stability of the test compounds in pooled liver microsomal incubations. Samples of the test compounds, exposed to pooled human and rat liver microsomes, were analyzed using UPLC LC- MS/MS detection.

Experimental Procedures:

1. Buffer A: 1.0 L of 0.1 M monobasic Potassium Phosphate buffer containing 1.0 mM EDTA.

Buffer B: 1.0 L of 0.1 M Dibasic Potassium Phosphate buffer containing 1.0 mM EDTA. Buffer C: 0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4 by titrating 700 mL of buffer B with buffer A while monitoring with the pH meter.

2. Reference compounds (Ketanserin) and test compounds spiking solution:

500 μΜ spiking solution: add 10 μL of 10 mM DMSO stock solution into 190 μL ΑCΝ.

For Ribociclib/Example 2/Example 4 1.5 μΜ spiking solution in microsomes (0.7S mg/mL): add l .S of 500 μΜ (Ribociclib/Example 2/Example 4) and 18.75 μL, of 20 mg/mL liver microsomes into 475.25 μL of Buffer C on ice.

3. Prepare NADPH stock solution (6 mM) by dissolving NADPH into buffer C.

4. Dispense 30 μL· of 1.5 μΜ spiking solution containing 0.75 mg/mL microsomes solution to the assay plates designated for different time points (0-, 5-, 15-, 30-, 45-min) on ice.

5. For 0-min, add 135 μL· of ACN containing IS to the wells of 0-min plate and then add 15 μL of NADPH stock solution (6 mM).

6. Pre-incubate all other plates at 37 °C for 5 minutes.

7. Add 15 μL of NADPH stock solution (6 mM) to the plates to start the reaction and timing.

8. At 5-min, 15-min, 30-min, and 45-min, add 135 μL of ACN containing IS to the wells of corresponding plates, respectively, to stop the reaction.

9. After quenching, shake the plates at the vibrator (IKA, MTS 2/4) for 10 min (600 rpm/min) and then centrifuge at 5594 g for 15 min (Thermo Mullifuge * 3R).

10. Transfer 50 μL of the supernatant from each well into a 96-weIl sample plate containing 50 μL· of ultra-pure water (Millipore, ZMQS50F01) for LC/MS analysis.

The positive control compound (Midazolam) was included in the assay. Any value of the compounds that was not within the specified limits was rejected and the experiment was repeated.

The negative control was used to exclude the misleading factor that resulted from instability of chemical itself.

The In peak area ratio (compound peak area/ internal standard peak area) was plotted against time and the gradient of the line was determined.

The elimination rate constant (k) = (- gradient)

Half-life (ti ₂ ) (minutes)

Results:

Table 1. Metabolic Stability in Human and Rat Liver Microsomes

The test compounds were evaluated in the human and rat liver microsome assay described above along with midazolam as a positive control and with no NADPH in reaction system as a negative control. The columns of Table 1 labeled "% remaining" refer to the percentage of each test compound remaining after 0, 5, 15, 30, and 45 minute intervals in the human.

As seen from Table 1, the deuterated analog (Example 2) of the invention displayed some stability over time with Ti ₂ extended to 74.63 min. from 72.34 min, providing a 3 % increase in human liver microsome; and to 54.61 min. from 52.98 min, providing a 3 % increase in rat liver microsome.

As seen from Table 1 , the deuterated analog (Example 4) of the invention displayed appreciable stability over time with T ₁ .2 extended to 83.5 min. from 72.34 min, providing a 15 % increase in human liver microsome; and to 69.99 min. from 52.98 min, providing a 32 % increase in rat liver microsome.

As seen from Table 1 , the deuterated analog (Example 4) of the invention also displayed appreciable clearance rate with Clint reduced to 20.82 mlJmin/kg from 24.03 mL/min/kg in human liver microsome and Clim reduced to 35.49 mL/min/kg from 46.88 mL/min/kg in rat liver microsome.

The results indicate that deuterated compounds according to the invention may exhibit beneficial properties when administered to patients, e.g., improved metabolic liability.

Example 12 NCI 60 cell one dose screen

The compound of Example 2 was screened Tor its anticancer activities at the Development Therapeutic Program (DTP), National Cancer Institute (NCI), USA, against full NCI 60 cell line panel (six cell lines of Leukemia, nine cell lines of Lung cancer, seven cell lines of Colon cancer, six cell lines of CNS cancer, nine cell lines of Melanoma, seven cell lines of Ovarian cancer, eight cell lines of Renal cancer, two cell lines of Prostate cancer and six cell lines of breast cancer) representing on full nine human systems as Leukemia, Melanoma and cancers of Lung, Colon, Brain, Breast, Ovary, Kidney and Prostate, according to their applied protocol. Primary in-vitro one dose anticancer assay was performed at a single dose (10 uM) against full NCI 60 cell lines panels. The one dose data reported as a mean graph of the percent growth of treated cells. The number reported for the single dose assay was growth relative to the no drug control and relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of -40 would mean 40% lethality. A value of -100 means all cells are dead. Results of one dose assay for the compound of Example 2 is shown in Table 2.

Table 2. Results of NCI 60 Cell One-Dose Screen.

The results indicate that the deuterated compound of Example 2 according to the invention significantly reduced the growth of the cell lines of leukemia RPMI-8226 (reducing to 1 1.5 %), MOLT-4 (reducing to 16.6 %), and HL-60 (TB) (reducing to 28.5 %); of breast cancer MCF7 (reducing to 47.9 %).

The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention.