RU2823603C2 - Co-crystals and pharmaceutical compositions containing thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to a method for potentiating a therapeutic regimen for treating cancer mediated by DNA-dependent protein kinase (DNA-PK) activity in a patient, comprising administering to said patient an effective amount of a co-crystal containing a compound of formula:

and a co-crystal former, where the co-crystal former is adipic acid, where each of R¹ and R² is deuterium, where the molar ratio of adipic acid to the compound of formula (I) is 1:2, where the compound has reflection peaks in the powder X-ray diffraction pattern at 6.46+/-0.2, 7.91+/-0.2, 11.92+/-0.2, 12.26+/-0.2, 12.99+/-0.2, 14.19+/-0.2, 18.68+/-0.2 and 19.07+/-0.2 °2-theta, where the cancer is breast cancer, gastroesophageal cancer or lung cancer, where the therapeutic regimen involves radiotherapy or chemotherapy, and where the chemotherapy is carried out with a therapeutic agent selected from etoposide, doxorubicin and bleomycin.

EFFECT: novel method for potentiating a therapeutic regimen for treating cancer is developed, involving administering to said patient an effective amount of a co-crystal containing a compound of formula (I).

3 cl, 31 dwg, 21 tbl, 14 ex

Description

Related applications

This application claims priority to U.S. Provisional Application No. 61/892002, filed October 17, 2013, which is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present invention relates to co-crystals of DNA-dependent protein kinase (DNA-PK) inhibitors. The present invention also relates to pharmaceutical compositions comprising them and methods of using the co-crystals and compositions in the treatment of cancer.

Prerequisites for creating the invention

Ionizing radiation (IR) causes a variety of DNA damage, of which double-strand breaks (DSBs) are the most cytotoxic. These DSBs can lead to cell death through apoptosis and/or mitotic catastrophe if they are not rapidly and completely repaired. In addition to IR, several chemotherapeutic agents, including topoisomerase II inhibitors, bleomycin, and doxorubicin, also provoke DSBs. These DNA lesions initiate a complex set of signals through the DNA damage response network, which acts to repair damaged DNA and maintain cell viability and genomic stability. In the case of mammalian cells, the dominant pathway for repair of DSBs is the nonhomologous end joining (NHEJ) pathway. This pathway operates independently of cell cycle phase and does not require a template to re-ligate the ends of broken DNA. NHEJ requires the coordination of many proteins and signaling pathways. The NHEJ core structure consists of a Ku70/80 heterodimer and a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which together encompass the active DNA-PK enzyme complex. DNA-PKcs is a member of the phosphatidylinositol 3-kinase-dependent kinase (PIKK) family of serine/threonine protein kinases, which also includes ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-dependent ataxia telangiectasia (ATR), mTOR, and four isoforms of PI3K. However, since DNA-PKcs is in the same family of protein kinases as ATM and ATR, these latter kinases act to repair DNA damage through the homologous recombination (HR) pathway and are restricted to the S and G ₂ phases of the cell cycle. While ATM is also recruited to sites of DSBs, ATR is recruited to sites of single-stranded DNA breaks.

NHEJ is believed to act through three key steps: recognition of DSBs, processing of DNA to remove unligated ends or other forms of end damage, and finally ligation of DNA ends. DSB recognition is accomplished by binding of the Ku heterodimer to dangling DNA ends, followed by the recruitment of two DNA-PKs molecules to adjacent DSB sites; this serves to protect the ends of the breaks until additional processing enzymes are recruited. Recent evidence supports the hypothesis that DNA-PKcs phosphorylates the processing enzyme, Artemis, as well as itself, before obtaining DNA ends for additional processing. In some cases, DNA polymerase may be needed to synthesize new ends prior to the ligation step. Autophosphorylation of DNA-PKcs is believed to cause a conformational change that opens the central DNA-binding cavity, releases DNA-PKcs from DNA, and facilitates eventual religation of DNA ends.

It has been known for some time that DNA-PK ^−/− mice are hypersensitive to IR exposure and that certain nonselective small molecule inhibitors of DNA-PKcs can radiosensitize multiple tumor cell types across a wide range of genetic backgrounds. While DNA-PK inhibition is thought to radiosensitize normal cells to some extent, this is observed to a lesser extent than in tumor cells, likely due to the fact that tumor cells have higher basal levels of endogenous replication stress and DNA damage ( tumor-induced replication stress) and DNA repair mechanisms are less efficient in tumor cells. Most importantly, an improved therapeutic window with greater sparing of normal tissue should be obtained from the DNA-PK inhibitor combination along with new improvements in precise delivery of focused IR, including image-directed radiotherapy (IGRT) and intensity-modulated radiotherapy (IMRT). ).

Inhibition of DNA-PK activity induces effects both in cycling cells and also in non-cycling cells. This is important because most cells in a solid tumor are not actively replicating at any given time, limiting the effectiveness of many cell cycle-targeting agents. Equally interesting are recent reports that suggest a strong link between inhibition of the NHEJ pathway and the ability to neutralize radioresistant cancer stem cells (CSCs). In some tumor cells, DSBs in dormant CSCs have been shown to predominantly activate DNA repair through the NHEJ pathway; It is believed that CSCs are usually present in the stationary phase of the cell cycle. This may explain why a proportion of cancer patients may experience local or distant tumor recurrence despite treatment, as current strategies fail to effectively target CSCs. DNA-PK inhibitors may have the ability to sensitize these potential metastatic progenitor cells to the effects of IR and select DSB-inducing chemotherapeutic agents.

With respect to the involvement of DNA-PK in DNA repair processes, DNA-PK inhibitory drugs may act as agents that enhance the effectiveness of both anticancer chemotherapy and radiotherapy. The present invention relates to crystalline compositions of DNA-PK inhibitors together with co-crystal formers (CCFs), that is, co-crystals. Compared to their free form(s), the co-crystals of the present invention are useful because these compounds have improved solubility, higher water solubility and higher physical stability in the solid state than amorphous dispersions. The co-crystals described in this context also provide a reduced volume of the dosage form and therefore a lower tablet weight, since these co-crystals also exhibit higher bulk densities relative to amorphous forms. Further, the co-crystals of this invention provide production advantages over amorphous forms that require spray drying, lyophilization or precipitation.

Brief description of graphic material

Figure 1 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and adipic acid (AA).

Figure 2 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 2 and adipic acid.

Figure 3 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and citric acid.

Figure 4 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and fumaric acid.

Figure 5 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and maleic acid.

Figure 6 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and succinic acid.

Figure 7 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and benzoic acid.

Figure 8 shows a thermogram of thermogravimetric analysis of the co-crystal formed between compound 1 and adipic acid.

Figure 9 shows a thermogram of thermogravimetric analysis of the co-crystal formed between compound 2 and adipic acid.

Figure 10 shows a differential scanning calorimetry thermogram of the co-crystal formed between compound 1 and adipic acid.

Figure 11 shows a differential scanning calorimetry thermogram of the co-crystal formed between compound 2 and adipic acid.

Figure 12 shows the NMR spectrum of the solid state co-crystal formed between compound 1 and adipic acid.

Figure 13 shows the NMR spectrum of the solid state co-crystal formed between compound 2 and adipic acid.

Figure 14 shows the X-ray powder diffraction pattern of the form A polymorph of the cocrystal formed between compound 1 and adipic acid.

Figure 15 shows an X-ray powder diffraction pattern of the form A polymorph of the cocrystal formed between compound 2 and adipic acid.

Figure 16 shows the NMR spectrum of polymorph Form A of the co-crystal in the solid state formed between compound 1 and adipic acid.

Figure 17 shows the NMR spectrum of polymorph Form A of the co-crystal in the solid state formed between compound 2 and adipic acid.

Figure 18 shows the NMR spectrum of the polymorph Form B of the co-crystal in the solid state formed between compound 2 and adipic acid.

Figure 19 shows a binary phase diagram of compound 2 and adipic acid.

Figure 20 shows a solubility diagram at the calculated pH value of the co-crystal formed between compound 2 and adipic acid (with excess adipic acid content) and compound 2 in free form.

Figure 21 shows two-phase dissolution profiles for: i) co-crystal of compound 1 :adipic acid obtained by melt extrusion (HME) and suspension crystallization (SC); ii) HME 65:35: co-crystal of compound 1 : adipic acid, obtained by using melt extrusion, with 65% wt. compounds 1 and 35% wt. adipic acid; iii) HME 75:25: co-crystal of compound 1 : adipic acid, obtained using melt extrusion, with 75% wt. compounds 1 and 25% wt. adipic acid; iv) HME 80:20: co-crystal obtained using melt extrusion, with 80% wt. compounds 1 and 20% wt. adipic acid; v) SC 80:20: crystallized from a co-crystal suspension of compound 2 : adipic acid with a final content of compound 2 of 79% wt. and 21% wt. adipic acid; and vi) free form: free form of compound 2 .

Figure 22 shows the predicted fraction absorbed on the co-crystal formed between compound 2 and adipic acid and the free form of compound 2 .

Figure 23 is a diagram summarizing the Bliss analysis of compound ( 2 ) in combination with a panel of cytotoxic and non-cytotoxic agents.

FIG. 24 is a chart summarizing the Bliss assay of compound ( 2 ) in combination with BMN-673 according to tumor type.

Figure 25 is a chart summarizing the Bliss analysis of compound ( 2 ) in combination with etoposide according to tumor type.

Figure 26 is a chart summarizing the Bliss analysis of compound ( 2 ) in combination with bleomycin according to tumor type.

Figure 27 is a chart summarizing the Bliss analysis of compound ( 2 ) in combination with erlotinib according to tumor type.

Figure 28 is a graph summarizing the Bliss analysis of compound ( 2 ) in combination with doxorubicin according to tumor type.

Figure 29 is a chart summarizing the Bliss analysis of compound ( 2 ) in combination with bleomycin according to tumor type.

Figure 30 is a chart summarizing the Bliss analysis of compound ( 2 ) in combination with carboplatin according to tumor type.

FIG. 31 is a graph summarizing the Bliss analysis of Compound 1 or Compound 2 and the standard of combinations of interest in primary tumor chemotherapy drug sensitivity tests in humans.

Summary of the invention

In a first aspect, this invention relates to a co-crystal containing a compound of formula I :

and a co-crystal former (CCF) selected from the group consisting of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid, wherein R ¹ and R ² are each hydrogen or deuterium.

In another aspect, the present invention relates to a pharmaceutical composition that includes a co-crystal of the compound of Formula I described above. In one embodiment, the pharmaceutical composition further includes a diluent, solvent, excipient or carrier.

In yet another aspect, this invention relates to a eutectic solid composition comprising: (a) a co-crystal containing a compound of formula ( I ) and a co-crystal former selected from adipic acid, wherein each of R ¹ and R ² is hydrogen or deuterium and wherein the molar ratio of the compound of formula I to adipic acid is from about 2 to 1; and (b) adipic acid. In yet another aspect, the present invention relates to a pharmaceutical composition comprising such a eutectic solid composition. In one embodiment, the pharmaceutical composition further includes a diluent, solvent, excipient or carrier.

Another aspect of the present invention relates to a method for preparing a co-crystal from a compound of Formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid. In one embodiment, the method includes: preparing a compound of formula I ; obtaining a co-crystal former; grinding, heating, co-sublimating, co-melting or contacting in solution a compound of formula I with a co-crystal former, under crystallization conditions, so as to form a co-crystal in the solid phase; and then, optionally, isolating the co-crystal thus formed. In another embodiment, the method involves mixing a compound of formula ( I ) with adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid, at an elevated temperature, until a co-crystal forms. In some embodiments, preparing a co-crystal from a compound of formula I and CCF involves providing a compound of formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid in a molar ratio of from about 1-1.2 to about 1 -3.6, respectively.

In yet another aspect, the present invention relates to a method of modulating chemical or physical properties of interest (such as melting point, solubility, dilution, hygroscopicity and bioavailability) of a co-crystal containing a compound of formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid. The method includes the steps of measuring the chemical or physical properties of a compound of Formula I and CCF of interest; determining the mole fraction of a compound of formula I and CCF that results in the desired modulation of the chemical or physical properties of interest; and obtaining a co-crystal with a mole fraction as determined.

The compositions and co-crystals of this invention can be used to treat diseases implicated in or associated with inhibition of DNA-PK. In particular, the present invention relates to a method of sensitizing a cell to an agent that causes DNA damage, comprising contacting the cell with a co-crystal of the present invention or a pharmaceutical composition containing the same.

The present invention further relates to methods of potentiating a therapeutic regimen for the treatment of cancer, comprising administering to an individual in need thereof an effective amount of a co-crystal of the present invention or a pharmaceutical composition containing the same. In one embodiment, a treatment regimen for treating cancer includes radiation therapy.

The present invention also provides methods for treating cancer in an animal which comprise administering to the animal an effective amount of a co-crystal or pharmaceutical composition of the present invention. The present invention further relates to methods of inhibiting the growth of cancer cells, including processes of cell proliferation, invasiveness and metastasis in biological systems. These methods include the use of such a co-crystal or pharmaceutical composition to inhibit the growth of cancer cells.

This invention relates to a method of inhibiting DNA-PK activity in a biological sample, which includes contacting the biological sample with a co-crystal or pharmaceutical composition according to this invention.

Also included within the scope of the present invention is a method of treating diseases disclosed herein, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a co-crystal of the present invention or a composition of the present invention.

Detailed description of this invention

In one aspect, this invention relates to co-crystals comprising a compound of formula I :

and a co-crystal former (CCF) selected from the group consisting of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid, wherein R ¹ and R ² are each hydrogen or deuterium.

In one embodiment, the compound of Formula I is (S)-N-methyl-8-(1-((2'-methyl-[4,5'-bipyrimidin]-6-yl)amino)propan-2-yl) Quinoline-4-carboxamide (Compound 1 ).

In another embodiment, the compound of Formula I is (S)-N-methyl-8-(1-((2'-methyl-4',6'-dideutero-[4,5'-bipyrimidin]-6-yl) amino)propan-2-yl)quinoline-4-carboxamide (Compound 2 ).

In one embodiment, this invention relates to a co-crystal that includes a compound of Formula I and adipic acid as the CCF. In a further embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks at approximately 6.46, 7.91, 11.92, 12.26, 12.99, 14.19, 18.68 and 19.07-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 1. In yet another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 2. In yet another further embodiment, a differential scanning calorimetry (DSC) thermogram of this crystal shows melting points of about 195°C and about 245°C.

In one embodiment, this invention relates to a co-crystal that includes a compound of formula I and citric acid as the CCF. In one embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflectance peaks at about 7.44, 8.29, 11.35, 13.26, 15.49, 21.55, and 23.57-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 3. In yet another embodiment, the compound of formula I and CCF are both in a solid state (eg, crystalline) and non-covalently bonded (ie, by hydrogen bonding).

In one embodiment, this invention relates to a co-crystal that includes a compound of formula I and fumaric acid as the CCF. In one embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflectance peaks at about 8.26, 10.11, 14.97, 16.61, 17.22, 25.20, and 26.01-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 4. In yet another embodiment, the compound of Formula I and CCF are both in a solid state (eg, crystalline) and non-covalently bonded (ie, by hydrogen bonding).

In one embodiment, this invention relates to a co-crystal that includes a compound of Formula I and maleic acid as the CCF. In one embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflectance peaks at about 6.21, 10.43, 11.28, 12.41, 13.26, 18.87, and 21.08-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 5. In yet another embodiment, the compound of formula I and CCF are both in a solid state (eg, crystalline) and non-covalently bonded (ie, by hydrogen bonding).

In one embodiment, this invention relates to a co-crystal that includes a compound of Formula I and succinic acid as the CCF. In one embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflectance peaks at about 8.02, 12.34, 14.78, 17.32, 19.56, and 20.06-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 6. In another embodiment, the compound of Formula I and CCF are both in a solid state (eg, crystalline) and non-covalently bonded (ie, by hydrogen bonding).

In yet another embodiment, the present invention relates to a co-crystal that includes a compound of Formula I and benzoic acid as the CCF. In one embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflectance peaks at about 8.70, 13.90, 15.62, 17.65, 18.15, 20.77, and 24.72-θ. In another embodiment, the X-ray powder diffraction pattern of this co-crystal shows reflection peaks as shown in FIG. 7. In another embodiment, the compound of formula I and CCF are both in a solid state (eg, crystalline) and non-covalently associated.

In one embodiment, the present invention provides co-crystals of the formula (Compound 1 ) _n :(AA) _m , where n is 1 and m is between 0.4 and 2.1. In one embodiment, n is 1 and m is between 0.9 and 3.1. In one embodiment, for co-crystals containing adipic acid, n is about 2 and m is about 1. In one embodiment, for co-crystals containing adipic acid, n is about 2 and m is about 1.

In another embodiment, the present invention provides co-crystals of the formula (Compound 2 ) _n :(AA) _m , where n is 1 and m is between 0.4 and 2.1. In one embodiment, for co-crystals containing adipic acid, n is about 2 and m is about 1.

In another embodiment, this invention relates to a co-crystal of a compound of formula I and CCF in the form of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid, where the co-crystal is in a solid state at room temperature and the compound formulas I and CCF interact through non-covalent bonds. In some embodiments, non-covalent bond interactions between a compound of Formula I and CCF include hydrogen bonding and van der Waals forces. In one embodiment, CCF is adipic acid.

In one embodiment, the present invention provides a co-crystal of compound ( 1 ) and adipic acid as a CCF, wherein the molar ratio of compound ( 1 ) to adipic acid is about 2:1.

In another embodiment, the present invention provides a co-crystal of compound ( 2 ) and adipic acid as a CCF, wherein the molar ratio of compound ( 2 ) to adipic acid is about 2:1.

In another embodiment, the co-crystal of compound ( 2 ) and adipic acid as the CCF (co-crystal of adipic acid with compound ( 2 )) is in polymorph form A or B. Polymorph forms A and B are two conformational polymorphs of co- adipic acid crystal with compound ( 2 ). In yet another embodiment, the co-crystal of compound ( 1 ) and adipic acid as CCF (co-crystal of adipic acid with compound ( 1 )) is in polymorph form A or B. Polymorph forms A and B are two conformational polymorphs with -crystal of adipic acid with compound ( 1 ) and their, in the solid state, ¹³ C nuclear magnetic resonance spectra are essentially the same as those for polymorphic forms A and B of compound ( 2 ).

In one particular embodiment, polymorph Form A is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 117.1, 96.8, 95.7, 27.6, 14.8 ppm. In another specific embodiment, polymorph Form A is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 161.6, 154.5, 117.1, 96.8, 95.7, 51.5, 50.2. 27.6, 25.6, 18.5 and 14.8 ppm. In yet another specific embodiment, polymorph Form A is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 179.4, 168.4, 161.6, 158.3, 154.5, 147.8, 145.7 , 143.2, 141.8, 124.6, 117.1, 96.8, 95.7, 51.5, 50.2, 31.2, 30.1, 27.6, 25.6, 18 .5 and 14.8 ppm. In yet another specific embodiment, polymorphic form A is characterized by ¹³ C peaks of solid state nuclear magnetic resonance spectroscopy, as shown in Fig. 16 or 17.

In one particular embodiment, polymorph Form B is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 117.9, 97.3, 94.0, 26.7 and 15.7 ppm. In another specific embodiment, polymorph Form B is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 161.7, 153.8, 117.9, 97.3, 94.0, 50.7, 25.3. 26.7, 18.8 and 15.7 ppm. In yet another specific embodiment, polymorph Form B is characterized by ¹³ C solid state nuclear magnetic resonance spectroscopy peaks at about 179.1, 168.3, 158.1, 147.2, 142.4, 125.8, 124.5 , 117.9, 97.3, 94.0, 32.3, 30.1, 26.7 and 15.7 ppm. In yet another specific embodiment, Form B is characterized by solid state nuclear magnetic resonance spectroscopy ¹³ C peaks, as shown in FIG. 17.

In yet another embodiment, the co-crystal of compound ( 2 ) and CCF as adipic acid (co-crystal of adipic acid with compound ( 2 )) is a mixture of polymorphs A and B. In yet another embodiment, the co-crystal of compound ( 1 ) and CCF, in the form of adipic acid (co-crystal of adipic acid with compound ( 1 )) is a mixture of polymorphic forms A and B.

The present invention covers co-crystals of the compound of formula I and CCF described above, in isolated pure form or as a mixture, as a solid composition when mixed with other substances, such as the free form of the compound of formula I or the free form of CCF. In one embodiment, the present invention provides pharmaceutically acceptable compositions comprising co-crystals of a compound of Formula I and CCF described above and additional free CCF. In one particular embodiment, the compositions include co-crystals of compound ( 1 ) or ( 2 ) and adipic acid as the CCF as described above, and additional adipic acid. In some specific embodiments, the total molar ratio of the compound of Formula I to CCF (both the co-crystal portion and free CCF, e.g., co-crystal adipic acid and free adipic acid) in such compositions ranges from about 1:0. 55 to about 1:100. In other specific embodiments, the overall molar ratio of compound of Formula I to CCF in such compositions ranges from about 1:0.55 to about 1:50. In other specific embodiments, the overall molar ratio of compound of Formula I to CCF in such compositions ranges from about 1:0.55 to about 1:10. In some specific embodiments, the total weight ratio of the compound of Formula I to CCF in such compositions is in the range of about 85 wt.%: 15 wt.%. to about 60% wt.:40% wt. In other specific embodiments, the total weight ratio of the compound of Formula I to CCF in such compositions is in the range of about 70 wt.%:30 wt.%. to about 60% wt.:40% wt. In yet other embodiments, the total weight ratio of the compound of Formula I to CCF in such compositions is about 65 wt%:35 wt%.

In another embodiment, this invention relates to eutectic solid compositions comprising: (a) a co-crystal containing a compound of formula ( I ) and a CCF selected from adipic acid, wherein each of R ¹ and R ² is hydrogen or deuterium and where the molar the ratio of the compound of formula I to adipic acid is from about 2 to 1; and (b) adipic acid. As used herein, the term "eutectic solid" means a solid resulting from a eutectic reaction known in the art. Without reference to a specific theory, the eutectic reaction is defined as follows:

In a eutectic reaction, one liquid phase and two solid phases all coexist at the same time and are in chemical equilibrium. After cooling, a superlattice or microstructure is formed, from which all its components are simultaneously released into a liquid mixture (melts) at a specific temperature (eutectic temperature).

In one embodiment, the total weight ratio of the compound of Formula I to adipic acid in eutectic solid compositions ranges from about 70 wt%:30 wt%. to about 60% wt.:40% wt. In yet another embodiment, the total weight ratio of the compound of formula I to adipic acid is in the range of about 65% wt.:35% wt. In yet another embodiment, the molar ratio in the co-crystal of the compound of Formula I to adipic acid is from about 1 to 1.03.

Pure form means that the particular co-crystal or polymorphic form contains greater than 95% (w/w), e.g., greater than 98% (w/w), greater than 99% (w/w), greater than 99 .5% (w/w) or above 99.9% (w/w).

More particularly, the present invention also provides pharmaceutically acceptable compositions wherein each of the co-crystals or polymorphs is in the form of a composition or mixture of the polymorph with one or more other crystalline form(s), solvate(s) , amorphous substance(s) or other polymorphic forms or combinations thereof. For example, in one embodiment, the compositions include co-crystal Form A of adipic acid and compound ( 2 ) together with one or more other polymorphic form(s) of compound ( 2 ), such as amorphous forms, hydrates, solvates and/or other forms or combinations thereof. In one particular embodiment, the compositions contain Form A of the adipic acid-compound co-crystal ( 2 ) together with Form B of the adipic acid-compound co-crystal ( 2 ). More specifically, the composition may contain from trace amounts up to 100% of a particular polymorphic form, or any amount, for example, in the range of 0.1 wt%. up to 0.5% wt., from 0.1% wt. up to 1% wt., from 0.1% wt. up to 2% wt., from 0.1% wt. up to 5% wt., from 0.1% wt. up to 10% wt., from 0.1% wt. up to 20% wt., from 0.1% wt. up to 30% wt., from 0.1% wt. up to 40% wt., from 0.1% wt. up to 50% wt., from 1% wt. up to 50% wt. or from 10% wt. up to 50% by weight, based on the total amount of compound of formula I in the composition. Alternatively, the composition may contain at least 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 97 wt%, 98 wt%. 99% wt., 99.5% wt. or 99.9% wt. specific polymorphic form, based on the total amount of compound of formula I in the composition.

In one embodiment, the compounds of the present invention are provided in the form of the individual enantiomer, at least 95%, at least 97%, and at least 99% free from the corresponding enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (+)-enantiomer, at least 95% free from the corresponding (-)-enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (+)-enantiomer, at least 97% free from the corresponding (-)-enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (+)-enantiomer, at least 99% free from the corresponding (-)-enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (-)-enantiomer that is at least 95% free from the corresponding (+)-enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (-)-enantiomer that is at least 97% free from the corresponding (+)-enantiomer.

In a further embodiment, the compounds of the present invention are in the form of a (-)-enantiomer that is at least 99% free from the corresponding (+)-enantiomer.

The present invention also relates to methods for preparing co-crystals described above. In one embodiment, the methods include grinding, heating, co-sublimating, co-melting or contacting, or (S)-N-methyl-8-(1-((2'-methyl-[4,5'-bipyrimidine]- 6-yl)amino)propan-2-yl)quinoline-4-carboxamide or (S)-N-methyl-8-(1-((2'-methyl-4',6'-dideutero-[4,5 '-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide with a co-crystal former, under crystallization conditions, to form a co-crystal in the solid phase, where the co-crystal former is selected from the group consisting of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid.

In another embodiment, the methods involve mixing a compound of formula ( I ) with a CCF selected from the group consisting of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid at an elevated temperature to form a co-crystal. A compound of formula ( I ) can be mixed with CCF to form a mixture of the compound and CCF, and then the mixture of the compound and CCF is heated at an elevated temperature until a co-crystal is formed. Alternatively, the mixing and heating steps can be carried out simultaneously.

In one specific embodiment, the CCF is adipic acid and the compound of formula ( I ) is mixed with the adipic acid at an elevated temperature, ranging from about 110°C to about 195°C, until a co-crystal is formed. In another specific embodiment, the elevated temperature is in the range of from about 130°C to about 180°C or in the range from about 140°C to about 160°C.

In another specific embodiment, CCF is adipic acid and is mixed at 10 wt%. up to about 85% wt. compound ( I ) and from about 90% of the mass. up to 15% wt. adipic acid. In yet another specific embodiment, compound ( I ) constitutes from about 30% of the mass. up to about 80% wt. and adipic acid constitutes from about 70% of the mass. to about 20% by weight. In another specific embodiment, compound ( I ) is from about 50% by weight. up to about 80% wt. and adipic acid constitutes from about 50% of the mass. up to about 20% wt. In yet another specific embodiment, compound ( I ) constitutes from about 60% of the mass. up to about 70% wt. and adipic acid constitutes from about 40% of the mass. up to about 30% wt. In yet another specific embodiment, compound ( I ) constitutes approximately 65% of the mass. and adipic acid makes up approximately 35% of the mass.

In yet another embodiment, the methods include: providing a compound of formula I ; provision of a co-crystal former; grinding, heating, co-sublimating, co-melting or contacting in solution a compound of formula I with a co-crystal former, under crystallization conditions, to form a co-crystal in the solid phase; and then, optionally, isolating the co-crystal thus formed. In some specific embodiments, preparing a co-crystal from a compound of formula I and CCF involves providing a compound of formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid in a molar ratio of from about 1-0.55 to about 1-3.6, respectively. In some specific embodiments, preparing a co-crystal from a compound of formula I and CCF involves providing a compound of formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid in a molar ratio of from about 1-1.2 to about 1-3.6, respectively.

In yet another embodiment, this invention relates to methods of modulating chemical or physical properties of interest (such as melting point, solubility, dilution, hygroscopicity and bioavailability) of a co-crystal containing a compound of formula I and adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid. These methods include: determining the chemical or physical properties of a compound of Formula I and CCF of interest; determining the mole fraction of a compound of formula I and CCF that results in the desired modulation of the chemical or physical properties of interest; and obtaining a co-crystal with a mole fraction as determined.

As used in this context, the following definitions apply unless otherwise noted. For the purposes of this invention, chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th ed., 1994. Additionally, the general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5th ed., Smith, M.B. and March, J., ed. John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

For designation of powder X-ray powder diffraction peaks, the term “about” means a range of +/- 0.2 relative to the specified value. For ¹³ C-NMR solid state nuclear magnetic resonance spectra, the term “about” means a range of +/- 0.1 relative to the specified value. Otherwise, the term “about” means +/- 10% of the specified value. When this term refers to a series of numerical values, it applies to each of the values in that series.

For compounds of this invention in which R ¹ or R ² is deuterium, the ratio of deuterium to hydrogen is at least 5 to 1. In some embodiments, the ratio of deuterium to hydrogen is at least 9 to 1. In others embodiments, the ratio of deuterium to hydrogen is at least 19 to 1.

Methods for preparing and characterizing co-crystals are well documented in the literature. See, for example, Trask et al., Chem. Commun., 2004, 890-891; and O. Almarsson and M. J. Zaworotko, Chem. Commun., 2004, 1889-1896. In general, these methods are also suitable for preparing and characterizing co-crystals according to this invention.

Examples of preparing co-crystals with the active pharmaceutical ingredient and CCF include melt extrusion, ball milling, reactor melting, solvent evaporation, conversion to suspension, mixing, sublimation or molding. In the case of the ball milling method, certain molar ratios of co-crystal components (eg, a compound of interest, such as the compound of formula I of the present invention and CCF) are mixed and ball milled. Optionally, a solvent such as methyl ethyl ketone, chloroform and/or water can be added to the ball milled mixture. After grinding, the mixture can be dried in vacuum or at room temperature or under heating conditions under which a powdered product is usually obtained. In the melting method, the components of the co-crystal (eg, CCF and a compound of Formula I ) are mixed, optionally with a solvent such as acetonitrile. The mixture is then placed in a lidded reaction block and then heated to an endothermic reaction. The resulting mixture is then cooled and the solvent, if used, is removed. In the case of the solvent evaporation method, each co-crystal component is first dissolved in a solvent (eg, a mixed solvent such as an azeotrope of methanol and dichloromethane or toluene and acetonitrile (eg, 50/50 by volume)), and the solutions are then mixed together. The mixture is then allowed to settle and the solvent is evaporated to dryness to obtain a co-crystal. In the case of the melt extrusion (HME) process, a new product (extrudate) is produced by extruding the melt through an orifice or die (extruder), under controlled conditions such as temperature, mixing, feed rate and pressure. An extruder typically includes a platform that supports a moving mechanism, an extruder barrel, a rotating screw located on the screw shaft, and an extruder head for shaping the product into a specific shape. Alternatively, the extruder head can be removed and the product can be formed in other ways. Typically, process parameters are controlled through communication with a central electronic control unit. The driving mechanism of an extruder typically includes a motor, gearbox, clutch and support section, where the drum and screw are typically used in a modular configuration. In the case of this invention, any suitable HME technologies known in the art can be used, for example, Gavin P. Andrews et al., “Hot-melt extrusion: an emerging drug delivery technology,” Pharmaceutical Technology Europe , Volume 21, Issue 1 (2009 ). In one embodiment, co-crystals according to this invention are produced by melt extrusion.

Examples of characterization methods include thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), solid state nuclear magnetic resonance spectroscopy (ss-NMR), solubility assays, dynamic vapor sorption, infrared absorption analysis, and suspension stability. TGA can be used to investigate the presence of residual solvents in a co-crystal sample and to identify the temperature at which degradation of each co-crystal sample occurs. DSC can be used to monitor the thermal transition occurring in a co-crystal sample as a function of temperature and determine the melting point of each co-crystal sample. Powder X-ray diffraction can be used to structurally characterize the co-crystal. Solubility analysis can be performed by reflecting changes in the physical state of each co-crystal sample. Suspension stability analysis can be used to determine the chemical stability of a co-crystal sample in a solvent.

Pharmaceutically acceptable salts

The present invention also covers co-crystals formed with pharmaceutically acceptable salts of the compounds of formula I. Also, the combination therapy of this invention, discussed below, includes the administration of compounds of formula I, and their pharmaceutically acceptable salts, and co-crystals thereof, as described herein. For treatment, the compounds of formula I may exist in free form or, if appropriate, in the form of a pharmaceutically acceptable salt.

The phrase "pharmaceutically acceptable salt" means any non-toxic salt of a compound of this invention which, when administered to a recipient, is capable of providing, either directly or indirectly, delivery of a compound of this invention or an inhibitory active metabolite or residue thereof. As used herein, the term “inhibitory active metabolite or residue thereof” means that the metabolite or residue thereof is also a DNA-PK inhibitor.

Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al . describe pharmaceutically acceptable salts in more detail in an article in J. Pharmaceutical Sciences , 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those prepared with suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds. Acid addition salts can be prepared 1) by reacting the purified compound in its free base form with a suitable organic or mineral acid and 2) by isolating the salt thus obtained.

Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods used in the art, such as ion exchange.

Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate , hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate , persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

Base addition salts can be prepared 1) by reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) by isolating the salt thus obtained. Salts prepared with appropriate bases include alkali metal salts (such as sodium, lithium and potassium), alkaline earth metal salts (such as magnesium and calcium), ammonium salts and N ⁺ (C _1-4 -alkyl) salts. ₄ . The present invention also covers the quaternization of any of the basic nitrogen-containing groups of compounds disclosed herein. Products that are soluble or dispersible in water or oil can be obtained by such quaternization.

Further, pharmaceutically acceptable salts include, as appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate. Other acids and bases, when not themselves pharmaceutically acceptable, can be used to prepare salts useful as intermediates in the preparation of the compounds of this invention and their pharmaceutically acceptable acid or base addition salts.

Use of co-crystals and pharmaceutical compositions according to this invention

An effective amount of the co-crystal or pharmaceutical composition of this invention can be used to treat diseases involved in or related to cancer. An effective amount is the amount required to provide a therapeutic effect to a subject being treated, eg a patient. As used in this context, the terms “subject” and “patient” are used interchangeably. The terms "subject" and "patient" refer to an animal (for example, a bird such as a chicken, quail or turkey, or a mammal), especially "mammal" includes non-primates (for example, a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog and mouse) and primates (eg monkey, chimpanzee and human), and more specifically humans. In one embodiment, the subject is a non-human animal, such as a farm animal (eg, a horse, cow, pig, or sheep) or a pet (eg, a dog, cat, guinea pig, or rabbit). In a preferred embodiment, subject means "person".

The exact amount of compound administered to a subject depends on the route of administration, the type and severity of the cancer, and characteristics of the subject, such as general health, age, gender, body weight, and drug tolerance. A qualified physician should be able to determine appropriate dosages based on these and other factors. When co-administered with other agents, for example, when co-administered with an anti-cancer drug, the "effective amount" of the second agent depends on the type of drug used. Suitable dosages are known for approved agents and can be determined by a qualified physician in accordance with the condition of the subject, the type(s) of condition(s) being treated, and the amount of the compound disclosed herein used. In cases where the amount is not specified, the effective amount must be assumed. In general, the dosage regimen may be selected according to a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound used; the specific composition used; the age, body weight, general health, gender and diet of the patient; time of administration, route of administration and rate of excretion of the particular compound used; the subject's renal and liver function; and the specific compound used, or its salt, the duration of treatment; drugs used in combination or combined with the particular compound used, and the like factors well known in medical practice. A skilled physician can readily determine and prescribe an effective amount of the compounds disclosed herein required to treat, prevent, inhibit (wholly or partially), or slow the progression of a disease.

An effective amount of a co-crystal or pharmaceutical composition according to this invention is from about 0.1 mg/kg to about 200 mg/kg body weight per day. In one embodiment, an effective amount of a co-crystal or pharmaceutical composition of this invention is from about 1 mg/kg to about 50 mg/kg body weight per day. In another embodiment, an effective amount of a co-crystal or pharmaceutical composition of this invention is from about 2 mg/kg to about 20 mg/kg body weight per day. Effective dosages may also vary, as will be known to one skilled in the art, depending on the route of administration, the excipient used, and the possibility of concomitant use with other therapies, including the use of other therapeutic agents and/or other therapy.

The co-crystals or pharmaceutical compositions of this invention can be administered to a subject in need thereof (eg, cells, tissue or patient (including animal or human)) by any route that allows delivery of a compound of formula I , for example, orally, intravenously or parenterally. For example, these compositions can be administered via pills, tablets, capsules, aerosols, suppositories, liquid dosage forms for ingestion or injection.

As described above, the pharmaceutically acceptable compositions of the present invention further contain a pharmaceutically acceptable carrier, excipient or excipient, which, as used herein, includes any and all solvents, diluents or other liquid excipients, dispersing or suspending agents, surfactants, active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, which are suitable for the particular dosage form desired. In Remington Manuals: The Science and Practice of Pharmacy , 21st ed., 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology , ed. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, the contents of each of which are incorporated herein by reference, disclose various carriers used in the formulated pharmaceutically acceptable compositions and known methods for their preparation. Except to the extent that any standard carrier environment is incompatible with the compounds of this invention, such as by producing any undesirable biological effect or otherwise interacting in a harmful manner with any other component(s) ) a pharmaceutically acceptable composition, its use is contemplated within the scope of this invention.

The pharmaceutically acceptable carrier may contain inert ingredients that do not unduly inhibit the biological activity of the compounds. Pharmaceutically acceptable carriers must be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or free from other adverse reactions or side effects upon administration to a subject. Standard methods for preparing pharmaceutical compositions can be used.

Some examples of substances that may serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins (such as human serum albumin), buffering agents (such as Twin 80, phosphates, glycine, sorbic acid or potassium sorbate), mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes , block polymers of polyethylene and polyoxypropylene, methylcellulose, hydroxypropylmethylcellulose, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-pyrogenic water; isotonic sodium chloride solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as dyes, release agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants may also be present in this compositions in accordance with the established recipe.

In one specific example, pharmaceutically acceptable compositions according to this invention contain methylcellulose, such as about 0.5 wt%. methylcellulose. In another specific example, pharmaceutically acceptable compositions according to this invention contain methylcellulose and benzoic acid, such as about 0.5 wt%. methylcellulose and about 0.2% wt. benzoic acid. In another specific example, pharmaceutically acceptable compositions contain methylcellulose and benzoic acid, such as about 0.5 wt%. methylcellulose, about 0.1% wt. benzoic acid, about 0.1% wt. sodium benzoate. In some embodiments, the pharmaceutical compositions further contain free adipic acid (free CCF that is not the CCF of the co-crystals of this invention). Such adipic acid is present in a concentration of, for example, about 5 mg/[g excipient] to about 10 mg/[g excipient], such as about 8.8 mg/[g excipient].

Any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions, can be used for oral administration. In the case of oral tablets, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are usually also added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When aqueous suspensions for oral administration are required, the active ingredient is combined with emulsifying and suspending agents. If desired, some sweeteners, flavoring agents or coloring agents may also be added.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, seed oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters , and mixtures thereof. Compared to inert diluents, oral compositions may also include auxiliary agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) excipients or diluents, such as starches, lactose, sucrose, glucose, mannitol or silicic acid, b) with binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and gum arabic, c) with humectants such as glycerin, d) with disintegrants such as agar-agar , calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate, f) with dissolution retarding agents such as paraffin, f) with absorption accelerators such as quaternary ammonium compounds, g) with wetting agents such as such as cetyl alcohol and glyceryl monostearate, h) with absorbents such as kaolin and bentonite clays, and i) with lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms in the form of tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings, such as enteric coatings and other coatings well known in the art of preparing finished dosage forms. They may optionally contain opalescent agents and may also be of such a composition that they release the active ingredient(s) only, or preferably in a certain part of the gastrointestinal tract, optionally in a sustained manner. Examples of potting compositions that can be used include polymeric substances and waxes. Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Microencapsulated forms with one or more excipient(s) as described above can also be used according to this invention. Solid dosage forms in the form of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the art of preparing finished dosage forms. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain, as is common practice, additional substances other than inert diluents, for example tableting lubricants and other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may also contain buffering agents. They may optionally contain opalescent agents and may also be of such a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a prolonged manner. Examples of potting compositions that can be used include polymeric substances and waxes.

Injectable dosage forms, for example, sterile aqueous or oleaginous injectable suspensions, can be prepared in accordance with the prior art using suitable dispersing or wetting agents and suspending agents. Sterile injectable dosage forms may also be a sterile injectable solution, suspension or emulsion, in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents and solvents that may be used include water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as a solvent or suspending medium. For this purpose, any soft, fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injection solutions.

The finished injectable dosage forms can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

Sterile injection forms can be an aqueous or oily suspension. These suspensions can be prepared according to methods known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable dosage forms may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as a solvent or suspending medium. For this purpose, any miscible fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are suitable for the preparation of injectable preparations in the form of natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylene versions. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersing agent, such as carboxymethylcellulose, or similar dispersing agents that are commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Also, for the purpose of preparing finished dosage forms, other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailable enhancing agents, which are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms, can be used.

In order to prolong the effect of administered active compounds, it is often desirable to delay the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor solubility in water. The rate of absorption of a compound then depends on its rate of dissolution, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of the parenterally administered form of the compound is achieved by dissolving or suspending the compound in an oily vehicle. "Depot" injection forms are prepared by molding microencapsulated matrices of the active compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active compound to polymer and the nature of the particular polymer used, the rate of release of the compound can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Slowly absorbed injectable dosage forms are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

If desired, the above-described finished dosage forms adapted to sustained release of the active ingredient can be used.

Compositions for rectal or vaginal administration are specific suppositories that can be prepared by mixing the active compound with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax that is solid at ambient temperature but liquid at body temperature, and therefore melts in the rectum or vaginal cavity and releases the active compound.

Dosage forms for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed, under sterile conditions, with a pharmaceutically acceptable carrier and any necessary preservative or buffer, if required. Ophthalmic dosage forms, ear drops and eye drops are also contemplated within the scope of this invention. Additionally, transdermal patches can also be used, which have the added benefit of providing controlled delivery of the compound into the body. Such dosage forms can be prepared by dissolving or distributing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of a compound through the skin. The rate can be controlled either by providing a controlled rate through the membrane or by dispersing the compound in a polymer matrix or gel.

Alternatively, the active compounds and pharmaceutically acceptable compositions containing them can also be administered by nasal aerosol or inhalation. Such compositions are prepared in accordance with methods well known in the art of preparing finished dosage forms, and they can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other standard solubilizing agents. or dispersing agents.

The co-crystals or pharmaceutical compositions of this invention can also be delivered by implantation (eg, surgery) using an implantation device. Examples of implantation devices include, but are not limited to, stents, pressure pumps, blood vessel implantable filters, and controlled release of compositions during implantation. Any implantable device may be used to deliver a compound of Formula I, as an active ingredient in co-crystals or pharmaceutical compositions of this invention, provided that 1) the device, the compound of Formula I , and any pharmaceutical composition including the compound are biocompatible and 2) that the device can deliver or release an effective amount of the compound to provide a therapeutic effect to the patient being treated.

Delivery of therapeutic agents through stents, pressure pumps (eg, mini-osmotic pumps), and other implantation devices are known in the art. See, for example, the article "Recent Developments in Coated Stents", Hofma et al., published in Current Interventional Cardiology Reports, 2001, 3: 28-36, the entire contents of which, including references therein, are incorporated herein by reference. Other descriptions of implantation devices such as stents can be found in US Patent Nos. 6,569,195 and 6,322,847, and PCT International Application Nos. WO 04/0044405, WO 04/0018228, WO 03/0229390, WO 03/0228346, WO 03 /0225450, WO 03/0216699 and WO 03/0204168, each of which (as well as other publications specified therein) are incorporated herein by reference in their entirety.

Active compounds and pharmaceutically acceptable compositions containing them can be used to obtain medicines in the form of standardized dosage forms. The term "unit dosage form" refers to physically discrete units suitable as a single dose for subjects being treated, with each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, optionally in combination with a suitable pharmaceutical carrier. The unit dosage form may be a single daily dose or one of multiple daily doses (eg, about 1-4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. The amount of active compound in unit dosage form varies depending, for example, on the host being treated and the particular route of administration, for example, from about 0.1 mg/kg to about 200 mg/kg body weight, per day.

In one embodiment, this invention relates to methods of potentiating a therapeutic regimen in the treatment of cancer. These methods include the step of administering to an individual in need thereof an effective amount of a co-crystal of the present invention or a pharmaceutical composition containing the same. Compounds of formula I and their co-crystals, without being bound by theory, can inhibit DNA-PK. DNA-PK plays an important role in the survival of cells, such as cancer cells, after DNA damage, through its activity in repairing double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Targeting DNA-PK may therefore improve outcomes in a cancer patient, particularly in cancer patients who receive therapies to induce DSBs in tumor cells, since DSBs in tumor cells cannot be repaired and quickly lead to to cell death. In some embodiments, the methods of this invention potentiate a therapeutic regimen to induce DSBs. Examples of such therapies include radiotherapy (RT) and some chemotherapy, such as topoisomerase I inhibitors (eg, topotecan, irinotecan/SN38, rubitecan and other derivatives), topoisomerase II inhibitors (eg, etoposide and doxil), DNA intercalators (eg, doxorubicin or epirubicin), radiomimetics (eg, bleomycin), PARP inhibitors (eg, BMN-673), DNA repair inhibitors (eg, carboplatin), DNA cross-linkers (eg, cisplatin), thymidylate synthase inhibitors (eg, fluorouracil (5-FU) ), mitotic inhibitors (eg, paclitaxel), EGFR inhibitors (eg, erlotinib), and EGFR monoclonal antibodies (eg, cetuximab).

In one specific embodiment, the above potentized therapeutic regimen for the treatment of cancer includes at least one chemotherapy selected from the group consisting of a topoisomerase I inhibitor, a topoisomerase II inhibitor, a DNA intercalator, a radiomimetic, a PARP inhibitor, a DNA repair inhibitor, a cross-linked DNA linkers, thymidylate synthase inhibitor, mitotic inhibitor, EGFR inhibitor, EGFR monoclonal antibody, or radiation. In another specific embodiment, a therapeutic regimen for treating cancer includes radiotherapy. The co-crystals or pharmaceutical compositions of the present invention are useful in cases where radiotherapy is indicated to enhance the therapeutic benefit of such treatment. In addition, radiotherapy is often indicated as an adjuvant to surgery in the treatment of cancer. In general, the goal of radiotherapy, when assisted, is to reduce the risk of recurrence and increase disease-free survival when the primary tumor is controlled. For example, adjuvant radiotherapy is indicated for cancers including, but not limited to, breast cancer, colorectal cancer, gastric cancer, fibrosarcoma, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, lung cancer, pancreatic cancer, and prostate cancer, as described below. In yet another specific embodiment, a therapeutic regimen for the treatment of cancer includes both radiotherapy and chemotherapy with at least one chemotherapeutic agent selected from the group consisting of topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalators, radiomimetics, PARP inhibitors , DNA repair inhibitors, DNA cross-linkers, thymidylate synthase inhibitors, mitotic inhibitors, EGFR inhibitors or EGFR monoclonal antibodies.

In another embodiment, this invention relates to methods of inhibiting or preventing DNA damage repair by homologous recombination in cancer cells. Another embodiment relates to methods of promoting cell death in the case of cancer cells. Yet another embodiment relates to methods for or prevention of cell repair of DNA damage in the case of cancer cells.

The present invention further relates to sensitization (eg, radiosensitization) of tumor cells by use of a co-crystal or pharmaceutical composition according to the present invention. Accordingly, such a co-crystal or pharmaceutical composition may "radiosensitize" a cell when administered to animals in a therapeutically effective amount to increase the sensitivity of cells to electromagnetic radiation and/or to promote the treatment of diseases that are treatable using electromagnetic radiation (for example, X-rays). . Diseases that can be treated with electromagnetic radiation include neoplasms, benign and malignant tumors, and cancer cells. In some embodiments, this invention further relates to sensitization of tumor cells using DNA damaging agents.

The present invention also provides methods for treating cancer in animals which comprise administering to the animal an effective amount of a compound of formula ( I ) or a co-crystal thereof or a pharmaceutical composition according to the present invention. The present invention further relates to methods of inhibiting the growth of cancer cells, including processes of cell proliferation, invasiveness and metastasis in biological systems. These methods include the use of such a co-crystal or pharmaceutical composition to inhibit the growth of cancer cells. Preferably, these methods are used to inhibit or reduce cancer cell growth, invasiveness, metastasis or extent of tumor growth in living animals, such as mammals. The methods of the present invention can also be readily adapted for use in systems assays, such as the analysis of cancer cell growth and properties, and for the identification of compounds that are detrimental to the growth of cancer cells.

Tumors or neoplasms involve the growth of tissue cells in which the increase in the number of cells is uncontrolled and progressive. Some such growth is benign, but other types of growth are called "malignant" and can lead to the death of the organism. Malignant neoplasms or "cancers" differ from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they can invade nearby tissues and form metastases. In addition, malignant neoplasms are characterized by the fact that they show a greater loss of differentiation (more than “dedifferentiation”) and their organization relative to others and their surrounding tissues. This property is also called "anaplasia".

Neoplasms treated according to the present invention also include solid tumors, that is, carcinomas and sarcomas.

Carcinomas include those malignant neoplasms originating from epithelial cells that penetrate (invade) surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue or from tissues that form recognizable glandular structures. Another broad category of cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance similar to embryonic connective tissue. The present invention also makes it possible to treat cancers of the myeloid or lymphoid systems, including leukemia, lymphomas and other cancers that are not typically present as a tumor mass but are distributed in the vascular or lymphoreticular systems.

DNA-PK activity may be associated with various forms of cancer, for example, adult and pediatric oncology, growth of solid tumors or malignancies, myxoid and round cell carcinomas, localized progressive tumors, metastatic cancer, human soft tissue sarcoma, including Ewing's sarcoma, cancer metastases, including lymph node metastases, squamous cell carcinoma, especially head and neck, esophageal squamous cell carcinoma, oral cavity carcinoma, blood cell malignancies, including multiple myeloma, leukemia, including acute lymphocytic leukemia, acute non-lymphocytic leukemia , chronic lymphocytic leukemia, chronic myelocytic leukemia and hairy cell leukemia, primary effusion lymphoma (lymphoma in the body cavity), thymic lymphoma, lung cancer, including small cell carcinoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, adrenal cortical cancer, ACTH-derived tumors, non-small cell cancer, breast cancer, including small cell carcinoma and ductal epithelial carcinoma, gastrointestinal cancer, including gastric cancer, colon cancer, colorectal cancer, polyps associated with colorectal neoplasia, pancreatic cancer, liver cancer, cancer of the urological system, including bladder cancer, including primary superficial tumor of the bladder, invasive transitional cell cancer of the bladder and muscle-invasive bladder cancer, prostate cancer, malignant tumors of the female genital tract, including ovarian cancer, primary peritoneal epithelial neoplasms, neck carcinoma, endometrial uterine cancer, vaginal cancer, vulvar cancer, uterine cancer and solid tumors in ovarian follicles, malignant tumors of the genital tract in men, including testicular and penile cancer, kidney cancer, including renal cell carcinoma, brain cancer, including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumors of invasive cells in the central nervous system, skeletal bone cancer, including osteomas and osteosarcomas, skin cancer, including malignant melanoma, advanced human skin keratinocyte tumor, squamous cell cancer, thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms tumors, gallbladder cancer, trophoblastic neoplasms, hemangiopericytoma and Kaposi's sarcoma. Thus, also within the scope of the present invention is a method of treating such diseases which comprises administering to a subject in need thereof a therapeutically effective amount of a co-crystal of the present invention or a pharmaceutical composition of the present invention.

In some embodiments, the present invention is used to treat lung cancer (eg, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or extensive small cell lung cancer (ED-SCLC)), breast cancer (eg, triple negative breast cancer ), prostate cancer, malignancies in the non-protein portion of hemoglobin (eg, acute myeloid leukemia (AML)), myeloma (eg, plasma cell myeloma (PCM)), gastroesophageal cancer (GEJ), ovarian cancer, colon cancer, cancer pharynx, pancreatic cancer, stomach cancer, esophageal cancer, lymphoma (eg diffuse large B-cell lymphoma (DLBL)) or lung fibroblast. In some specific embodiments, the present invention is used to treat lung cancer (eg, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or extensive small cell lung cancer (ED-SCLC)), breast cancer (eg, triple negative breast cancer). gland), prostate cancer, acute myeloid leukemia, myeloma, gastroesophageal cancer (GEJ) or ovarian cancer. In some specific embodiments, the present invention is used to treat lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer, such as extensive small cell lung cancer (ED-SCLC). In some specific embodiments, the present invention is used to treat breast cancer, such as triple negative breast cancer. In some specific embodiments, the present invention is used for the treatment of gastroesophageal cancer (GEJ). In some specific embodiments, the present invention is used for the treatment of acute myeloid leukemia (AML).

This invention also relates to a method of inhibiting DNA-PK activity in a biological sample, which includes contacting the biological sample with a co-crystal or pharmaceutical composition according to this invention. The term "biological sample" as used herein means a sample of the external part of a living organism and includes, without limitation, cultures or extracts of cells of that organism; biopsied material obtained from a mammal, or extracts thereof; and blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof. Inhibition of kinase activity, particularly DNA-PK activity, in a biological sample is useful for a variety of purposes known to one skilled in the art. An example includes, but is not limited to, inhibition of DNA-PK in a biological test. In one embodiment, the method of inhibiting DNA-PK activity in a biological sample is limited to non-therapeutic methods.

The term "biological sample" as used herein includes, without limitation, cultures or extracts of cells from an organism; biopsied material obtained from a mammal, or extracts thereof; blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

Combination therapies

The present invention also relates to the combination of chemotherapy with a compound or composition according to this invention, or with a combination of other anti-cancer therapy, such as an anti-cancer agent or radiotherapy (or radiotherapy). In some embodiments, the compounds of Formula I and co-crystals thereof are used in combination with other anticancer therapy, such as an anticancer drug or radiotherapy. As used herein, the terms “in combination” or “co-administration” can be used interchangeably to refer to the use of more than one therapy (eg, one or more prophylactic and/or therapeutic agent(s) ov)). The use of the terms is not limited to the order in which therapies (eg, prophylactic(s) and/or therapeutic agent(s)) are administered to a subject.

In some embodiments, the above other anticancer therapy is an anticancer agent. In other embodiments, the above other anti-cancer therapy is a DNA damaging agent. In yet other embodiments, the above other anticancer therapy is selected from radiotherapy. In another specific embodiment, the radiotherapy is ionizing radiation.

Examples of DNA damaging agents that can be used in combination with compounds of formula I and their co-crystals include, but are not limited to, platinized agents such as carboplatin, nedaplatin, satraplatin and other derivatives; topoisomerase I inhibitors such as topotecan, irinotecan/SN38, rubitecan and other derivatives; antimetabolites such as the folic acid family (methotrexate, pemetrexed and related forms); purine antagonists and pyrimidine antagonists (thioguanine, fludarabine, cladribine, cytarabine, gemcitabine, 6-mercaptopurine, 5-fluorouracil (5-FU) and related forms); alkylating agents such as nitrogen mustards (cyclophosphamide, melphalan, chlorambucil, mechlorethamine, ifosfamide and related forms); nitrosoureas (for example, carmustine); triazines (dacarbazine, temozolomide); alkylsulfonates (eg bisulfane); procarbazine and aziridines; antibiotics such as hydroxyurea, anthracyclines (doxorubicin, daunorubicin, epirubicin and other derivatives); anthracenediones (mitoxantrone and related forms); Streptomyces family (bleomycin, mitomycin C, actinomycin); and ultraviolet radiation.

Other therapies or anticancer agents that can be used in combination with the agents of the present invention include surgery, radiotherapy (but, in some examples, ionizing radiation (IR), gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy , brachytherapy and systemic radioactive isotopes, to name a few), endocrine therapy, biological response modifiers (interferons, interleukins, and tumor necrosis factor (TNF), to name a few), hyperthermia and cryotherapy, agents to reduce any adverse effects (eg, antiemetics) and other approved chemotherapy drugs, including, but not limited to, DNA damaging agents listed herein, spindle poisons (vinblastine, vincristine, vinorelbine, paclitaxel), podophyllotoxins (etoposide, irinotecan, vopotecan), nitrosoureas (varmustine, lomustine), inorganic ions (cisplatin, carboplatin), enzymes (vsparaginase) and hormones (tamoxifen, leuprolide, flutamide and megestrol), Gleevec™, Adriamycin, dexamethasone and cyclophosphamide.

Additional examples of therapeutic agents for co-therapy according to this invention include: abarelix (Plenaxis depot®); aldesleukin (Prokine®); aldesleukin (Proleukin®); alemtuzumab (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bevacizumab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); intravenous busulfan (Busulfex®); oral busulfan (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with polypheprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leusstatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); darbepoetin alfa (Aranesp®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); denileukin-diftitox (Ontak®); dexrazoxane (Zinecard®); docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (dromostanolone®); dromostanolone propionate (masterone injection®); Elliott's B Solution®; epirubicin (Ellence®); epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®); exemestane (Aromasin®); filgrastim (Neupogen®); floxuridine (intra-arterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine (Gemzar®); gemtuzumab-ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implant®); hydroxyurea (Hydrea®); ibritumomabtiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinibmesylate (Gleevec®); interferon alpha 2a (Roferon A®); interferon alpha 2b (Intron A®); irinotecan (Camptosar®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®); leuprolide acetate (Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); mechlorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate®; methoxalene (Uvadex®); mitomycin C (Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®); nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); nofetumomab (Verluma®); oprelvequin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); protein-bound particles of paclitaxel (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); sodium porfimer (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®); rasburicase (Elitek®); rituximab (Rituxan®); sargramostim (Leukine®); sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinibmaleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); tositumomab (Bexxar®); tositumomab/I-131 tositumomab (Bexxar®); trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); uracil mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); zoledronate (Zometa®) and vorinostat (Zolinza®).

For a comprehensive discussion of modified cancer treatments, see nci.nih.gov, the FDA list of approved cancer drugs at fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual, 17th ed., 1999.

Some embodiments include administering to said patient an additional therapeutic agent selected from a DNA damaging agent, wherein said additional therapeutic agent is suitable for the disease being treated, and said additional therapeutic agent is administered together with the above compound, in a single dosage form, or separately from the above. compounds, as part of a multiple dosage form.

In some embodiments, the above DNA damaging agent is selected from at least one of radiation (e.g., ionizing radiation), radiomimetic neocarcinostatin, platinating agent, topoisomerase I inhibitor, topoisomerase II inhibitor, antimetabolite, alkylating agent, alkyl sulfonates, antimetabolite, inhibitor PARP or antibiotic. In other embodiments, the above DNA damaging agent is selected from at least one of ionizing radiation, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a PARP inhibitor, or an antibiotic.

Examples of platinating agents include cisplatin, oxaliplatin, carboplatin, nedaplatin, satraplatin and other derivatives. Other platinating agents include lobaplatin and triplatin. Other platinating agents include tetranitrate, picoplatin, satraplatin, prolindac, and aroplatin.

Examples of topoisomerase I inhibitors include camptothecin, topotecan, irinotecan/SN38, rubitecan and other derivatives. Other topoisomerase I inhibitors include belotecan.

Examples of topoisomerase II inhibitors include etoposide, daunorubicin, doxorubicin, mitoxantrone, aclarubicin, epirubicin, idarubicin, amrubicin, amsacrine, pyrarubicin, valrubicin, zorubicin and teniposide.

Examples of antimetabolites include members of the folate family, the purine family (purine antagonists), or the pyrimidine family (pyrimidine antagonists). Examples of the folic acid family include methotrexate, pemetrexed and related forms; examples of the purine family include thioguanine, fludarabine, cladribine, 6-mercaptopurine and related forms; examples of the pyrimidine family include cytarabine, gemcitabine, 5-fluorouracil (5FU) and related forms.

Some other specific examples of antimetabolites include aminopterin, methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil, capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, and hydroxyurea.

Examples of alkylating agents include nitrogen mustards, triazenes, alkyl sulfonates, procarbazine and aziridines. Examples of nitrogen mustards include cyclophosphamide, melphalan, chlorambucil and related forms; examples of nitrosoureas include carmustine; examples of triazenes include dacarbazine and temozolomide; examples of alkyl sulfonates include busulfan.

Other specific examples of alkylating agents include mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquion, thiotepa. , triaziquion, triethylenemelamine, procarbazine, dacarbazine, temozolomide, altretamine, mitobronitol, actinomycin, bleomycin, mitomycin and plicamycin.

Examples of antibiotics include mitomycin, hydroxyurea; anthracyclines, anthracenediones, Streptomyces family.

Examples of anthracyclines include doxorubicin, daunorubicin, epirubicin and other derivatives; examples of anthracenediones include mitoxantrone and related forms; examples of the Streptomyces family include bleomycin, mitomycin C and actinomycin.

Examples of PARP inhibitors include PARP1 and PARP2 inhibitors. Specific examples include olaparib (also referred to as AZD2281 or KU-0059436), iniparib (also referred to as BSI-201 or SAR240550), veliparib (also referred to as ABT-888), rucaparib (also referred to as PF-01367338), CEP-9722 , INO-1001, MK-4827, E7016, BMN-673 or AZD2461. In other embodiments, the agent that inhibits or modulates PARP1 and PARP2 is veliparib (also referred to as ABT-888) or rucaparib. In other embodiments, the agent that inhibits or modulates PARP1 and PARP2 is BMN-673.

In some embodiments, the above platinating agent is cisplatin or oxaliplatin; the above topoisomerase I inhibitor is camptothecin; the above topoisomerase II inhibitor is etoposide; and the above antibiotic is mitomycin. In other embodiments, the above platinating agent is selected from the group consisting of cisplatin, oxaliplatin, carboplatin, nedaplatin or satraplatin; the above topoisomerase I inhibitor is selected from the group consisting of camptothecin, topotecan, irinotecan/SN38, rubitecan; the above topoisomerase II inhibitor is selected from etoposide; the above antimetabolite is selected from the group consisting of the folic acid family, the purine family or the pyrimidine family; the above alkylating agent is selected from the group consisting of nitrogen mustards, nitrosoureas, triazenes, alkylsulfonates, procarbazine or aziridines; and said antibiotic is selected from the group consisting of hydroxyurea, anthracyclines, anthracenediones or the Streptomyces family.

In some embodiments, the additional therapeutic agent is radiation (eg, ionizing radiation). In other embodiments, the additional therapeutic agent is cisplatin or carboplatin. In still other embodiments, the additional therapeutic agent is etoposide. In still other embodiments, the additional therapeutic agent is temozolomide.

In some embodiments, additional therapeutic agents are selected from those that inhibit or modulate base excision repair protein. In a specific embodiment, the base excision repair proteins are selected from the group consisting of UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glycosylase); ARE1, AREX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III); XRCC1 (extra LIG3); PNK, PNKP (polynucleotide kinase and phosphatase); PARP1, PARP2 (poly(ADP-ribose) polymerase); PolB, PolG (polymerases); FEN1 (endonuclease) and aprataxin. In another specific embodiment, the base excision repair protein is selected from PARP1, PARP2 or PolB. In yet another embodiment, the base excision repair protein is selected from PARP1 or PARP2.

In some embodiments, this method is applied to a cancer cell having defects in the ATM signaling cascade. In some embodiments, the above defect alters the expression or activity of one or more of the following: ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, or SMC1. In other embodiments, the above defect alters the expression or activity of one or more of the following: ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX. In another embodiment, the cell is a cancer cell expressing DNA damaging oncogenes. In some embodiments, the above cancer cell alters the expression or activity of one or more of the following: K-Ras, N-Ras, H-Ras, Raf, Myc, Moc, E2F, Cdc25A, CDC4, CDK2, cyclin E, cyclin A, and Rb .

According to another embodiment, the method is applied to a cancer, a cancer cell, or a cell having a defect in a protein involved in base excision repair (“base excision repair protein”). There are many methods known in the art to determine whether a tumor is defective in base excision repair. For example, sequencing of either the genomic DNA or mRNA products of each base excision repair gene (eg, UNG, PARP1, or LIG1) can be performed on a tumor sample to determine whether there are mutations predicted to modulate the function or expression of the gene product (Wang et al., Cancer Research 52:4824 (1992)). In addition to mutational inactivation, tumor cells can be modulated by a DNA repair gene by overmethylation of its promoter region, resulting in decreased gene expression. This is generally assessed using methylation-specific polymerase chain reaction (PCR) to determine methylation levels at the promoters of base excision repair genes of interest. The base excision repair gene promoter assay is commercially available (eg, sabiosciences.com/dna_methylation_product/HTML/MEAH-421A).

Expression levels of base excision repair genes can be assessed by directly quantifying the mRNA levels and protein products of each gene using standard techniques such as quantitative reverse transcriptase-linked polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively (Shinmura et al., Carcinogenesis 25: 2311 (2004); Shinmura et al., Journal of Pathology 225:414 (2011)).

In some embodiments, the base excision repair protein is UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glycosylases); ARE1, AREX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III); XRCC1 (extra LIG3); PNK, PNKP (polynucleotide kinase and phosphatase); PARP1, PARP2 (poly(ADP-ribose) polymerase); PolB, PolG (polymerases); FEN1 (endonuclease) and aprataxin.

In some embodiments, the base excision repair protein is PARP1, PARP2, or PolB. In other embodiments, the base excision repair protein is PARP1 or PARP2.

In certain embodiments, the additional therapeutic agent is selected from one or more of the following: cisplatin, carboplatin, gemcitabine, etoposide, temozolomide, or ionizing radiation.

In other embodiments, the additional therapeutic agents are selected from one or more of the following: gemcitabine, cisplatin or carboplatin, and etoposide. In still other embodiments, the additional therapeutic agents are selected from one or more of the following: cisplatin or carboplatin, etoposide, and ionizing radiation. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

In some embodiments, anticancer therapies for combination therapy according to this invention include DNA damaging agents such as topoisomerase inhibitors (eg, etoposide and doxil), DNA intercalators (eg, doxorubicin, daunorubicin and epirubicin), radiomimetics (eg, bleomycin), PARP inhibitors (eg, BMN-673), DNA repair inhibitors (eg, carboplatin), DNA cross-linkers (eg, cisplatin), thymidylate synthase inhibitors (eg, fluorouracil (5-FU)), mitotic inhibitors (eg, paclitaxel), EGFR inhibitors (eg, erlotinib) and EGFR monoclonal antibodies (eg, cetuximab) and radiation (eg, ionizing radiation). Specific examples include etoposide, doxil, gemcitabine, paclitaxel, cisplatin, carboplatin, 5-FU, etoposide, doxorubicin, daunorubicin, epirubicin, bleomycin, BMN-673, carboplatin, erlotinib, cisplatin, carboplatin, fluorouracil, cetuximab, and radiation (eg, ionizing radiation). In some embodiments, compounds of Formula I and co-crystals thereof are used in combination with at least one anticancer drug selected from the group consisting of etoposide, doxil, gemcitabine, paclitaxel, cisplatin, carboplatin, 5-FU, etoposide, doxorubicin, daunorubicin, epirubicin, bleomycin, BMN-673, carboplatin, erlotinib, cisplatin, carboplatin, fluorouracil or cetuximab, and with or without radiation exposure. In certain particular embodiments, compounds of Formula I and co-crystals thereof are used in combination with etoposide and cisplatin, with or without radiation (eg, ionizing radiation). In certain specific embodiments, compounds of Formula I and co-crystals thereof are used in combination with paclitaxel and cisplatin, with or without radiation (eg, ionizing radiation). In certain specific embodiments, compounds of Formula I and co-crystals thereof are used in combination with paclitaxel and carboplatin, with or without radiation (eg, ionizing radiation). In certain specific embodiments, compounds of Formula I and co-crystals thereof are used in combination with cisplatin and 5-FU, with or without radiation (eg, ionizing radiation).

Specific examples of cancers for combination therapy are as described above. In some embodiments, the present invention is used to treat lung cancer (eg, non-small cell lung cancer (NSCLC), extensive small cell lung cancer (ED-SCLC)), breast cancer (eg, triple negative breast cancer), prostate cancer, acute myeloid leukemia, myeloma, esophageal cancer (eg gastroesophageal cancer (GEJ)), ovarian cancer, colon cancer, pharyngeal cancer, pancreatic cancer, lung fibroblast and gastric cancer. In some specific embodiments, the present invention is used to treat lung cancer (e.g., non-small cell lung cancer (NSCLC), extensive small cell lung cancer (ED-SCLC)), breast cancer (e.g., triple negative breast cancer), prostate cancer, acute myeloid leukemia, myeloma, gastroesophageal cancer (GEJ), pancreatic cancer and ovarian cancer.

In certain specific embodiments, this invention relates to co-therapy using compounds of Formula I and co-crystals thereof, in combination with a standard therapeutic agent (eg, doxorubicin, etoposide, paclitaxel and/or carboplatin), with or without radiation (eg , ionizing radiation), for the treatment of lung cancer, such as non-small cell lung cancer (NSCLC) or extensive small cell lung cancer (ED-SCLC).

In certain specific embodiments, this invention relates to co-therapy using compounds of formula I and co-crystals thereof, in combination with a standard therapeutic agent (eg, cisplatin, 5-FU, carboplatin, paclitaxel and/or etoposide), with or without radiation (eg, ionizing radiation), for the treatment of gastroesophageal cancer (GEJ).

In certain specific embodiments, this invention relates to co-therapy using compounds of Formula I and co-crystals thereof, in combination with a standard therapeutic agent (e.g., doxorubicin and/or vincristine), with or without radiation (e.g., ionizing radiation) , for the treatment of acute myeloid leukemia or chronic lymphocytic leukemia.

In certain specific embodiments, this invention relates to co-therapy using compounds of Formula I and co-crystals thereof, in combination with a standard therapeutic agent (e.g., doxorubicin and/or epirubicin), with or without radiation (e.g., ionizing radiation) , for the treatment of breast cancer, such as triple negative breast cancer.

In some specific embodiments, this invention relates to combination therapy using compounds of Formula I and co-crystals thereof, in combination with radiation (or ionizing radiation); cisplatin, etoposide, paclitaxel, doxorubicin, or cetuximab, with or without radiation exposure (eg, ionizing radiation); cisplatin and etoposide, with or without radiation exposure (eg, ionizing radiation); or cisplatin and paclitaxel, with or without radiation (eg, ionizing radiation), for the treatment of lung cancer, such as non-small cell lung cancer (NSCLC), small cell lung cancer, or extensive small cell lung cancer (ED-SCLC).

In some specific embodiments, this invention relates to combination therapy using compounds of Formula I and co-crystals thereof, in combination with radiation (or ionizing radiation); cisplatin, with or without radiation exposure (eg, ionizing radiation); etoposide, with or without radiation exposure (eg, ionizing radiation); carboplatin, with or without radiation exposure (eg, ionizing radiation); 5-FU, with or without radiation exposure (eg, ionizing radiation); cisplatin and paclitaxel with or without radiation exposure (eg, ionizing radiation); cisplatin and 5-FU with or without radiation exposure (eg, ionizing radiation); or carboplatin and paclitaxel with or without radiation exposure (eg, ionizing radiation); for the treatment of gastroesophageal cancer (GEJ).

In certain specific embodiments, this invention relates to combination therapy using compounds of Formula I and co-crystals thereof, in combination with doxorubicin or epirubicin, with or without radiation (eg, ionizing radiation), for the treatment of breast cancer, such as triple negative breast cancer.

Another embodiment relates to a method of treating breast cancer using compounds of formula I and co-crystals thereof, in combination with a platinating agent, with or without radiation (eg, ionizing radiation). In some embodiments, the breast cancer is triple negative breast cancer. In other embodiments, the platinating agent is cisplatin.

In certain specific embodiments, the present invention relates to combination therapy using compounds of Formula I and co-crystals thereof, in combination with cetuximab, with or without radiation (eg, ionizing radiation); or cisplatin with or without radiation (eg, ionizing radiation), for the treatment of pharyngeal cancer.

In some specific embodiments, this invention relates to combination therapy using compounds of formula I and co-crystals thereof, in combination: with cisplatin with or without radiation (eg, ionizing radiation); with etoposide, with or without radiation (eg, ionizing radiation); with cisplatin and 5-FU with or without radiation exposure (eg, ionizing radiation); or with paclitaxel with or without radiation (eg, ionizing radiation), for the treatment of pulmonary fibroblast.

In some specific embodiments, this invention relates to combination therapy using compounds of formula I and co-crystals thereof, in combination with: radiation (eg, ionizing radiation); with bleomycin, doxorubicin, cisplatin, carboplatin, etoposide, paclitaxel, or 5-FU, with or without radiation (eg, ionizing radiation), for the treatment of lung cancer such as NSCLC, pancreatic cancer, esophageal cancer, or gastric cancer.

Another embodiment relates to methods of treating pancreatic cancer by administering a compound described herein in combination with another known treatment for pancreatic cancer. One aspect of the present invention involves administering a compound as described herein in combination with gemcitabine.

Co-administration in the case of combination therapies covers the administration of first and second amounts of compounds/therapies when administered together substantially simultaneously (such as in the form of a single pharmaceutical composition, e.g., a capsule or tablet having a fixed ratio of the first and second amounts, or multiple times , in the form of separate capsules or tablets for each administration), or sequentially, in any order.

When coadministration involves separately administering a first amount of a compound of this invention and a second amount of an additional therapeutic agent/therapy, they are administered sufficiently closely in time to achieve the desired therapeutic effect. The present invention can also be practiced by including another anti-cancer chemotherapeutic agent in a therapeutic regimen for treating cancer, with or without radiation therapy. The combination of a co-crystal or pharmaceutical composition of this invention with such other agents can potentiate the chemotherapeutic protocol. For example, the inhibitor compound of this invention can be administered with etoposide, bleomycin, doxorubicin, epirubicin, daunorubicin or analogs thereof, agents known to cause DNA strand breaking.

In some embodiments, the compounds of Formula I and their co-crystals are used in combination with a DNA damaging agent (eg, etoposide, radiation) and the compounds of Formula I and their co-crystals are administered after administration of the DNA damaging therapy. Specific examples of DNA damaging agents are described above.

In some embodiments, the above-mentioned one or more additional anticancer agent(s) or therapy(ies) are administered with compound ( 1 ) or a pharmaceutically acceptable salt thereof. In some embodiments, the above-mentioned one or more additional anti-cancer agent(s) or therapy(ies) are used with compound ( 2 ) or a pharmaceutically acceptable salt thereof. In some embodiments, the above-mentioned one or more additional anticancer agent or therapy is used in conjunction with a co-crystal of adipic acid and compound ( 1 ) (e.g., in a 2:1 ratio of compound ( 1 ) to adipic acid). In some embodiments, the above-mentioned one or more additional anticancer agent or therapy is used in conjunction with a co-crystal of adipic acid and compound ( 2 ) (e.g., in a 2:1 ratio of compound ( 2 ) to adipic acid).

In some embodiments, the aforementioned one or more additional anticancer agent or therapy is used in conjunction with the pharmaceutical compositions of this invention described above.

Examples of the preparation and characterization of co-crystals according to this invention are described below, which are intended to be illustrative only and not to limit the present invention in any way.

Example 1: Preparation of compounds according to this invention

As used in this context, all abbreviations, symbols and terms are consistent with those used in contemporary scientific literature. See, for example, Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authors and Editors , 2nd ed., Washington, DC: American Chemical Society, 1997. The following definitions define the terms and abbreviations used in in this context:

BPin pinacolboronate Brine saturated solution of NaCl in water DCM dichloromethane DIEA diisopropylethylamine DMA dimethylacetamide DME dimethoxyethane DMF dimethylformamide DMSO (DMSO) dimethyl sulfoxide EtDuPhos (2R,5R)-1-[2-[(2R,5R)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane ESMS electrospray mass spectrometry Et ₂ O diethyl ether EtOAc ethyl acetate EtOH ethanol HPLC high performance liquid chromatography IPA isopropanol LC-MS liquid chromatography with mass spectrometry Me methyl MeOH methanol MTBE methyl tert-butyl ether NMP N-methylpyrrolidine PdCl ₂ [P(cy) ₃ ] ₂ dichlorobis(tricyclohexylphosphoranyl)palladium Ph phenyl RT or rt room temperature TBME tert-butyl methyl ether tBu tert-butyl THF tetrahydrofuran TEA triethylamine TMEDA tetramethylethylenediamine

Example A. Preparation of 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-4,6-d ₂ (Compound 1003 )

Scheme 1

As shown in step 1-i of Scheme 1, to a stirred solution of 4,6-dichloro-2-methylpyrimidin-5-amine (14.04 g, 78.88 mmol), in methanol-d ₄ (140.4 ml), add formic acid-d ₂ (7.77 g, 161.7 mmol) and palladium black (765 mg, 7.19 mmol, soaked in methanol-d ₄ ), then triethylamine (16.36 g, 22.53 ml, 161.7 mmol). The reaction mixture is hermetically sealed in a test tube and stirred at room temperature overnight. The mixture is then filtered and concentrated under reduced pressure. Et ₂ O (250 ml) was added and the mixture was stirred for 1 hour at room temperature. The resulting solids are filtered and washed with Et ₂ O (2 times). The filtrate was concentrated under reduced pressure to give 4,6-dideutero-2-methylpyrimidin-5-amine (Compound 1001 , 5.65 g, 65% yield) as a light yellow solid. ¹ H-NMR (300 MHz, DMSO-d ₆ ): δ 5.25 (s, 2H), 2.40 (s, 3H). This compound is used in subsequent steps without further purification.

As shown in step 1-ii of Scheme 1, to 4,6-dideutero-2-methylpyrimidin-5-amine (5.35 g, 48.14 mmol), in CH ₃ CN (192.5 ml), add copper dibromide (16.13 g, 3.38 mL, 72.21 mmol), then tert-butyl nitrite (8.274 g, 9.54 mL, 72.21 mmol). After 1 hour, the reaction mixture is filtered through diatomaceous earth along with dichloromethane. The filtrate was washed with water/saturated sodium chloride solution (1:1), the organic layer was separated, the aqueous layer was extracted with dichloromethane (2 times), and the combined organic layers were filtered through diatomaceous earth and concentrated under reduced pressure. The crude product is purified by silica gel column chromatography, medium pressure (0-10% EtOAc/hexanes) to give 5-bromo-4,6-dideutero-2-methylpyrimidine (Compound 1002 , 4.1 g, 49% yield) . ^1H -NMR (300 MHz, methanol- _d4 ): δ 2.64 (s, 3H).

As shown in step 1-iii of Scheme 1, a mixture of 5-bromo-4,6-dideutero-2-methylpyrimidine (8.5 g, 48.57 mmol), bis(pinacolato)diboron (13.57 g, 53.43 mmol) and COAc (14.30 g, 145.7 mmol), in 2-methyltetrahydrofuran (102.0 ml), degassed by purging with nitrogen. Dichlorobis(tricyclohexylphosphoranyl)palladium (PdCl ₂ [P(cy) ₃ ] ₂ , 1.01 g, 1.364 mmol) is added to this mixture and the reaction mixture is stirred in a sealed tube overnight at 100°C. The mixture was filtered and the filtrate was stirred with Silabond® DMT silica (SiliCycle, Inc., 0.58 mmol/g, 3.53 g) for 1 hour. The mixture is filtered and concentrated under reduced pressure to give 2-methyl-4,6-dideutero-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (Compound 1003 , 13 .6 g, purity 72%, the main contaminant is pinacol), in the form of a light yellow oil. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 2.75 (s, 3H), 1.30 (s, 12H). This compound is used in subsequent steps without further purification.

Example B: Preparation of (S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl)-N-methylquinoline-4-carboxamide (Compound 1013 )

Scheme 2

As shown in step 2-i of Scheme 2, 2-bromoaniline (520 g, 3.02 mol) was melted at 50° C. in an incubator and then added to a reaction vessel containing stirred acetic acid (3.12 L). Methanesulfonic acid (871.6 g, 588.5 ml, 9.07 mol) was then added over 15 minutes. The reaction mixture was heated to 60°C and methyl vinyl ketone (377 ml, 1.5 eq.) was added over 5 minutes and the reaction mixture was stirred for 1 hour at 90°C. After this time, another 50 ml (0.2 eq.) of methyl vinyl ketone was added and the reaction mixture was stirred for an additional 16 hours. The resulting dark brown solution is cooled in an ice-water bath and, in portions, poured into a stirred aqueous solution of 50% w/w. NaOH (3.894 L, 73.76 mol) and ice (1 kg), also cooled using an ice-water bath. Add additional ice, if required, during addition in order to maintain the reaction temperature below 25°C. After addition is complete, the reaction mixture (pH > 10) is stirred for 30 minutes while cooled in an ice/water bath. A precipitate forms, which is collected by filtration, washed with water (3 times 2 L) and dissolved in DCM (4 L). The organic products are washed with water (2 L) and the aqueous phase is again extracted with DCM (1 L). The combined organics were dried over Na ₂ SO ₄ , filtered through a pad of silica gel (about 2 L), eluted with DCM and then with 3% EtOAc/DCM until all product had passed through the silica gel pad. The volatiles from the filtrate are removed under reduced pressure and the residue is triturated in hexanes (approximately 500 ml). The resulting solid was collected by filtration, washed with hexanes (4 x 500 mL) and dried in vacuo to give 8-bromo-4-methylquinoline (Compound 1004 , 363 g, 54% yield) as a light tan solid. . LC-MS = 222.17 (M+H); ¹ H-NMR (300 MHz, CDCl ₃ ): δ 8.91 (d, J=4.3 Hz, 1H), 8.06 (d, J=7.4 Hz, 1H), 7.99 (d , J=8.4 Hz, 1H), 7.42 (t, J=7.9 Hz, 1H), 7.30 (d, J=4.2 Hz, 1H), 2.73 (s, 3H ).

As shown in step 2-ii of Scheme 2, selenium dioxide (764.7 g, 6.754 mol) is dissolved in 3.25 L of dioxane and 500 ml of water. The stirred solution is heated to 77° C. and 8-bromo-4-methylquinoline (Compound 1004 , 500 g, 2.251 mol) is added in one portion. The reaction mixture is stirred at reflux for 30 minutes and then cooled in a water bath to a temperature of approximately 45°C, at which precipitation is observed. The suspension is filtered through diatomaceous earth, which is then washed with hot THF to dissolve any residual solids. The filtrate is concentrated to a minimum volume under reduced pressure and 2 M NaOH solution (2.81 L, 5.63 mol) is added to achieve pH=8-9. The reaction mixture is stirred at this pH value for 30 minutes. A precipitate forms, which is collected by filtration and air-dried overnight to give 8-bromoquinoline-4-carbaldehyde (Compound 1005 ) as a yellowish solid. LC-MS = 236.16 (M+H); ¹ H-NMR (300 MHz, CDCl ₃ ): δ 10.52 (s, 1H), 9.34 (d, J=4.2 Hz, 1H), 9.05 (dd, J=8.5, 1.2 Hz, 1H), 8.18 (dd, J=7.5, 1.3 Hz, 1H), 7.88 (d, J=4.2 Hz, 1H), 7.60 (dd, J=8.5, 7.5 Hz, 1H). This product is used in subsequent reactions.

As shown in step 2-iii of Scheme 2, to a stirred suspension of 8-bromoquinoline-4-carbaldehyde (531.4 g, 2.25 mol), in THF (4.8 L), add water (4.8 L) and monosodium phosphate (491.1 g, 4.05 mol). The mixture is cooled to 5°C and, while maintaining the reaction temperature below 15°C, sodium chlorite (534.4 g, 4.727 mol) as a solid is slowly added in portions over about 1 hour. After addition is complete, the reaction mixture is stirred at 10°C for 1 hour, then 1N Na ₂ S ₂ O ₃ solution (1.18 L) is added in portions while maintaining the temperature below 20°C. The reaction mixture is stirred at room temperature, then THF is removed under reduced pressure. The resulting aqueous solution containing the precipitate is treated with a saturated solution of NaHCO ₃ (approximately 1 l) until pH = 3-4 is achieved. This mixture is stirred for an additional 15 minutes and the solid is collected by filtration, washed with water (2 x 1 L), washed with tert-butyl methyl ether (2 x 500 ml) and dried in a convection oven at 60°C for 48 hours. Further drying under high vacuum provided 8-bromoquinoline-4-carboxylic acid (Compound 1006 , 530.7 g, 94% yield based on Compound 1004 ) as a tan solid. LC-MS = 252.34 (M+H); ¹ H-NMR (300 MHz, DMSO-d ₆ ): δ 14.09 (s, 1H), 9.16 (d, J=4.4 Hz, 1H), 8.71 (dd, J=8, 6, 1.2 Hz, 1H), 8.25 (dd, J=7.5, 1.2 Hz, 1H), 8.03 (d, J=4.4 Hz, 1H), 7.64 ( dd, J=8.6, 7.5 Hz, 1H).

As shown in step 2-iv of Scheme 2, to a suspension of 8-bromoquinoline-4-carboxylic acid (Compound 1006 , 779.4 g, 3.092 mol), in DCM (11.7 L), add anhydrous DMF (7.182 ml, 92 .76 mmol). The reaction mixture was cooled to 10°C and oxalyl chloride (413 ml, 4.638 mol) was added dropwise over 30 minutes. The reaction mixture is stirred for an additional 30 minutes, after addition is complete, transferred to an evaporation flask and the volatiles are removed under reduced pressure. Anhydrous THF (2 L) was added and the volatiles were removed again under reduced pressure to remove any residual oxalyl chloride. Anhydrous THF was added to the residue under a nitrogen atmosphere, and the resulting suspension of 8-bromoquinoline-4-carboxylic acid chloride intermediate was stored for future use.

Separately, the initial reaction flask was purged thoroughly with nitrogen gas to remove any remaining oxalyl chloride and anhydrous THF (1.16 L) was introduced into the flask. After cooling to 5° C., add aqueous methylamine (2.14 L 40% w/w MeNH ₂ /water, 24.74 mol), then add additional THF (1.16 L). To this solution, a suspension of the acid chloride intermediate is added in portions over 1 hour, maintaining the temperature of the reaction mixture below 20°C during the addition. The evaporation vessel used to store the acid chloride is rinsed with anhydrous THF and aqueous MeNH ₂ solution (500 ml) and these rinsing liquids are added to the reaction mixture, which is allowed to settle at room temperature for 16 hours. Organic volatiles are removed under reduced pressure and the remaining mostly aqueous suspension is diluted with water (1.5 L). The solids are collected by filtration, washed with water until the pH of the filtrate is less than 11, washed with MTBE (2 x 800 ml) and dried in a convection oven at 60°C to obtain 8-bromo -N-methylquinoline-4-carboxamide (Compound 1007 , 740.4 g, 90% yield), as a light brown solid. LC-MS = 265.04 (M+H); ¹ H-NMR (300 MHz, DMSO-d ₆ ): δ 9.08 (d, J=4.3 Hz, 1H), 8.78 (d, J=4.7 Hz, 1H), 8.21 (dd, J=7.5, 1.2 Hz, 1H), 8.16 (dd, J=8.5, 1.3 Hz, 1H), 7.65 (d, J=4.3 Hz, 1H), 7.58 (dd, J=8.5, 7.5 Hz, 1H), 2.88 (d, J=4.6 Hz, 3H).

As shown in step 2-v of Scheme 2, 8-bromo-N-methylquinoline-4-carboxamide (Compound 1007 , 722 g, 2.723 mol) and tert-butyl-N-[2-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)allyl]carbamate (Compound 1008 , 925.4 g, 3.268 mol) was combined in a reaction flask. Na ₂ CO ₃ (577.2 g, 5.446 mol) was added, then water (2.17 L) was added. The mixture was stirred for 5 minutes, 1,4-dioxane (5.78 L) was added and the mixture was deoxygenated by bubbling a stream of nitrogen for 30 minutes. Pd(dppf)Cl ₂ /DCM (44.47 g, 54.46 mmol) was added and deoxygenation continued as before for an additional 30 minutes. The reaction mixture was stirred at reflux for 16 hours, allowed to cool to 70° C. and water (5.42 L) was added. The mixture is further cooled in an ice-water bath and continued stirring at <10°C for 2 hours. A precipitate is formed, which is collected by filtration, washed with water (3 times 1 L) and washed with TBME (2 times 1 L). The resulting filter cake is divided into two equal portions. Each portion is dissolved in THF/DCM (4 L) and poured onto a layer of Florisil® (3 L filter funnel with approximately 1.5 L of Florisil, using DCM to wet the layer). This layer is then washed with MeTHF until it is determined by thin layer chromatography analysis that no product remains in the filtrate. The filtrates from both portions of the filter cake are combined and concentrated under reduced pressure to give an orange solid. Add TBME (1 L) and the resulting suspension is filtered. The collected solid was washed with 800 ml TBME and dried under high vacuum overnight to give tert-butyl (2-(4-(methylcarbamoyl)quinolin-8-yl)allyl)carbamate (Compound 1009, 653 g, 70% yield ), as an off-white solid. LC-MS = 342.31 (M+H); ¹ H-NMR (300 MHz, CDCl ₃ ): δ 8.93 (d, J=4.3 Hz, 1H), 8.17 (dd, J=8.4, 1.6 Hz, 1H), 7 ,68-7.53 (m, 2H), 7.41 (d, J=4.3 Hz, 1H), 6.09 (br.s, 1H), 5.54 (s, 1H), 5, 28 (s, 1H), 5.10 (br.s, 1H), 4.33 (d, J=6.0 Hz, 2H), 3.11 (d, J=4.8 Hz, 3H), 1.38 (s, 9H). Additional product (34.9 g, total yield 74%) was obtained by concentrating the filtrate under reduced pressure, dissolving the residue in THF, filtering the solution through a pad of Florisil® as before, washing the layer with MeTHF, concentrating the filtrate under reduced pressure, adding 250 mg TBME, stirring for 0.5 hours, collecting the resulting precipitate after filtration, washing the solid with EtOAc (40 ml), acetonitrile (50 ml), and drying the solid under high vacuum overnight.

As shown in step 2-vi of Scheme 2, to a stirred suspension of tert-butyl(2-(4-(methylcarbamoyl)quinolin-8-yl)allyl)carbamate (Compound 1009 , 425 g, 1.245 mol), in EtOH (4. 25 L), add a 5.5 M solution of HCl in iPrOH (1.132 L, 6.225 mol). The reaction mixture is stirred at reflux (internal temperature is 76°C) for 30 minutes, and then left to stand for 90 minutes to cool to a temperature of 40°C. EtOAc (2.1 L) was added and the mixture was stirred for an additional 2 hours. The solid was collected by filtration, washed with EtOAc and dried under high vacuum to give 8-(3-acetamidoprop-1-en-2-yl)-N-methylquinoline-4-carboxamide (Compound 1010 , 357.9 g, yield 91%), in the form of a yellow-brown solid. LC-MS = 242.12 (M+H); ¹ H-NMR (300 MHz, methanol- _d4 ): δ 9.07 (d, J=4.6 Hz, 1H), 8.27 (dd, J=8.5, 1.5 Hz, 1H) , 7.89 (dd, J=7.2, 1.5 Hz, 1H), 7.81-7.72 (m, 2H), 5.85 (s, 1H), 5.75 (s, 1H ), 4.05 (s, 2H), 3.04 (s, 3H).

As shown in step 2-vii of Scheme 2, under nitrogen, 8-(3-acetamidoprop-1-en-2-yl)-N-methylquinoline-4-carboxamide (12.4 g, 43.77 mmol) and cyclooct -1,5-diene/(2R,5R)-1-[2-[(2R,5R)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane: rhodium cation (+1) - trifluoromethanesulfonate (Ph(COD)(R,R)-Et-DuPhos-OTf, 316.3 mg, 0.4377 mmol) is combined in methanol (372.0 ml) and heated to a temperature of 35-40 ° C until solid substances will not dissolve. The reaction mixture was introduced into a hydrogenation apparatus, the atmosphere was replaced with hydrogen, and the mixture was shaken at 100 psi hydrogen at 50° C. for 14 hours. After cooling to room temperature, the mixture is filtered through a pad of Florisil®, which is then washed with MeOH (2 x 50 ml). The filtrate is concentrated under reduced pressure and any trace amounts of water are removed by azeotropic distillation with DCM under reduced pressure. The residue was triturated with 20% DCM in TBME (2 x 100 ml) to give (S)-8-(1-acetamidopropan-2-yl)-N-methylquinoline-4-carboxamide (Compound 1011 , 11.0 g, yield 88%, 96% e.u.), as an off-white solid. ¹ H-NMR (300 MHz, DMSO-d ₆ ): δ 8.97 (d, J=4.3 Hz, 1H), 8.67 (d, J=4.7 Hz, 1H), 7.97 (dd, J=8.1, 1.5 Hz, 1H), 7.88 (t, J=5.6 Hz, 1H), 7.73-7.54 (m, 2H), 7.52 ( d, J=4.3 Hz, 1H), 4.31 (dd, J=14.3, 7.1 Hz, 1H), 3.55-3.32 (m, 3H), 2.86 (d , J=4.6 Hz, 3H), 1.76 (s, 3H), 1.28 (d, J=7.0 Hz, 3H). Enantiomeric excess (e.u.) is determined using chiral HPLC (ChiralPac IC, 0.46 cm × 25 cm), flow rate is 1.0 ml/min, for 20 minutes, at 30°C (20: 30:50, methanol/ethanol/hexanes and 0.1% diethylamine), with a retention time for the (R)-enantiomer of 5.0 minutes and for the (S)-enantiomer of 6.7 minutes.

As shown in step 2-viii of Scheme 2, (S)-8-(1-acetamidopropan-2-yl)-N-methylquinoline-4-carboxamide (11.0 g, 38.55 mmol), in 6 M aqueous solution HCl (192.7 ml, 1.156 mol), heat to 60°C. After stirring for 2 days at this temperature, the reaction mixture is cooled and an additional 20 ml of 6 M aqueous HCl solution is added. Continue stirring for an additional 2 days at 70°C. The reaction mixture is cooled in an ice bath and the pH is adjusted to approximately 11 with 6 M NaOH (aq). The aqueous mixture is extracted with 5% MeOH/DCM, and the combined organic extracts are washed with water (60 ml), saturated sodium chloride solution (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product, in in the form of a yellow-brown solid. This solid is suspended in EtOAc (200 ml), cooled to 3°C in an ice bath, and 6 M HCl/i-PrOH (30 ml) is added in portions until a white precipitate is obtained, which is collected by filtration. The solid was washed with EtOAc (100 ml) and dried under high vacuum to give (S)-8-(1-aminopropan-2-yl)-N-methylquinoline-4-carboxamide dihydrochloride [Compound 1012 , 7.8 g, yield 61%, 95% purity (5% compound 1011 )], as a white solid. This product is used in subsequent reactions.

As shown in step 2-ix of Scheme 2, 8-[(1S)-2-amino-1-methylethyl]-N-methylquinoline-4-carboxamide hydrochloride (Compound 1012 , 24.0 g, 72.86 mmol) was dissolved in THF (230 ml) and water (40 ml) and stir for 5 minutes. Sodium carbonate (15.44 g, 145.7 mmol) was added to 100 ml of water and the reaction mixture was stirred for 10 minutes. 4,6-dichloropyrimidine (12.18 g, 80.15 mmol) was added and the reaction mixture was refluxed at 66°C for 2 hours. The reaction mixture is cooled to room temperature, diluted with 200 ml EtOAc, the organic layer is separated and the aqueous layer is extracted with 100 ml EtOAc. The combined organic layers were washed with water (60 ml), saturated sodium chloride solution (100 ml), dried over Na ₂ SO ₄ , filtered through a pad of silica gel (100 g) and concentrated under reduced pressure. The remaining crude product is triturated with 20% DCM in MBTE (200 ml) then MBTE (200 ml) to give (S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl )-N-methylquinoline-4-carboxamide (Compound 1013 , 23.15 g, 88% yield), as a white solid. ¹ H-NMR (300 MHz, DMSO-d ₆ , 70°C): δ 8.97 (d, J=4.3 Hz, 1H), 8.38 (s, 1H), 8.20 (s, 1H), 8.03 (d, J=8.5 Hz, 1H), 7.71 (d, J=6.8 Hz, 1H), 7.66-7.55 (m, 1H), 7, 52 (d, J=4.2 Hz, 2H), 6.63 (s, 1H), 4.46 (dd, J=14.1, 7.1 Hz, 1H), 3.67 (s, 2H ), 2.90 (d, J=4.6 Hz, 3H), 1.40 (d, J=7.0 Hz, 3H); [α] _D ²⁴ = 44.77 (c = 1.14, MeOH).

Example C. Preparation of (S)-N-methyl-8-(1-((2'-methyl-[4,5'-bipyrimidine]-6-yl-4',6'-d ₂ )amino)propan-2-yl)quinoline-4-carboxamide (Compound 2 )

Scheme 3

As shown in step 3-i of Scheme 3, (S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl)-N-methylquinoline-4-carboxamide (Compound 1013 , 2.542 g, 7.146 mmol), 2-methyl-4,6-dideutero-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (Compound 1003 , 2.204 g, 7.146 mmol, 72 wt%), Na ₂ CO ₃ (10.72 mL 2 M aqueous solution, 21.44 mmol) and Silacat® DPP Pd (SiliCycle, Inc., 1.429 g, 0.3573 mmol) are dissolved in dioxane ( 30.00 ml), the solution was purged with nitrogen gas for 5 minutes and the reaction mixture was stirred at 90°C for 16 hours. The mixture is filtered through diatomaceous earth, concentrated under reduced pressure, dissolved in DMSO and purified by reverse phase chromatography (10-40% CH ₃ CN/H ₂ O, 0.1% TFA). The product fractions are combined and DCM and MeOH are added, followed by 1 N NaOH until the pH is greater than 7. The product solution is extracted with DCM (2 times) and the combined extracts are dried over Na ₂ SO ₄ and filtered. and concentrated under reduced pressure to give (S)-N-methyl-8-(1-((2'-methyl-4',6'-dideutero-[4,5'-bipyrimidin]-6-yl)amino) propan-2-yl)quinoline-4-carboxamide (Compound 2 , 181 mg, 28% yield), as an off-white solid. ¹ H-NMR (300 MHz, DMSO-d ₆ , 70°C): δ 8.95 (d, J=4.2 Hz, 1H), 8.47 (s, 1H), 8.35 (s, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.74 (d, J=7.1 Hz, 1H), 7.59 (t, J=7.8 Hz, 1H) , 7.50 (d, J=4.3 Hz, 1H), 7.30 (s, 1H), 7.03 (s, 1H), 4.51 (g, J=7.2 Hz, 1H) , 3.78 (m, 2H), 2.88 (d, J=4.6 Hz, 3H), 2.68 (s, 3H), 1.41 (d, J=7.0 Hz, 3H) . When 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine is used in this reaction instead of deuterated Compound 1003 , Compound 1 is obtained: LCMS = 414.40 (M+N); ¹ H-NMR (300 MHz, DMSO-d ₆ , 70°C): δ 9.14 (s, 2H), 8.95 (d, J=4.3 Hz, 1H), 8.47 (s, 1H), 8.34 (br.s, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.74 (d, J=7.3 Hz, 1H), 7.59 ( t, J=7.8 Hz, 1H), 7.50 (d, J=4.3 Hz, 1H), 7.28 (br.s, 1H), 7.04 (s, 1H), 4, 52 (g, J=7.0 Hz, 1H), 3.83-3.66 (m, 2H), 2.88 (d, J=4.4 Hz, 3H), 2.68 (s, 3H ), 1.42 (d, J=6.9 Hz, 3H).

Example 2: General method for preparing co-crystals of the compound of formula I and CCF selected from the group consisting of adipic acid, citric acid, fumaric acid, maleic acid, succinic acid or benzoic acid

In general, co-crystals according to this invention can be obtained by crystallization from a suspension or crystallization from NME.

In one particular example, either Compound 1 or Compound 2 was weighed into test tubes and mixed with CCF in a ratio of approximately 1:1.2, respectively, and mixed in a suitable solvent for 2 weeks. At the end of this time, X-ray powder diffraction analysis revealed new crystal structures. Table 1 summarizes the compound ratios and concentrations for the formation of co-crystals of Compound 1 .

Table 1 Co-educator Weight CCF
(mg) Weight of Connection 1
(mg) Solvent Volume
(µl) adipic acid 6.12 14.0 CH ₃ CN 500 succinic acid 5.45 14.9 CH ₃ CN 500 maleic acid 5.14 15.0 EtOAc 500 fumaric acid 5.33 15.0 CH ₃ CN 500 lemon acid 7.45 12.8 EtOAc 500 benzoic acid 5.25 14.8 water 500

Example 3: Preparation of co-crystal from compounds 1 And 2 and adipic acid

Add compound 1 (36.04 g, 0.087 mol, 1.000 eq.), adipic acid (16.65 g, 0.114 mol, 2.164 eq.), 1-propanol (321 .00 g, 5.342 mol, 122.564 eq.) and the suspension was stirred at 750 rpm. A co-crystal seed (co-crystal seed 0.5%) is added and the reaction mixture is stirred at 25°C. Co-crystal formation is monitored by aliquoting and analyzing by Raman spectroscopy. After 114 hours, the co-crystal formation was determined to be complete. Filter the suspension using a 600 ml medium porosity glass plate funnel until the solvent level is uniform, with a wet cake on the filter. The mother liquor is separated, indexed and analyzed for content. The wet filter cake was then washed with 1-propanol (270.0 mL, 7.49 vol). The wet filter cake solids are weighed and dried in a vacuum oven at 50°C. The final yield of the co-crystal of compound 1 and adipic acid is 21.7 g. A similar procedure is also carried out for the co-crystal of compound 2 and adipic acid. HPLC analyzes show a stoichiometry of approximately 2:1 for Compound 1 or Compound 2 relative to adipic acid.

Alternatively, co-crystals of adipic acid and compound ( 1 ) are also prepared by HME crystallization. NME crystallization is carried out on a 20 g scale on a 16 mm extruder. Compound ( 1 ) in free form and pure adipic acid are extruded under high shear stirring and at elevated temperatures (eg 144°C or 155°C) until a co-crystal is formed.

Some physical properties of compound ( 2 ) in free base form and its co-crystal with adipic acid are summarized in Table 2 below.

table 2
Physical properties of the free form of the compound ( 2 ) and its co-crystal with adipic acid Substance assessment Free form Co-crystal with adipic acid (80% Compound 2:20% AA) (w/w)
Solvent crystallization method Co-crystal with adipic acid (80% Compound 2:20% AA) (w/w) Melt extrusion method Co-crystal with adipic acid (75% Compound 2:25% AA) (w/w) Melt extrusion method Bulk Density 0.33 g/cm ² 0.14 g/ ^cm2 0.43 g/cm ² 0.62 g/cm ² Shake density 0.47 g/cm ² 0.25 g/cm ² 0.60 g/cm ² 0.70 g/cm ²

Example 4: Powder X-ray Characterization

X-ray powder diffraction spectra for co-crystals according to this invention (see figures 1-7) were recorded at room temperature, by reflection, using a Bruker D8 Advance diffractometer equipped with a sealed-in-tube Cu source and a Vantec PSD detector (Bruker AXS, Madison, WI). The X-ray generator operated at a voltage of 40 kV and a current of 40 mA. The powdered sample was placed on a silicone or PMM holder. Data were recorded over the range 4° - 45° 2θ, with a step size of 0.0140° and a dwell time of 1 sec. one step. Fixed deflection slits of 0.2 mm were used.

Powder X-ray diffraction patterns for the co-crystals of Form A and Form B according to this invention (see Figures 14 and 15) were recorded at room temperature, by transmission, using a PANanalytical Empyrean diffractometer equipped with a sealed-in-tube Cu source and a PIXCel 1D detector. The X-ray generator operated at a voltage of 45 kV and a current of 40 mA. The powdered sample was placed on a transmission holder and held in place using Mylar thin films. Data were recorded over the range 4° - 40° 2θ, with a step size of 0.007° and a dwell time of 1549 sec. one step. The diffractometer was a device with 0.02° solar slits, fixed 1/2° anti-scattering slits relative to the incident beam, and 1/4° anti-scattering slits relative to the diffracted side. Two scans were accumulated.

Figure 1 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and adipic acid. The X-ray powder diffraction pattern shows that the co-crystal is a mixture of forms A and B. Some specific reflectance peaks of the X-ray powder diffraction spectrum are summarized below.

Table 3 No. Position [°2 θ] Relates. int. [%] 1 6.540282 61.33 2 7.858682 60.04 3 11.92977 52.67 4 12.2278 23.87 5 13.03317 29.49 6 14.22935 100 7 18.75679 59.81 8 19.0885 36.36

Figure 2 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 2 and adipic acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 4 No. Position [°2 θ] Relates. int. [%] 1 6.459033 55.29 2 7.911365 51.42 3 11.91567 45.41 4 12.25639 24.61 5 12.98715 34.47 6 14.19256 100 7 18.67692 38.85 8 19.06727 28.68

Figure 3 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and citric acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 5 No. Position
[°2θ] Relates. int. [%] 1 7.435926 50.1 2 8.291282 19.41 3 11.35154 21.73 4 13.26446 100 5 15.49248 47.42 6 21.55281 20.72 7 23.57031 30.18

Figure 4 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and fumaric acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 6 No. Position
[°2θ] Relates. int. [%] 1 8.264997 97.26 2 10.1077 23.4 3 14.97012 35.06 4 16.60917 41.79 5 17.21781 100 6 25.1975 67.75 7 26.01104 24.39

Figure 5 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and maleic acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 7 No. Position
[°2θ] Relates. int. [%] 1 6.205335 15.27 2 10.43158 20.84 3 11.28478 40.95 4 12.41363 34.13 5 13.26101 19 6 18.86924 43.52 7 21.08017 31.35

Figure 6 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and succinic acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 8 No. Position
[°2θ] Relates. int. [%] 1 8.01725 26.29 2 12.33839 42.72 3 14.77709 37.21 4 17.31539 12.09 5 19.56132 13.66 6 20.05503 100

Figure 7 shows the X-ray powder diffraction pattern of the co-crystal formed between compound 1 and benzoic acid. Some specific X-ray powder diffraction reflection peaks are summarized below.

Table 9 No. Position
[°2θ] Relates. int. [%] 1 8.699594 88.63 2 13.90495 68.65 3 15.6186 80.96 4 17.6481 100 5 18.15049 41.75 6 20.76838 39 7 24.72293 67.36

Example 5: Thermogravimetric Analysis

Thermogravimetric analyzes (TGA) were performed on a TA Instruments model Q5000 thermogravimetric analyzer. Approximately 1-4 mg of solid sample was placed in a platinum sample cup and heated to 300°C, 10°C/min, under a nitrogen flow of 90 ml/min. All thermograms were analyzed using V4.4A TA Instruments Universal Analysis 2000 software.

Thermogravimetric analysis curves for co-crystals of compound ( 1 ) and adipic acid and for co-crystals of compound ( 2 ) and adipic acid are presented in Figures 8 and 9, respectively. The figures show the loss of adipic acid starting at approximately 150°C for both co-crystals.

Example 6: Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed on a TA Instruments model Q2000 calorimetric analyzer. Approximately 1-4 mg of the solid sample was placed in a pinhole corrugated aluminum dish and heated to 300°C, 10°C/min, under a nitrogen flow of 50 ml/min. All data were analyzed using V4.4A TA Instruments Universal Analysis 2000 software.

Typical differential scanning calorimetry thermograms are presented in Fig. 10 and Fig. 11 for co-crystals of compound ( 1 ) and adipic acid and for co-crystals of compound ( 2 ) and adipic acid, respectively.

Example 7: Solid State Nuclear Magnetic Resonance Spectroscopy

Solid state nuclear magnetic resonance (ss-NMR) spectra were acquired on a Bruker-Biospin, Advance III, 400 MHz spectrometer equipped with a 4 mm HFX head. Approximately 70 mg of each sample was compacted to the full volume of 4 mm Bruker-Biospin ZrO ₂ rotors. Magic angle rotation speed (MAS) was used, typically at 12.5 kHz. The temperature of the measuring head was up to 275°K to minimize the effect of frictional heating during rotation. A relaxation delay of 30 seconds was used for all experiments. The CP contact time of ^{the 13} C-CPMAS experiment was up to 2 milliseconds. A CP proton pulse with a linear slope (50% to 100%) was used. The Hartmann-Hahn fit was optimized using an external reference sample (glycine). SPINAL 64 splitting was used, with a field strength of approximately 100 kHz. The chemical shift was related to the external standard adamantane, with its upfield resonance found at 29.5 ppm.

After solvent washing, ss-NMR was used to study co-crystal complexes of compound 1 and compound 2 with adipic acid. See Figures 12 and 13, respectively. The absence of reflection peaks typical of free compound 1 , compound 2 , or adipic acid indicates a pure co-crystal.

Example 8: Preparation of polymorphic forms A and B of co-crystals of adipic acid and compounds ( 1 ) And ( 2 )

A. Preparation of polymorphic form A of the co-crystal of adipic acid and compound ( 1 )

Polymorph Form A of the cocrystal of adipic acid and compound ( 1 ) can be obtained by melt extrusion of compound ( 1 ) and adipic acid. A specific example of obtaining Form A by melt extrusion is described below.

Adipic acid was jet milled using a Jet-O-Mizer Fluid Energy Model 00 using the following settings:

Parameter Pressure [psi] Air supply 100 Grinding attachment 60 Pushing nozzle 80

Compound ( 1 ) was sieved through a No. 18 mesh sieve. Compound ( 1 ) and jet-milled adipic acid were weighed to prepare binary mixtures of approximately 80 wt.%, 75 wt.%. and 65% wt. : mass of connection ( 1 ). The initial mixtures were prepared by passing through a No. 30 sieve and then mixing in a cylindrical mixer for 5 minutes.

The mixtures were extruded using a Leistritz Nano 16 twin screw extruder with three temperature zones and equipped with a plunger doser. The screw design contained conveyor delivery, surging and 30° and 60° plasticizing elements. All experiments were carried out outside the head mounted on the extruder. Temperature, screw speed and temperature are listed in the table below. The temperature was set and controlled at the same value for all three heating elements. During extrusion, the torque was monitored and the screw speed was increased when the screw was in danger of “sticking.”

Parameter Installation Feed rate [ml/min] 1.5
3.75
5 Auger speed [rpm] from 20 to 150 Temperature [°C] 110
130
144
155

The transmission X-ray powder diffraction pattern and ¹³ C-NMR spectrum of Form A co-crystal of adipic acid and compound ( 1 ) are presented in Figures 14 and 16, respectively. Some peaks observed in the ¹³ C NMR spectrum are summarized below.

Table 10 Peak Offset ±0.1 [ppm] Intensity [% of max.] 1 117.1 47.6 2 96.8 28.2 3 95.7 26.2 4 27.6 48.1 5 14.8 32.7 6 161.6 36.5 7 154.5 33.4 8 51.5 24.7 9 50.2 24.3 10 25.6 99.2 eleven 18.5 33.7 12 179.4 54.4 13 168.4 55.9 14 158.3 83.5 15 147.8 46.5 16 145.7 27.9 17 143.2 44.1 18 141.8 43.2 19 124.6 100.0 20 31.2 31.7 21 30.1 35.2

B. Preparation of polymorphic form A of the co-crystal of adipic acid and compound ( 2 )

Form A co-crystal of adipic acid and compound ( 2 ) was prepared by suspension in acetone. 322 mg of a mixture of Form A and Form B of the co-crystal of compound 2 :adipic acid, prepared as described in example 3, and 221 mg of adipic acid were stirred in 9.8 g of acetone, at a temperature of 20°C to 30°C, for 30 days. Approximately 50 mg of solid was isolated by filtration centrifugation through a membrane filter using a centrifugal filter device and dried under vacuum at 20°C to 30°C for approximately 2 hours. Solid state NMR spectra were obtained as described in Example 7, except that the amount of sample was approximately 50 mg and the relaxation delay was up to 5 seconds. ^{The 13} C-NMR spectrum of Form A of the co-crystal of adipic acid and compound ( 2 ) (see Figure 17) is essentially the same as that of Form A of the co-crystal of adipic acid and compound ( 1 ). Some peaks observed in the ¹³ C NMR spectrum are summarized below.

Table 11 Peak Offset ±0.1 [ppm] Intensity [% of max.] 1 116.9 48.3 2 96.6 27.4 3 95.6 23.9 4 27.5 45.6 5 14.7 36.7 6 161.4 32.9 7 153.9 15.9 8 51.3 22.5 9 49.9 22.2 10 25.4 100.0 eleven 18.3 35.8 12 179.2 55.6 13 168.2 49.5 14 158.2 48.2 15 147.6 46.0 16 145.5 27.1 17 143.1 45.7 18 141.6 44.6 19 124.4 91.9 20 31.0 30.9 21 29.9 33.4

C. Preparation of polymorphic form B of a co-crystal of adipic acid and compound ( 2 )

Polymorph Form B of the co-crystal of adipic acid and compound ( 2 ) can be prepared by using spray drying. A specific example is described below.

A solvent spray drying mixture is prepared by weighing 50 g of methanol and 117.5 g of dichloromethane in a glass flask and shaking. A mixture of 500 mg of compound ( 2 ), 176.2 mg of adipic acid and 19.3 g of a mixture of methanol and dichloromethane is weighed into a clear glass vial and stirred until all solids are dissolved. The solution is spray dried using a Buchi B-290 mini spray dryer using the following setup:

Parameter Installation Inlet temperature 99°С Aspirator 100% Pump 40% Capacitor -5°С Nozzle 1 mm Spray 35 mm Filter pressure -60 mbar

The isolated substance completely recrystallizes within 2 months, at room temperature, to form B co-crystal of compound ( 2 ): adipic acid.

The X-ray powder diffraction pattern and ^13C -NMR spectrum of Form B of the co-crystal of adipic acid and compound ( 2 ) are presented in Figures 15 and 18, respectively. Some peaks observed in the ¹³ C NMR spectrum are summarized below.

Table 12 Peak Offset ±0.1 [ppm] Intensity [% of max.] 1 117.9 42.2 2 97.3 25.6 3 94.0 19.6 4 26.7 64.6 5 15.7 32.7 6 161.7 45.0 7 153.8 14.5 8 50.7 37.0 9 25.3 84.7 10 18.8 31.8 eleven 179.1 61.6 12 168.3 54.1 13 158.1 67.2 14 147.2 31.5 15 142.4 44.9 16 124.5 100.0 17 32.3 30.7 18 30.1 31.2 19 125.8 70.3

Example 9: Binary phase diagram of a co-crystal compound ( 2 )/adipic acid

Figure 18 shows an approximate phase diagram consistent with the temperature data: _TAA : melting temperature of adipic acid, T _CoX : melting temperature of the co-crystal of compound ( 2 ): adipic acid, T _P : peritectic temperature, T _CMPD2 : melting temperature of the compound 2 , T _E1 : eutectic melt temperature, P: peritectic point, E1: eutectic point, S _AA : solid adipic acid, L: liquid, S _CoX : solid co-crystal compound ( 2 ): adipic acid, S _CMPD2 : solid compound ( 2 ), T _mE : metastable eutectic melt temperature, mE: metastable eutectic point.

The binary phase diagram was examined using differential scanning calorimetry with respect to mixtures of compound ( 2 ) and adipic acid and mixtures of compound ( 2 ):adipic acid and co-crystal. The stoichiometric composition of the co-crystal in % wt:wt of the compound ( 2 ) was calculated from the molar stoichiometry. A typical differential scanning calorimetry thermogram is shown in Fig. 11. The thermogram of the co-crystal of compound ( 2 ):adipic acid shows a melting endotherm at 196°C ± 2°C, followed by a recrystallization ectotherm, followed by a broad dissolution endotherm. Melting of compound ( 2 ) was observed at a temperature of 256°C ± 2°C, when adipic acid completely decomposes and volatilizes. The observed differential scanning calorimetry thermogram depends on the composition, that is, the solid phases that are present in the substance, and is explained by means of a binary phase diagram. In addition, it depends on other experimental details. The eutectic melt endotherm was observed when excess adipic acid was present in addition to the co-crystal, at a temperature of 138°C ± 2°C. The binary phase diagram of compound ( 2 ) and adipic acid is consistent with the observed differential scanning calorimetry curves for the co-crystal of compound 1 :adipic acid and mixtures of compound 1 :adipic acid with adipic acid; an example is shown in Fig. 10.

Some measured points of the phase diagram of Fig. 19 are summarized below:

Table 13 Dot Temperature [°C]±2 Composition
[%wt:wt] Compound 2 E1 T _E1 =138 65±5 P or E2 T _P or T _E2 =196 Undefined _TAA 153 0 _TCMPD1 256 100

Example 9: Biopharmaceutical Analysis

The pH solubility curve for compound ( 2 ), co-crystal compound ( 2 ):adipic acid and co-crystal compound ( 2 ):adipic acid in the presence of excess adipic acid was calculated from the pK _a values of compound ( 2 ) and adipic acid, values K _sp of the co-crystal of compound ( 2 ): adipic acid, binding constants of compound ( 2 ) and adipic acid in aqueous buffer and self-association constants of compound ( 2 ) in aqueous buffer and solubility of the free form of compound ( 2 ). The solubility of the co-crystal of adipic acid and compound ( 2 ) was dependent on the pH value and the concentration of excess adipic acid. In general, when the concentration of adipic acid increased, the apparent solubility of the co-crystal decreased. At low pH, the solubility of the co-crystal was less than that of compound ( 2 ) in the free base form, however, within the pH range of the fasting small intestine, the co-crystal is much more soluble than compound ( 2 ) in the free form (or in free base form) as shown in FIG. 20. Simulated oral dosing resulted in almost complete absorption of the adipic acid co-crystal at doses up to 1.5 g, and at doses greater than 800 mg, the negative effect of adipic acid on co-crystal solubility did not significantly reduce exposure (data not shown).

Example 10 Dissolution Analysis

In vitro two-phase dissolution experiments using simulated intestinal and gastric fluids were used to evaluate and predict the in vivo effects of compounds ( 1 ) and ( 2 ) and their co-crystals with adipic acid. Most commonly, drug absorption may occur in the upper intestine and high solubility usually indicates high in vivo bioavailability after addition of simulated intestinal fluid in two-phase experiments for drugs with solubility-limited bioavailability. Figure 21 shows two-phase dissolution profiles for: i) co-crystal of compound 1 :adipic acid obtained by melt extrusion (HME) and suspension crystallization (SC); ii) HME 65:35: co-crystal of compound 1 : adipic acid, obtained by using melt extrusion, with 65% wt. compounds 1 and 35% wt. adipic acid; iii) HME 75:25: co-crystal of compound 1 : adipic acid, obtained using melt extrusion, with 75% wt. compounds 1 and 25% wt. adipic acid; iv) HME 80:20: co-crystal obtained using melt extrusion, with 80% wt. compounds 1 and 20% wt. adipic acid; v) SC 80:20: crystallized from a co-crystal suspension of compound 2 : adipic acid with a final content of compound 2 of 79 wt%. and 21% wt. adipic acid; and vi) free form: free form of compound 2 . As presented in FIG. 21, the two-phase dissolution data for the Compound 1 :adipic acid co-crystal and the Compound 2 :adipic acid co-crystal showed higher concentrations of Compound 1 and Compound 2 than Compound 1 or Compound 2 in free form, respectively. Also, the concentration of compound 1, in the case of a co-crystal of compound 1 :adipic acid, obtained by melt extrusion from compound 1 and adipic acid, with 65% wt. and 35% wt., was higher than in the case of co-crystal compound 2 :adipic acid crystallized from a suspension or co-crystal compound 1 :adipic acid obtained by melt extrusion from compound 1 and adipic acid, with 75% wt. and 25% wt., and a co-crystal of compound 1 : adipic acid obtained by melt extrusion from compound 1 and adipic acid, with 80% wt. and 20 wt.%, respectively. Without being bound by a particular theory, this is possible due to the microstructure that is obtained for the eutectic solid.

Two-phase dissolution experiments were performed at least in duplicate. Fasting simulated gastric fluid (FaSSGF) was equilibrated for 30 minutes with stirring at 37°C in a 100 mL clear glass vial using a water bath consisting of a temperature-controlled jacketed reservoir. Compound 1 :adipic acid co-crystal and Compound 2 :adipic acid co-crystal were added and the suspension was stirred at approximately 130 rpm. and at a temperature of 37°C, respectively. Aliquots (0.5 ml) were withdrawn at time points 5, 15, 30 and 60 min. Solids were separated by filtration centrifugation using centrifugal filter units with 0.45 μm membrane and rotation at 5000 rpm. for 5 min, on an Eppendorff centrifuge, model 5418. The pH value of dissolved samples was measured after their collection at time points of 15 min and 60 min. The supernatants of the filtered samples were diluted 10-fold using HPLC diluent. At time point 65 minutes, simulated fasting intestinal fluid was added to the suspension and the suspension continued to be stirred at 130 rpm. Aliquots (0.5 ml) were removed at time points 75, 90, 120 and 180 min. Solids were separated by filter centrifugation using centrifugal filter units with 0.45 µm membrane and spinning at 5000 rpm for 5 min on an Eppendorff centrifuge model 5418. The pH of dissolved samples was measured after collection at time 75, 90 and 180 min. The supernatants of the filtered samples were diluted 10-fold using HPLC diluent. The amounts of substance and simulated liquids used are summarized below:

Substance Weight [mg] FaSSGF Volume FaSSIF volume HME 65:35 43.5, 44.5 10 16 HME 75:25 40.8, 40.3 10 16 HME 80:20 38.3, 38.1 10 16 SC 80:20 31.7, 32.0, 31.6 8 12 Free form 24.7, 25.1, 26.6 8 12

The concentrations of compounds 1 and 2 were measured using the following HPLC method, respectively:

Column "Xterra, phenyl, 4.6 × 50 mm, 5.0 µm" Column temperature 30°C Volume flow rate 1.5 ml/min Dispenser volume 10 µl Automatic dispenser temperature 25°С Total analysis time 3.0 min Detector wavelength 240 nm Needle cleaning solution methanol Sample injection rate 1 per sample Data collection time 3 Mobile phase A 0.1% TFA in water Mobile phase B 0.1% TFA in acetonitrile Gradient 85% mobile phase A,
15% mobile phase B

Typical simulated liquid preparations were used for two-phase dissolution experiments: FaSSIF was prepared by adding approximately 1.80 g sodium hydroxide granules, 2.45 g maleic anhydride, 6.37 g sodium chloride, 1.61 g sodium taurocholate, and 618.8 mg lecithin to 800 ml water. The solution was stirred until all substances were completely dissolved. The pH was then adjusted to 6.5 using 1.0 N HCl and 50% NaOH while the solution was stirred. Water was added to a final volume of 1 L.

FaSSGF was prepared by adding 50.0 ml of 1.0 N HCl, about 1.0 g of “800-2500 U/mg” pepsin, 43 mg of sodium taurocholate, 2.0 g of sodium chloride (NaCl) to 800 ml of water. Water was added to a final volume of 1 L. The final pH value was typically 1-2.

Example 12 Bioavailability of co-crystals according to this invention

The oral bioavailability of the co-crystal of Compound 2 :adipic acid and the free form of Compound 2 in humans was predicted based on the calculated pH solubility curves presented in Figure 20 using GastroOlus software, version 8.5.0002 Simulations Plus, Inc. . A small intestinal permeability of 1.67e-4 cm/s and a particle radius of 10 microns were used. All other parameters were set by default in the software. The simulations predict a 100% fraction absorbed for oral doses up to 1500 mg of compound 2 :adipic acid co-crystal and compound 2 :adipic acid co-crystal with the presence of additional adipic acid, whereas the predicted oral absorbable fraction of compound 2 decreases sharply with increasing doses. As presented in Figure 22, simulations show that the co-crystal of compound 2 :adipic acid has superior oral bioavailability compared to the free form of compound 2, while achieving sufficient exposure according to human safety studies, for doses up to, but not limited to this, 1500 mg, and, as a result, this may lead to wider safety limits for compound 2 . In addition, high oral bioavailability may reduce the oral dose required to achieve effective blood levels. Similar results are expected for compound 1 based on the similarity in the observed physical properties of compound 1 and compound 2 .

Example 13. Biological effectiveness of the co-crystal of compound 2/adpipic acid

Example A: DNA-PK Kinase Inhibition Test.

The co-crystal of adipic acid and compound 2 was screened for its ability to inhibit DNA-PK using a standard radiometric assay. Briefly, this kinase assay examines the transfer of the terminal ^33P -phosphate to ^33P -ATP to a peptide substrate. The test was performed on 384-well plates with a final volume of 50 μl per well containing approximately 6 nM DNA-PK, 50 μ m HEPES (pH = 7.5), 10 mM MgCl ₂ , 25 mM NaCl, 0.01% DSA, 1 mM DTT, 10 µg/ml fragmented double-stranded DNA (obtained from Sigma), 0.8 mg/ml DNA-PK-peptide (Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp -Leu-Trp-Lys-Lys-Lys, obtained from American Peptide) and 100 µM ATP. Accordingly, the compounds of the present invention were dissolved in DMSO to obtain stock 10 mM solutions. Serial dilutions were then made in DMSO to obtain final test solutions. An aliquot of 0.75 μl of DMSO or inhibitor in DMSO was added to each well, then an ATP substrate solution containing ³³ P-ATP (available from Perkin Elmer) was added. The reaction was initiated by adding DNA-PK, peptide and ds-DNA. After 45 minutes, the reaction mixture was quenched with 25 μl of 5% phosphoric acid. The reaction mixture was transferred to 384-well MultiScreen HTS PH plates (available from Millipore), allowed to bind for one hour and washed three times with 1% phosphoric acid. After adding 50 μl of Ultima Gold ^™ High Efficiency Scintillator (available from Perkin Elmer), samples were counted on a Packard TopCount NXT microplate scintillation and luminescence counter (Packard BioScience). K _i values were calculated using Microsoft Excel Solver macros to fit the data to a kinetic model for competitive tight binding inhibition. The co-crystal of adipic acid and compound ( 2 ) has a Ki _of approximately 2 nM.

Example B: Efficiency of connections ( 1 ) And ( 2 ) in combination with total body irradiation.

The in vivo efficacy of compounds ( 1 ) and ( 2 ) in combination with total body irradiation was studied in the OD26749 xenograft model of primary NSCLC (non-small cell lung cancer) and in xenograft models of the OE-19 GEJ cell line. The results are summarized in Tables 14 and 15. In these studies, compounds ( 1 ) and ( 2 ) were mixed with 16% captisol, 1% polyvinylpyrrolidone, 1% hydroxypropyl methylcellulose E5, pH 2.

IN 1. Connection efficiency ( 1 ) in combination with radiation on the OD26749 NSCLC xenograft model.

The in vivo efficacy of compound ( 1 ) was assessed in the OD26749 subcutaneous xenograft model of primary NSCLC. Compound ( 1 ), administered at 100 mg/kg three times daily, on day 1 significantly enhanced the radiation effect of a single dose of 2 Gy total body irradiation in this model (% T/C 26 for the combination compared with % T/C 80 for one exposure, P<0.001). Efficacy was assessed using regimens in which total body irradiation with a dose of 2 Gy was performed twice, one week apart. Compound ( 1 ) was administered orally (three times daily at time points 0, 3 and 7 hours), at 100 mg/kg, alone or together with one dose of 2 Gy total body irradiation, for about 3.25 hours. Seven days later, the same regimens were repeated. Compound ( 1 ), in combination with total body irradiation with a dose of 2 Gy, induced significant tumor regression (% T/Ti -75; P <0.01) compared with irradiation alone.

Compound ( 1 ) individually and irradiation individually did not cause significant (P>0.05) inhibition of tumor growth compared to control media (% T/C 74 and 64, respectively). In this primary tumor model, both groups showed some degree of weight loss (maximum loss of 6.7% and 8.7% on day 2 or day 9, for the radiation alone and combination groups, respectively), which was repeated over the course of the study. . The addition of a second 2 Gy dose of radiation, in combination with compound ( 1 ), resulted in a significant increase in time to tumor doubling (TTD), with a daily TTD of 33.4 in the combination group compared with only two to three days for the single agent groups : environment, irradiation and connection ( 1 ).

AT 2. Summary study: compounds ( 1 ) And ( 2 ) in combination with two cycles of total body irradiation in a xenograft model of primary NSCLC (OD26749) in nude mice

The efficacy of compounds ( 1 ) and ( 2 ), in combination with total body irradiation (2 Gy), was assessed in the OD26749 xenograft model of primary NSCLC, at a dose level of compound ( 1 ) of 100 mg/kg, orally, twice daily (times 0 and 4 hours) and compound dose levels ( 2 ) of 50 mg/kg and 100 mg/kg, orally, twice daily (times 0 and 4 hours). Two cycles of total body irradiation (2 Gy) were performed 15 minutes after administration of the first compound (0.25 hours). Control animals were administered vehicle orally, twice daily (time points 0 and 4 hours). Two treatment cycles were carried out on day 0 and day 7.

Two cycles of total body irradiation (2 Gy) individually did not inhibit tumor growth compared to vehicle-treated tumors (% T/C = 106). However, the efficacy was significantly enhanced when compounds ( 1 ) and ( 2 ) were combined with radiation, as mean tumor volumes for all combination groups were significantly smaller than those for the radiation alone group (P<0.001). In addition, combinations with compounds ( 1 ) and ( 2 ) (100 mg/kg twice daily) groups showed very similar antitumor activity (% T/C = 4.80 and 7.80, respectively), blood exposure ( AUC 65.8 and 58.2 mcg*hour/ml) and tolerance (maximum change in body weight -2.4% and 2.70%). In addition, the mean tumor volume of the 50 mg/kg combination group was statistically different from that of the compound ( 1 ) and ( 2 ) 100 mg/kg combination groups (P<0.001).

AT 3. Connection efficiency ( 1 ) And ( 2 ), in combination with irradiation on a xenograft model of gastroesophageal cancer (GEJ) OE-19

A xenograft model of the OE-19 cell line was used to evaluate the effectiveness of compounds ( 1 ) and ( 2 ), individually and in combination with irradiation. Two treatment cycles were carried out (day 0 and day 7) as performed above for model OD26749. Cycles of total body irradiation (2 Gy) individually showed minimal effects on tumor growth compared to vehicle control (% T/C = 60.0), indicating that this tumor model is relatively resistant to radiation. In contrast, the combination of compound ( 2 ) and a dose of 2 Gy total body irradiation resulted in significant inhibition of tumor growth compared to vehicle control with % T/C = 8.00 (P < 0.001). The combination group also showed significant inhibition of tumor growth compared with the radiation alone group (P<0.001). Compound ( 1 ) in combination with total irradiation at a dose of 2 Gy also significantly inhibited tumor growth in this model.

AT 4. Connection efficiency ( 1 ) And ( 2 ) on a xenograft model of primary NSCLC

The in vivo efficacies of compounds ( 1 ) and ( 2 ) were assessed individually and in combination with three consecutive days of focused irradiation in a subcutaneous xenograft model of primary NSCLC LU-01-0030. The dose-dependent antitumor activity of compound ( 2 ), individually and in combination with a focused beam of ionizing radiation, was assessed in the LU-01-0030 model. In this model, ionizing radiation treatment individually resulted in significant tumor regression, however, tumor regrowth was observed approximately 20 days after the last day of treatment. At day 34, the compound ( 2 ) combination groups demonstrated statistically significant (P<0.001) antitumor activity compared to the vehicle and radiation-only groups, with %T/Ti values of -96.3, -67.1, -96. 9, and 1.6%, for the 50 mg/kg and 25 mg/kg, twice daily, and 50 mg/kg and 25 mg/kg, every day groups. Mice in the combination treatment groups were monitored (mice without treatment) until 90 days, as tumor burden was not confirmed in some mice. For all experimental groups, treatments were generally well tolerated, as evidenced by maximum weight losses ranging from -1.11% to -6.93%, on days 1 to 9 after the start of treatment.

AT 5. Connection efficiency ( 1 ) And ( 2 ), in combination with radiation in the case of a xenograft model of primary GEJ cancer

The in vivo activities of compounds ( 1 ) and ( 2 ) were compared in combination with focused beam ionizing radiation in a subcutaneous xenograft model of primary gastric cancer. In model ST 02 0004, focused ionizing radiation was administered on three consecutive days individually and in combination with compound ( 1 ) or ( 2 ). Radiation treatment individually resulted in a slight reduction in tumor growth approximately 7 days after the last day of treatment. The combination with compounds ( 1 ) and ( 2 ) groups demonstrated statistically significant (P<0.001) antitumor activity compared to the vehicle and radiation-only groups with a %T/Ti value of -2.8%, for the combination with compound ( 1 ). at a dose of 100 mg/kg group and %T/C-values of 9.2 and 17.4 for groups combined with compound ( 2 ) at a dose of 100 mg/kg and 25 mg/kg, respectively. For all experimental groups, treatment was generally well tolerated, as evidenced by maximum weight losses ranging from -8.06% to -10.0%, on days 10 to 48 after initiation of treatment.

The antitumor activity of compound ( 2 ), in combination with focused ionizing radiation and the standard of treatment agents, paclitaxel and carboplatin, was also evaluated in the ST 02 0004 model. Treatment with paclitaxel, carboplatin and radiation was carried out once a week, for three weeks, alone or in combination with a compound ( 2 ). Treatment with paclitaxel/carboplatin had no effect on tumor growth, nor did treatment with the combination of paclitaxel/carboplatin and 50 mg/kg compound ( 2 ). However, on day 45, 25 mg/kg and 50 mg/kg of compound ( 2 ), in combination with paclitaxel/carboplatin and radiation, demonstrated a statistically significant difference (P<0.001) in antitumor activity compared with the %T vehicle group. /C-values of 2.5 and 11.1, for the combination groups of compound ( 2 ), at a dose of 50 mg/kg and 25 mg/kg, paclitaxel/carboplatin, radiation. Further, the 50 mg/kg and 25 mg/kg compound ( 2 ), paclitaxel/carboplatin, and radiation combination groups were statistically different (P<0.05) from the paclitaxel/carboplatin, paclitaxel/carboplatin/50 mg/kg compound ( 2) groups. ) and paclitaxel/carboplatin/irradiation. Blood exposures to compound ( 2 ) were 9.3 and 27 μg*h/mL for the 25 mg/kg and 50 mg/kg compound ( 2 ) dose groups, twice daily, respectively.

In Tables 14 and 15, for example, the indication PO bid (0.4h) means that compound ( 2 ) is administered twice (twice daily) at time 0 and then 4 hours later; indications IR (0.25h) qdx3 mean that irradiation is carried out 15 minutes (0.25 hours) after administration of compound ( 2 ) (0 hours) and once a day for 3 days (qdx3); q7dx2 directions mean once a week, for two weeks; qod indications mean twice, on every other day (for example, day 1 and day 3); and the indications paclitaxel q7dx3 (-0.25h), carboplatin q7dx3 (-0.25h) mean administration of paclitaxel and carboplatin 15 minutes before administration of compound ( 2 ), followed by administration of compound ( 2 ), 4 hours after the first administration of compound ( 2 ). In one specific example, the instructions are “5 mg/kg paclitaxel q7dx3 (-0.25h), 25 mg/kg carboplatin q7dx3 (-0.25h), 2 Gy IR qdx3 (0.25h), PO 50 mg/kg bid ( 0.4h) qdx3” means that 5 mg/kg paclitaxel and 25 mg/kg carboplatin are administered 15 minutes before the first administration of the compound ( 2 ); carry out the first introduction of the compound ( 2 ); irradiation is carried out 15 minutes after the first administration of the compound ( 2 ); and then a second administration of the compound ( 2 ) is performed 4 hours after the first administration of the compound ( 2 ) .

Table 14
Summary of in vivo efficacy studies of the compound ( 1 ) Tumor model, DNA damaging agent Study groups results OD26749
(Primary NSCLC)
Whole body irradiation %T/C (Day 20) %T/Ti (Day 20) maximum body weight loss (%) 2 Gy of radiation, every day - 1 time orally 80 - -6.90 (Day 2) 100 mg/kg, three times a day (0, 3, 7 hours), every day - 1 time orally 101 - -2.40 (Day 2) 100 mg/kg, three times a day (0, 3, 7 hours), every day - 1 time, 2 Gy of radiation, every day - 1 time (3.25 hours) 26.0 - -9.70 (Day 2) OD26749
(Primary NSCLC)
Whole body irradiation %T/C (Day 16) %T/Ti (Day 16) maximum body weight loss (%) 2 Gy of radiation, every 7th day twice, orally 64 - -6.7 (Day 2) 100 mg/kg, three times a day (0, 3, 7 hours) every 7th day twice, orally, 74 - weight gain 100 mg/kg, three times a day (0, 3, 7 hours) every 7th day twice, 2 Gy of radiation, every 7th day twice (3.25 hours) -75 - -8.7 (Day 9) OD26749
(Primary NSCLC extension study)*
Whole body irradiation %T/C (Day 22) %T/Ti (Day 22) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 106 - -0.90 (Day 1) 100 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 3rd day 4.8 - -2.40 (Day 1) OD26749
(Primary NSCLC)
Whole body irradiation %T/C (Day 29) %T/Ti (Day 29) maximum body weight loss (%) 2 Gy of radiation, every 7th day twice, orally 42 - -3.50 (Day 1) 200 mg/kg, every day, 2 Gy of radiation (0.25 h), every 7th day twice, orally 6.5 - -6.10 (Day 1) 100 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 7th day twice, orally -3.1 -3.70 (Day 8) 50 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 7th day twice, orally 11.7 - -5.50 (Day 8) 25 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 7th day twice 25.6 - -7.70 (Day 8) LU-01-0030
(Primary NSCLC)
focused irradiation %T/C (Day 30) %T/Ti (Day 30) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 14.8 - -4.0 (Day 4) 100 mg/kg, three times a day (0, 3, 7 hours), every 5th day, orally 79.1 - -0.63 (Day 6) 100 mg/kg, three times a day (0, 3, 7 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally, -90.6 - -1.58 (Day 4) 100 mg/kg, three times a day (0, 3, 7 h), every 5th day, 2 Gy of radiation (0.25 h), every 3rd day, orally, -91.6 - -1.68 (Day 4) 100 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day -85.6 - -1.42 (Day 4) LU-01-0030
(Primary NSCLC)
focused irradiation %T/C (Day 27) %T/Ti (Day 27) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 16.1 - -7.44 (Day 3) 100 mg/kg, every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -76.5 - -3.68 (Day 2) 100 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -90.1 - -2.87 (Day 3) 50 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -87.8 -5.70 (Day 3) 25 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day -80.3 - -5.81 (Day 2) LU-01-0030
(Primary NSCLC)
focused irradiation %T/C (Day 27) %T/Ti (Day 27) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 16.1 - -7.44 (Day 3) 50 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -76.5 -3.68 (Day 2) 50 mg/kg, twice a day (0.4 h), every 2nd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -90.1 - -2.87 (Day 3) 25 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -87.8 - -5.70 (Day 3) 10 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day -80.3 - -5.81 (Day 2) LU-01-0030
(Primary NSCLC)
focused irradiation %T/C (Day 31) %T/Ti (Day 31) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 49.3 - -4.46 (Day 2) 10 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), orally -5.3 - -3.33 (Day 3) 50 mg/kg, every 3rd day, 2 Gy of radiation (0.25 h), orally 4.5 - -2.07 (Day 1) 50 mg/kg, twice a day (0.4 h), every day - 1 time, 2 Gy of radiation (0.25 h), orally 7.2 - -0.59 (Day 1) 50 mg/kg, twice a day (0.4 h), every 2nd day, 2 Gy of radiation (0.25 h), orally -1.7 - -2.11 (Day 1) 50 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h) -14.1 - -0.94 (Day 3) LU-01-0030
(Primary NSCLC)
focused irradiation %T/C (Day 24) %T/Ti (Day 24) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 26.7 - -0.40 (Day 2) 10 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation, every 3rd day (0.25 h), orally -29.8 - -1.46 (Day 4) 25 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation, every 3rd day (0.25 h), orally -75.2 - -2.03 (Day 4) 50 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation, every 3rd day (0.25 h), orally, -87.6 - -1.19 (Day 4) 50 mg/kg, twice a day (0.4 h), every 2nd day, 2 Gy of radiation, every 3rd day (0.25 h) -79.9 - -1.59 (Day 4) OE-19 (GEJ cell line)
Whole body irradiation %T/C (Day 18) %T/Ti (Day 18) maximum body weight loss (%) 2 Gy of radiation, every 7th day twice, orally 86.0 - -1.90 (Day 1) 100 mg/kg, twice a day (0.4 hours), every 7th day twice, orally 79.0 - -1.70 (Day 8) 100 mg/kg, twice a day (0.4 h), every 7th day twice, 2 Gy of radiation, every 7th day twice (0.25 h) 24.0 - -3.50 (Day 1) ST-02-0004
(GEJ Primary Tumor - Additional Study)*
focused irradiation %T/C (Day 34) %T/Ti (Day 34) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 59.6 - -8.06 (Day 48) 100 mg/kg, every 3rd day, orally 95.6 - -6.31 (Day 14) 100 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h) -2.8 - -10.0 (Day 10)

Table 15
Summary of in vivo studies on the effectiveness of the compound ( 2 ) Tumor model, DNA damaging agent Study groups results OD26749
(Primary NSCLC - additional study) %T/C (Day 22) %T/Ti (Day 22) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 106 - -0.90 (Day 1) 100 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 3rd day, orally 7, - -2.70 (Day 1) 50 mg/kg, twice a day (0.4 h), 2 Gy of radiation (0.25 h), every 3rd day 27.2 - -2.10 (Day 1) LU-01-0030
(Primary NSCLC) %T/C (Day 34) %T/Ti (Day 34) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 16.9 - -4.93 (Day 3) 50 mg/kg, twice a day (0.4 hours), every 3rd day, orally 98.3 - -1.11 (Day 9) 50 mg/kg, twice a day (0.4 hours), every 3rd day, 2 Gy radiation (0.25 h), every 3rd day, orally -96.3 - -6.93 (Day 3) 25 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -67.1 - -6.59 (Day 3) 50 mg/kg, every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day, orally -96.9 - -4.66 (Day 3) 25 mg/kg, every 3rd day, 2 Gy of radiation (0.25 h), every 3rd day -1.6 - -4.62 (Day 1) OE-19 (GEJ cell line - additional study) %T/C (Day 21) %T/Ti (Day 21) maximum body weight loss (%) 2 Gy of radiation, every 7th day twice, orally 60.0 - -0.80 (Day 1) 100 mg/kg, twice a day (0.4 h), every 7th day twice, 2 Gy of radiation, every 7th day twice (0.25 h) 8.0 - -6.50 (Day 7) ST-02-0004
(GEJ primary tumor) %T/C (Day 34) %T/Ti (Day 34) maximum body weight loss (%) 2 Gy of radiation, every 3rd day, orally 56.9 - -8.06 (Day 48) 100 mg/kg, twice a day (0.4 hours), every 3rd day, orally 67.6 - -7.61 (Day 34) 100 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation, every 3rd day (0.25 h), orally, 9.2 - -9.15 (Day 14) 25 mg/kg, twice a day (0.4 h), every 3rd day, 2 Gy of radiation, every 3rd day (0.25 h) 17.4 - -6.73 (Day 48) ST-02-0004
(Primary tumor GEJ - with SOC) %T/C (Day 45) %T/Ti (Day 45) maximum body weight loss (%) 5 mg/kg paclitaxel, every 7th day three times (0 hours) 98.0 - -8.93 (Day 45) 25 mg/kg carboplatin, every 7th day three times (0 hours) 5 mg/kg paclitaxel, every 7th day three times (-0.25 hours), 25 mg/kg carboplatin, every 7th day three times (-0.25 hours), orally, 50 mg/ kg, twice a day (0.4 h), every 3rd day 95.4 - -10.1 (Day 45) 5 mg/kg paclitaxel, every 7th day three times, 25 mg/kg (-0.25 h), carboplatin, every 7th day three times (-0.25 h), 2 Gy of radiation, every 3rd day (0 h) 34.9 - -10.0 (Day 3) 5 mg/kg paclitaxel, every 7th day three times (-0.25 h), 25 mg/kg carboplatin, every 7th day three times (-0.25 h), 2 Gy of radiation, every 3rd day ( 0.25 h), orally, 50 mg/kg, twice a day (0.4 h), every 3rd day 2.5 - -9.20 (Day 10) 5 mg/kg paclitaxel, every 7th day three times (-0.25 h), 25 mg/kg carboplatin, every 7th day three times (-0.25 h), 2 Gy of radiation, every 3rd day ( 0.25 h), orally, 25 mg/kg, twice a day (0.4 hours), every 3rd day 11.1 - -8.21 (Day 3)

Example 11. Connection combination ( 1 ) or connections ( 2 ) with standard drugs or radiation, in the case of cancer cell lines

Cell-based experiments and analyzes were performed with each molecule, but not always with both. Compounds ( 1 ) and ( 2 ) are generally very similar in these assays and experiments. Analysis of the combination experiments was carried out using two methods: the Bliss-additivity model method and the mixture method to determine the degree of synergism, additivity or antagonism. Using the Bliss method, a matrix of Bliss scores was created for each cell line and treatment and the sum of the Bliss scores was calculated relative to the range of combination concentrations tested. The average Bliss score (Bliss sum divided by the number of points of all data) was then used to categorize the cell line and treatment as follows: greater than 10 indicates strong synergy, greater than 5 indicates synergism, between 5 and -5 indicates Additivity less than -5 means antagonism and less than -10 means strong antagonism. Larger Bliss scores indicate stronger significance in the reported synergism, and smaller scores indicate stronger significance in the reported antagonism. In the case of the mixture formulation method, the components to be combined were added in a range of optimal ratios using design of experiment (DOE) software (DX-8 from STAT-EASE); cells were irradiated with a dose of 2 Gy as required. Synergism was determined using statistical data analysis (ANOVA) to indicate linear (additivity) or statistically significant (p < 0.1) non-linear (antagonism or synergism) mixtures of combined components.

Some cancer cell lines and their tumor types are listed in Table 16.

Table 16
List of cancer cell lines Cell line Tumor type DOHH-2 B cell lymphoma DU-4475 breast EOL-1 leukemia Farage non-Hodgkin lymphoma B-cell lymphoma GRANTA-519 mantle cell lymphoma HBL-1 B cell lymphoma HCC2935 lungs - NSCLC HCC95 lungs - NSCLC HH T cell lymphoma HT-115 rectum JHH-2 liver KARPAS-299 non-Hodgkin lymphoma B-cell lymphoma KARPAS-422 non-Hodgkin lymphoma B-cell lymphoma KARPAS-620 multiple myeloma KASUMI-1 leukemia, AML KE-97 stomach KELLY neuroblastoma KG-1 leukemia, AML KG-1a leukemia, AML KMS-20 multiple myeloma KMS-21-BM multiple myeloma KMS-34 multiple myeloma LC-1F lungs - NSCLC LCLC-103H lungs - NSCLC LU-134-A lungs - SCLC LU-139 lungs - SCLC MDST8 rectum ML-1 thyroid MOLM-13 leukemia CML MV-4-11 leukemia NCI-H1048 lungs - SCLC NCI-H1650 lungs - NSCLC NCI-H1694 lungs - SCLC NCI-H1944 lungs - NSCLC NCI-H1993 lungs - NSCLC NCI-H2126 lungs - NSCLC NCI-H2141 lungs - SCLC NCI-H2171 lungs - SCLC NCI-H2228 lungs - NSCLC NCI-H446 lungs - SCLC NCI-H820 lungs - NSCLC NCI-H841 lungs - SCLC NCI-H929 multiple myeloma NOMO-1 leukemia, AML OCI-Ly3 B cell lymphoma OCI-Ly7 B cell lymphoma OPM-2 B cell lymphoma OVK18 B cell lymphoma PC-3 prostate PC-9 lungs - NSCLC R.L. B cell lymphoma RPMI-8226 B cell lymphoma SU-DHL-10-epst B cell lymphoma TE-1 esophagus TE-14 esophagus THP-1 leukemia, AML U-2932 B cell lymphoma WM-266-4 leather WSU-NHL B cell lymphoma ZR-75-1 breast

A. Double combinations

Compound ( 2 ) was tested against a panel of 60 cancer cell lines (see Table 16) individually and in combination with a panel of cytotoxic and non-cytotoxic SOC agents. The 60 Cancer cell lines are lines derived from breast cancer, prostate cancer, acute myeloid leukemia (AML), myeloma and other cancers. Cells were removed from liquid nitrogen storage, thawed, and expanded in appropriate culture media. Once they had expanded in volume, the cells were seeded into 384-well tissue culture-treated plates at 500 cells per well. After 24 hours, cells were treated for either 0 hours or treated for 144 hours with compound ( 2 ), in combination with genotoxin:bleomycin (radiomimetic), doxorubicin (topoisomerase II inhibitor), etoposide (topoisomerase II inhibitor), carboplatin (cross-linker DNA), BMN-673 (PARP inhibitor) and Tarseva (EGFR inhibitor). At the end of either 0 hours or 144 hours, cellular status was analyzed using ATPLite (Perkin Elmer) to assess the biological response of cells to drug combinations.

Compound ( 2 ) showed strong synergy with several of the agents tested: etoposide (topoisomerase inhibitor), doxorubicin (DNA intercalator) and bleomycin (radiomimetic) (Figure 23). Some synergy was observed when combined with BMN-673 (a PARP inhibitor) and carboplatin (a DNA repair inhibitor). Additivity was observed with erlotinib (EGFR inhibitor) (Figure 23). When analyzed according to cancer cell line type, compound ( 2 ) and BMN-673 showed extremely potent anti-AML activity. The combination of compound ( 2 ) and etoposide, although highly active against most lines, was particularly active against non-small cell lung cancer lines, like compound ( 2 ) and doxorubicin (see below). Bliss data on the synergism of the compound ( 2 ), in the case of various tumor types (acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), non-small cell lung cancer (NSCLC), multiple myeloma (MSM), large cell lung cancer ( SCLC)) are presented in Figs. 24-30:

combination of compound ( 2 ) with BMN-673 in Fig. 24;

combination of compound ( 2 ) with etoposide in Fig. 25;

combination of compound ( 2 ) with bleomycin in Fig. 26;

combination of compound ( 2 ) with erlotinib in Fig. 27;

combination of compound ( 2 ) with doxorubicin in Fig. 28;

combination of compound ( 2 ) with bleomycin in Fig. 29;

combination of compound ( 2 ) with carboplatin in Fig. 30.

Combinations of compound ( 2 ) and doxorubicin or epirubicin (a DNA intercalator) were tested against breast cancer cell lines (Tables 17 and 18), with comparisons between wild-type and mutant lines being the focus of the study. Regardless of seeding density, individual doxorubicin sensitivity, or BRCA status, the combination of doxorubicin and compound ( 2 ) was highly synergistic in all five cell lines and at both concentrations of compound ( 2 ) tested (Bliss assay). The more than threefold change in IC ₅₀ of the combination of doxorubicin and compound ( 2 ), compared with doxorubicin individually, also indicates a high degree of synergy. A similar experiment using doxorubicin or epirubicin, in combination with compound ( 2 ), in the case of breast cancer line DU4475, showed strong synergy (Bliss analysis) (Table 18).

The combination of compound ( 2 ) and doxorubicin or epirubicin was highly synergistic in all triple-negative breast cancer cell lines evaluated, regardless of BRCA status or seeding density.

Table 17
Summation of combinations with compound ( 2 ) and doxorubicin, in the case of triple-negative breast cancer cell lines Cell line Seeding density Status
BRCA Average Bliss Score Doxorubicin IC ₅₀ (µM) Max. IC shift ₅₀
(once) HCC-1395 5000 mutant 11.1 0.5 4.6 HCC-1599 unknown mutant 14.3 0.2 4.3 HCC-1937 5000 mutant 16.6 0.2 3.7 HCC-1937 20000 mutant 15.6 0.6 3.9 MDA-MB-436 5000 mutant 14.5 0.7 9.1 MDA-MB-436 20000 mutant 14.4 0.3 4.7 MDA-MB-468 5000 wild type 23.1 0.02 19 MDA-MB-468 20000 wild type 24.7 0.04 13

Table 18
Summation of combinations with compound ( 2 ) and doxorubicin or epirubicin, in the case of DU4475 cells Medicine Average Bliss Score Doxorubicin 31.9 Epirubicin 33.3

B. Double and triple combinations with irradiation (2 Gy) or without irradiation

The following SOC agents were tested in dual combinations with compound ( 1 ): etoposide (a topoisomerase inhibitor that induces DSBs), cisplatin (DNA cross-linker), carboplatin (DNA cross-linker), fluorouracil (5-Fu, an antimetabolite that inhibits thymidylate synthase), paclitaxel (a mitotic inhibitor that binds to tubulin), cetuximab (an EGFR monoclonal antibody), and radiation. Other than the combination of irradiation and compound ( 1 ), the strongest interaction, in the case of double combination studies, was exhibited by the combination of etoposide and compound ( 1 ) in A549 cells (Table 19) and compound ( 1 ) and etoposide in ESJ26 (Table 20) . These findings supported the use of the Bliss additivity model (Table 21). Other combinations demonstrated additivity with rare examples of antagonism. While there is no complete agreement on the various lines tested (as detailed in other sections), the agreements and conclusions reached are general for each case. The same experiment, carried out using OE19 cells, showed more complex patterns of interaction in the absence of irradiation. A significantly enhanced effect was observed when irradiation was added to the combinations, enhancing the strong relationship between DNA damage (DSB and SSB) and DNA-PK inhibition. Table 19 shows cancer cell lines: ESO26 - gastroesophageal cancer, OE19 - gastroesophageal cancer, DMS-53-SCLC, A549 - lung cancer, colo205 - colon cancer, H460 - lung cancer, H2009 - lung cancer, FaDu - pharyngeal cancer, Mipaca2 - pancreatic cancer, HEL1 - human fetal lung fibroblast.

The combination of compound ( 2 ) and doxorubicin or epirubicin was highly synergistic in all three triple-negative cancer cell lines evaluated, regardless of BRCA status or seed density.

In triple SOC combination experiments, synergy was observed with the combination of etoposide, cisplatin and compound ( 1 ) in the DMS-53 and A549 cell lines. A significant driver for this synergy was the combination of etoposide with a compound ( 1 ). The combination of paclitaxel, cisplatin and compound ( 1 ) was additive in the case of the A549 cell line, while the combination of cisplatin, 5-Fu and compound ( 1 ) was synergistic in the case of the Colo205 cell line. A very significant decrease in cell viability was observed after adding irradiation to these combinations, mainly due to the contribution of the compound ( 1 ). Compound ( 2 ) demonstrated similar combination results in terms of cell viability with the SOC components of the combination (using a smaller set of cancer cell lines) compared to compound ( 1 ).

Table 19
Effect of compound ( 1 ) in combination with genotoxic agents on the viability of cancer cell lines ND = not determined, N/A = not applicable: etoposide is a radiomimetic, Plus = increased exposure to radiation. * = reduction in viability occurs primarily due to compound ( 1 ) plus irradiation.

Table 20
Effect of compound ( 2 ) in combination with genotoxic agents on the viability of the cancer cell line ESO26 (GEJ) (mixture analysis) 1. Combinations with Compound 2 2. No radiation 3. Plus radiation Cisplatin, 5-FU Synergy; Connection 2 with 5-FU Significant reduction in cell survival under the influence of Compound 2 and radiation Carboplatin,
Paclitaxel Full additivity Significant reduction in cell survival under the influence of Compound 2 and radiation Etoposide Significant synergies Not applicable

Based on the _IC50 , for 20 µM compound 2 , 50 µM carboplatin, 1.5 µM cisplatin, 3 µM paclitaxel, 0.6 µM etoposide and 20 µM 5-FU. Not applicable: etoposide is a radiomimetic.

Table 21
Effect of compound ( 2 ) in combination with etoposide on the viability of cancer cell lines (Bliss assay) Cell line Average Bliss Score A549 27.3 (n=1) ESO26 43.2 ± 6.5 (n=3) HFL1 8.7 ± 5.6 (n=3)

C. Effect of a combination of compounds ( 1 ) or ( 2 ) and SOC in primary tumor chemotherapy sensitivity tests (TCAs)

In vitro tested human primary tumors may provide a better indicator of the effectiveness of DNA-PK inhibition than immortalized cancer cell lines due to their increased heterogeneity and closer proximity to the patient tumor from which they originate. A panel of primary human tumors (NSCLC, pancreatic, esophageal, gastric, etc.) were tested using compound ( 1 ) to determine the effectiveness of DNA-PK inhibition, in combination with radiation, bleomycin (a radiomimetic that causes DSBs), doxorubicin (DNA intercalator), cisplatin, carboplatin, etoposide, paclitaxel or 5-FU.

Compound ( 1 ) (10x and 30x _IC50 ) was administered in combination with a range of doses of bleomycin or radiation. Dissociated cells from passivated mouse tumors were cultured for 6 days after exposure to the combination and then assessed for viability using the Cell Titer-Glo assay. The Bliss additivity statistical model was used to determine the degree of synergism, additivity, or antagonism relative to each combination treatment.

The combination of compound ( 1 ) and bleomycin or radiation was additive or synergistic in all tumors tested (29/29) (see Fig. 31). In addition, strong synergy was observed in approximately a third of two tumors treated with bleomycin (9/29) and radiation (3/8) in combination with compound ( 1 ). Similarly, compound ( 2 ) was tested in combination with a range of bleomycin doses in a smaller subpopulation of tumors (gastric, pancreatic). The combination of compound ( 2 ) and bleomycin was additive or synergistic in all tumors tested (20/20) and highly synergistic in a subpopulation (3/20) (see FIG. 31). These data suggest that a DNA-PK inhibitor in combination with radiotherapy may be more broadly effective than standard of care treatment alone.

A set of primary tumors were also treated with compound ( 1 ), in combination with a variety of chemotherapeutic agents (gemcitabine, paclitaxel, cisplatin, carboplatin, 5-FU, etoposide) commonly used in the treatment of the tumor types tested. Additivity was observed in the largest number of tumors treated with compound ( 1 ) and either gemcitabine (2/4), paclitaxel (1/5), 5-FU (5/5), or doxorubicin (1/1). However, antagonism was observed in the same tumors when using demcitabine (2/4) and paclitaxel (1/5). Synergism or additivity was observed in almost all tumors using both carboplatin (5/5) and cisplatin (9/10), however, one tumor showed antagonism with cisplatin. The combination of compound ( 1 ) and etoposide showed strong synergy in all tumors tested (4/4). These TCA results were consistent with combination data obtained using cancer cell lines. Overall, these data suggest that a selective DNA-PK inhibitor may provide additional benefit to cancer patients receiving standard-of-care treatment alone in a variety of clinical applications.

Example 14: Impact of connection ( 1 ) on clonal-derived survival of irradiated cancer cell lines

The clonal cell survival test measures the ability of cells to proliferate indefinitely, thereby maintaining their self-renewing ability to form colonies (i.e., clones). This test has been a mainstay in radiation oncology for decades and has been used to determine the effect of a compound ( 1 ) on the clonal origin of a set of cell lines through subsequent irradiation of numerous tumor types. Compound ( 1 ) in combination with irradiation was found to be very effective in reducing the clonal origin of all cancer cell lines tested with dose enhancement factors (DEF, difference in colony count at 0.1 survival fraction) varying from 2.5 to >5. Miapaca2 cells showed the lowest DEF (2.5), whereas, in the case of FaDu cells, the combination of compound ( 1 ) and irradiation completely eliminated colony formation at a low dose of irradiation, such as 0.5 Gy, and showed a DEF >8 . DEF greater than 1.5 is generally considered clinically significant; therefore, by these standards, compound ( 1 ) should be characterized as a strong radio enhancer. These data are consistent with previous data regarding cell viability, suggesting that a broad responder population may be expected in cancer patients treated with compound ( 1 ) in combination with radiation.

Although the above invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, one skilled in the art, in light of the teachings of the present invention, it should be readily apparent that, in addition, certain changes and modifications may be made, without departing from the scope or spirit of the appended claims.

All references given in this context are incorporated in their entirety into this context by reference. As used in this context, all abbreviations, symbols and conventions are the same as those used in contemporary scientific literature. See, for example, the guide ed. Janet S. Dodd, “The ACS Style Guide: A Manual for Authors and Editors,” 2nd edition, Washington, D.C.: American Chemical Society, 1997.

Claims (10)

1. A method of potentiating a therapeutic regimen for the treatment of cancer mediated by DNA-dependent protein kinase (DNA-PK) activity in a patient, comprising administering to said patient an effective amount of a co-crystal containing a compound of the formula: and a co-crystal former, wherein the co-crystal former is adipic acid, wherein R ¹ and R ² are each deuterium, where the molar ratio of adipic acid to the compound of formula (I) is 1:2, where the compound has X-ray powder diffraction reflection peaks at 6.46+/-0.2, 7.91+/-0.2, 11.92+/-0.2, 12.26+/-0.2, 12 .99+/-0.2, 14.19+/-0.2, 18.68+/-0.2 and 19.07+/-0.2 °2-theta, wherein the cancer is breast cancer, gastroesophageal cancer or lung cancer, where the therapeutic regimen includes radiotherapy or chemotherapy, and wherein chemotherapy is administered with a therapeutic agent selected from etoposide, doxorubicin and bleomycin. 2. The method of claim 1, wherein the compound has a DSC peak in its DSC thermogram at a temperature of about 195°C and about 245°C. 3. The method according to claim 1, where the compound is administered in the form of a pharmaceutical composition.