Substituted (3-hydroquinazolin-4-one) derivatives and pharmaceutical compositions thereof use as potent & selective poly [ADP ribose] polymerase 1(p ARP-1) inhibitors for the treatment of cancer

Abstract

Disclosed is a compound of formula (X), a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof, wherein R1, R2, R3, and A are as disclosed herein. Also disclosed are a process for preparation of such compounds, compositions containing such compounds and use thereof. The compounds disclosed herein are potent and selective PARP-1 inhibitors, and can be used in the treatment of different diseases. Formula (X)

Description

SUBSTITUTED (3-HYDROQUINAZOLIN-4-ONE) DERIVATIVES AND PHARMACEUTICAL COMPOSITIONS THEREOF USE AS POTENT & SELECTIVE POLY [ADP RIBOSE] POLYMERASE 1 (P ARP-1 ) INHIBITORS FOR THE TREATMENT OF CANCERCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to US Provisional Patent Application No. 63 / 680908, filed on August 8th, 2024, and having the title “POTENT & SELECTIVE POLY [ADP RIBOSE] POLYMERASE 1 (PARP-1 ) INHIBITORS”. The content of the above-noted patent application is hereby expressly incorporated by reference into the detailed description hereof.FIELD

[0002] The specification relates to potent and selective poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors, their process of preparation, formulations containing them and the use thereof.BACKGROUND

[0003] Cancer is a group of diseases involving abnormal cell growth with the potential to migrate to and affect other different parts of the body. Due to the broad range of diseases that are classified as cancer, a wide variety of different approaches are required for its treatment. Typical cancer treatments can include radiation therapy and chemotherapeutic drugs such as alkylating agents, anti-metabolites and topoisomerase inhibitors, which are designed to damage DNA, halt cellular division and influence apoptosis of cancer cells.

[0004] It is postulated that the efficacy of chemotherapy and radiation therapy is dampened by the activity of DNA repair enzymes (Ganesan, S. MYC, PARP1 , and Chemoresistance: BIN There, Done That? Sci. Signal., 2011 , 4, 15, incorporated herein by reference). Two specific enzymes that are involved in the detection and repair of DNA damage are poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-2 and members of the PARP family of enzymes (Herceg Z, Wang ZQ. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res. 2001 Jun 2;477(1-2):97-110, incorporated herein by reference). Inhibitorsthat target PARP enzymes and the mechanism of DNA repair are a new class of drugs that have recently received a large degree of attention and are of clinical importance with regard to various forms of cancer (Jagtap, P.; Szabo, C. Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors, Nat. Rev. Drug Discov. 2005, 4, 421 ; Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier, G. G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer, 2010, 10, 293; Cepeda, V.; Fuertes, M. A.; Castilla, J.; Alonso, C.; Quevedo, C;. Soto, M.; Perezb, M. Poly(ADP-Ribose) Polymerase-1 (PARP-1) inhibitors in cancer chemotherapy, Recent Patents on Anti- Cancer Drug Discovery, 2006, 1 , 39; Ferraris, D. V. Evolution of Poly(ADP-ribose) Polymerase-1 (PARP-1) Inhibitors. From Concept to Clinic. J. Med. Chem. 2010, 53, 4561 , all incorporated herein by reference).

[0005] Development of PARP-1 inhibitors has gone through several fluctuations. In the past, failure of several advanced clinical PARP-1 inhibitors dampened the enthusiasm for PARP inhibitors; however, it is notable that PARP-1 inhibitors have shown great potential to target cancers such as high-grade ovarian cancers and triple-negative breast cancers that are resistant to current treatments. Today, there are now a variety of PARP inhibitors that have been approved by the FDA for the treatment of certain kinds of cancer including Olaparib, Talazoparib, and others (Rudolpha, J., Junga, K., Lugera, K. Inhibitors of PARP: Number crunching and structure gazing. Proc. Natl. Acad. Sci. U.S. A. 2022, 119 (11):e2121979119, incorporated herein by reference).

[0006] Studies have also discussed that PARP-2 is required to maintain hematopoiesis (Fames et al., Blood, 2013, 122(1), 44, incorporated herein by reference). Pilie (Pilie et al., Clin. Cancer Res., 25(13), 2019, 3759, incorporated herein by reference) studied the similarities and differences between individual PARP inhibitors and their implications, and discuss data that supported the extending benefit of PARP inhibitors beyond BRCA-mutant cancers. While LaFargue (LaFargue et al., Lancet Oncol., 2019, 20, e15-28, incorporated herein by reference) explores and compares the adverse events between PARP inhbitors. In view of such studies, research has focused on design and development of selective inhibitors that PARP-1 over PARP-2 (Liu et al., J. Med. Chem., 2023, 66, 16464; Zhang et al., J. Med. Chem., 2024, 67, 8877, both incorporated herein by reference). Such work has led to the development of AZD5305, a selective inhibitor of PARP-1 over PARP-2 (Johannes etal., J. Med. Chem., 2021, 64, 14498; and Zheng et al., Front. Pharmacol. 2023, 13, 979873, both incorporated herein by reference).

[0007] PCT publication number WO 2018 / 125961 (incorporated herein by reference) discloses compounds that can be used for treating a subject with a disease which can be ameliorated by inhibition of poly(ADP-ribose)polymerase (PARP). Prior to the filing of the priority application of the WO 2018 / 125961 publication, the inventors of the subject application had been working on compounds of similar structure. Further, PCT publication number WO 2018 / 125961 fails to disclose, teach or suggest selective targeting of PARP-1 over PARP-2 inhibitors. Moreover, the compounds disclosed in PCT publication number WO 2018 / 125961 fail to show high potency and selectivity of PARP-1 over PARP-2.

[0008] There is a need in the art for potent and selective PARP-1 inhibitors. In addition, there is a need in the art for a process for preparation of potent and selective PARP-1 inhibitors. Further, there is a need in the art for a composition containing potent and selective PARP-1 inhibitors. Moreover, there is a need in the art for a method of treatment of a disease using potent and selective PARP-1 inhibitors. Furthermore, there is a need in the art for use of potent and selective PARP-1 inhibitors for treatment of a disease.

[0009] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.SUMMARY

[0010] In one aspect, the specification relates to a compound of formula (X)(X)

[0011] a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof,

[0012] wherein

[0013] when R1is OH, R2and R3each is H, and A is C;

[0014] when R2is OH, R1and R3each is H, and A is N; and

[0015] when R3is OH, R1and R2each is H, and A is N; and

[0016] when R1, R2and R3each is H, A is C.

[0017] In a second aspect, the specification relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, as disclosed herein, and at least one pharmaceutically acceptable carrier.

[0018] In a third aspect, the specification relates to a product comprising the compound, its pharmaceutically acceptable salt, pro-drug or isotopic thereof, as disclosed herein, and one or more chemotherapeutic agents.

[0019] In a fourth aspect, the specification relates to a method for preventing or treating a disease mediated by PARP-1 protein, the method comprising the step of administering to a mammal in need thereof an effective amount of the compound, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, the pharmaceutical composition, or the product, as disclosed herein.

[0020] In a fifth aspect, the specification discloses use of the compound, its pharmaceutically acceptable salt or pro-drug thereof, or the product, as disclosed herein, for treating cancer.

[0021] In a sixth aspect, the specification relates to an in vitro method for selectively inhibiting PARP-1 protein activity, the method comprising the step of contacting a PARP-1 protein with an effective amount of the compound, its pharmaceutically acceptable salt, pro-drug or isotope thereof, as disclosed herein.

[0022] In a seventh aspect, the specification relates to a compound of formula 127127

[0023] a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof.

[0024] In an eighth aspect, the specification relates to a process for preparation of the compound of formula 127, a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof, the process comprising the step of:

[0025] carboxylating the compound of formula 124 to form the compound of formula124 125

[0026] 125 126

[0027] converting the carboxylic acid of the compound of formula 126 to the amide to form the compound of formula 127126 127BRIEF DESCRIPTION OF DRAWINGS

[0028] Reference will now be made, by way of example, to the accompanying drawings which show example embodiments of the present application, and by which the present application can be further understood from the following description with reference to the Figures. The present application includes drawings, wherein:

[0029] Figure 1 shows results from assessing the metabolic stability of compound 1 (for comparison to compound 19, Figure 2) over time in the presence of liver microsomes activated by NADPH;

[0030] Figure 2 shows results from assessing the metabolic stability of compound 19 (for comparison to compound 1 , Figure 1) over time in the presence of liver microsomes activated by NADPH;

[0031] Figure 3 shows results from assessing the metabolic stability of compound 1 (for comparison to compound 20, Figure 4) over time in the presence of liver microsomes activated by NADPH;

[0032] Figure 4 shows results from assessing the metabolic stability of compound 20 (for comparison to compound 1 , Figure 3) over time in the presence of liver microsomes activated by NADPH;

[0033] Figure 5 shows results from assessing the metabolic stability of compound 1 (for comparison to compound 21 , Figure 6) over time in the presence of liver microsomes activated by NADPH; and

[0034] Figure 6 shows results from assessing the metabolic stability of compound 21 (for comparison to compound 1 , Figure 5) over time in the presence of liver microsomes activated by NADPH.DESCRIPTION OF EXAMPLE EMBODIMENTS

[0035] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the typical materials and methods are described herein. In describing and claiming the present invention, the common terminology generally used is described herein below. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0036] Many patent applications, patents, and publications are referred to herein to assist in understanding the aspects described. Each of these references are incorporated herein by reference in their entirety.

[0037] When introducing elements disclosed herein, the articles “a”, “an”, “the”, and “said” are intended to mean that there may be one or more of the elements.

[0038] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. It will be understood that any embodiments described as “comprising” certain components may also “consist of” or “consist essentially of,” these components, wherein “consisting of” has a closed-ended or restrictive meaning and “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effects described herein. For example, a composition defined using the phrase “consisting essentially of” encompasses any known acceptable additive, excipient, diluent, carrier, and the like, suitable for the composition described herein. Typically, a composition consisting essentially of a set of components will comprise less than5% by weight, typically less than 3% by weight, more typically less than 1 % by weight of non-specified components.

[0039] It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[0040] In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0041] Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0042] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0043] The phrase “at least one of” is understood to be one or more. The phrase “at least one of... and...” is understood to mean at least one of the elements listed or a combination thereof, if not explicitly listed. For example, “at least one of A, B, and C” is understood to mean A alone or B alone or C alone or a combination of A and B or a combination of A and C or a combination of B and C or a combination of A, B, and C.

[0044] The specification relates to selective and potent inhibitors of the nuclear enzyme poly(adenosine 5'-diphospho-ribose) polymerase 1 , which may also be referred to as poly(ADP-ribose) polymerase 1 , PARP-1 , NAD+ADP-ribosyl transferase 1 and poly(ADP-ribose) synthase 1 , and discloses compounds and compositions containing the disclosed compounds. Moreover, the specification discloses a method for treatment of a subject by administration of the disclosed PARP-1 inhibitors. In a particular embodiment, the disclosed PARP-1 inhibitors are used to treat cancer.

[0045] The inventors of the subject application have investigated PARP inhibitors having a 4-quinazolinone moiety for a number of years. In 2018, PCTinternational publication number WO2018 / 125961 to Mitobridge, Inc. (incorporated herein by reference) disclosed compounds targeting the PARP-1 and PARP-2 enzymes. The inventors of the subject application had been working on compounds having a similar structure prior to the priority date of the WO 2018 / 125961 publication, and recognized the importance of compounds having a high potency and selectivity towards PARP-1 over PARP-2 protein, and in particular, compounds that have a 4- quinazolinone moiety. The compounds disclosed herein are based on these findings, and that have a PARP-1 IC50activity of ≤7nM and a selectivity of PARP-1 to PARP-2 of about 100, 150, 200 or more.

[0046] In one aspect, the specification relates to a compound of formula (X)

[0047] a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof,

[0048] wherein

[0049] when R1is OH, R2and R3each is H, and A is C;

[0050] when R2is OH, R1and R3each is H, and A is N;

[0051] when R3is OH, R1and R2each is H, and A is N; and

[0052] when R1, R2and R3each is H, A is C.

[0053] The term, pharmaceutically acceptable salt, as disclosed herein is not particularly limited and should be known to a skilled worker, or can be determined. There are no particular limitations on the pharmaceutically acceptable salt so long as the compound disclosed herein, and salt are formed, whether inorganic acid salt, inorganic base salt, organic base salt or organic acid salt. For example and without limitation, the salt can be hydrochloric acid salt, sulfuric acid salt, citrate, hydrobromic acid salt, hydroiodic acid salt, nitric acid salt, bisulfate, phosphoric acid salt, isonicotinic acid salt, acetic acid salt, lactic acid salt, salicic acid salt, tartaric acid salt,pantotenic acid salt, ascorbic acid salt, succinic acid salt, maleic acid salt, fumaric acid salt, gluconic acid salt, saccharinic acid salt, formic acid salt, benzoic acid salt, glutaminic acid salt, methanesulfonic acid salt (also referred to as mesylic acid salt), ethanesulfonic acid salt, benzenesulfonic acid salt, p-toluenesulfonic acid salt, pamoic acid salt (pamoate), sodium salt, potassium salt, calcium salt, magnesium salt, arginine salt, diethanolamine, ethanolamine, ethylenediamine, histidine salt, triethylamine salt and so on.

[0054] The term, pro-drug, as used herein is not particularly limited and should be known to a person of ordinary skill in the art. A prodrug is a compound that is administered in a pharmacologically inactive form which is then converted to an active form through a normal metabolic process, such as hydrolysis of an ester. In other words, a pro-drug is a precursor chemical compound of an active pharmaceutical ingredient. In addition, a pro-drug of the compound disclosed herein can be determined or formed by a person of ordinary skill in the art. There are no particular limitations on the pro-drug of the compounds disclosed herein.

[0055] The term, isotope, as used herein is not particularly limited and should be known or understood by a person of skill in the art. Isotopes are distinct nuclear species (or nuclides) of the same chemical element. They have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), but differ in nucleon numbers (mass numbers) due to different numbers of neutrons in their nuclei. While all isotopes of a given element have similar chemical properties, they have different atomic masses and physical properties.

[0056] In determining the potent and selective compounds disclosed herein, a large number of compounds were prepared and studied. A small subset of the compounds studied having different substituents on different positions in the compound of formula (X’) that has a 4-quinazolinone moiety, is disclosed in Table 1 below. Other studied compounds having different substituents, that have a 4- quinazolinone moiety, which did not meet the potency and selectivity criteria have not been disclosed in the specification.Table 1 : PARP-1 and PARP-2 activity screening results for selected compounds of formula (X’).

[0057] The selected compounds of the current invention are highly potent for PAPR-1 with IC50average values in the sub-nanomolar range. In addition, these compounds are highly selective for PARP-1 over PARP-2. The potency and selectivity of these PARP-inhibitors are discussed below in more detail and a comparison with other PARP inhibitors including approved PARP inhibitors has also been made.

[0058] During the course of this work, it was discovered that certain structural features are responsible for the observed high potency and high selectivity for PARP- 1 . The inventors provide their theory and understanding, however, the invention is not limited by this theory or understanding. These structural features mentioned earlier, include (i) presence and position of a hydroxyl group in aryl moiety of the quinazolinone structure (i.e., hydroxyl group in the leftmost aryl ring), (ii) preference for a C atom (over a N atom) in the aryl moiety on the rightmost aryl ring, and (iii) presence of a nitrile group (-CN) on the right-hand side aryl moiety, in the compound of formula (X) (please also see Table 1). Further discussion about these features is noted herein.

[0059] (i) Presence and position of a hydroxyl group in aryl moiety of the quinazolinone structure (i.e., hydroxyl group in the leftmost aryl ring)

[0060] It has been observed in numerous examples of structures of formula (X) that a hydroxyl group on the left most aryl ring, preferably in the R1position, was required for high potency and high selectivity for PARP-1. Entry 1 in Table 1 with an average IC50value of 0.03 nM for PARP-1 illustrates this point. When this hydroxyl group is absent as in entry 17 (1.4 nM for PARP-1 ), there is a reduction in PARP-1 potency of up to 50 times. It can also be seen from these two entries (1 and 17) with all other functionalities kept identical, that when the hydroxyl group is absent, selectivity for PARP-1 over PARP-2 is reduced by a factor of 9.

[0061] When the hydroxyl group in the R1position was replaced with a different group such as F or OMe, both PARP-1 potency and selectivity reduced drastically.Compare the IC50average value of 0.03 nM for PARP-1 (entry 1) to an IC50average value of 5.6 nM, when the OH group in R1position was replaced by F (entry 11), while keeping all other functionalities unchanged. Changing only the OH into F here, resulted in the loss of PARP-1 activity by a factor of more than 186 and PARP-1 selectivity over PARP-2 was reduced from 933 times to only 27 times. The same trend is also observed when the hydroxyl group in the R1position (entry 1) was replaced by a methoxy group (entry 9). Changing only the OH into OMe here, resulted in the loss of PARP-1 activity by a factor of more than 1000 and PARP-1 selectivity over PARP- 2 was reduced from 933 times to only 4 times. Similar observations can be made when entries 2 and 10 (OH to OMe) and I or entries 2 and 12 (OH to F) are compared in Table 1.

[0062] Moving the hydroxyl group to R2or R3positions resulted in PARP-1 potency of 5.2 nM and 3 nM, respectively. Although, these compounds are still highly potent, the selectivity for PARP-1 over PARP-2 was about 93 times when the hydroxyl group was moved to the R2position and 193 times when the hydroxyl group was moved to the R3position for compounds of formula (X), and where A is nitrogen (N). Where A is carbon, and only the -OH group is moved from R1to R2(Entry 13 and 15), a significant drop in both PARP-1 potency, and selectivity of PARP-1 over PARP-2 can be observed.

[0063] (ii) Preference for a C atom (over a N atom) in the aryl moiety on the rightmost aryl ring

[0064] Another observation that has been made using numerous examples of compounds of formula (X), is the impact of the presence or absence of a nitrogen atom in the right-most aryl ring (A is N or C). It has been observed that compounds of formula (X) in which the hydroxyl group is in the R1position, are more potent and more selective for PARP-1 over PARP-2 whenever the right-most aryl ring is a simple phenyl ring and not a pyridine ring. A closer look at entries 1 and 2 in Table 1 reveals that the presence of one nitrogen atom is the only difference between the structures and yet, the structure with a pyridine ring (entry 2) was found to be 23 times less active for PARP-1 . In addition, the selectivity for PARP-1 over PARP-2 changes from 21 times (entry 2) to 933 times by just removing the nitrogen from the right-most aryl ring (entry 1).

[0065] (iii) Presence of a nitrile group (-CN) on the right-hand side aryl moiety

[0066] Different functionalities on the right-hand side ring have been explored including cyano, amide, fluoro, methyl, ester, and a carboxylic acid group to see the effect, if any, such functionalities could have on PARP-1 potency and I or selectivity of compounds of formula (X). Comparing PARP-1 potency I selectivity of compounds of formula (X) when the cyano group (entry 1) was replaced by an amide group (entry 4) while keeping every other functionalities unchanged, PARP-1 activity reduced by a factor of more than 143, from 0.03 nM to 4.3 nM. In addition, PARP-1 selectivity over PARP-2 reduced from 933 times to only 5 times. This same observation was even more pronounced when the cyano group (entry 1) was replaced by a methyl group (entry 5) while keeping every other functionalities unchanged. Here, PARP-1 activity reduced by a factor of more than 6666, from 0.03 nM to 200 nM. In addition, PARP-1 selectivity over PARP-2 drastically reduced by a factor about 1555, from 933 times to only 0.6 times. The effect of this cyano functionality can also be seen when comparisons of entries 2 and 6 (methyl ester) and entries 2 and 8 (fluoro) are made as shown in Table 1.

[0067] Comparison of PARP-1 activity data for selected examples and top WO 2018 / 125961 A1 compounds

[0068] Table 2 and Table 3 below illustrate activity data for PARP-1 , PARP-2, and corresponding PARP-1 selectivity values for selected compounds disclosed herein and top WO 2018 / 125961 A1 compounds respectively. As it can be seen from the PARP-1 activity data in Tables 2 and 3, there is a clear superiority of the subset of compounds disclosed herein over all reported examples in WO 2018 / 125961 A1. For example, Entries 1, 19, 20 and 21 have been shown to have IC50average values of 0.03 nM, 0.04 nM, 0.03 nM, and 0.04 nM respectively. One of the most active compounds disclosed in WO 2018 / 125961 A1 is example 32, with a reported IC50average value of 5 nM for PARP-1. A direct comparison between PARP-1 activity of example 32 (5 nM) and PARP-1 activity of species such as Entries 1 and / or 20 (0.03 nM) shows that this subset of compounds disclosed herein are up to 167 times more active than the best example that is reported in WO 2018 / 125961 A1.

[0069] This superiority in potency is very important and needs to be emphasized because with every other conditions being similar, a cancer patient will be required to take a much lower dose of such potent and selective compounds (for example and without limitation, Entry 1 ), but a much higher dosage of example 32 (disclosed in WO 2018 / 125961 A1 , incorporated herein by reference) to elicit a similar biological response. There are numerous advantages associated with low drug dosages some of which can, for example and without limitation, include: lower doses can help to prevent adverse side effects, drug diversion, and poisonings especially for drugs with narrow therapeutic ranges; lower doses can also help in empowering the patients by encouraging self-titration of that low but effective dose which in turn promotes better compliance and ultimately achieve therapeutic goals more reliably (Daughton, C. G., Ruhoy, I. S., Lower-dose prescribing: Minimizing “side effects” of pharmaceuticals on society and the environment. Science of the Total Environment, 2013, 443, 324 - 337, incorporated herein by reference). In addition, lower dosages translate into lower environmental loadings of excreted drug residues and more importantly, lower doses can lower the cost of healthcare.Table 2: PARP-1 and PARP-2 activity screening results for selected compounds disclosed herein, Olaparib, and AZD5305

[0070] Comparison of PARP-1 over PARP-2 selectivity for selected compounds and top WO 2018 / 125961 A1 compounds

[0071] The role of PARP inhibitors is to target cancer cells based on their inherent deficiencies, while appearing to avoid normally functioning cells. As this is not always the case, PARP inhibitors may be associated with some level of toxicity (Zheng, J., Li, Z., Min, W. Current status and future promise of next generation poly (ADP-Ribose) polymerase 1 -selective inhibitor AZD5305. Front. Pharmacol. 2023, 13, 979873, incorporated herein by reference). PARP inhibitors inhibit both PARP-1 and PARP-2 (as well as other PARP family members) and thus present a class of undesirable adverse events including hematological effects, gastrointestinal effects, renal toxicities, fatigue and others (Dellavedova, G., Decio, A., Anna, S., Leo, E., Giavazzi, R., and Rosa Bani, M. The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer. Mol. Cancer Ther 2021 , 20, 217, incorporated herein by reference). These adverse effects common to PARP inhibitors though usually mild can be serious and in rare cases, leading to dose discontinuation (Zheng, J., Li, Z., Min, W. Current status and future promise of next generation poly (ADP-Ribose) polymerase 1 -selective inhibitor AZD5305. Front. Pharmacol. 2023, 13, 979873, incorporated herein by reference). Currently approved PARP inhibitors lack good selectivity for PARP-1 over PARP-2 and several other PARP family members, and it has recently been hypothesized that this contributes to toxicity (Dellavedova, G., Decio, A., Anna, S., Leo, E., Giavazzi, R., and Rosa Bani, M. The next generation PARP inhibitor AZD5305 is active in a broad range of pre- clinical models of ovarian cancer. Mol. Cancer Ther 2021, 20, 217, incorporated herein by reference). In addition, PARP-2 is particularly linked with the hematological toxicities, which means that it is now more important than ever to develop the next- generation PARP inhibitors with improved selectivity for PARP-1 .

[0072] The data in Table 2 shows values of PARP-1 over PARP-2 selectivity to range from 93-fold to 1867-fold for the selected compounds (disclosed herein) compared to values in Table 3 ranging from 6-fold to 145-fold fortop WO 2018 / 125961 A1 examples, incorporated herein by reference. It is important to point out that example 23 in WO 2018 / 125961 A1 , though not included in Table 3 (because its PARP-1 activity IC50value is 35 nM), is reported to have a selectivity of 294-fold.

[0073] This superiority in PARP-1 selectivity can translate into less or no observed PARP-2 associated toxicity for the selected compounds disclosed herein incomparison to compounds disclosed in WO 2018 / 125961 A1 or other currently approved PARP inhibitors.Table 3: PARP-1 and PARP-2 reported results for WO 2018 / 125961 A1 (to Mitobridge) examples having a PARP-1 IC50value of 15 nM or better.* for structures of the compounds in the column, please see WO 2018 / 125961 A1(Mitobridge), incorporated herein by reference.

[0074] Comparison between selected inhibitors (disclosed herein) with FDA approved PARP inhibitors

[0075] Recently, Rudolpha and coworkers provided a perspective on the reported potencies of the most studied PARP inhibitors (Veliparib, Niraparib, Olaparib, Talazoparib, and Rucaparib). These five inhibitors have been approved as drugs for various oncologic indications. The PARP 1 and PARP-2 numbers for these are reported in Table 4 (Rudolpha, J., Junga, K., Lugera, K. Inhibitors of PARP: Number crunching and structure gazing. Proc. Natl. Acad. Sci. U.S. A. 2022, 119 (11):e2121979119, incorporated herein by reference). Some important observationscan be made when a head-to-head comparison between the compounds disclosed herein (Table 2) and each of the approved drugs in Table 4 is made. It can be seen from the results in these two tables that the compounds of the subject invention are highly potent against PARP-1 compared to each of the approved drugs. This subset of compounds (disclosed herein) appears to be at least 13 to 167 times more potent. It can also be seen from these results that the selectivity of this subset of compounds is unmatched by the selectivity of any of the drugs in Table 4. For example, compound 20 with a PARP-1 over PARP-2 selectivity of 1867-fold (Table 2) is about 9335 times more selective than Olaparib with a PARP-1 over PARP-2 selectivity of 0.2 (Table 4). Notice that this subset of compounds disclosed herein were tested on the same plate with Olaparib for both PARP-1 and PARP-2. In our hands, Olaparib showed an IC50average value of 2 nM for PARP-1 and 0.7 nM for PARP-2 resulting in a 0.35-fold selectivity for PARP-1 over PARP-2; a number that agrees with a 0.2-fold selectivity reported in the literature and in Table 4.Table 4: Literature values for approved PARP Inhibitors’ potencies.

[0076] In addition, this subset of compounds herein was shown to be up to 60 times more potent for PARP-1 than AZD5305 (Dellavedova, G., Decio, A., Anna, S., Leo, E., Giavazzi, R., and Rosa Bani, M. The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer. Mol. Cancer Ther. 2021 , 20, 217, incorporated herein by reference) (Table 2) when tested on the same plate in our hands. Also, the PARP-1 over PARP-2 selectivity for the compounds disclosed herein appear to be up to 156-fold that of AZD5305.

[0077] In summary, there is a clear and undeniable superiority of the species reported herein over current approved inhibitors like Olaparib and / or previouslyreported inhibitors such as AZD5305 and others. Furthermore, this subset of compounds disclosed herein is highly selective for PARP-1 over PARP-2 and this is expected to translate into highly potent drugs with negligible PARP-2 associated toxicity in comparison to currently approved drugs.

[0078] PARP-1 percent inhibition comparison

[0079] Another criteria that can be used to compare compounds disclosed herein with the FDA-approved Olaparib and AZD5305 is the percent inhibition for these species at different concentrations. Here, the percent inhibition studies / calculations for all the compounds reported in Table 5 were performed on the same plates, in order to get a real head-to-head comparison. Concentrations ranging from 1000 nM to 0.1 nM were looked at for all the compounds in Table 5. All the compounds show high percentage inhibitions for concentrations of 10 nM and above.Table 5: PARP-1 percent inhibition data for AZD5305, Olaparib, and selected compounds disclosed herein.

[0080] However, the percent inhibitions for AZD5305 and Olaparib were observed to be reduced significantly at concentrations lower than 10 nM. For example, at 0.1 nM, AZD5305 and Olaparib were observed to have percent inhibitions of 7% and 8% respectively (Table 5). On the other hand, compounds such as 1, 19, 20 and 21 were all observed to have percent inhibitions of at least 60% or better even at 0.1 nM concentrations (Table 5). The result in Table 5 is yet another indication that the compounds reported herein are of higher potency for PARP-1 compared to previously approved and reported inhibitors.

[0081] Stability studies and comparison

[0082] For stability studies, a representative compound 1 was selected and analyzed together with AZD5305 and Olaparib for comparison purposes. The results are shown in Table 6. The results here show that species such as 1 are stable with a half-life of 66 minutes (Human) and 45 minutes (Mouse, CD-1). Having established the stability of 1 in comparison to other stable inhibitors such as AZD5305 and Olaparib, the stabilities of other compounds disclosed herein such as 19, 20 and 21 relative to the stability of 1 were also determined using head-to-head screening assays between 1 and each of 19, 20 and 21. It can be seen from the microsomal studies results at the end, as disclosed herein, that all the compounds 19, 20 and 21 screened were shown to have almost identical half-lives to that of 1 in all head-to-head comparisons.Table 6: Microsomal stability work comparing 1 with Olaparib and AZD5305.

[0083] In a second aspect, the specification relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound, the pharmaceutically acceptable salt thereof, or the pro-drug thereof, as disclosed herein, or a combination thereof, and at least one pharmaceutically acceptable carrier.

[0084] The term, pharmaceutical composition, as used herein is not particularly limited and should be known or understood by a person of skill in the art. Pharmaceutical composition means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure can encompass any composition made by admixing at least one compound of the present disclosure and a pharmaceutically acceptable carrier.

[0085] The term, therapeutically effective amount or effective amount, as used herein is not particularly limited and should be understood by a person of skill in the art. The therapeutically effective amount of a compound described herein, a pharmaceutically acceptable salt, tautomer, prodrug, or deuterated analog thereof means an amount sufficient to effect treatment when administered to a subject, to provide a therapeutic benefit such as amelioration of symptoms or slowing of disease progression. For example, a therapeutically effective amount may be an amount sufficient to decrease a symptom of a disease or condition responsive to PARP-1 inhibitors. The therapeutically effective amount may vary depending on the subject, and disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the manner of administering, which can be determined by a person of skill in the art.

[0086] The term, pharmaceutically acceptable carrier, as used herein is not particularly limited and should be known or understood by a person of skill in the art. Pharmaceutically acceptable carrier can include one or more excipients or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are not deleterious to the disclosed compound or use thereof. The use of such carriers and agents to prepare compositions of pharmaceutically active substances is well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker s C.T. Rhodes, Eds., both incorporated herein by reference)

[0087] Non-limiting examples of pharmaceutically acceptable carriers or diluents include water, sodium chloride (NaCI), normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with or interfere with the activity of the compounds provided herein. One of ordinary skill in the art will recognize that other pharmaceutical excipients are suitable for use with disclosed compounds.

[0088] Conventional procedures and ingredients for the selection and preparation of suitable pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Sciences (2003 -20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999 (both incorporated herein by reference). The one or more carriers are "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0089] In another aspect, the specification provides compounds, a pharmaceutically acceptable salt, an isotope or pro-drug thereof, which inhibit in vitro and / or in vivo polymerase activity of poly(ADP-ribose) polymerase (PARP), and compositions containing the disclosed compounds, or a pharmaceutically acceptable salt or pro-drug thereof.

[0090] In a further aspect, the specification provides methods to inhibit, limit and / or control the in vitro and / or in vivo polymerase activity of poly(ADP-ribose) polymerase (PARP) in solutions cells, tissues, organs or organ systems. In one embodiment, the present specification provides methods of limiting or inhibiting PARP activity in a mammal, such as a human, for example and without limitation, either through local or systemic administration.

[0091] In another further aspect, the specification provides a chemosensitization method for treating cancer, the method containing the step ofcontacting the cancer cells with a cytotoxicity-potentiating quinazolinone compound of Formula (X), a pharmaceutically acceptable salt, an isotope or a pro-drug thereof, and further contacting the tumor or cancer cells with an anticancer agent.

[0092] In a still another aspect, the specification provides a chemosensitization method wherein a first dose of at least one compound of Formula (X), a pharmaceutically acceptable salt, an isotope or a pro-drug thereof, is administered singly or repeatedly to a patient in need thereof, and wherein subsequently a second dose of at least one chemotherapeutic agent is administered singly or repeatedly to said patient after a time period to provide an effective amount of chemosensitization.

[0093] An aspect of the present specification provides a pharmaceutical formulation comprising the compound of Formula (X) in a form, for example and without limitation, pharmaceutically acceptable free base, salt, hydrate, ester, solvate, stereoisomer, isotope and mixtures thereof. According to a further aspect, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier or diluent, and, optionally, a chemotherapeutic agent. The following embodiments are for illustrative purposes only and are not intended to limit in any way the scope of the present specification. In one embodiment, a pharmaceutical formulation, as disclosed herein, comprises a compound, as disclosed herein, in a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation, as disclosed herein, comprises a pharmaceutically acceptable salt of a compound, as disclosed herein, in a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation, as disclosed herein, comprises a compound, as disclosed herein, and one or more chemotherapeutic agents in a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation, as disclosed herein, comprises a pharmaceutically acceptable salt of a compound, as disclosed herein, and one or more chemotherapeutic agents in a pharmaceutically acceptable carrier. Non-limiting examples of such chemotherapeutic agents are noted below.

[0094] In another further aspect in accordance with the specification, the chemosensitizing compound and the chemotherapeutic agent can be administered essentially simultaneously. In another further aspect in accordance with the specification, the chemosensitizing compound and the chemotherapeutic agent can be administered essentially sequentially.

[0095] In a particular embodiment, the chemotherapeutic agent is, for example and without limitation, temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon-alpha, interferon-beta, interferon- gamma, interleukin 2, irinotecan, paclitaxel, a taxoid, dactinomycin, danorubicin, 4'- deoxydoxorubicin, bleomycin, pilcamycin, mitomycin, neomycin and gentamycin, etoposide, 4-OH cyclophosphamide, a platinum coordination complex, topotecan, therapeutically effective analog or derivative of the same, or a mixture thereof. In a particular embodiment, the chemotherapeutic agent is temozolomide.

[0096] In another aspect, the present specification provides a method of treating the effect of cancer and / or to radiosensitize cancer cells to render the cancer cells more susceptible to radiation therapy and thereby to prevent the tumor cells from recovering from potentially lethal damage of DNA after radiation therapy, the method containing the step of administering to a subject an effective amount of a compound of Formula (X), a pharmaceutically acceptable salt, an isotope or a pro-drug thereof. In a particular embodiment, the method is directed to radiosensitizing cancer cells rendering the cancer cells more susceptible to radiation therapy than non-tumor cells.

[0097] In still further aspect, the specification provides a method of treatment of cancer in a subject in need thereof containing the step of administering to the subject a therapeutically effective amount of a compound of Formula (X), a pharmaceutically acceptable salt, an isotope or a pro-drug thereof, wherein the cancer cells have a defect in repair of double-stranded DNA scission. In one embodiment, the defect in repair of double-stranded DNA scission is a defect in homologous recombination. In another embodiment, the cancer cells have a phenotype selected from, for example and without limitation, a BRCA-1 defect, a BRCA-2 defect, a BRCA-1 and BRCA-2 defect, or Fanconi anemia.

[0098] In one embodiment, the present specification provides methods of treating BRCA1 / 2-associated breast cancer containing the step of administering a compound of Formula (X), a pharmaceutically acceptable salt, an isotope or a pro- drug thereof.

[0099] In accordance with another aspect, the specification discloses a compound for use in the chemosensitization method disclosed herein, the radiosensitization method disclosed herein, or the treatment of cancer wherein thecancer cells have a defect in repair of double-stranded DNA scission method, where the compound is selected from Formula (X), a pharmaceutically acceptable salt, an isotope or a pro-drug thereof.

[0100] In a seventh aspect, the specification relates to a compound of formula 127

[0101] a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof.

[0102] In an eighth aspect, the specification relates to a process for preparation of the compound of formula 127, a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof, the process comprising the step of:

[0103] carboxylating the compound of formula 124 to form the compound of formula 125;124 125

[0104] deprotecting the amine to form the compound of formula 126; and

[0105] converting the carboxylic acid of the compound of formula 126 to the amide to form the compound of formula 127126 127

[0106] The step of carboxylation or carboxylating the compound of formula 124 to form the compound of formula 125 is not particularly limited, and can be determined by a person of skill in the art. In an embodiment, for example and without limitation, the step involves reacting the compound of formula 124 with a strong organolithium base to remove a deuterium atom from the aromatic ring (metalation by lithium- deuterium exchange), followed by reaction with carbon dioxide (CO2) to form the compound of formula 125. In the reaction, the organometallic base used is not particularly limited and can be determined by a person of skill in the art, so long as the base can remove a deuterium atom from the aromatic ring and induce ortho- metalation. It is believed that the presence of the carboxyl group in the amide group present at the ortho-position helps direct the carboxylation and improve selectivity. In one embodiment, for example and without limitation, the base is sec-butyl lithium (sec- BuLi or sBuLi).

[0107] The step of deprotecting the amine to form the compound of formula 126 is not particularly limited and can be determined by a person of skill in the art. In one embodiment, for example and without limitation, where the amine protecting group is pivaloyl, an acid is used to deprotect the amine and form the compound of formula 126. The acid used is not particularly limited and can be determined by a person of skill in the art, and in one embodiment, for example and without limitation, is hydrochloric acid (HCI).

[0108] The step of conversion of the carboxylic acid to the amide is not particularly limited and can be determined by a person of skill in the art. In one embodiment, for example and without limitation, the step involves activation of the carboxylic acid with an activating agent and reaction with ammonia or ammoniumhydroxide to form the amide of formula 127. The activating agent used for forming the amide is not particularly limited and should be known to a person of skill in the art, and in one embodiment is, for example and without limitation, N,N'-carbonyldiimidazole (CDI).

[0109] The compound of formula 127 can then be used in general procedures A and B, as disclosed herein, to form intermediates that can react with an amine to form a penultimate intermediate for forming the compound of formula (X), that has a methoxy protecting group on the aromatic ring. The methoxy protecting group on the aromatic ring can be removed by treatment with boron tribromide to form the compound of formula (X). Given the harsh conditions required in the process for preparation of the deuterated compounds, wherein the deuterium is present on the aromatic ring on the left hand side of the compound of formula (X), the inventors uncovered that a methoxy group provides a better alternative than other protecting groups.

[0110] To prepare the compound of formula 124, 2-aminophenol can be reacted with hydrogen in deuterium oxide (source of the deuterium-hydrogen exchange) in the presence of platinum to replace the hydrogen atoms on the aromatic ring with deuterium and form the compound of formula 122. The process for forming the compound of 122 is not particularly limited and can be determined by a person of skill in the art based on common general knowledge.122

[0111] The amine in the compound of formula 122 can be protected with an amine protecting group to form the compound of formula 123. The amine protecting group used is not particularly limited and can be determined by a person of skill in the art based on common general knowledge and / or routine experimentation. In one embodiment, for example and without limitation, the amine protecting group is pivaloyl.122 123

[0112] The compound of formula 123 can be methylated to form the compound of formula 124. The step of methylation is not particularly limited, and should be known or can be determined by a person of skill in the art based on common general knowledge or routine experimentation. In one embodiment, for example and without limitation, the compound of formula 123 is reacted with a methylating agent to form the compound of formula 124. The methylating agent used is not particularly limited and should be known or can be determined by a person of skill in the art based on common general knowledge or non-inventive routine experimentation. In one embodiment, for example and without limitation, the methylating agent is methyl iodide.

[0113] The specification is further illustrated by the selected examples whose preparation is shown below.EXAMPLES

[0114] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient.Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the constructs of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the typical aspects of the present invention and are not to be construed as limiting in any way in the remainder of the disclosure. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0115] General Methods

[0116] 1H NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer operating at 299.992 MHz using the solvent resonances as secondary standards. 2-amino-3-hydroxybenzoic acid, benzyl chloride, and other reagents used herein were purchased from Sigma Aldrich or Combi Blocks and were used as received. N,N-diisopropylethylamine, trimethylamine, tetrahydrofuran, acetonitrile, dichloromethane, diethyl ether, diisopropyl ether, acetone, hexanes, ethyl acetate, and methanol were purchased from Sigma Aldrich and used without further drying. All reactions were carried out under a nitrogen or Argon atmosphere. Compounds were visualized / located by spraying the TLC plate with Ninyhydrin solution, KMnO4solution, or a solution of 2 % ceric ammonium sulfate in 0.5 M H2SO4followed by heating on a hot plate until color developed.

[0117] Description of Scheme 1

[0118] General procedure for synthesis of compounds 103

[0119] Hydroxy-2-aminobenzoic acid starting material (1 equiv.) was dissolved in dimethyl formamide (DMF), then benzyl chloride (2.4 equiv.) and K2CO3(3 equiv.) were added, and the mixture was heated at 50 °C for 18h. After cooling down to room temperature, ethyl acetate (EtOAc) was added, and the mixture was filtered and concentrated to a dark brown residue. The residue was redissolved in dichloromethane (DCM), washed with saturated aqueous NaHCO3, dried over Na2SO4, filtered and concentrated to a brown solid or paste. This residue was further purified by DCM / methanol (MeOH) trituration and precipitation at room temperature. After filtration and wash with MeOH, the off-white solid was dried to yield benzyl ester 101 (60-80% yield).

[0120] Benzyl ester 101 (1 equiv.) was dissolved in 1 ,4-dioxane I MeOH mixture (1 / 1 , v / v). NaOH solid (1.5 equiv.) was added, and the mixture was heated at 70 °C until thin layer chromatograph (TLC) showed complete reaction (16-24h). Volatiles were removed under vacuum and the resulting solid redissolved in water (at about 1 M). At 0 °C the solution was acidified to pH = 3 with 2N HCI. The resulting precipitate was filtered, washed with cold water and dried under vacuum to yield benzoic acid 102 as an off-white solid (>90% yield).

[0121] Benzoic acid 102 (1 equiv.) was dissolved in DMF and carbonyldiimidazole (CDI) (1.2 equiv.) was added. The mixture was heated at 70 °C for2h. After cooling down to 0 °C, concentrated (28-30%) aqueous NH4OH (30 equiv.) was added slowly, then stirring maintained at 0 °C for 30 min, then room temperature for 1 h, and eventually the mixture was heated at 50 °C for 18h. The mixture was concentrated to remove DMF and yield a yellow solution. The residue was diluted with water and pH was adjusted to 10 with aqueous 2N NaOH. The product was extracted from the aqueous layer with EtOAc multiple times. The pooled organic layers were subsequently washed multiple times with saturated aqueous Na2CO3, a pH~6 mixture of aqueous NaHCO3 / NH4CI and brine. The organic layer was then dried over Na2SO4, filtered and concentrated to yield benzamide 103 as a beige to light-yellow solid (50- 80% yield).

[0122] General procedure A

[0123] Aniline amide (1 equiv.) and succinic anhydride (1.25 equiv.) were suspended in DCM I acetic acid (2 / 1 , V / V) and the resulting clear solution was stirred under argon at RT. Sometimes there is formation of a precipitate product after a few minutes but stirring is continued until all starting materials have reacted (1 to 12 hrs). DCM was removed and the solid product was collected using suction filtration and washed several times using diethyl ether (Et20) I hexanes mixture. No further purification is required. The open acid product is a colorless solid (80 - 95% yield).

[0124] General procedure B

[0125] The open acid was suspended in ethanol (EtOH) at room temperature (RT) and 3M NaOH solution (~ 5 Equiv.) was added slowly. After a few minutes, all solids dissolved, and a clear solution was observed. The clear solution was stirred for a further 2 to 5 hours when completion consumption of the open acid was confirmed. EtOH was removed and the resulting residue was dissolved in a minimum amount of water and acidified slowly to pH~3-4. The resulting precipitated product was collected using suction filtration and dried to give a colorless solid (85 - 95% yield) which required no further purification.

[0126] General procedure C

[0127] The acid (1 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCI) (1.5 equiv.) was suspended in DMF and diisopropylethhylamine (DIPEA) (4 equiv.) was slowly added at RT under argon. The clear solution was stirred at RT for 30 min. when the amine dissolved in a minimum amount of DMF was added into the mixture via syringe. The mixture was stirred overnight, and the solvent was removed under vacuum. Aqueous NaHCO3solution was added, and the mixture was stirred for 15 minutes after which the solid benzylated amide product (80 - 90% yield) was filtered off and dried. No further purification was required.

[0128] General procedure D

[0129] The benzylated species was dissolved in a minimum amount of a suitable solvent. A catalytic amount of Pd / C was added, and the reaction vessel was evacuated and backfilled with hydrogen twice. The mixture was stirred under a hydrogen atmosphere for 5 to 12 hrs until complete consumption of the startingmaterial. The mixture was filtered using celite and the celite cake was washed using MeOH. The combined filtrates were concentrated and optional purification using column chromatography gives a colorless solid product (90 - 96% yield).Scheme 2: Preparation of Inhibitor 1.

[0130] 4-(4-(3-(8-(benzyloxy)-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)benzonitrile (106a)

[0131] The benzylated species 106a was prepared according to general procedure C using acid 105a and 4-(piperazin-1-yl)benzonitrile as the amine.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.23 (s, 1 H), 7.65 (dd, 1 H), 7.59 (d, 2H), 7.46 (d, 2H), 7.40-7.22 (m, 5H), 6.96 (d, 2H), 5.22 (s, 2H), 3.64-3.49 (m, 4H), 3.34-3.25 (m, 4H), 2.90 (s, 4H). LR ESI MS: m / z calcd for C29H26N5O3[M-Hp, 492.2 found 492.9.

[0132] 4-(4-(3-(8-hydroxy-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)benzonitrile (1 )

[0133] The hydroxyl-terminated inhibitor 1 was prepared from the benzylated species intermediate 106a according to general procedure D.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.16 (br s, 1 H), 7.60 (d, 2H), 7.49 (d, 1 H), 7.26 (t, 1 H), 7.15 (d, 1 H), 7.02 (d, 2H), 3.76-3.65 (m, 2H), 3.64-3.54 (m, 2H), 3.49-3.31 (m, 4H), 3.01 (t, 2H), 2.88 (t, 2H). LR ESI MS: m / z calcd for C22H20N5O3[M-Hp, 402.2 found 402.9.Scheme 3: Preparation of Inhibitor 16.

[0134] 6-(4-(3-(6-(benzyloxy)-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)nicotinonitrile (106b)

[0135] The benzylated species 106b was prepared according to general procedure C using acid 105b and 6-(piperazin-1-yl)nicotinonitrile as the amine.1H NMR (300 MHz, DMSO): δ 2.83-2.90 (m, 4H), 3.51 - 3.83 (m, 8H), 5.21 (s, 2H), 6.89 - 6.98 (m, 1 H), 7.28 - 7.60 (m, 8H), 6.28 - 6.46 (m, 1 H), 7.84 - 7.93 (m, 1 H), 8.48 - 8.53 (m, 1 H), 12.14(s, 1 H); LR ESI MS: m / z calcd for C28H26N6O3[M + H]+, 494.5, found 495.5.

[0136] 6-(4-(3-(6-hydroxy-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)nicotinonitrile (16)

[0137] The hydroxyl-term inated inhibitor 16 was prepared from the benzylated species intermediate 106b according to general procedure D.1H NMR (300 MHz,DMSO): 6 2.81-2.87 (m, 4H), 3.51 - 3.81 (m, 8H), 6.89 - 6.98 (m, 1 H), 7.13 - 7.23 (m, 1 H), 7.31 - 7.37 (m, 1 H), 6.28 - 6.46 (m, 1 H), 7.37 - 7.45 (m, 1 H), 7.84 - 7.93 (m, 1 H), 8.48 - 8.54 (m, 1 H), 9.97 - 1 .21 (m, 1 H), 11 .64 - 12.25 (m, 1 H); LR ESI MS: m / z calcd for C21H20N6O3[M + H]+, 404.4, found 404.5.Scheme 4: Preparation of Inhibitor 14

[0138] 6-(4-(3-(7-(benzyloxy)-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)nicotinonitrile (106c)

[0139] The benzylated species 106c was prepared according to general procedure C using acid 105c and 6-(piperazin-1-yl)nicotinonitrile as the amine.1H NMR (300 MHz, DMSO): δ 2.75 - 2.93 (m, 4H), 3.46 - 3.85 (m, 8H), 5.20 (s, 2H), 6.79 - 7.12 (m, 3H), 7.23 - 7.53 (m, 5H), 7.78 - 8.03 (m, 2H), 8.50 (s, 1 H), 11.59 - 12.38 (br, 1 H). LR ESI MS: m / z calcd for C28H26N6O3[M - H]_, 494.21 found 494.0

[0140] 6-(4-(3-(7-hydroxy-4-oxo-3,4-dihydroquinazolin-2- yl)propanoyl)piperazin-1 -yl)nicotinonitrile (14)

[0141] The hydroxyl-term inated inhibitor 14 was prepared from the benzylated species intermediate 106c according to general procedure D.1H NMR (300 MHz, DMSO): δ 2.75 - 2.93 (m, 4H), 3.49 - 3.87 (m, 8H), 6.69 - 7.06 (m, 3H), 7.77 - 8.02 (m, 2H), 8.43 - 8.57 (s, 1 H), 11.23 - 12.36 (br, 2H). LR ESI MS: m / z calcd for C21H20N6O3[M + H]+, 404.16 found 405.8.Scheme 5: Preparation of Inhibitor 19

[0142] 4-[2-(Benzyloxy)-6-carbamoylphenylamino]-4-oxo(2H4)butyric acid (108)

[0143] The deuterated open acid 108 was prepared following general procedure A with deuterated (2Hs)-succinic anhydride.1H NMR (300 MHz, CD3OD): δ (ppm) 7.50-7.43 (m, 2H), 7.43-7.24 (m, 4H), 7.24-7.18 (m, 2H), 5.16 (s, 2H). LR ESI MS: m / z calcd for C18H132H4N2O5[M-H]’, 345.1 found 345.8.

[0144] 3-[8-(Benzyloxy)-4-oxo-3H-quinazolin-2-yl](2H4)propionic acid (109)

[0145] The deuterated acid 109 was prepared from compound 108, following general procedure B.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.25 (s, 2H), 7.66 (dd, 1 H), 7.52 (d, 2H), 7.43-7.28 (m, 5H), 5.28 (s, 2H). LR ESI MS: m / z calcd for C18H112H4N2O4[M-H]; 327.1 found 327.7.

[0146] p-(4-{3-[8-(Benzyloxy)-4-oxo-3H-quinazolin-2-yl](2H4)propionyl}-1 - piperazinyl)benzonitrile (110)

[0147] The benzylated species 110 was prepared following general procedure C with acid 109 and 4-(piperazin-1-yl)benzonitrile as the amine.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.23 (s, 2H), 7.65 (dd, 1 H), 7.59 (d, 2H), 7.46 (d, 2H), 7.39-7.23 (m, 5H), 6.96 (d, 2H), 5.22 (s, 2H), 3.64-3.51 (m, 4H), 3.33-3.25 (m, 4H). LR ESI MS: m / z calcd for C29H222H4N5O3[M-H]; 496.2 found 496.9.

[0148] p-{4-[3-(8-Hydroxy-4-oxo-3H-quinazolin-2-yl)(2H4)propionyl]-1 - piperazinyl}benzonitrile (19)

[0149] The hydroxyl-term inated species 19 was prepared from compound 110 following general procedure D.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.16 (s, 1 H), 9.26 (s, 1 H), 7.60 (d, 2H), 7.49 (dd, 1 H), 7.26 (t, 1 H), 7.15 (dd, 1 H), 7.03 (d, 2H), 3.75-3.65 (m, 2H), 3.64-3.54 (m, 2H), 3.49-3.40 (m, 2H), 3.39-3.32 (m, 2H). LR ESI MS: m / z calcd for C22H162H4N5O3[M-H]; 406.2 found 406.9.Scheme 6: Preparation of Inhibitor 20

[0150] General procedure E:

[0151] tert-Butyl 4-(p-cyanophenyl)(2Hs)-1 -piperazinecarboxylate (113)

[0152] To a stirred solution of methyl p-bromobenzoate (0.55 mmol) in anhydrous toulene (2 mL) was added tert-Butyl (2,2,3,3,5,5,6,6-2H8)-1 - piperazinecarboxylate (1 equiv.), tris(dibenzylideneacetone)dipalladium(0) (Pd(dba)3) (10 mol %), XantPhos (10 mol%), CsCO3(3 equiv.) at RT. The resulting suspension was stirred and heated to 100 °C for 16 hours. The reaction mixture was cooled to RT, diluted with EtOAc, and filtered. The filtrate was concentrated to give a crude material which was purified using column chromatography (eluting 0 to 30% ethyl acetate inhexane) to afford compound 113 as a colorless solid in quantitative yield.1H NMR (300 MHz, CDCI3): δ (ppm) 7.50 (d, 2H), 6.84 (s, 2H), 1.47 (s, 9H).

[0153] General procedure F

[0154] p-[(2,2,3,3,5,5,6,6-2H8)-1 -Piperazinyl]benzonitrile (114)

[0155] Boc-protected compound 113 was dissolved in a mixture ofDCM / trifluoroacetic acid (TFA) (2 / 1 , v / v) and the mixture was stirred at RT under argon for 2 to 3 hrs. Concentration under vacuum gave the TFA salt 114 as an off-white solid.

[0156] p-(4-{3-[8-(Benzyloxy)-4-oxo-3H-quinazolin-2- yl](2H4)propionyl}(2Hs)-1 -piperazinyl)benzonitrile (115)

[0157] The benzylated species 115 was prepared following general procedureC with acid 109 and amine salt 114.1H NMR (300 MHz, CDCI3): δ (ppm) 7.83-7.76 (m, 1 H), 7.51-7.41 (m, 4H), 7.38-7.27 (m, 4H), 7.22-7.16 (m, 1 H), 6.82-6.73 (m, 2H), 5.28 (s, 2H). LR ESI MS: m / z calcd for C29H142H12N5O3[M-H]; 504.63 found 504.9.

[0158] p-{4-[3-(8-Hydroxy-4-oxo-3H-quinazolin-2-yl)(2H4)propionyl](2H8)-1 - piperazinyl}benzonitrile (20)

[0159] The hydroxyl-term inated species 20 was prepared from 115 following general procedure D.1H NMR (300 MHz, CDCI3): δ (ppm) 12.2 (s, 1 H), 9.3 (s, 1 H), 7.69-7.62 (m, 2H), 7.58-7.52 (m, 1 H), 7.35-7.27 (m, 1 H), 7.24-7.17 (m, 1 H), 7.11-7.04 (m, 2H). LR ESI MS: m / z calcd for C22H82H12N5O3[M-H]; 414.51 found 414.7.Scheme 7: Preparation of Inhibitor 21

[0160] tert-Butyl 4-[p-cyano(2H4)phenyl]-1 -piperazinecarboxylate (117)

[0161] Compound 117 was prepared following general procedure E using p- bromo(2H4)benzonitrile and tert-Butyl 1 -piperazinecarboxylate.1H NMR (300 MHz,DMSO-d6): δ (ppm) 3.61-3.53 (m, 4H), 3.33-3.26 (m, 4H), 1.47 (s, 9H).

[0162] p-(1 -Piperazinyl)(2H4)benzonitrile (118)

[0163] The TFA salt 118 was prepared from compound 117 following general procedure F.1H NMR (300 MHz, CDCI3): δ (ppm) 9.76 (s, 1 H), 3.65-3.50 (m, 4H), 3.43-3.27 (m, 4H).

[0164] p-(4-{3-[8-(Benzyloxy)-4-oxo-3H-quinazolin-2-yl]propionyl}-1 - piperazinyl)(2H4)benzonitrile (119)

[0165] The benzylated species 119 was prepared following general procedure C with acid 105a and amine salt 118. LR ESI MS: m / z calcd for C29H222H4N5O3[M-H]- , 496.58 found 496.9.

[0166] p-{4-[3-(8-Hydroxy-4-oxo-3H-quinazolin-2-yl)propionyl]-1 - piperazinyl}(2H4)benzonitrile (21 )

[0167] The hydroxyl-term inated species 21 was prepared from 119 following general procedure D.1H NMR (300 MHz, CDCI3): δ (ppm) 12.20 (s, 1 H), 9.33 (S, 1 H), 7.53-7.46 (m, 1 H), 7.31-7.23 (m, 1 H), 7.20-7.13 (m, 1 H), 3.75-3.51 (m, 4H), 3.50-3.39(m, 4H), 3.07-2.83 (m, 4H). LR ESI MS: m / z calcd for C22H162H4N5O3[M-Hp, 406.4 found 406.6.Scheme 8: Preparation of Inhibitor 17

[0168] 4-(o-Carbamoylphenylamino)-4-oxobutyric acid (120)

[0169] The open acid 120 was prepared following general procedure A with o- aminobenzamide and succinic anhydride.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.16 (s, 1 H), 11.73 (s, 1 H), 8.45 (d, 1 H), 8.26 (s, 1 H), 7.79 (d, 1 H), 7.73 (s, 1 H), 7.48 (t, 1 H), 7.10 (t, 1 H), 3.34 (br s, 1 H), 2.62-2.49 (m, 4H).

[0170] 3-(4-Oxo-3H-quinazolin-2-yl)propionic acid (121 )

[0171] The acid 121 was prepared from the acid 120 following general procedure B.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.21 (s, 2H), 8.07 (d, 1 H), 7.76 (t, 1 H), 7.57 (d, 1 H), 7.45 (t, 1 H), 3.34 (br s, 1 H), 2.91-2.80 (m, 2H), 2.80-2.69 (m, 2H). LR ESI MS: m / z calcd for C11H9N2O3[M-Hp, 217.1 found 217.8.

[0172] p-{4-[3-(4-Oxo-3H-quinazolin-2-yl)propionyl]-1 - piperazinyl}benzonitrile (17)

[0173] Compound 17 was prepared from acid 121 following general procedureC with 4-(piperazin-1-yl)benzonitrile as the amine.1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.18 (s, 1 H), 8.07 (d, 1 H), 7.74 (t, 1 H), 7.61 (d, 2H), 7.54 (d, 1 H), 7.44 (t, 1 H), 7.03 (d, 2H), 3.73-3.64 (m, 2H), 3.64-3.53 (m, 2H), 3.50-3.40 (m, 2H), 3.40-3.30 (m, 2H), 2.89 (m, 4H). LR ESI MS: m / z calcd for C22H20N6O2[M-Hp, 386.2 found 386.8.Scheme 9: Preparation of deuterated acid intermediates 129 and 131

[0174] 2-Amino-(2H4)-phenol (122)

[0175] To solution of o-aminophenol in D2O under argon atmosphere was added 5% Pt / C. The reaction mixture was stirred at 100 °C under hydrogen atmosphere for 2 h and then diluted with EtOAc. The mixture was filtered through a pad of celite. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4and concentrated in reduced pressure to give product as brown solid. A second reaction cycle was performed under the same reaction condition to get the desired product 122. ESI MS: m / z calcd for C6H32H4NO. [M + H]+114.1 .

[0176] 2-(Pivaloyl-amino)-(2H4)-phenol (123)

[0177] o-Amino(2H4) phenol (122) (1 equiv.) was dissolved in THF and triethylamine (1.1 equiv.) was added. The mixture was cooled to 0 °C and pivaloyl chloride (1.1 equiv.) was added in portions. After 1 h stirring at 0 °C the mixture wasallowed to warm up to 20 °C and continue to stir for 16h. The mixture was filtered, and the organic phase evaporated in reduced pressure and crude residue was purified by chromatography using EtOAc / Hexane. The product was isolated as colorless liquid.1H NMR (300 MHz, CDCI3): δ (ppm) 1.2 (s, 9H); ESI MS: m / z calcd for C11H112H4NO2[M - H]+196.8.

[0178] N-Pivaloyl-2-methoxy-(2H4)-aniline (124)

[0179] To a suspension of hydroxy compound 123 (1 equiv.) and potassium carbonate (2 equiv.) in acetonitrile, iodomethane (1.1 equiv.) was added dropwise. The mixture was stirred at 60 °C for 7 h. The solid was separated by filtration, washed with EtOAc and the filtrate was evaporated in reduced pressure to yield crude product which was purified by column chromatography using EtOAC / Hexane to get methoxy compound 124.1H NMR (300 MHz, MeOH-d4): δ (ppm) 1.18 (s, 9H), 3.76 (s, 3H); ESI MS: m / z calcd for C12H132H4NO2[M + H]+212.4.

[0180] 2-(Pivaloyl-amino)-3-methoxy-(2H3)-benzoic acid (125)

[0181] To a solution of compound 124 (1 equiv.) in THF was added sec-BuLi (0.8 equiv.) at -78 °C, dropwise over 30 minutes, under argon atmosphere. The mixture was stirred at -78 °C for 3 h and then a large amount of crushed dry ice was added to the reaction mixture. The temperature of reaction mixture was brought up to room temperature. The mixture was diluted with water, aqueous 2M NaOH was added and extracted with Et2O. The aqueous layer was acidified with 1 M HCI to pH 4 and extracted with EtOAc. The solvent was evaporated in reduced pressure and the crude residue was purified by column chromatography using MeOH / DCM (5-25 %) to yield compound 125.1H NMR (300 MHz, MeOH-d4): δ (ppm) 1.21 (s, 9H), 3.75 (s, 3H); ESI MS: m / z calcd for C13H142H3NO4, [M - H]+253.8.

[0182] 2-Amino-3-methoxy-(2H3)-benzoic acid (126)

[0183] A mixture of Pivaloyl-protected aminobenzoic acid 125 (1 equiv.) and concentrated HCI (5 equiv.) in D2O / acetonitrile (10+5 mL) was heated at 110 °C for 16 h. The solvent was evaporated in reduced pressure and the residue was dissolved in water, neutralized with saturated aqueous solution of NaHCO3to pH 7-9. The aqueous layer was extracted with EtOAc, dried (anhydrous Na2SO4) and evaporated to yield benzoic acid 126.1H NMR (300 MHz, MeOH-d4): δ (ppm) 4.20 (s, 3H); ESI MS: m / z calcd for C8H62H3NO3, [M+H]+171.1.

[0184] 2-Amino-3-methoxy-(2H3)-benzamide(127)

[0185] To a solution of benzoic acid 126 (1 equiv.) in DMF was added CDI (1 .2 equiv.). The mixture was heated at 60 °C for 1 h under nitrogen atmosphere. The mixture was cooled to room temperature and 33% NH4CI aqueous solution (15 equiv.) was added. The reaction flask was sealed and kept at room temperature for 16 h then the solvent was evaporated. The solid was dissolved in EtOAc and washed with water, brine, dried (anhydrous Na2SO4) and evaporated to yield benzamide 127.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.86 (s, 3H); ESI MS: m / z calcd for C8H72H3N2O2, [M+H]+170.1.

[0186] 4-(2-Carbamoyl-6-(2H3)-anisidino)-4-oxobutyric acid (128)

[0187] Compound 128 was prepared following General procedure A, from compound 127 and Succinic anhydride.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.95 (s, 3H), 2.60-2.75 (m, 4H); ESI MS: m / z calcd for C12H112H3N2O5[M - H]+268.1 .

[0188] 3-(8-Methoxy-4-oxo-3H-(5,6,7-2H3)-quinazolin-2-yl)propionic acid (129)

[0189] Compound 129 was prepared following General procedure B, from compound 128.1H NMR (300 MHz, MeOH-d4): δ (ppm) 4.20 (s, 3H), 3.3-3.4 (m, 4H), 1 H); ESI MS: m / z calcd for C12H92H3N2O4[M - H]+250.7.

[0190] 4-(2-Carbamoyl-6-(2H3)-anisidino)-4-oxo-(2H4)-butyric acid (130)

[0191] Compound 130 was prepared following General procedure A, from compound 127 and2Hs-Succinic anhydride.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.85 (s, 3H); ESI MS: m / z calcd for C12H72H7N2O5[M+H]+274.6.

[0192] 3-(8-Methoxy-4-oxo-3H-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propionic acid (131)

[0193] Compound 131 was prepared following General procedure B, from compound 130.1H NMR (300 MHz, MeOH-d4): δ (ppm) 4.20 (s, 3H); ESI MS: m / z calcd for C12H52H7N2O4[M+H]+256.6.Scheme 10: Preparation of Inhibitors 22-24

[0194] General Procedure G

[0195] The 8-methoxyquinazolin-4(3 / 7)-one protected species (1 equiv.) was dissolved in anhydrous 1 ,2-dichloroethane (DCE) and excess BBr3solution (1 .0 M, 2-4 equiv.) was added under nitrogen. The mixture was stirred at reflux overnight. The solvent was removed, and purification was achieved using silica gel flash chromatography (eluting with a gradient of MeOH / DCM) and / or semi-preparative HPLC (Cis column, eluting with a gradient of MeOH vs 0.1 % Formic Acid in water), yielding the corresponding 8-hydroxyquinazolin-4(3 / 7)-one product.

[0196] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)piperazin-1 -yl)benzonitrile (132)

[0197] Compound 132 was prepared following General procedure C, from compound 129 and p-(1-Piperazinyl)benzonitrile.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.57 (d, J=9.3Hz, 2H), 7.05 (d, J=8.8Hz, 2H), 3.92 (s, 3H), 3.88-3.81 (m, 2H),3.80-3.72 (m, 2H), 3.54-3.48 (m, 2H), 3.43-3.37 (m, 2H), 3.10-3.00 (m, 2H); ESI MS: m / z calcd for C23H202H3N5O3[M + H]+421 .1 .

[0198] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)piperazin-1 -yl)benzonitrile (22)

[0199] Inhibitor 22 was prepared from compound 132 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.44 (d, J=9 Hz, 2H), 6.09 (d, J=8.8Hz, 2H), 3.75-3.59 (m, 4H), 3.42-3.34 (m, 2H), 3.31-3.21 (m, 2H); ESI MS: m / z calcd for C22H182H3N5O3[M - H]+405.0.

[0200] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)piperazin-1 -yl)-(2H4)-benzonitrile (133)

[0201] Compound 133 was prepared following General procedure C, from compounds 129 and 118.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.94 (s, 3H) 3.89- 3.71 (m, 4H), 3.55-3.36 (m, 4H), 3.10-3.00 (m, 4H); ESI MS: m / z calcd for C23H162H7N5O3[M+H]+425.0.

[0202] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)piperazin-1 -yl)-(2H4)-benzonitrile (23)

[0203] Inhibitor 23 was prepared from compound 133 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.74-3.59 (m, 4H), 3.38-3.23 (m, 4H), 3.00-2.90 (m, 4H); ESI MS: m / z calcd for C22H142H7N5O3[M+H]+411.0.

[0204] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)-(2Hs)-piperazin-1 -yl)benzonitrile (134)

[0205] Compound 134 was prepared following General procedure C, from compounds 129 and 114.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.44 (d, J=8.3Hz, 2H), 6.90 (d, J=8.7Hz, 2H), 3.81 (s, 3H), 2.95-2.90 (m, 4H); ESI MS: m / z calcd for C23H122H11N5O3[M+H]+429.0.

[0206] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2- yl)propanoyl)-(2Hs)-piperazin-1 -yl)benzonitrile (24)

[0207] Inhibitor 24 was prepared from compound 134 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.43 (d, J=8.6Hz, 2H), 6.95 (d, J=8.7Hz, 2H), 2.99-2.90 (m, 4H); ESI MS: m / z calcd for C22H1O2H11N5O3[M+H]+415.0.Scheme 11 : Preparation of Inhibitors 25-27

[0208] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propanoyl)piperazin-1 -yl)benzonitrile (135)

[0209] Compound 135 was prepared following General procedure C, from compound 131 and p-(1-Piperazinyl)benzonitrile.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.57 (d, J=9.8 Hz, 2H), 7.06 (d, J=9.1 Hz, 2H), 3.94 (s, 3H), 3.89-3.71 (m, 4H), 3.55-3.37 (m, 4H); ESI MS: m / z calcd for C23H162H7N5O3[M+H]+425.9.

[0210] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)- (2H4)-propanoyl)piperazin-1-yl)benzonitrile (25)

[0211] Inhibitor 25 was prepared from compound 135 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.44 (d, J=8.8 Hz, 2H), 7.36 (d, J=8.9 Hz, 2H), 3.75-3.59 (m, 4H), 3.39-3.20 (m, 4H); ESI MS: m / z calcd for C22H142H7N5O3[M+H]+411 .0.

[0212] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propanoyl)piperazin-1 -yl)-(2H4)-benzonitrile (136)

[0213] Compound 136 was prepared following General procedure C, from compounds 131 and 118.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.81 (s, 3H), 3.75- 3.59 (m, 4H), 3.41 -3.23 (m, 4H); ESI MS: m / z calcd for C23H122H11N5O3[M+H]+429.0.

[0214] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propanoyl)piperazin-1 -yl)-(2H4)-benzonitrile (26)

[0215] Inhibitor 26 was prepared from compound 136 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 3.74-3.58 (m, 4H), 3.39-3.24 (m, 4H); ESI MS: m / z calcd for C22H1O2H11N5O3[M+H]+415.0.

[0216] 4-(4-(3-(8-Methoxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propanoyl)-(2Hs)-piperazin-1 -yl)benzonitrile (137)

[0217] Compound 137 was prepared following General procedure C, from compounds 131 and 114.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.44 (d, J=9.1 Hz, 2H), 6.91 (d, J=9.1 Hz, 2H), 3.82 (s, 3H); ESI MS: m / z calcd for C23H82H15N5O3[M+H]+433.0.

[0218] 4-(4-(3-(8-Hydroxy-4-oxo-3,4-dihydro-(5,6,7-2H3)-quinazolin-2-yl)-(2H4)-propanoyl)-(2H8)-piperazin-1 -yl)benzonitrile (27)

[0219] Inhibitor 27 was prepared from compound 137 following General procedure G.1H NMR (300 MHz, MeOH-d4): δ (ppm) 7.56 (d, J=8.6Hz, 2H), 7.06 (d, J=9.2Hz, 2H); ESI MS: m / z calcd for C22H62H15N5O3[M+H]+419.0.

[0220] IN VITRO PARP-1 INHIBITION ASSAY

[0221] All the compounds herein were screened against PARP-1 and PARP-2 in order to determine the selectivity of inhibitors for PARP-1 over PARP-2.

[0222] Activities of compounds against PARP-1 were determined using the PARP1 Colorimetric Assay Kit (Cat.# 80580) supplied by BPS Bioscience, USA.

[0223] PARP-1 Inhibitor Assay Design

[0224] All samples and controls were performed in duplicates. The assay included a “Blank, a “Positive control”, and control inhibitor compound AZD5305 (CAS Number [2589531-76-8]). AZD5305 will inhibit the activity of PARP-1 at a wide range of concentrations from 1 nM to 10 μM. Serially dilute the stock AZD5305 or PARP inhibitor(s) with 1X PARP Buffer and add to designated wells.

[0225] Step 1 : Coat histone solution using a 96-well transparent plate

[0226] 1) Dilute 5x histone mixture 1 :5 with Phosphate-Buffered Saline (PBS) to make 1x histone mixture

[0227] 2) Add 50 μL of histone mixture to each well and incubate at 4°C overnight

[0228] 3) Wash the plate three times using 200 μL of PBST buffer (1x PBS containing 0.05% Tween 20) per well.

[0229] 4) Tap the plate onto clean paper towel to remove the liquid.

[0230] 5) Block the wells by adding 200 μL of Blocking buffer 3 to every well.Incubate at room temperature for at least 90 minutes.

[0231] 6) Wash the plate three times with 200 μL / well of PBST buffer.

[0232] 7) Tap the plate onto clean paper towel to remove the liquid.

[0233] Step 2: Ribosylation reaction

[0234] 1) Prepare a fresh solution of 10 mM Dithiothreitol (DTT) in water.

[0235] 2) Dilute Activated deoxyribonucleic acid (DNA) 1 :32 with PBS.

[0236] 3) Prepare the Master Mix (25 μL / well): N wells x (2.5 μL of 10x PARP buffer + 5 μL of PARP Substrate Mixture 1 + 5 μL of diluted Activated DNA + 10 μL of water + 2.5 μL of 10 mM fresh DTT).

[0237] 4) Add 25 μL of Master Mix to every well.

[0238] 5) Prepare 1x PARP buffer with DTT. Dilute 10x PARP assay buffer to1x PARP assay buffer containing DTT by adding 1 volume of 10x PARP assay buffer + 1 volume of 10 mM DTT + 8 volumes of water.

[0239] 6) Add 5 μL of Test Inhibitor to each well labeled as “Test Inhibitor.”

[0240] For the “Positive Control" and “Blank,” add 5 μL of the same diluent solution used to dilute the inhibitor, but without inhibitor (Diluent Solution).

[0241] 7) Thaw PARP-1 enzyme on ice. Briefly spin the tube containing the enzyme to recover the full content of the tube. Calculate the amount of PARP-1 required for the assay and dilute enzyme to 0.23 ng / μL with 1x PARP buffer with DTT.The final concentration of PARP1 will be 1 nM. Aliquot the remaining undiluted PARP- 1 enzyme into aliquots and store at -80 °C.

[0242] 8) Initiate the reaction by adding 20 μL of diluted PARP-1 enzyme to the wells designated “Positive Control” and "Test Inhibitor."

[0243] To the wells designated as "Blank," add 20 μL of 1x PARP buffer with DTT.

[0244] Incubate at room temperature for 1 hour.

[0245] 9) Wash the plate three times with 200 μL PBST buffer and tap the plate onto clean paper towel as described above.

[0246] Step 3: Detection

[0247] 1) Dilute Streptavidin-HRP (Streptavidin conjugated to HorseRadishPeroxidase enzyme) 1 :50 in Blocking buffer 3.

[0248] 2) Add 50 μL of diluted Streptavidin-HRP to each well. Incubate for 30 minutes at room temperature.

[0249] 3) Wash three times with 200 μL PBST buffer and tap the plate onto clean paper towel.

[0250] 4) Add 100 μL of the colorimetric HRP substrate to each well and incubate the plate at room temperature until blue color is developed in the positive control well. For PARP-1 , it normally takes 15-20 min to fully develop the color. However, the optimal incubation time may vary, and should be determined empirically by the user.

[0251] 5) After the blue color is developed, add 100 μL of 2 M sulfuric acid to each well. Read the absorbance at 450 nm using UVA / is spectrophotometer microplate reader. The negative control-blank well should be - 0.05 absorbance at 450 nm. Alternatively, the plate may be read at 650 nm without adding 2 M sulfuric acid, but the Signal-to-Background ratio will be decreased.

[0252] IN VITRO PARP-2 INHIBITON ASSAY:

[0253] Activities of compounds against PARP-2 were determined using the PARP-2 Colorimetric Assay Kit (Cat.# 80581 ) supplied by BPS Bioscience, USA.

[0254] PARP-2 Inhibitor Assay Design

[0255] All samples and controls were performed in duplicates. The assay included a “Blank, a “Positive control”, and control inhibitor compound AZD5305 (CAS Number [2589531-76-8]). AZD5305 will inhibit the activity of PARP-2 at a wide range of concentrations from 1 nM to 10 μM. Serially dilute the stock AZD5305 or PARP inhibitor(s) with 1X PARP Buffer and add to designated wells.

[0256] Step 1 : Coat histone solution using a 96-well transparent plate

[0257] 1) Dilute 5x histone mixture 1 :5 with PBS to make 1x histone mixture

[0258] 2) Add 50 μL of histone mixture to each well and incubate at 4°C overnight

[0259] 3) Wash the plate three times using 200 μL of PBST buffer (1x PBS containing 0.05% Tween 20) per well.

[0260] 4) Tap the plate onto clean paper towel to remove the liquid.

[0261] 5) Block the wells by adding 200 μL of Blocking buffer 3 to every well.Incubate at room temperature for at least 90 minutes.

[0262] 6) Wash the plate three times with 200 μL / well of PBST buffer.

[0263] 7) Tap the plate onto clean paper towel to remove the liquid.

[0264] Step 2: Ribosylation reaction

[0265] 1) Prepare a fresh solution of 10 mM DTT in water.

[0266] 2) Dilute Activated DNA 1 :32 with PBS.

[0267] 3) Prepare the Master Mix (25 μL / well): N wells x (2.5 μL of 10x PARP buffer + 5 μL of PARP Substrate Mixture 1 + 5 μL of diluted Activated DNA + 10 μL of water + 2.5 μL of 10 mM fresh DTT).

[0268] 4) Add 25 μL of Master Mix to every well.

[0269] 5) Prepare 1x PARP buffer with DTT. Dilute 10x PARP assay buffer to1x PARP assay buffer containing DTT by adding 1 volume of 10x PARP assay buffer + 1 volume of 10 mM DTT + 8 volumes of water.

[0270] 6) Add 5 μL of Test Inhibitor to each well labeled as “Test Inhibitor.”

[0271] For the “Positive Control" and “Blank,” add 5 μL of the same diluent solution used to dilute the inhibitor, but without inhibitor (Diluent Solution).

[0272] 7) Thaw PARP-2 enzyme on ice. Briefly spin the tube containing the enzyme to recover the full content of the tube. Calculate the amount of PARP-2 required for the assay and dilute enzyme to 0.23 ng / μL with 1x PARP buffer with DTT. The final concentration of PARP-2 will be 1 nM. Aliquot the remaining undiluted PARP- 2 enzyme into aliquots and store at -80 °C.

[0273] 8) Initiate the reaction by adding 20 μL of diluted PARP-2 enzyme to the wells designated “Positive Control” and "Test Inhibitor."

[0274] To the wells designated as "Blank," add 20 μL of 1x PARP buffer with DTT.

[0275] Incubate at room temperature for 1 hour.

[0276] 9) Wash the plate three times with 200 μL PBST buffer and tap the plate onto clean paper towel as described above.

[0277] Step 3: Detection

[0278] 1) Dilute Streptavidin-HRP 1 :50 in Blocking buffer 3.

[0279] 2) Add 50 μL of diluted Streptavidin-HRP to each well. Incubate for 30 minutes at room temperature.

[0280] 3) Wash three times with 200 μL PBST buffer and tap the plate onto clean paper towel.

[0281] 4) Add 100 μL of the colorimetric HRP substrate to each well and incubate the plate at room temperature until blue color is developed in the positive control well. For PARP-2, it normally takes 15-20 min to fully develop the color. However, the optimal incubation time may vary, and should be determined empirically by the user.

[0282] 5) After the blue color is developed, add 100 μL of 2 M sulfuric acid to each well. Read the absorbance at 450 nm using UVA / is spectrophotometer microplate reader. The negative control-blank well should be - 0.05 absorbance at 450 nm. Alternatively, the plate may be read at 650 nm without adding 2 M sulfuric acid, but the Signal-to-Background ratio will be decreased.Table 7: PARP-1 and PARP-2 activity screening results for selected compounds disclosed herein, along with Olaparib and AZD5305

[0283] IN VITRO PARP-3 INHIBITION ASSAY:

[0284] Activities of compounds against PARP-3 were determined using thePARP-3 Chemiluminescent Assay Kit (Cat.# 80553-1) supplied by BPS Bioscience, USA.

[0285] PARP-3 Inhibitor Assay Design

[0286] All samples and controls were performed in duplicates. The assay included a “Blank, a “Positive control”, and control inhibitor compound Olaparib (CAS Number [763113-22-0]). Olaparib will inhibit the activity of PARP-3 at a wide range of concentrations from 20 nM to 10 μM. Serially dilute the stock Olaparib or PARP inhibitor(s) with 1X PARP Buffer and add to designated wells.

[0287] Step 1 : Coat histone solution using a 96-well transparent plate

[0288] 1 . Dilute 5x Histone Mixture 5-fold with PBS. This makes 1x HistoneMixture (50 pl / well).

[0289] 2. Add 50 μL of 1x Histone Mixture to each well.

[0290] 3. Incubate at 4°C overnight.

[0291] 4. Wash the plate three times using 200 μL of PBST Buffer per well.

[0292] 5. Tap the plate onto clean paper towel to remove the liquid.

[0293] 6. Block the wells by adding 200 μL of Blocking Buffer 3 to every well.

[0294] 7. Incubate at Room Temperature (RT) for at least 90 minutes.

[0295] 8. Wash the plate three times with 200 μL of PBST Buffer per well.

[0296] 9. Tap the plate onto clean paper towel to remove the liquid.

[0297] Step 2: Ribosylation reaction

[0298] 1 . Dilute Activated DNA 32-fold with PBS.

[0299] 2. Dilute 0.5 M DTT 25-fold with distilled water. This makes a 20 mMDTT solution. 3. Prepare a Master Mix (25 μL / well): N wells x (2.5 μL of 10x PARP Assay Buffer + 10 μL of PARP Substrate Mixture 1 + 5 μL of diluted Activated DNA + 5 μL of distilled water + 2.5 μL of 20 mM DTT solution).

[0300] 4. Add 25 μL of Master Mix to every well.

[0301] 5. Prepare 1x PARP Assay Buffer by adding 1 volume of 10x PARPAssay Buffer and 1 volume of 20 mM DTT solution to 8 volumes of distilled water.

[0302] 6. Prepare the Test Inhibitor (5 μL / well): for a titration prepare serial dilutions at concentrations 10-fold higher than the desired final concentrations. The final volume of the reaction is 50 μL.

[0303] 7. Add 5 μL of Test Inhibitor to each well labeled as “Test Inhibitor”.

[0304] 8. Add 5 μL of Diluent Solution to the “Positive Control” and “Blank” wells.

[0305] 9. Thaw PARP-3 enzyme on ice. Briefly spin the tube containing the enzyme to recover its full content.

[0306] 10. Dilute PARP-3 enzyme to 1.1 ng / μL with 1x PARP Assay Buffer (20 μL / well).

[0307] 11 . Initiate the reaction by adding 20 μL of diluted PARP-3 enzyme to the wells designated “Positive Control” and "Test Inhibitor."

[0308] 12. Add 20 μL of 1x PARP Assay Buffer to the “Blank” wells.

[0309] 13. Incubate at RT for 1 hour.

[0310] Step 3: Detection

[0311] 1. Dilute Streptavidin-HRP 50-fold in Blocking Buffer s (50 μL / well).

[0312] 2. Add 50 μL of diluted Streptavidin-HRP to every well.

[0313] 3. Incubate for 30 minutes at RT.

[0314] 4. Wash the plate three times with 200 μL of PBST Buffer per well and tap the plate onto clean paper towel.

[0315] 5. Just before use, mix 1 volume of ELISA ECL (Enzyme-LinkedImmunoSorbent Assay - ElectroChemiLuminescence) Substrate A and 1 volume of ELISA ECL Substrate B (100 μL of mix / well).

[0316] 6. Add 100 μL of mix to every well.

[0317] 7. Immediately read the plate in chemiluminescence.

[0318] 8. The “Blank” value should be subtracted from all other values.

[0319] IN VITRO PARP-6 INHIBITON ASSAY:

[0320] Activities of compounds against PARP-6 were determined using the PARP-6 Chemiluminescent Assay Kit (Cat.# 80556) supplied by BPS Bioscience, USA.

[0321] PARP-6 Inhibitor Assay Design

[0322] All samples and controls were performed in duplicates. The assay included a “Blank, a “Positive control”, and control inhibitor compound Olaparib (CAS Number [763113-22-0]). Olaparib will inhibit the activity of PARP-6 at a wide range of concentrations from 700 nM to 10 μM. Serially dilute the stock Olaparib or PARP inhibitor(s) with 1X PARP Buffer and add to designated wells.

[0323] Step 1 : Coat histone solution using a 96-well transparent plate

[0324] 1 . Dilute 5x Histone Mixture 5-fold with PBS. This makes 1x HistoneMixture (50 μL / well).

[0325] 2. Add 50 μL of 1x Histone Mixture to each well.

[0326] 3. Incubate at 4°C overnight.

[0327] 4. Wash the plate three times using 200 μL of PBST Buffer per well.

[0328] 5. Tap the plate onto clean paper towel to remove the liquid.

[0329] 6. Block the wells by adding 200 μL of Blocking Buffer 3 to every well.

[0330] 7. Incubate at Room Temperature (RT) for at least 90 minutes.

[0331] 8. Wash the plate three times with 200 μL of PBST Buffer per well.

[0332] 9. Tap the plate onto clean paper towel to remove the liquid.

[0333] Step 2: Ribosylation reaction

[0334] 1. Dilute 0.5 M DTT 50-fold with distilled water. This makes a 10 mMDTT solution. 2. Prepare a Master Mix (25 μL / well): N wells x (2.5 μL of 10x PARP Assay Buffer + 10 μL of PARP Substrate Mixture 2 + 10 μL of distilled water + 2.5 μL of 10 mM DTT solution).

[0335] 3. Add 25 μL of Master Mix to every well.

[0336] 4. Prepare 1x PARP Assay Buffer by adding 1 volume of 10x PARPAssay Buffer and 1 volume of 10 mM DTT solution to 8 volumes of distilled water.

[0337] 5. Prepare the Test Inhibitor (5 μL / well): for a titration prepare serial dilutions at concentrations 10-fold higher than the desired final concentrations. The final volume of the reaction is 50 μL.

[0338] 6. Add 5 μL of Test Inhibitor to each well labeled as “Test Inhibitor”.

[0339] 7. Add 5 μL of Diluent Solution to the “Positive Control” and “Blank” wells.

[0340] 8. Thaw PARP-6 enzyme on ice. Briefly spin the tube containing the enzyme to recover its full content.

[0341] 9. Dilute PARP-6 enzyme to 12 ng / μL with 1x PARP Assay Buffer (20 μL / well).

[0342] 10. Initiate the reaction by adding 20 μL of diluted PARP-6 enzyme to the wells designated “Positive Control” and "Test Inhibitor."

[0343] 11 . Add 20 μL of 1x PARP Assay Buffer to the “Blank” wells.

[0344] 12. Incubate at RT for 1 hour.

[0345] Step 3: Detection

[0346] 1 . Dilute Streptavidin-HRP 50-fold in Blocking Buffer 3 (50 μL / well).

[0347] 2. Add 50 μL of diluted Streptavidin-HRP to every well.

[0348] 3. Incubate for 30 minutes at RT.

[0349] 4. Wash the plate three times with 200 μL of PBST Buffer per well and tap the plate onto clean paper towel.

[0350] 5. Just before use, mix 1 volume of ELISA ECL Substrate A and 1 volume of ELISA ECL Substrate B (100 μL of mix / well).

[0351] 6. Add 100 μL of mix to every well.

[0352] 7. Immediately read the plate in chemiluminescence.

[0353] 8. The “Blank” value should be subtracted from all other values.Table 8: PARP-1 , PARP-2, PARP-3 and PARP-6 activity screening summary forCompound 1 compared to known reference compounds

[0354] PARP microsomal stability study design

[0355] Solutions required

[0356] Mouse microsome vial 20 mg / mL

[0357] NADPH (Nicotinamide Adenine Dinucleotide Phosphate) 20 mM

[0358] Phosphate buffer pH 7.4 at a concentration of 100 mM

[0359] Time points: 5, 15, 30, 45 and 60 minutes X3Table 9: Microsomal stability work comparing 1 with Olaparib and AZD5305

[0360] Microsomal stability work comparing compound 1 with compound 19

[0361] Test compounds: 1 and 19

[0362] Test compound concentration: 2 μM

[0363] Microsome species: Mouse (CD-1) Microsomes

[0364] Supplier / lot# Gibco™ / MSMCPL / MS059

[0365] Temperature: 37 °C

[0366] Time points: 5, 15, 30, 45, 60 minutes

[0367] Replicates: 3

[0368] Aim

[0369] To determine the metabolic stability of test compound over time in the presence of liver microsomes activated by NADPH.

[0370] Test System Preparation

[0371] Assay to be run in a 96 well plate. To each well, the following was added:

[0372] The assay was initiated by addition of 10 μL of 20 mM NADPH solution. At the specified time points, 100 μL acetonitrile was added to the appropriate wells to stop metabolic activity and the plate returned to the incubator. After the final time point, the contents of the wells will be mixed by repeated pipetting and a 150 μL sample placed into an Eppendorf tube for analysis.

[0373] Sample preparation and analysis

[0374] Microsomal sample preparation• 200 μL of acetonitrile was added to 150 μL of sample• Sample was vortexed for 1 minute• Centrifuged at 19000 RCF (Relative Centrifugal Force) for 20 mins• Supernatant (5 μL) injected to LC / MS for analysis.• Standard sample concentration 2000 nM

[0375] Results

[0376] Calculations

[0377] Half-life (T1 / 2) was calculated by dividing 0.693 by k; where k = slope of the In concentration vs time plot (Figures 1 and 2).

[0378] V = incubation volume (μL) / protein (mg)

[0379] Intrinsic clearance (CLint) = (μL / min / mg protein) = V * In (2) / T 1 / 2

[0380] T1 / 2for 1 : 37.1 minutes

[0381] T1 / 2for 19: 37.5 minutes

[0382] Microsomal stability work comparing compound 1 with compound 20

[0383] Test compounds: 1 and 20

[0384] Test compound concentration: 2 μM

[0385] Microsome species: Mouse (CD-1) Microsomes

[0386] Supplier / lot# Gibco™ / MSMCPL / MS059

[0387] Temperature: 37 °C

[0388] Time points: 5, 15, 30, 45 minutes

[0389] Replicates: 1

[0390] Aim

[0391] To determine the metabolic stability of test compound over time in the presence of liver microsomes activated by NADPH.

[0392] Test System Preparation

[0393] Assay to be run in a 96 well plate. To each well, the following was added:

[0394] The assay was initiated by addition of 10 μL of 20 mM NADPH solution. At the specified time points, 100 μL acetonitrile was added to the appropriate wells to stop metabolic activity and the plate returned to the incubator. After the final time point, the contents of the wells will be mixed by repeated pipetting and a 150 μL sample placed into an Eppendorf tube for analysis.

[0395] Sample preparation and analysis

[0396] Microsomal sample preparation:

[0397] • 200 μL of acetonitrile was added to 150 μL of sample

[0398] • Sample was vortexed for 1 minute

[0399] • Centrifuged at 19000 RCF for 20 mins

[0400] • Supernatant (5 μL) injected to LC / MS for analysis.

[0401] • Standard sample concentration 2000 nM

[0402] Results (Figures 3 and 4)

[0403] Calculations

[0404] Half-life (T1 / 2) was calculated by dividing 0.693 by k; where k = slope of the In concentration vs time plot.

[0405] V = incubation volume (μL) / protein (mg)

[0406] Intrinsic clearance (CLint) = (μL / min / mg protein) = V * In (2) / T 1 / 2

[0407] T 1 / 2 for compound 1 : 29.7 minutes

[0408] T1 / 2for compound 20: 29.4 minutes

[0409] Microsomal stability work comparing compound 1 with compound 21

[0410] Test compounds: 1 and 21

[0411] Test compound concentration: 2 μM

[0412] Microsome species: Mouse (CD-1) Microsomes

[0413] Supplier / lot# Gibco™ / MSMCPL / MS059

[0414] Temperature: 37 °C

[0415] Time points: 5, 15, 30, 45 minutes

[0416] Replicates: 1

[0417] Aim

[0418] To determine the metabolic stability of test compound over time in the presence of liver microsomes activated by NADPH.

[0419] Test System Preparation

[0420] Assay to be run in a 96 well plate. To each well, the following was added:

[0421] The assay was initiated by addition of 10 μL of 20 mM NADPH solution. At the specified time points, 100 μL acetonitrile was added to the appropriate wells to stop metabolic activity and the plate returned to the incubator. After the final time point, the contents of the wells will be mixed by repeated pipetting and a 150 μL sample placed into an Eppendorf tube for analysis.

[0422] Sample preparation and analysis

[0423] Microsomal sample preparation:

[0424] • 200 μL of acetonitrile was added to 150 μL of sample

[0425] • Sample was vortexed for 1 minute

[0426] • Centrifuged at 19000 RCF for 20 mins

[0427] • Supernatant (5 μL) injected to LC / MS for analysis.

[0428] • Standard sample concentration 2000 nM

[0429] Results (Figures 5 and 6)45 528 493

[0430] Calculations

[0431] Half-life (T1 / 2) was calculated by dividing 0.693 by k; where k = slope of the In concentration vs time plot.

[0432] V = incubation volume (μL) / protein (mg)

[0433] Intrinsic clearance (CLint) = (μL / min / mg protein) = V * In (2) / T1 / 2

[0434] T1 / 2for compound 1 : 22.3 minutes

[0435] T1 / 2for compound 21 : 21.5 minutes.

[0436] All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0437] Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims

1. WE CLAIM:1 . A compound of formula (X)(X) a pharmaceutically acceptable salt, a pro-drug or an isotopic form thereof, wherein when R1is OH, R2and R3each is H, and A is C; when R2is OH, R1and R3each is H, and A is N; when R3is OH, R1and R2each is H, and A is N; and when R1, R2and R3each is H, A is C.

2. The compound of formula (X) of claim 1 , the pharmaceutically acceptable salt, the pro-drug or the isotopic form thereof, wherein R1is OH, R2and R3each isH, and A is C.

3. The compound of formula (X) of claim 1 , the pharmaceutically acceptable salt, the pro-drug or the isotopic form thereof, wherein R2is OH, R1and R3each isH, and A is N4. The compound of formula (X) of claim 1 , the pharmaceutically acceptable salt, the pro-drug or the isotopic form thereof, wherein R3is OH, R1and R2each isH, and A is N.

5. The compound of formula (X) of claim 1 , the pharmaceutically acceptable salt, the pro-drug or the isotopic form thereof, wherein when R1, R2and R3each isH, A is C.

6. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.

7. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.

8. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.

9. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.

10. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.11 . The compound of formula (X) of claim 1 wherein the com ound isits pharmaceutically acceptable salt or pro-drug thereof.

12. The compound of formula (X) of claim 1 wherein the com ound isits pharmaceutically acceptable salt or pro-drug thereof.

13. The compound of formula (X) of claim 1 wherein the compound isits pharmaceutically acceptable salt or pro-drug thereof.

14. The compound of formula X of claim 1 wherein the com ound isits pharmaceutically acceptable salt or pro-drug thereof.

15. A pharmaceutical composition comprising a therapeutically effective amount of the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, and at least one pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15 for use in anticancer therapy.

17. A product comprising the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, and one or more chemotherapeutic agents.

18. The product of claim 17, wherein the compound, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, and the one or morechemotherapeutic agents are for simultaneous, separate or sequential administration.

19. The product of claim 17 or 18, where the product is for anticancer therapy.

20. A method for preventing or treating a disease mediated by PARP-1 protein, the method comprising the step of administering to a mammal in need thereof• an effective amount of the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof,• the pharmaceutical composition of claim 15, or• the product of claim 17 or 18.21 . The method of claim 20, wherein the mammal is human.

22. Use of• an effective amount of the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof,• the pharmaceutical composition of claim 15, or• the product of claim 17 or 18, for prevention or treatment of a disease mediated by PARP-1 protein.

23. Use of an effective amount of the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotopic form thereof, in the preparation of a medicament, for prevention or treatment of a disease mediated by PARP-1 protein.

24. The method of claim 20, or use of claim 22 or 23, wherein the disease is cancer.

25. An in vitro method for selectively inhibiting PARP-1 protein activity, the method comprising the step of contacting a PARP-1 protein with an effective amount of the compound as defined in any one of claims 1 to 14, its pharmaceutically acceptable salt, pro-drug or isotope thereof.

26. The compound of formula 127a pharmaceutically acceptable salt, or a pro-drug thereof.

27. A process for preparation of the compound of formula 127, a pharmaceutically acceptable salt, or a pro-drug thereof, the process comprising the step of: carboxylating the compound of formula 124 to form the compound of formula 125, wherein PGNis an amine protecting group;124 125 deprotecting the amine to form the compound of formula 126; and125 126 converting the carboxylic acid of the compound of formula 126 to the amide to form the compound of formula 127126 12728. The process of claim 27, further comprising the steps of: deuteration of 2-aminophenol to form the compound of formula 122122 protection of the amino functional group in the compound of formula 122 to form the compound of formula 123122 123 wherein PGNis amino protecting group; methylation of the hydroxy functional group in the compound of formula 123 to form the compound of formula 124123 124

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