WO2020202232A1

WO2020202232A1 - Compounds having anti-cdk4/6 and anti-cdk9 enzymatic activities for inhibiting cancer proliferation and related screening method for their detection

Info

Publication number: WO2020202232A1
Application number: PCT/IT2020/050079
Authority: WO
Inventors: Franco Lori; Davide De Forni; James G. Chafouleas
Original assignee: Virostatics Srl
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2020-10-08
Also published as: IT201900004737A1

Abstract

The present invention describes the surprising finding that only certain CDK inhibitors, those possessing a certain balance between the enzymatic activities against CDK4/6 and CDK9 protein complexes (for example an optimal ratio between 1:2 and 1:5) showed the highest efficacy in inhibiting cancer proliferation.

Description

COMPOUNDS HAVING ANTI-CDK4/6 AND ANTI-CDK9 ENZYMATIC ACTIVITIES FOR INHIBITING CANCER PROLIFERATION AND RELATED SCREENING METHOD FOR THEIR DETECTION

Background of invention

Cyclins and cyclin-dependent protein kinases (CDKs) are important regulatory components that are required for cell cycle progression and gene transcription. In humans, there are 20 members of this family: CDKs 1 , 2, 3, 4, 5 and 6 regulate cell cycle progression, while CDKs 7 through 13 regulate gene transcription. CDKs 14-20 are less understood but have widespread cellular activities. The levels of the cell cycle CDKs are controlled by cyclins, whose levels oscillate during each cell cycle. Additional CDK family members showed significant roles in a wide range of activities including the control of gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the first“G1” phase of the cell cycle produce cyclins of the D type that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein (“Rb”). Then CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2 transcription factor (“E2F”), which in turn lead to the generation of several proteins required for cell cycle progression. Cells then pass through the G1 -restriction point and are committed to complete cell division. CDK2-cyclin A, CDK1 -cyclin A, and CDK1 -cyclin B are required for S, G2, and M-phase progression. Increased cyclin or CDK expression or decreased levels of endogenous CDK inhibitors such as INK4 or CIP/KIP have been observed in various cancers. Given their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Commercially available drugs abemaciclib, ribociclib, and palbociclib target CDK4/6 with IC50 values in the low nanomolar range. Palbociclib and other CDK inhibitors bind in the cleft between the small and large lobes of the CDKs and inhibit the binding of ATP. CDK antagonists are in clinical trials for the treatment of a variety of malignancies. Abemaciclib, ribociclib and palbociclib have been approved by the FDA for the treatment of hormone-receptor positive/human epidermal growth factor receptor-2 negative breast cancer.

CDK9 and its partner Cyclin T comprise the positive transcription elongation factor b (P-TEFb), which promotes transcription elongation by phosphorilating Serine-2 of the RNA Polymerase II (Pol II) C-terminal domain (CTD) heptapeptide repeat, which in turn recruits other factors necessary for productive elongation, and also phosphorilating subunits of Negative Elongation Factor (NELF) and DRB (5,6-Dichloro-1 -p-D- ribofuranosylbenzimidazole) Sensitivity Inducing Factor (DSIF), inhibiting NELF’s ability to pause Pol II in a non-elongation competent state and convert DSIF into a positive elongation factor. Therefore, CDK9 has a crucial role in promoting transcription. Several observations have stimulated interest in the development of CDK9 inhibitors for clinical use. First, CDK9 promotes Pol II pause release, a rate-limiting step in normal transcriptional regulation that is frequently dysregulated in cancer. Second, modified natural products, such as flavopiridol, potently induce apoptosis in cancer cells, due in part to the loss of short-lived anti-apoptotic mRNA transcripts following CDK9 inhibition.

The inventors of the present patent application are developing CDK4/6/9 inhibitors to overcome existing disease class limitations. Palbociclib from Pfizer, abemaciclib from Lilly and ribociclib from Novartis, all approved for breast cancer, appear to be quite versatile and their initial indication (breast cancer) is presently expanding towards new indications. In addition to those, there are other CDKi materials targeting CDK9, which are only in early clinical or even preclinical development (e.g. at Bayer and at National Center for Tumor Diseases Heidelberg - NCT).

The presently claimed Example 2 clearly differentiates from prior art materials, because it exhibits the activity against three different CDK materials. Combined anti-CDK4, -CDK6 and -CDK9 activities within the same molecule is synergistic, because their combined effect is greater than would be predictable considering their individual potencies. Such synergistic effect has been demonstrated within a widely used statistical model [Prichard Antimicrob Agents Chemother 1993] and proved to be relevant in vivo according to the model assumptions. Such synergy provides the basis for the clear superiority of the CDK4/6/9 inhibitor Example 2 over the CDK4/6 inhibitor palbociclib and the CDK9 inhibitor LDC067 observed in all tumor cell lines tested so far.

Example 2 was not only active in vitro (human tumor cell lines, see Table 5, below) but also has demonstrated efficacy towards inhibition of cancer proliferation for the following tumors in vivo (xenograft mice models): a) Breast cancer resistant to CDK4/6 inhibitors: CDK4/6-Rb axis is important particularly in estrogen-receptor-positive breast cancer; anti- apoptotic proteins, BCL2 and MCL1 , and oncoprotein c-MYC are implicated in evasion of apoptosis and resistance to chemotherapy in both triple negative breast cancer (TNBC) and estrogen receptor-positive breast cancer (ER+ BC). Transcription of these oncogenic factors requires cyclin dependent kinase 9 (CDK9). CDK9 phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAPII) and acts as a rate-limiting step of transcription.

Approved CDK4/6 inhibitors (palbociclib, abemaciclib, ribociclib) represent, in combination with endocrine therapy, a standard of care for patients with advanced HR-positive/HER2-negative breast cancer. However, resistance to CDK4/6 inhibitors is now the major emerging consideration in preclinical and clinical drug development and development of resistance is inevitable. In trials reported so far, that have led to FDA and EMA approval, at least 1 /3 of patients recurred on CDK4/6 inhibitors within 2 years, and in the PALOMA-2 trial >70% of patients treated with the palbociclib plus letrozole had progressive disease by 40 months. Example 2 was active in vivo in a mouse xenograft model of breast cancer resistant to CDK4/6 inhibitors.

b) Malignant pleural mesothelioma (MPM): Malignant mesothelioma is a deadly and a rare type of cancer that affects the mesothelium, a membrane that forms a thin lining of the body’s internal organs (pleurae, peritoneum, and pericardium). Mesothelioma still lacks a molecular targeted treatment option, local tumor recurrence is common and represents the most relevant unmet medical need in mesothelioma-stricken patients even after complete surgical resection, with a life expectancy of 4-12 months. P16INK4A tumor suppressor encoding CDKN2A gene is often downregulated in MPM. P16INK4A is a CDK4/6 inhibitor and is involved in the regulation of cell cycle machinery.

Example 2 proved to be effective against MPM and also to MPM become resistant to CDK4/6 inhibitors.

c)Lung cancer: Lung cancer growth (specifically Non-Small Cell Lung Cancer, NSCLC) is driven by overexpression of cyclin D [Sherr N Engl J Med 2016]; furthermore, inactivation of Rb pathway is seen in more than 70% of NSCLC. This results in continuous activation of CDK4/6 complexes and therefore in uncontrolled cell proliferation [Dhakal J Targeted Ther 2017], thus representing a strong rationale to develop Example 2 for the treatment of lung cancer.

d)Pancreatic cancer. CDK4/6 is an important and actionable target in pancreatic cancer [Caldas Nature Genetics 1994; Schutte Cancer Research 1997; Wilentz Cancer Research 1998]. Also, CDK9 represents a good target in pancreatic cancer as its inhibition can synergize with DNA damaging agents [Nepomuceno Cell cycle 2017] In fact, Example 2 has shown synergy when tested in association with DNA damaging compounds such as Abraxane, SN-38 and oxaliplatin. In addition, high CDK9 expression in pancreatic tumor tissue is associated with significantly shortened survival. Inhibitors of CDK9 drastically reduced cell viability in pancreatic cancer cells and potently suppressed long-term survival, by inducing apoptosis and cell cycle arrest in a time-dependent manner by suppression of anti-apoptotic proteins [Kretz Tumor Biology 2017] Therefore, Example 2, by inhibiting CDK4/6 and CDK9, represents a novel and promising therapeutic approach for pancreatic cancer.

e)Lymphomas and Leukemias: Deregulation of CDK4/6/9 expression is found in lymphomas [Peyressatre Cancers 2015] which could thus become possible targets of CDKi. More specifically, inhibition of CDK9 downregulates transcription of genes involved in cell cycle progression and anti-apoptotic factors [Rajput Oncotarget 2016; Lam J Biol Chem 2001 ; Gojo Clin Cancer Res 2002; MacCallum Cancer Res 2005; Santo Oncogene 2010]. CDK9 inhibition thus affects cell proliferation by affecting cell cycle progression at the G2/M transition and induces apoptosis by reducing levels of anti-apoptotic proteins such as XIAP (X-linked inhibitor of apoptosis protein) and Mcl-1 (Myeloid Cell Leukemia 1 ).

Example 2 is active on diffuse large B cell lymphoma, follicular lymphoma, PEL, primary central nervous system lymphoma, mantle cell lymphoma and Kaposi sarcoma as well as on other hematological malignancies (i.e. leukemias).

Consistently with the observed synergy of the CDK4/6 and CDK9 pathways, Example 2 showed greater efficacy compared to anti-CDK4/6 or anti-CDK9 only drugs in solid and liquid tumor cell lines.

While screening a proprietary library of CDK inhibitors, including compounds listed in the present invention, against other materials such as palbociclib, abemaciclib and LDC067, it was unexpectedly found that there was no direct correlation between anti-CDK4/6 or anti-CDK9 enzymatic activity and the anticipated antitumoral efficacy. Instead, a balanced ratio between the two enzymatic activities was found to be necessary to yield optimal synergy and thus efficacy in inhibiting cancer proliferation.

Field of the invention

The invention therefore encompasses the discovery of the need of a certain balance between enzymatic activities against the different CDK proteins in order to optimize anti-cancer efficacy of CDK inhibitors.

The discovery required the experimental testing for the anti-cancer activity of the individual compounds, because their properties could not be deduced a priori just by examination of either their structure or their enzymatic activity against CDK4/cyclin D1 , CDK6/cyclin D1 and CDK9/cyclin T1 proteins.

According to the present invention, an improved method of inhibiting cancer proliferation has been found, the steps comprising selecting a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT 1 complexes is between about 1 :1 to 1 :10 or about 1 :1 to 10:1 , and administering an effective amount of said compound to a system containing cancer cells. A more preferred ratio is about 1 :1 to 1 :5 or about 1 :1 to 10:1. The most preferred ratio is about 1 :2 to 1 :5.

Another aspect of the present invention is an improved method of screening cancer compounds for inhibition of cancer proliferation, the steps comprising obtaining data relating to enzymatic activities against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes, comparing said activities, and selecting compounds having the desired activity ratios.

A key advantage of the present invention is that synergistic activity exists among a group of metabolic pathways. Another advantage is that it allows smaller amounts of a given medicament to be used.

Particularly preferred compounds tested and used in this invention include:

(Example 1 ) N^*1 ^*-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-yl]-4- methoxy-benzene-1 ,3-diamine;

(Example 2) 4-Fluoro-5-[6-(5-fluoro-2-methoxy-phenyl)-pyrimidin-4- ylamino]-2-methoxy-benzamide;

(Example 3) 2-methoxy-5-(6-(2-methoxyphenyl)pyrimidin-4- ylamino)phenol;

(Example 4) 4-Fluoro-5-[6-(4-fluoro-2-methoxy-phenyl)-pyrimidin-4- ylamino]-2-methoxy-benzamide;

(Example 5) 5-[6-(2-Ethoxy-4-fluoro-phenyl)-pyrimidin-4-ylamino]-4- fluoro-2-methoxy-benzamide;

(Example 6) 5-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-ylamino]- 2-methoxy-benzamide;(Example 7) 4-Fluoro-2-methoxy-5-[6-(2-methoxy- phenyl)-pyrimidin-4-ylamino]-benzamide.

Most preferred compound is (Example 2) 4-Fluoro-5-[6-(5-fluoro-2- methoxy-phenyl)-pyrimidin-4-ylamino]-2-methoxy-benzamide

The structures of the preferred compounds of examples 1 -7 are shown in Table 1. Table 1

Description of related art

In the context of the current invention, the following references present the general state of the art:

WO201 1077171 A1 by Greff, et. al, discloses certain 4-phenylamino- pyrimidine derivatives having protein kinase inhibitor activity. In particular, the invention described in WO201 1077171 A1 relating to novel 4,6- disubstituted aminopyrimidine compounds, has a main element of novelty based on arylaminopyrimidines that have a substituted aryl part containing -(CH2)n-W substituents (with n being preferably 1 to 3). Preferred meanings for W include methanesulfonamide, thiophene, indolyl or isoindolyl, phthalimido or benzoimidazo-1 -yl groups.

The compounds in the patent represent a library of compounds linking their CDK9/cyclin T 1 -inhibition activity with protein kinase-related diseases such as infectious diseases and cell proliferative diseases. Their structure is different from the compounds claimed in this patent application, because the compounds in patent WO201 1077171 A1 are only mono substituted pyrimidin-4-ylaminophenyl derivatives.

WO2014/031937 A1 by Lori, et al. discloses certain 4,6-disubstituted aminopyrimidine derivatives with anti-proliferative and anti-viral properties, possessing anti-CDK9/cyclin T1 activity. The inventors disclose that, unexpectedly, the results of the activity tests highlighted the lack of any direct correlation between CDK9 inhibitory activity and the anticipated anti- HIV activity or anti-proliferative activity, as compounds with potent CDK9- related IC50 exhibited poor anti-HIV profiles and vice versa because the disclosed compounds act by an as yet unknown mechanism. This patent is incorporated here by reference, as if set forth in full.

The compounds in the patent only refer to treatment of viral infections, not cancer. No claims nor experimental data on anti-cancer activity are inserted in such application. As hyperproliferation of the immune system has a key role in the pathogenesis of HIV, the anti-proliferative characteristic of the compounds has been described but only related to the effect on the cells of immune system, specifically of T cells, which are normal cells derived from healthy donors and are not cancer cells.

Moreover, in WO2014/031937 A1 there is no mentioning of anti- CDK4 or anti-CDK9 activity.

Benson et al.,“A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib(CYC2020; R-Roscovitine), administered twice daily for 7 days every 21 days” (British J Cancer 2006), is related to the use of Roscovitine in patients with tumors. Roscovitine is not known to possess at the same time anti-CDK4/6 and anti-CDK9 activity, i.e. the unique inhibition profile of the compounds described in the present application, and therefore no correlation between anti-CDK4/6 and anti-CDK9 enzymatic activity ratio and anti-tumoral activity can be drawn, that is the subject of the present application.

J.J. Luke et al.,”The cyclin-Dependent Kinase Inhibitor Flavopiridol Potentiates Doxorubicin Efficacy in Advanced Sarcomas: Preclinical Investigations and Results of a Phase I Dose-Escalation Clinical Trial” (Clinical Cancer Research 2012), discuss the use of flavopiridol in patients with advanced sarcomas (i.e. cancer). Flavopiridol is shown to be a pan- CDK inhibitor that specifically inhibits CDK2, CDK4, CDK6 and CDK9 at nanomolar concentrations (no exact IC50 value is provided for enzymatic activity) i.e. it does not inhibit CDK4/6 and CDK9 only. The paper does not disclose or discuss any correlation between anti-CDK4/CDK6 and anti- CDK9 enzymatic activity ratio and anti-tumoral activity.

G. Nemeth et al., "Novel selective CDK9 inhibitors for the treatment of H IV infection” (Current Medicinal Chemistry 201 1 ), is disclosing compounds with anti-CDK9 activity. The inventors of the present application tested the materials of the Nemeth reference and found no correlation between CDK9/CycT1 inhibition and H IV inhibition. Certain compounds in the paper have been characterized also for activity on other CDK/cyc complexes (including CDK4/CycD1 and CDK6/CycD1 ) to understand their selectivity towards CDK9, therefore the inventors were able to derive the ratio between CDK4/6 and CDK9 inhibition (see Table 2).

Table 2

To calculate the proportion between CDK4/6 inhibition activity and CDK9 inhibition activities, a mean of CDK4 and CDK6 inhibition was obtained.

Only compounds 57 (4-(2-methoxyphenyl)-6-[3- (sulfamoylamino)anilino]pyrimidine), 72 (N1 -[6-(2- methoxyphenyl)pyrimidin-4-yl]-4-(4-methylpiperazin-1 -yl)-N3- (propylsulfamoyl)benzene-l ,3-diamine), 78 (tert-butyl N-[[5-[[6-(2- methoxyphenyl)pyrimidin-4-yl]amino]-2-morpholino- phenyl]sulfamoyl]carbamate), 100 84-[[6-(2-methoxyphenyl)pyrimidin-4- yl]amino]-N-(2-pyrrolidin-1 -ylethyl)benzenesulfonamide) and 101 (N-(2- dimethylaminoethyl)-4-[[6-(2-methoxyphenyl)pyrimidin-4- yl]amino]benzenesulfonamide) have a ratio between 1 :3 and 1 :10. Such compounds are claimed to possess anti-HIV activity (though very low, i.e. less than 10% inhibition compared to control at the tested concentration, Fig. 5 of the paper) and not anti-cancer activity. Other compounds in the table do exhibit much higher ratios though having similar (again, very low) anti-HIV activity, therefore no correlation between ratio and anti-HIV activity can be derived from the paper. Such compounds are not claimed in the present application.

WO2018/005533 A1 (G1 Therapeutics Inc) discloses compounds which have different chemical structures from the ones disclosed in the present application. For certain compounds of document WO2018/005533 enzymatic activities against CDK4/CDK6 and CDK9 are shown and data are summarized in the following Table 3.

Table 3

Compounds 20 (1 -(2-{[5-(4-Methylpiperazin-1 -yl)pyridin-2- yl]amino}pyrimidin-4-yl)-1 ,4-diazaspirol[5.5]undecan-3-one) and 32 (4-2- {[5-(4-Methylpiperazin-1 -yl)pyridin-2-yl]amino}pyrimidin-4-yl)piperazin-2- one) have a ratio of 1 :1. Compounds 6 (1 -(2-{[5-(4-Methylpiperazin-1 - yl)pyridin-2-yl]amino}pyrimidin-4-yl)-1 ,3-diazaspirol[4.5]decan-2-one) and 16 (1 -(2-{[5-(4-Methylpiperazin-1 -yl)pyridin-2-yl]amino}pyrimidin-4- yl)imidazolin-2-one) have a ratio between 2:1 and 3:1. However, there is no mentioning of anti-cancer activity for these compounds. Therefore, such patent provides no guidance for the inventors’ area of interest as no correlation between ratio and anti-cancer activity is described.

Brief summary of the invention

As discussed above, according to the present invention it has now surprisingly been found that only certain CDK inhibitors, those possessing a certain balance between the enzymatic activities against CDK4/6 and CDK9 protein complexes (an optimal ratio between 1 :1 and 1 :10), showed the highest efficacy in inhibiting cancer proliferation.

According to the present invention, when a library of proprietary CDK inhibitors was experimentally tested, a number of compounds with optimal efficacy against tumor cells was identified, in particular Example 2 was identified as a clinical candidate which showed an efficacy superior to that of CDK inhibitors already in the market (palbociclib, ribociclib, abemaciclib) or in preclinical or clinical stage of development.

The Compounds identified according to the present invention, possessing these particular characteristics, exert a potent stand-alone anti cancer activity, irrespective of their activity against each individual CDK/cyclin.

Particularly the preferred compounds (and physiologically acceptable salts thereof) of the present invention and their ratio between the enzymatic activities against CDK4/6 and CDK9 protein are shown in Table 4. These compounds were initially disclosed and claimed as raw materials in the inventor’s application WO2014/031937 A1 , which is incorporated herein as if set forth in full.

Table 4

Chemical structures for the preferred compounds include, for Examples 2, 6, 7, 4, 4,6-disubstituted aminopyrimidines represented by formula (I), having the following general formula:

Where X, Y and Z are H, F, or Cl

R\si is 2’-OCH3 and R\S3 is OCH3 and R\S2 is CONH2

Another preferred structure is Example 5, a compound comprising a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Ring 1 Ring 2 Ring 3

(I)

Where X is 4’-F, Y is H and Z is 2’-F,

R\si is 2’-OCH2CH₃, R\S₃ is OCH₃ and R\s₂ is CONH2

Yet another preferred compound is Example 1 , a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula: Where X is 4’-F, Y and Z are H,

R\si is 2’-OCH₃, R\S3 is OCH₃ and R\s₂ is NH2

A further preferred compound is Example 3, a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Ring 1 Ring 2 Ring 3

(I)

Where X, Y and Z are H,

R\si is 2’-OCH3, R\S3 is OCH3 and R\S2 is OH.

It is, therefore, a specific object of the present invention a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 , for use in the treatment of cancer by inhibiting cancer proliferation.

The present invention concerns also a non-therapeutic use of a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 , for inhibiting cancer proliferation in a system containing cancer cells, for example in an in vitro system.

The compounds for use according to the present invention do not comprise the following compounds: flavopiridol, abemaciclib, roscovitine.

According to a further embodiment, the compound of the invention for the therapeutic use mentioned above or non-therapeutic use of the compound of the invention can be characterized by the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes ranging from about 1 :1 to 1 :5 or from 1 :1 to 1 :3, from 1 :1 to 1 :2, from 1 :2 to 1 :5, from 1 :2 to 1 :3 or about 1 :1 to 5:1.

According to an embodiment, the present invention concerns a method of inhibiting cancer proliferation, the steps comprising selecting a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 , and administering an effective amount of said compound to a system containing cancer cells.

In particular, the invention concerns a method of inhibiting cancer proliferation, the steps comprising selecting a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :5, from 1 :1 to 1 :3, from 1 :1 to 1 :2, from 1 :2 to 1 :5, from 1 :2 to 1 :3 or from about 1 :1 to 5:1 , and administering an effective amount of said compound to a system containing cancer cells.

According to the present invention, the compound for the above mentioned therapeutic or non-therapeutic uses can be:

- a compound, such as the compounds indicated above as EXAMPLE 2, EXAMPLE 6, EXAMPLE 7, EXAMPLE 4, comprising a 4,6- disubstituted aminopyrimidine represented by formula (I), having the following general formula: Where X, Y and Z are H, F, or Cl,

R\si is 2’-OCH₃, R\S3 is OCHs and R\s₂ is CONH₂;

- a compound, such as the compound indicated above as EXAMPLE 5, comprising a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Where X, Y and Z are H, F, or Cl,

R\si is 2’-OCH2CH₃, R\S₃ is OCHs and R\s₂ is CONH2;

- a compound, such as the compound indicated above as EXAMPLE

1 , comprising a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Ring 1 Ring 2 Ring 3

(I)

Where X, Y and Z are H, F, or Cl,

R\si is 2’-OCH₃, R\S₃ is OCH₃ and R\S2 is NH2; - a compound, such as the compound indicated above as EXAMPLE 3, comprising a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Ring 1 Ring 2 Ring 3

(I)

Where X, Y and Z are H, F, or Cl,

R\si is 2’-OCH3, R\S3 is OCH3 and R\S2 is OH.

Particularly, the compounds can be selected among the following compounds:

(Example 1 ) N^*1 ^*-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-yl]-4- methoxy-benzene-1 ,3-diamine

(Example 2) 4-Fluoro-5-[6-(5-fluoro-2-methoxy-phenyl)-pyrimidin-4- ylamino]-2-methoxy-benzamide

(Example 3) 2-methoxy-5-(6-(2-methoxyphenyl)pyrimidin-4-ylamino)phenol (Example 4) 4-Fluoro-5-[6-(4-fluoro-2-methoxy-phenyl)-pyrimidin-4- ylamino]-2-methoxy-benzamide

(Example 5) 5-[6-(2-Ethoxy-4-fluoro-phenyl)-pyrimidin-4-ylamino]-4-fluoro- 2-methoxy-benzamide

(Example 6) 5-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-ylamino]-2- methoxy-benzamide

(Example 7) 4-Fluoro-2-methoxy-5-[6-(2-methoxy-phenyl)-pyrimidin-4- ylamino]-benzamide, preferably, 4-Fluoro-5-[6-(5-fluoro-2-methoxy- phenyl)-pyrimidin-4-ylamino]-2-methoxy-benzamide.

According the present invention, cancer is selected among solid and liquid cancers, such as breast cancer, such as breast cancer resistant to CDK4/6 inhibitors, malignant mesothelioma such as of pleurae, peritoneum and pericardium, lung cancer such as non-small cell lung cancer and squamous cell lung cancer, pancreatic cancer, lymphomas and leukemias, large B cell lymphoma, follicular lymphoma, primary effusion lymphoma, primary central nervous system lymphoma, mantle cell lymphoma, sarcoma such as Kaposi sarcoma, as well as on other hematological malignancies, glioblastoma, melanoma, ovary cancer, gastric cancer, head/neck cancer, colon cancer, endometrial cancer, cervix cancer, prostate cancer; preferably breast cancer, mesothelioma, lung cancer, pancreatic cancer, lymphomas and leukemias.

The present invention concerns also a method of screening cancer compounds for inhibition of cancer proliferation, the steps comprising obtaining data relating to enzymatic activities against CDK4/cyclinD1 - CDK6/cyclinD1 and CDK9/cyclinT1 complexes, comparing said activities, and selecting compounds having activity ratios from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 .

According to an embodiment of the present invention, the method of screening cancer compounds for inhibition of cancer proliferation, can comprise the steps of obtaining data relating to enzymatic activities against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes, comparing said activities, and selecting compounds having activity ratios from about 1 :1 to 1 :5, from 1 :1 to 1 :3, from 1 :1 to 1 :2, from 1 :2 to 1 :5, from 1 :2 to 1 :3 or from about 1 :1 to 5:1 .

The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

Figure 1 shows a synergy plot showing regions of synergy when combining CDK4/6 and CDK9 inhibition, together with statistical analysis performed at the 99.9% confidence.

EXAMPLE: in vitro study

Methods and results

In Vitro CDK4/CyclinD1, CDK6/CyclinD1, CDK9/CyclinT Assays

A radiometric protein kinase assay was used for measuring the kinase activity of the protein kinases. All kinase assays were performed in 96-well FlashPlatesTM from Perkin Elmer (Boston, MA, USA) in a 50 pi reaction volume. The reaction cocktail was pipetted in 4 steps in the following order:

• 10 mI of non-radioactive ATP solution (in H2O)

• 25 mI of assay buffer/ [y-33P]-ATP mixture

• 5 mI of test sample in 10% DMSO

· 10 mI of enzyme/substrate mixture

The assay for all protein kinases contained 70 mM HEPES-NaOH pH7.5, 3mM MgCI2, 3 mM MnCI2, 3 mM Na-orthovanadate, 1.2 mM DTT, ATP (variable amounts, corresponding to the apparent ATP-Km of the respective kinase), [y-33P]-ATP (approx. 8 x 1005 cpm per well), protein kinase (variable amounts), and substrate.

All protein kinases were expressed in Sf9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins (the preparation and use of protein kinases have been done in Germany by ProQinase company according to the German legislation: “Gentechnik Gesetz (GenTG)”; "Gentechnik Sicherheitsverordung (GenTSV)").

All kinases were produced from human cDNAs proprietary to ProQinase. Kinases were purified by either GSH-affinity chromatography or immobilized metal affinity chromatography. Affinity tags were removed from a number of kinases during purification. The purity of the protein kinases was examined by SDS-PAGE/Coomassie staining, the identity was checked by mass spectroscopy.

CDK4/6/9 IC50 values (nM) of the compounds are shown in Table 5.

Table 5

Example 8 (WO2014031937A1 ) is A/-(4-methoxy-3-

((methylamino)methyl)phenyl)-6-(2-methoxyphenyl)pyrimidin-4-amine, example 9 (WO201107717A1 ) is N-{3-[6-(2-Ethoxy-phenyl)-pyrimidin-4- ylamino]-benzyl}-methanesulfonamide.

To calculate the proportion between CDK4/6 inhibition activity and CDK9 inhibition activities, a mean of CDK4 and CDK6 inhibition was obtained. Ratio between anti-CDK4/6 and anti-CDK9 activity has been calculated using known methodologies, the screening method is not intended to guide the synthesis of novel compounds but rather to identify those ones with the optimal ratio, which is predictive of high anti-cancer activity. The method of screening according to the present invention can be applied on compounds that are already known to have anti-CDK4/6 and anti-CDK9 activity. Alternatively, a compound can be tested in order to know if it has anti-CDK4/6 and anti-CDK9 activity and then the method of the invention can be applied. Tested compounds showed different proportions between anti-CDK4/6 and anti-CDK9 enzymatic activities. A proportion between anti-CDK4/6 and anti-CDK9 enzymatic activities of 1 :2 was most commonly observed among compounds claimed in this patent application.

Biological Assays

The biological activity of the compounds described in the present invention was evaluated in the following assays:

Synergy between CDK4/6 and CDK9 cell inhibition activity pathways

Synergy of the CDK4/6 and CDK9 pathways was demonstrated by assessing the antitumoral effect of a CDK4/6 inhibitor in combination with a CDK9 inhibitor in K562 cells (from ATCC CCL-243), using the well- established Bliss Independence Model (BIM) and MacSynergy II Software for data analysis (99% Confidence)(Prichard, Antivir Res, 1990). BIM assumes that the relative effect of a drug at a given concentration is independent of the presence of the other drug (additivity) and calculates any deviation from this as either synergism or antagonism. Antitumoral activity data obtained at non-cytotoxic concentrations were used to determine synergy and antagonism values. Statistical analysis was performed to determine significant synergy and/or antagonism at the levels of 95%, 99% and 99.9% confidence.

Table 6 illustrates the regions (volumes) of synergy (cluster of data cells significantly above zero) obtained when combining a CDK4/6 inhibitor and a CDK9 inhibitor at the different concentrations. According to the model, a log volume value > 2 is indicative of significant synergy.

Table 6

A region of synergy or antagonism is identified by a cluster of data cells significantly above or below zero and the concentrations of drug responsible for this effect can be read from the data sheet (volumes of synergy and antagonism in units of mM²%).

The inventors found that there is a synergy between the CDK4/6 and the CDK9 inhibition pathways, however, the synergy is found only at discrete concentration of CDK4/6 and CDK9 inhibitors, indicating that a discrete proportion of CDK4/6 and the CDK9 inhibitory activity is required for optimal synergy (Figure 1 ). Anticancer activity in vitro against tumor cell lines

The anticancer activity of the selected compounds was tested in several cancer cell lines (Rec-1 from ATCC CRL-3004, Granta 519 from Mario Negri Institute, Mino-1 from ATCC CRL-3000, BC-3 from ATCC CRL- 2277, BCP-1 from ATCC CRL-2294, MV-4-1 1 from ATCC CRL-9591 , THP-

1 from ATCC TIB-202, HL60 from ATCC CCL-240, K562 from ATCC CRL- 3344, Jurkat from ATCC TIB-152, H1299 from ATCC CRL-5803, BXPC-3 from ATCC CRL-1687, A2780 from Mario Negri Institute) using standardized MTS cytotoxicity assay. Briefly, exponentially growing cells were seeded into 96-well plates at their optimal density in complete medium.

72 hours later cells were treated or not with different concentrations (ranging 100 - 0.0001 mM) of the selected compounds for 72 hours using 2 replicates for each concentration and the anticancer effect was evaluated through MTS Colorimetric Assay (Promega). Dilutions of the compounds were performed in complete medium. The IC50 values were interpolated after constructing a dose-response curve. At least three experiments were performed for each cell line.

In all the biological assays, commercially available CDK inhibitors were also included in the analysis: palbociclib, ribociclib, abemaciclib and LDC067.

The inventors tested inhibition of cancer proliferation (by determining “IC50”) of compounds in several types of cancer. IC50 values, together with CDK4/6 and CDK9 inhibition proportions, are reported in the following table. Lower IC50 values indicate higher potency. “N.A.” means“Not available”.

Inhibition of cancer proliferation (expressed as IC50, mM) of the compounds in MTS proliferation assay is shown in Table 7, wherein antitumor activity is indicated using a scale going from very high (++++) to low (-).

When compounds with anti-CDK4/6 or anti-CDK9 activity were tested, these test results were compared to those of the commercially available compounds palbociclib, abemaciclib and LDC067. Unexpectedly, it was not found a direct correlation between CDK4/6 or CDK9 inhibition, if analyzed separately, and inhibition of cancer proliferation. For instance, Example 2, bearing low anti-CDK4/6 and anti-CDK9 activity, that was 2,970, 2,640 and 1 ,255 nanomolar, respectively, (see Table 3) was more potent than palbociclib, bearing high anti-CDK4/6 activity and moderate anti-CDK9 activity, that was 6, 3, and 860 nanomolar, respectively, (see Table 5) and also more potent than abemaciclib, bearing high anti-CDK4/6 activity and anti-CDK9 activity, that was 2, 10, and 57 nanomolar, respectively (see Table 5).

Instead, it was unexpectedly found that the balance between anti- CDK4/6 and anti-CDK9 activity was necessary for maximal inhibition of cancer proliferation. For instance, Example 2, having a proportion between anti-CDK4/6 and anti-CDK9 enzymatic activities of 1 :2, was 10- to 1 ,000- fold more potent than CDK4/6 inhibitors and CDK9 inhibitors having an unbalanced proportion between the two enzymatic activities (e.g. palbociclib=200:1 ; LDC067=1 :200). Compared to other CDK4/6 inhibitors, abemaciclib possesses a higher anti-CDK9 activity and a better balance between CDK4/6/9 inhibition pathways (proportion between anti-CDK4/6 and anti-CDK9 enzymatic activities of 10:1 ). Abemaciclib also shows intermediate potency in inhibiting cancer proliferation, less than each of the Examples of the present invention.

For the above reasons it can be derived that a 1 :1 ratio would be indicative of even higher anti-cancer activity.

Through the screening according to the present invention it was found that balancing CDK4/6 and CDK9 enzymatic inhibition results in a logarithmic increase of inhibition of cancer proliferation. CDK4/6 and CDK9 enzymatic activities (IC50) must be balanced to achieve optimal synergy.

Anti-cancer activity of certain Examples has been evaluated in a larger number of tumor types, test results were compared to those of the commercially available compounds palbociclib, abemaciclib and LDC067, as shown in the following Table 8 A-D.

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The results show that the compounds 1 -7 of the present invention have higher effectiveness in comparison to the compounds of the state of the art.

Claims

1 . Compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT 1 complexes is from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 , for use in the treatment of cancer by inhibiting cancer proliferation, with the proviso that said compound is not Flavopiridol or abemaciclib.

2. Non-therapeutic use of a compound having both CDK4/6 and CDK9 activity, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :10 or from about 1 :1 to 10:1 , for inhibiting cancer proliferation in a system containing cancer cells, with the proviso that said compound is not Flavopiridol or abemaciclib.

3. Compound according to claim 1 for use according to claim 1 or non- therapeutic use of the compound according to claim 2, wherein the ratio of enzymatic activity against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes is from about 1 :1 to 1 :5, from 1 :1 to 1 :3, from 1 :1 to 1 :2, from 1 :2 to 1 :5, from 1 :2 to 1 :3 or from about 1 :1 to 5:1 .

4. Compound according to anyone of claims 1 , 3 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-3, wherein the compound comprises a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Where X, Y and Z are H, F, or Cl

R\si is 2’-OCH3 and R\S3 is OCH3 and R\S2 is CONH2

5. Compound according to anyone of claims 1 , 3 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-3, wherein the compound comprises a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Where X, Y and Z are H, F, or Cl

R\si is 2’-OCH2CH₃, R\S₃ is OCH₃ and R\s₂ is CONH2

6. Compound according to anyone of claims 1 , 3 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-3, wherein the compound comprises a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Where X, Y and Z are FI, F, or Cl

R\si is 2’-OCH₃, R\S3 is OCH₃ and R\s₂ is NH2

7. Compound according to anyone of claims 1 , 3 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-3, wherein the compound comprises a 4,6-disubstituted aminopyrimidine represented by formula (I), having the following general formula:

Where X, Y and Z are H, F, or Cl

R\si is 2’-OCH3, R\S3 is OCH3 and R\S2 is OH

8. Compound according to anyone of claims 1 , 3-7 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-7, wherein the compound is selected from:

N^*1 ^*-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-yl]-4-methoxy- benzene-1 ,3-diamine

4-Fluoro-5-[6-(5-fluoro-2-methoxy-phenyl)-pyrimidin-4-ylamino]-2- methoxy-benzamide

2-methoxy-5-(6-(2-methoxyphenyl)pyrimidin-4-ylamino)phenol

4-Fluoro-5-[6-(4-fluoro-2-methoxy-phenyl)-pyrimidin-4-ylamino]-2- methoxy-benzamide

5-[6-(2-Ethoxy-4-fluoro-phenyl)-pyrimidin-4-ylamino]-4-fluoro-2- methoxy-benzamide

5-[6-(4-Fluoro-2-methoxy-phenyl)-pyrimidin-4-ylamino]-2-methoxy- benzamide

4-Fluoro-2-methoxy-5-[6-(2-methoxy-phenyl)-pyrimidin-4-ylamino]- benzamide, preferably 4-Fluoro-5-[6-(5-fluoro-2-methoxy-phenyl)- pyrimidin-4-ylamino]-2-methoxy-benzamide.

9. Compound according to anyone of claims 1 , 3-8 for use according to claim 1 or non-therapeutic use of the compound according to anyone of claims 2-8, wherein the cancer is selected among solid and liquid cancers, such as breast cancer, such as breast cancer resistant to CDK4/6 inhibitors, malignant mesothelioma such as of pleurae, peritoneum and pericardium, lung cancer such as non-small cell lung cancer and squamous cell lung cancer, pancreatic cancer, lymphomas and leukemias, large B cell lymphoma, follicular lymphoma, PEL, primary central nervous system lymphoma, mantle cell lymphoma, sarcoma such as Kaposi sarcoma, as well as on other hematological malignancies, glioblastoma, melanoma, ovary cancer, gastric cancer, head/neck cancer, colon cancer, endometrial cancer, cervix cancer, prostate cancer, preferably breast cancer, mesothelioma, lung cancer, pancreatic cancer, lymphomas and leukemias.

10. Method of screening cancer compounds for inhibition of cancer proliferation, the steps comprising obtaining data relating to enzymatic activities against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes, comparing said activities, and selecting compounds having activity ratios from about 1 :1 to 1 :10 or from about 1 :1 to 10:1.

1 1. Method of screening cancer compounds for inhibition of cancer proliferation, the steps comprising obtaining data relating to enzymatic activities against CDK4/cyclinD1 -CDK6/cyclinD1 and CDK9/cyclinT1 complexes, comparing said activities, and selecting compounds having activity ratios from about 1 :1 to 1 :5, from 1 :1 to 1 :3, from 1 :1 to 1 :2, from 1 :2 to 1 :5, from 1 :2 to 1 :3 or about 1 :1 to 5:1.