WO2014005182A1

WO2014005182A1 - Phosphoinositide 3-kinase (pi3k) inhibitors

Info

Publication number: WO2014005182A1
Application number: PCT/AU2013/000728
Authority: WO
Inventors: Philip Evan Thompson; Jo-Anne PINSON; Ian George JENNINGS; Zhaohua ZHENG
Original assignee: Monash University
Priority date: 2012-07-04
Filing date: 2013-07-04
Publication date: 2014-01-09

Abstract

The invention relates to compounds, and uses of the compounds, that are inhibitors of the enzyme phosphoinositide 3-kinase (PI3K). More particularly the compounds are selective inhibitors of one or more isoforms of PI3K. In particular embodiments the compounds are selective inhibitors of one isoform of PI3K.

Description

PHOSPHOINOSITIDE 3-KINASE (PI3K) INHIBITORS FIELD OF THE INVENTION

BACKGROUND

The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases.

Four distinct PI3K subfamilies exist - commonly referred to as class I, II, III and IV - based upon their substrate specificities, primary structures, modes of regulation and domain content. Of these, it is the class I isoforms, pi 10a, ρΐ ΐθβ, ρΐ ΐ θγ and ρΐ ΐθδ, together with the class IV PI3K-related kinase (PIKK), mTOR, which have been the most intensively examined targets in the small molecule therapeutic arena.

The class I PI3K/AKT pathway is dysregulated in a tissue-diverse range of tumours. The PI3K signaling pathway is frequently altered in human cancers as a result of (i) activating mutations of one PI3K and/or (ii) alterations upstream (e.g. RTKs) or downstream of PI3Ks (e.g. Akt and PTEN), which promote cell proliferation, resistance to apoptosis, anabolism, invasiveness and.angiogenesis. The gene encoding for pi 10a (PIK3CA) is one of the most frequently mutated oncogenes in human tumors. The phosphatase PTEN, the negative regulator of PI3K, is one of the most commonly mutated proteins in human malignancy. More particularly, the gene encoding for the pi 10a subunit, PIK3CA, is amplified, overexpressed and frequently mutated in many cancers. On the other hand, to date, no genetic alterations have been found in the genes encoding for ρΐ ΐθβ, γ and δ, however increased expression of ρΐ ΐθβ and ρΐ ΐθδ occurs in glioblastomas, colon and bladder tumors. Indeed, overexpression of wild-type pi 10 β, γ and δ is sufficient to induce an oncogenic phenotype in cultured cells, ρΐ ΐ θγ has also been found overexpressed in pancreatic cancer, where it is required for cell proliferation, as shown by reduced cell growth in the lack of pi 10γ lipid kinase activity.

A greater understanding of the specific roles of the pi 10a and ρΐ ΐθβ isoforms in tumourigenesis has recently been established: it has been shown that pi 10a is critical for the growth of tumours driven by PIK3CA mutations as well as oncogenic receptor tyrosine kinases and RAS, whilst ρΐ ΐθβ is the principal isoform involved in mediating PTEN- deficient tumourigenesis. pi 10β has been pursued as a target for antithrombotic therapy, and there is also growing evidence that pi 10β inhibitors could have significant therapeutic potential in autoimmune diseases, ρΐ ΐθγ has been reported to play an important role in mast cell, eosinophil and neutrophil function. In addition, pi 105 has also emerged as a key therapeutic target for haematological malignancies, notably acute myeloid leukaemia (AML), and there is also some evidence that this isoform is upregulated in melanoma and breast cancer, and is overexpressed in neuroblastoma. The ρΐ ΐ θδ subtype has also been shown to play a central function in the recruitment and activation of a range of immune and inflammatory cells.

The ability to selectively inhibit one isoform, or even two isoforms, without significant inhibition of the other isoforms represents a challenge to the development of effective therapeutics, but would provide significant therapeutic advantage. Isoform-selective or isoform-specific inhibitors have the potential to retain their therapeutic effect while limiting adverse effects and thus increasing tolerability. There is emerging potential for selective ρΐ ΐ θβ and dual selective ρ1 10β/δ inhibitors for the treatment of immune- inflammatory diseases and cancer and also of selective pi 10γ inhibitors in the treatment of cancer.

Consequently, selective inhibition of PI3K isoforms pi 10a, ρΐ ΐ θβ, ρΐ ΐθγ and/or ρΐ ΐθδ represents an important strategy for the development of novel therapeutics, including cancer therapeutics and therapeutics for cardiovascular diseases such as arterial thrombosis and also inflammatory diseases such as arthritis. SUMMARY OF THE INVENTION

It has now been discovered that a particular class of compounds is not only able to inhibit one or more isoforms of PI3K but is able to selectively inhibit one or more isoforms of PI3K, without significant inhibition of one or more other isoforms of PI3K.

Accordingly in one aspect the present invention provides a compound of formula:

wherein:

R¹ is selected from optionally substituted C6-i₄aryl, optionally substituted Ci.gheteroaryl, optionally substituted -(C]-6alkylene)-C_6-i₄aryl, optionally substituted -(C|.6alkylene)-C|.₉heteroaryl, optionally substituted -(C_2-6alkyenylene)-C6-i₄aryl and optionally substituted -(C_2-6alkyenylene)-Ci.9heteroaryl;

q is 0, 1 , 2, 3 or 4;

where present, each R²- is independently selected from optionally substituted Cugalkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^U) wherein R^{1 1} is hydrogen or optionally substituted Ci_-6alkyl; and R⁹ is optionally substituted Chalky! or where present two R² groups together form a methylene, 1 ,2-ethylene or 1 ,3- propylene group;

r is 0, 1, 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted Cj. ₈alkyl, optionally substituted C_2-8alkenyl, optionally substituted C_2-8alkynyl, optionally substituted Ci_-6alkoxy, optionally substituted Ci^alkylthio, optionally substituted C|. 6alkylamino, optionally substituted di-[Ci.6alkyl]amino, optionally substituted C|. 6alkoxycarbonyl, optionally substituted N-Ci.₆alkylcarbamoyl, optionally substituted N,N- di-[Ci.6alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C₂. ₆alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1,2-ethylene or 1 ,3 -propylene group;

m, n and p are each independently selected from 0 or 1 with the proviso that at least one of m, n or p is 1 ;

R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted Ci_ ₆alkyl, optionally substituted Q^alkenyl, optionally substituted Ce-uaryl, optionally substituted Ci-₉heterocycloalkyl, optionally substituted Ci-₉heteroaryl, optionally substituted Ci.ncycloalkyl, optionally substituted Ci^alkyloxy, optionally substituted C_6- naryloxy, optionally substituted

optionally substituted Ci-₉heteroaryloxy, optionally substituted Cj.gheterocycloalkyloxy, optionally substituted Ci-ncycloalkyloxy, amino, optionally substituted Ci-ealkylamino, optionally substituted Ci-₆alkenylamino, optionally substituted Ce-uarylamino, optionally substituted C|.₉heteroary]amino, optionally substituted Ci-ncycloalkylamino and optionally substituted Cj. ₉heterocycloalkylamino;

or any two of R³, R⁴ and R^4a together with the carbon atoms to which they are attached form a 3 to 7 membered optionally substituted ring that is aromatic or non-aromatic and may contain one or more heteroatoms,

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R ^a forms 1 ,2-ethylene or 1 ,3-propylene;

wherein, where present, the or each optional substituent is independently selected from: optionally substituted C|.6alkyl; optionally substituted C_6-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(C_)-6alkyl); -NHCOO(Ci_-6alkyl); -NH-COR³ wherein R^a is H or Ci_-6alkyl; -NR^aR^b wherein R^a is H or C_1-6alkyl and R^b is H or Ci^alkyl; -C(0)NR^aR^b, wherein R^a is H or Ci-₆alkyl and R^b is H, C,_-6alkyl; -C(0)R^a wherein R^a is H or C^alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(C|_-6alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(Ci.₆alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC( H)NH-, or absent, and

Q is selected from: optionally substituted Ce-ioaryl; optionally substituted 5-10 membered

optionally substituted 3-10 membered Ci_ 9heterocyclyl; optionally substituted C₃.iocycloalkyl; optionally substituted C|_-6alkyl; optionally substituted C_2-6alkenyl; optionally substituted C₂. 6alkynyl; and hydrogen;

with the proviso that when the moiety:

is ^N 2 then R¹ is selected from:

wherein: R is selected from hydrogen, halo and hydroxy and R is selected from wherein z is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

In another aspect the present invention provides a compound of formula:

wherein:

Xi, X₂ and X₃ are each independently selected from C(Ci-8alkyl), CH and N with the proviso that at least one of Xi, X₂ and X₃ is N;

R¹ is selected from optionally substituted C6-i₄aryl, optionally substituted Ci-₉heteroaryl, optionally substituted -(Ci.6alkylene)-C6.i₄aryl, optionally substituted -(C[.6alkylene)-C₁.9heteroaryl, optionally substituted -(C_2-6alkyenylene)-C₆-i₄aryl and optionally substituted -(C_2-6alkyenylene)-Ci.₉heteroaryl;

q is 0, 1 , 2, 3 or 4;

where present, each R² is independently selected from optionally substituted Ci_-galkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(Rⁿ ) wherein R^{1 1} is hydrogen or optionally substituted Ci.₆alkyl; and R⁹ is optionally substituted Ci-6alkyl or where present two R² groups together form a methylene, 1 ,2-ethylene or 1 ,3- propylene group;

r is 0, 1 , 2, 3 or 4; where present, each R is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted Q. galkyl, optionally substituted C2.₈alkenyl, optionally substituted C2.salkynyl, optionally substituted Ci-ealkoxy, optionally substituted Ci-6alkylthio, optionally substituted Ci. 6alkylamino, optionally substituted di-[Ci-6alkyl]amino, optionally substituted C|. 6alkoxycarbonyl, optionally substituted N-Ci.6alkylcarbamoyl, optionally substituted N,N- di-[Ci.6alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C₂. ₆alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1,2-ethylene or 1,3 -propylene group;

R³, R⁴ and R ^a are each independently selected from: hydrogen, phenylmethyl, 4- hydroxyphenylmethyl, 1-methylpropyl, 2-methylpropyl, 2-methylthioeth-l-yl, 4-aminobut- 1-yl, 2-carboxamidoeth-l-yl, 2-carboxyeth-l-yl;

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R^4a forms 1,2-ethylene or 1,3-propylene;

with the proviso that when the moiety:

do not represent CH;

with the proviso that when the moiety:

then X² and X³ do not represent CH;

with the proviso that when the moiety:

IS X NH₂ then R¹ is selected from:

wherein: R⁷ is selected from hydrogen, halo and hydroxy and R⁸ is selected from CH_ZF₃.₇. wherein z is 0, 1 or 2;

wherein, where present, the or each optional substituent is independently selected from: optionally substituted Ci^alkyl; optionally substituted Ce-io ryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COOCCi-ealkyl); -NHCOO(C_I-6alkyl); -NH-COR³ wherein R^a is H or C,.₆alkyl; -NR^aR wherein R^a is H or C,_-6alkyl and R^b is H or C,.₆alkyl; -C(0)NR^aR^b, wherein R^a is H or Ci_-6alkyl and R^b is H, Ci_-6alkyl; -C(0)R^a wherein R^a is H or C,_-6alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(C,.₆alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C,.₆alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C6-ioaryl; optionally substituted 5-10 membered Ci^heteroaryl; optionally substituted 3-10 membered Ci. 9heterocyclyl; optionally substituted C3-iocycloalkyl; optionally substituted C|.₆alkyl; optionally substituted C_2-6alkenyl; optionally substituted C₂. 6alkynyl; and hydrogen;

and pharmaceutically acceptable salts thereof. compound of formula:

wherein:

Xi is selected from C(C]-8alkyl), CH and N,

1 is selected from:

wherein: R⁷ is selected from hydrogen, halo, NH₂, NHR³⁰, NR³⁰R³¹ and hydroxy, wherein R³⁰ and R³¹ are each independently selected from Ci_-6alkyl and R is selected from CH_ZF3_-Z wherein z is 0, 1 or 2, hydroxy Ci_-6alkyl, NHR wherein R¹² represents hydrogen, or Ci_-6alkoxy;

q is 0, 1 , 2, 3 or 4;

where present, each R² is independently selected from optionally substituted Ci_-8alkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R") wherein R^{1 1} is hydrogen or optionally substituted

and R⁹ is optionally substituted Ci-salkyl or where present two R groups together form a methylene, 1 ,2-ethylene or 1,3- propylene group;

r is 0, 1 , 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted C|. ₈alkyl, optionally substituted C_2-8alkenyl, optionally substituted C_2-8alkynyl, optionally substituted

optionally substituted C|. ealkylamino, optionally substituted di-[Ci-6alkyl]amino, optionally substituted C_\. ealkoxycarbonyl, optionally substituted N-Ci_-6alkylcarbamoyl, optionally substituted N,N- di-[Ci-₆alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C₂. ₆alkanoyloxy, optionally substituted C₂.₆alkanoylamino, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1,3-propylene group;

R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted C_\. ₆alkyl, optionally substituted Ci^alkenyl, optionally substituted C6-i₄aryl, optionally substituted

optionally substituted Ci- heteroaryl, optionally substituted Ci.ncycloalkyl, optionally substituted Ci.6alkyloxy, optionally substituted C_6- naryloxy, optionally substituted Ci-ealkenyloxy, optionally substituted Ci.gheteroaryloxy, optionally substituted

optionally substituted C|.i₄cycloalkyloxy, amino, optionally substituted Ci.₆alkylamino, optionally substituted Ci-ealkenylamino, optionally substituted C6-i₄arylamino, optionally substituted Ci-9heteroarylamino, optionally substituted Ci.ucycloalkylamino and optionally substituted C;. gheterocycloalkylamino;

R^J and R° are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R^4a forms 1 ,2-ethylene or 1,3 -propylene, wherein, where present, the or each optional substituent is independently selected from: optionally substituted Ci^alkyl; optionally substituted C6-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(Ci_-6alkyl); -NHCOO(Ci.₆alkyl); -NH-COR³ wherein R^a is H or C_{l -6}alkyl; -NR^aR^b wherein R^a is H or C_1-6alkyl and R^b is H or Ci_-6alkyl; -C(0)NR^aR^b, wherein R^a is H or Ci_-6alkyl and R^b is H, Ci_-6alkyl; -C(0)R^a wherein R^a is H or Cu₆alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(C,.₆alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C,.₆alkyl)s -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC( H)NH-, or absent, and

Q is selected from: optionally substituted C6-ioaryl; optionally substituted 5-10 membered Ci-₉heteroaryl; optionally substituted 3-10 membered C_\. 9heterocyclyl; optionally substituted C_3-iocycloalkyl; optionally substituted Ci-6alkyl; optionally substituted C₂.₆alkenyl; optionally substituted C₂. 6alkynyl; and hydrogen;

and pharmaceutically acceptable salts thereof. now been discovered that the moiety depicted below:

has a significant influence on the degree of inhibition of one or more isoforms of PI3 . by the compounds of the invention.

For example, in those embodiments bearing stereocentres at one or more of the positions marked in the following moiety with an asterix (*), it is believed that the¹ stereochemistry of the stereocentre(s) has an influence on the nature of the selectivity of the inhibition of one or more isoforms of PI3K:

Without wishing to be bound by theory, in some embodiments, particularly those compounds where any two of n, m and p = 0, it is believed that the selectivity of the compounds may be "switched" between selectively inhibiting the β isoform of PI3K and the δ isoform of PI3K over the other isoforms of PI3K through choice of the stereochemistry about either of these stereocentres. For example, where the above moiety has the stereochemistry and substituents as shown below (which may be derived from L- phenylalanine):

the compound displayed selective inhibition of the β over the δ isoform of 35 fold, (as shown in Example 2) and significantly greater selectivity for the β isoform over the a and γ isoforms.

On the other hand, where the above moiety has the same substituents but opposite stereochemistry as depicted below (as shown in Example 1 1, which may be derived from -phenylalanine):

the compound displayed the opposite selectivity, instead selectively inhibiting the δ isoform over the β isoform by 16 fold, and displayed significantly greater selectivity for the δ isoform over the a and γ isoforms.

Accordingly compounds of the invention may be used moreover not only as inhibitors of PI3K, but may also be used as selective inhibitors of one or more isoforms of P13K, without significant inhibition of one or more other isoforms of PI3K. In this latter respect, compounds of the invention may be used as dual isoform inhibitors (such as β/δ dual inihibors) or as isoform selective inhibitors (such as β selective inhibitors or δ selective inhibitors).

In another aspect the invention provides the use of the compounds of the invention as inhibitors of PI3K. For example the invention provides the use of the compounds of the invention in the manufacture of a medicament for the inhibition of PI3 . The invention also provides a method for inhibiting PI3K comprising administering a compound of the invention to a subject in need thereof. The invention further provides the compounds of the invention for use as inhibitors of PI3 .

The present invention advantageously provides compounds which may be used as selective inhibitors of one or more isoforms of PI3 . In this respect compounds of the following formula may be used as selective inhibitors of one or more isoforms of PI3 :

wherein:

Xi, X₂ and X₃ are each independently selected from C(Ci.₈alkyl), CH and N with the proviso that at least one of Xi, X₂ and X₃ is N;

R¹ is selected from optionally substituted Ce-nar l, optionally substituted Cj.gheteroaryl, optionally substituted -(C).₆alkylene)-C₆-i₄aryl, optionally substituted -(Ci-6alkylene)-Ci-₉heteroaryl, optionally substituted -(C₂.₆alkyenylene)-C₆.i₄aryl and optionally substituted -(C₂-₆alkyenylene)-C|.9heteroaryl;

q is O, 1 , 2, 3 or 4;

where present, each R² is independently selected from optionally substituted Ci.galkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^! ') wherein R^{1 1} is hydrogen or optionally substituted Ci_-6alkyl; and R⁹ is optionally substituted C alkyl or where present two R² groups together form a methylene, 1,2-ethylene or 1 ,3- propylene group;

r is 0, 1, 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted Ci. galkyl, optionally substituted C_2-8alkenyl, optionally substituted C_2-gaIkynyl, optionally substituted Ci_-6alkoxy, optionally substituted

optionally substituted Ci. 6alkylamino, optionally substituted di-[Ci_-6alkyl]amino, optionally substituted Ci- 6alkoxycarbonyl, optionally substituted N-C]_-6alkylcarbamoyl, optionally substituted N,N- di-[Ci-6alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C₂. ₆alkanoyloxy, optionally substituted C2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1^2-ethylene or 1,3 -propylene group;

R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted C|. ₆alkyl, optionally substituted Ci-ealkenyl, optionally substituted C6-i₄aryl, optionally substituted Ci.gheterocycloalkyl, optionally substituted Ci-ciheteroaryl, optionally substituted Ci-i₄cycloalkyl, optionally substituted

optionally substituted C_6- aryloxy, optionally substituted Ci^alkenyloxy, optionally substituted Ci.gheteroaryloxy, optionally substituted Ci_-9heterocycloalkyloxy, optionally substituted Ci-ucycloalkyloxy, amino, optionally substituted C].6alkylamino, optionally substituted Ci^alkenylamino, optionally substituted C6-i₄arylamino, optionally substituted Ci^heteroarylamino, optionally substituted Ci.ncycloalkylamino and optionally substituted C|. ₉heterocycloalkylamino;

or any two of R³, R⁴ and R^4a together with the carbon atoms to which they are attached forrn a 3 to 7 membered optionally substituted ring that is aromatic or non-aromatic and may contain one or more heteroatoms,

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R^4a forms 1 ,2-ethylene or 1,3 -propylene;

wherein, where present, the or each optional substituent is independently selected from: optionally substituted C|.₆alkyl; optionally substituted C_6-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(C,.₆alkyl); -NHCOO(C_1-6alkyl); -NH-COR³ wherein R^a is H or C,_-6alkyl; -NR^aR^b wherein R^a is H or Cealkyl and R^b is H or C,.₆alkyl; -C(0)NR^aR^b, wherein R^a is H or Ci.₆alkyl and R^b is H, Ci_-6alkyl; -C(0)R^a wherein R^a is H or Ci_-6alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(Ci_-6alkyl , -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C_l-6alkyi)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(0)-, -NHC(NH)NH-, or absent, and

Q is selected from:

membered

gheterocyclyl; optionally substituted C3-iocycloalkyl; optionally substituted

optionally substituted C₂. 6alkynyl; and hydrogen;

or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use as a selective inhibitor of one or more isoforms of PI3K.

In some embodiments the invention provides the use of these compounds in the manufacture of a medicament for the selective inhibition of one or more isoforms of PI3K. The invention also provides a method for selectively inhibiting one or more isoforms of PI3K comprising administering at least one of these compounds to a subject in need thereof. The invention provides these compounds for use as selective inhibitors of one or more isoforms of PI3 .

Inhibition of PI3K, or selective inhibition of one or more isoforms of PI3K, may be used in the prevention and/or treatment of a range of symptoms, diseases or conditions including cancer, inflammation and cardiovascular diseases. For example, inhibition of the a isoform of PI3K is considered advantageous in the treatment of cancers bearing activating mutations in the PI 3CA gene, frequently observed in breast and colon cancers. Inhibition of the β isoform of PI3 is considered adavantageous in the treatment of cancers bearing inactivating mutations in the PTEN gene, such as breast and prostate cancers. Inhibition of the γ and δ isoforms of PI3K, alone or in combination is considered advantageous in the treatment of immunoinflammatory diseases. Inhibition of the β isoform of PI3K is considered advantageous in the treatment of cardiovascular diseases such as arterial thrombosis but also inflammatory diseases such as arthritis. These examples are not exhaustive but exemplify the various isoforms and multiplicity of isoforms for which inhibition might deliver a therapeutic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a dose-response curve for compound 18 of the invention (Example 18) against α, β, γ and δ isoforms of PI3K

Figure 2 shows the results of an assay of Akt phosphorylation in MB-MDA-468 cells where the blots were detected by Odyssey infrared imaging system (LI-COR) and the bands were quantified by Image-J of cell lysates of MB-MDA-468 cells. ■

Fig. 3 shows ADP induced platelet (washed) aggregation. (A). Dose-dependent effects of Compound 2 (10 nM, 100 nM & 1 μΜ) on washed platelet aggregation. Representative of three different donor responses.

Figure 4 shows stationary adhesion of platelets to vWF-coated microcapillary tubes. Adhesion is inhibited by compound 2 and TGX221.

Figure 5 shows that compound 2 prevents thrombus formation and arterial occlusion in an electrolytic model of thrombosis in vivo in mice.

DETAILED DESCRIPTION OF THE INVENTION

The language "PI3K-modulating compound", "modulator of PI3K" or "PI3K inhibitor" refers to compounds that modulate, e.g., inhibit, or otherwise alter, the activity of PI3K. Examples of PI3 -modulating compounds include compounds of the invention and pharmaceutically acceptable salts thereof, as well as stereoisomers thereof. The term "selectively" or "selective" in the context of inhibition of one or more isoforms of PI3K refers to inhibition of the activity of the one or more isoforms of PI3 without significant inhibition of one or more other isoforms of PI3K. In some embodiments the compounds of the invention selectively inhibit one or two isoforms of PI3K without significant inhibition of one or more other isoforms of PI3K. In still further embodiments the compounds of the invention selectively inhibit the β and/or δ isoforms of PI3K without significant inhibition of the a and/or γ isoforms of PI3K. In particular embodiments the compounds of the invention selectively inhibit the β and/or δ isoforms of PI3K without significant inhibition of the a isoform of PI3 . In still more particular embodiments the compounds of the invention selectively inhibit the β isoform of PI3K without significant inhibition of the α, γ and/or δ isoforms of PI3K, or selectively inhibit the δ isoform of PI3K without significant inhibition of the α, β and/or γ isoforms of PI3K.

The skilled person will recognise that there are a number of ways of determining the degree of inhibition of a particular isoform of PI3 . One such measure is determining the IC50 value, such as using the assay detailed herein. In some embodiments the difference in inhibition between the inhibited isoform(s) and the inhibition of the other isoform(s) may be at least 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold or more. In some embodiments the difference in inhibition between the inhibited isoform(s) and the inhibition of the other isoform(s) may be approximately one, two, three, or more order(s) of magnitude.

In some embodiments R¹ is selected from optionally substituted C6-i4aryl, optionally substituted

optionally substituted -(Ci-6alkylene)-C₆.|4aryl, optionally substituted -(C).6alkylene)-C|.9heteroaryl, optionally substituted -(C_2-6alkyenylene)-C6- i₄aryl and optionally substituted -(C_2-6alkyenylene)-Ci.₉neteroaryl.

In other embodiments R¹ is selected from optionally substituted C6-i₄aryl and optionally substituted Ci.9heteroaryl.

In further embodiments R¹ is selected from optionally substituted phenyl and optionally substituted Cvheteroaryl, such as benzimidazolyl. n still further embodiments R¹ is selected from:

wherein: R⁷ is selected from hydrogen, halo, NH₂, NHR³⁰, NR³⁰R³¹, hydroxy and hydroxymethyl, wherein R³⁰ and R³¹ are each independently selected from Ci^alkyl and R⁸ is selected from CH_ZF3_-Z wherein z is 0, 1 or 2, hydroxy

NHR¹² wherein R¹² represents hydrogen, Ci-6alkyl or Ci_-6alkoxy. In yet further embodiments R⁷ is selected from hydrogen, halo or hydroxy, and R⁸ is selected from CH₂F3._Z wherein z is 0, I or 2,

1 may be:

In some embodiments q is 0, 1, 2, 3 or 4. In other embodiments q is 0 or 1. In further embodiments q is 0.

In some embodiments, where present, each R² is independently selected from optionally substituted C|_-galkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^U) wherein R¹¹ is hydrogen or optionally substituted C|.6alkyl; and R⁹ is optionally substituted Ci^alkyl or where present two R² groups together form a methylene, 1,2-ethylene or 1,3-propylene group.

In other embodiments, where present, each R² is independently selected from optionally substituted Ci_-galkyl, or independently selected from optionally substituted C^alkyl.

In some embodiments r is 0, 1, 2, 3 or 4. In other embodiments r is 0 or 1. In further embodiments r is 0.

In some embodiments, where present, each R is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted Ci.₈alkyl, optionally substituted C_2-8alkenyl, optionally substituted C_2-8alkynyl, optionally substituted Ci.₆alkoxy, optionally substituted C|.₆alkylthio, optionally substituted

optionally substituted d-ealkoxycarbonyl, optionally substituted N-Ci-6alkylcarbamoyl, optionally substituted

optionally substituted C_2-6alkanoyl, optionally substituted C_2-6alkanoyloxy, optionally substituted C₂^alkanoylamino, or two R¹⁰ groups together form a methylene, 1,2-ethylene or 1,3-propylene group.

In other embodiments, where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, hydroxy, amino, carboxy, carbamoyl, optionally substituted C|.₈alkyl, optionally substituted C].6alkoxy, optionally substituted C|.₆alkylamino, optionally substituted di-[Ci_-6alkyl]amino, optionally substituted

optionally substituted N-Ci.₆alkylcarbamoyl, optionally substituted N_jN-di-fCi-ealkyljcarbamoyl, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1 ,3-propylene group.

In some embodiments R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted Ci_-6alkyl, optionally substituted Ci^alkenyl, optionally substituted C6.|4aryl, optionally substituted Ci.gheterocycloalkyl, optionally substituted Ci- heteroaryl, optionally substituted Ci-i₄cycloalkyl, optionally substituted

optionally substituted C6-i₄aryloxy, optionally substituted Ci_-6alkenyloxy, optionally substituted C|. 9heteroaryloxy, optionally substituted Ci.gheterocycloalkyloxy, optionally substituted C|. ncycloalkyloxy, amino, optionally substituted Ci-₆alkylamino, optionally substituted C|. 6alkenylamino, optionally substituted Ce-narylamino, optionally substituted C|. gheteroarylamino, optionally substituted Ci-ncycloalkylamino and optionally substituted Ci.gheterocycloalkylamino, or any two of R³, R⁴ and R^4a together with the carbon atoms to which they are attached form a 3 to 7 membered optionally substituted ring that is aromatic or non-aromatic and may contain one or more heteroatoms, or R⁵ together with any one of R³, R⁴ or R ^a forms 1 ,2-ethylene or 1 ,3-propylene, with the proviso that when the moiety:

wherein: R⁷ is selected from hydrogen, halo and hydroxy and R⁸ is selected from CH_ZF3_-Z wherein z is 0, 1 or 2;.

In other embodiments R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted Ci-ealkyl, optionally substituted

optionally substituted Ce-naryl, optionally substituted C_t-gheterocycloalkyl, optionally substituted Ci^heteroaryl, optionally substituted Cuncycloalkyl, optionally substituted Ci-ealkyloxy, optionally substituted Ci.6alkylamino, or R⁵ together with any one of R³, R⁴ or R^4a forms 1,2-ethylene proviso that when the moiety:

^NH2 then R¹ is selected from:

wherein: R is selected from hydrogen, halo and hydroxy and R is selected from CH_ZF_3-Z wherein z is 0, 1 or 2.

In further embodiments R³, R⁴ and R ^a are each independently selected from: hydrogen, optionally substituted Ci_-6alkyl, optionally substituted Q-i-iaryl, optionally substituted Ci_ 9heteroaryl or R⁵ together with any one of R³, R⁴ or R ^a form s 1,2-ethylene or 1,3- propylene, with the proviso that when the moiety:

wherein: R⁷ is selected from hydrogen, halo and hydroxy and R⁸ is selected from CH_ZF3_-Z wherein z is 0, 1 or 2.

In still further embodiments R³, R⁴ and R ^a are each independently selected from: hydrogen, phenylmethyl, 4-hydroxyphenylmethyl, 1-methylpropyl, 2-methylpropyl, 2- methylthioeth-l-yl, 4-aminobut-l-yl, 2-carboxamidoeth-l-yl, 2-carboxyeth-l-yl, or R⁵ together with any one of R³, R⁴ or R^4a forms 1,2-ethylene or 1,3 -propylene, with the proviso that when the moiety:

IS then R¹ is selected from:

wherein: R⁷ is selected from hydrogen, halo and hydroxy and R⁸ is selected from CH_ZF_3-Z wherein z is 0, 1 or 2.

In some embodiments R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl or R⁵ together with either R³ or R⁴ forms 1,2-ethylene or 1,3-propylene. In other embodiments R⁵ and R⁶ are each independently selected from hydrogen and methyl or R³ together with either R³ or R⁴ forms 1 ,2-ethylene or 1,3-propylene. In further embodiments R⁵ is hydrogen or together with either R³ or R⁴ forms 1,2-ethylene or 1,3-propylene and R⁶ is hydrogen.

As used herein m, n and p are each independently selected from 0 and 1 with the proviso that at least one of m, n or p is 1. In some embodiments, one of m, n and p is 0. In yet further embodiments, two of m, n and p are 0.

In some embodiments X¹, X² and X³ are each independently selected from C(Ci_-8alkyl), CH and N with the proviso that at least one of X¹, X² and X³ is N. In other embodiments X¹ and X² are each independently selected from C(C|.₈alkyl), CH and N and X³ is N. In further embodiments X² and X³ are N, such as where each of X¹, X² and X³ is N.

As used herein, the term "optionally substituted" typically refers to where a hydrogen atom on a group has been substituted with a non-hydrogen group as detailed below. Any optionally substituted group may bear one, two, three or more optional substituents.

In some embodiments the optional substituents are selected from: optionally substituted C|. ₆alkyl; optionally substituted C₆-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(C^alkyl); -NHCOO(C,.₆alkyl); -NH-COR³ wherein R^a is H or C_1-6alkyl; -NR^aR^b wherein R^a is H or Ci_-6alkyl and R^b is H or C|.₆alkyl; -C(0)NR^aR^b, wherein R^a is H or Q. ₆alkyl and R^b is H, C_r-6alkyl; -C(0)R^a wherein R^a is H or Ci.₆alkyl; or -Y-Q wherein: Y is selected from: -0-, -NH-, -N(C,.₆alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C,.₆alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C₆-ioaryl; optionally substituted 5- 10 membered Ci^heteroaryl; optionally substituted 3-10 membered C_\. 9heterocyclyl; optionally substituted C_3-iocycloalkyl; optionally substituted C|_-6alkyl; optionally substituted C_2-6alkenyl; optionally substituted C_2- ₆alkynyl; and hydrogen.

In other embodiments the optional substituents are selected from: optionally substituted C]. ₆alkyl; optionally substituted C₆-ioaryl; halogen; -OH; -NH₂; -COOH; -COO(C|_-6alkyl); -NHCOO(C,.₆alkyl); -NR^aR^b wherein R^a is H or C^alkyl and R^b is H or C_1-6alkyl; -NH- COR^a wherein R^a is H or C,_-6alkyl; -C(0)NR^aR^b, wherein R^a is H or C_1-6alkyl, and R^b is H, Ci_-6alkyl; C(0)R^a wherein R^a is H or C^alkyl; or -Y-Q, wherein:

Y is selected from: -0-, -NH-, -N(C,_-6alkyl)-, -NHCONH-, -S-, -C(0)NH-_>

-C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C6-ioaryl; optionally substituted 5-10 membered Ci.₉heteroaryl; optionally substituted 3-10 membered C|. 9heterocyclyl; optionally substituted C_3-iocycloalkyl; optionally substituted Ci-6alkyl; optionally substituted C_2-6alkenyl; optionally substituted C_2- ₆alkynyl; and hydrogen.

In other embodiments the optional substituents are selected from: optionally substituted C|. ₆alkyl; optionally substituted C₆-ioaryl; halogen; -OH; -NH₂; -COOH; -COO(Ci_-6alkyl); -NR^aR^b wherein R^a is H or Cealkyl and R^b is H or C_{1 -6}alkyl; -NH-COR³ wherein R^a is H or Ci_-6alkyl; -C(0)NR^aR^b, wherein R^a is H or C,.₆alkyl, and R^b is H, C,.₆alkyl; C(0)R^a wherein R^a is H or Ci.₆alkyl; or -Y-Q, wherein:

Y is selected from: -0-, -NH-, -N(C,.₆alkyl)-, -NHCONH-, -S-, -C(0)NH-,

-C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: C_6-ioaryl optionally substituted with -OH; 5-10 membered C|. gheteroaryl; 3-10 membered C|_-9heterocyclyl; C_3-iocycloalkyl; Ci_-6alkyl; C_2- ₆alkenyl; C_2-6alkynyl; and hydrogen.

The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.). The expression "Cx-_yalkyl", wherein x is 1-5 and y is 2-10 indicates an alkyl group (straight- or branched- chain) containing the specified number of carbon atoms. For example, the expression Ci^alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

In one embodiment, a straight chain or branched chain alkyl has 10 or fewer carbon atoms (ie Ci-io). In some embodiments a straight chain or branched chain alkyl has 6 or fewer carbon atoms (ie C|_-6).

The term "cycloalkyl" includes saturated cyclic aliphatic groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl). The term C₃.₆cycloalkyl includes, but is not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. Likewise, preferred cycloalkyls have from 4- 7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. As used herein the term "heterocycloalkyl" refers to a cycloalkyl group containing one or more endocyclic heteroatoms.

The term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.) and branched-chain alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groups containing 2 to 6 carbon atoms. The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term "alkynyl" includes straight-chain alkynyl groups {e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.) and branched -chain alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, Ci-C₆ for branched chain). The term C₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

The term "amine" or "amino" should be understood as being broadly applied to both a molecule, or a moiety or functional group, as generally understood in the art, and may be primary, secondary, or tertiary. The term "amine" or "amino" includes compounds where a nitrogen atom is covalently bonded to at least one carbon, hydrogen or heteroatom.

The term "amides" "amido" or "aminocarbonyl" includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group.

The term "aryl" refers to aromatic monocyclic (eg phenyl) or polycyclic groups e.g., tricyclic, bicyclic, e.g., naphthalene, anthryl, phenanthryl. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin, methylenedioxyphenyl).

The term "heteroaryl", as used herein, represents a monocyclic or bicyclic ring, typically of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include but are not limited to: benzimidazole (otherwise known as benzoimadazole), acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indoiyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively.

The term "heterocycle" or "heterocyclyl" as used herein is intended to mean a 5- to 10- membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. "Heterocyclyl" therefore includes the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof. Further examples of "heterocyclyl" include, but are not limited to the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indoiyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoiine, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyt, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyI, pyrrolidinyl, mocpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. As referred to herein "heterocycloalkyl" refers to a saturated heterocyclyl group.

The term "acyl" includes compounds and moieties which contain the acyl radical (CH₃CO-) or a carbonyl group. The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups and may include cyclic groups such as cyclopentoxy.

The term "carbonyl" or "carboxy" includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom, and tautomeric forms thereof. Examples of moieties that contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc. The term "carboxy moiety" or "carbonyl moiety" refers to groups such as "alkylcarbonyl" groups wherein an alkyl group is covalently bound to a carbonyl group, "alkenyl carbonyl" groups wherein an alkenyl group is covalently bound to a carbonyl group, "alkynylcarbonyl" groups wherein an alkynyl group is covalently bound to a carbonyl group, "arylcarbonyl" groups wherein an aryl group is covalently attached to the carbonyl group. Furthermore, the term also refers to groups wherein one or more heteroatoms are covalently bonded to the carbonyl moiety. For example, the term includes moieties such as, for example, aminocarbonyl moieties, (wherein a nitrogen atom is bound to the carbon of the carbonyl group, e.g., an amide), aminocarbonyloxy moieties^', wherein an oxygen and a nitrogen atom are both bond to the carbon of the carbonyl group (e.g., also referred to as a "carbamate"). Furthermore, aminocarbonylamino groups (e.g., ureas) are also include as well as other combinations of carbonyl groups bound to heteroatoms (e.g., nitrogen, oxygen, sulfur, etc. as well as carbon atoms). Furthermore, the heteroatom can be further substituted with one or more alkyl, alkenyl, alkynyl, aryl, aralkyl, acyl, etc. moieties.

The term "thiocarbonyl" or "thiocarboxy" includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom. The term "thiocarbonyl moiety" includes moieties that are analogous to carbonyl moieties. For example, "thiocarbonyl" moieties include aminothiocarbonyl, wherein an amino group is bound to the carbon atom of the thiocarbonyl group, furthermore other thiocarbonyl moieties include, oxythiocarbonyls (oxygen bound to the carbon atom), aminothiocarbonylamino groups, etc. The term "ester" includes compounds and moieties that contain a carbon or a heteroatom bound to an oxygen atom that is bonded to the carbon of a carbonyl group. The term "ester" includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxy carbonyl, etc. The alkyl, alkenyl, or alkynyl groups are as defined above.

The term "hydroxy" or "hydroxyl" includes groups with an -OH. /

The term "halogen" includes fluorine, bromine, chlorine, iodine, etc. The term "perhalogenated" generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.

The terms "polycyclyl" or "polycyclic radical" include moieties with two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.

The term "heteroatom" includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. Particularly preferred heteroatoms are nitrogen and oxygen.

Additionally, the phrase "any combination thereof implies that any number of the listed functional groups and molecules may be combined to create a larger molecular architecture. For example, the terms "phenyl," "carbonyl" (or "=0"), "-0-," "-OH," and Cialkyl and C₃alkylene (i.e., -CH₃ and -CH₂CH₂CH₂-) can be combined to form a 3- methoxy-4-propoxybenzoic acid substituent. It is to be understood that when combining functional groups and molecules to create a larger molecular architecture, hydrogens can be removed or added, as required to satisfy the valence of each atom. It is to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, double bonds and/or hydrogen atoms are typically added to provide the following number of total bonds to each of the following types of atoms: carbon: four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur: two, four or six bonds.

It is also to be understood that definitions given to the variables of the generic formulae described herein will result in molecular structures that are in agreement with standard organic chemistry definitions and knowledge, e.g., valency rules.

It will be noted that the structures of some of the compounds of this invention include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates) are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Compounds described herein may be obtained through art recognized synthesis strategies. It will also be noted that the substituents of some of the compounds of this invention include isomeric cyclic structures. It is to be understood accordingly that constitutional isomers of particular substituents are included within the scope of this invention, unless indicated otherwise. For example, the term "tetrazole" includes tetrazole, 2H-tetrazole, 3H-tetrazole, 4H-tetrazole and 5H-tetrazole.

The skilled person will appreciate that there are a range of techniques available to produce the compounds of the invention in racemic, enantioenriched or enantiopure forms. For example, enantioenriched or enantiopure forms of the compounds may be produced through stereoselective synthesis and/or through the use of chromatographic or selective recrystallisation techniques. In some embodiments the compounds of the invention may be prepared by appending a natural or unnatural amino acid to the piperazinyl ring system. Accordingly, a racemic mixture of amino acids may be used to prepare a racemic mixture of a compound of the invention, an enantioenriched amino acid may be used to prepare an enantioenriched mixture of a compound of the invention and an enantiopure amino acid may be used to prepare an enantiopure compound of the invention.

Accordingly in one or more aspects the present invention provides compounds of any of the formulae:

wherein R , R , R , R , R , R R , X¹, X², X³, q, r, m and p have the meanings defined within with the proviso that R³ is not hydrogen;

wherein R , R , R , R , R , R , R , q, r, m and p have the meanings defined within with the proviso that R is not hydrogen;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R¹⁰, X¹, X², X³, q, r, m and p have the meanings defined

wherein R¹, R², R³, R⁴, R⁵, R⁶, R¹⁰, q, r, m and p have the meanings defined within with the proviso that R³ is not hydrogen;

wherein R , R , R% R , R , R^b, R , X¹, X^, X% q, r and p have the meanings defined within

wherein R¹, R², R³, R⁴, R⁵, R⁶, R , q, r and p have the meanings defined within with the proviso that R⁴ is not hydrogen;

r and p have the meanings defined within with the proviso that R⁴ is not hydrogen;

wherein R , R , R , R , R , R , R , q, r and p have the meanings defined within with the proviso that R is not hydrogen;

wherein R , R , R and m have the meanings defined within with the proviso that R is not hydrogen;

wherein R¹, R³, R⁴ and m have the meanings defined within with the proviso that R³ is not hydrogen;

wherein R¹, R³ and R⁴ have the meanings defined within with the proviso that R⁴ is not hydrogen;

N N

O R₄

wherein R and, R have the meanings defined within with the proviso that R is not hydrogen;

wherein R¹ and R³ have the meanings defined within with the proviso that R³ is not hydrogen.

The compounds and methods of the present invention may be used in the treatment and/or prevention of a range of symptoms, diseases or conditions susceptible to modulation (particularly inhibition) of PI3K activity, including thrombosis, cancer and/or inflammatory disease. As used herein, treatment may include alleviating or ameliorating the symptoms, diseases or conditions associated with PI3 activity, including reducing the severity and/or frequency of the diseases or conditions. As used herein, prevention may include preventing or delaying the onset of, inhibiting the progression of, or halting or reversing altogether the onset or progression of the particular symptoms, disease or condition associated with PI3K activity.

The term "subject" is intended to include organisms such as mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from a PI3K-associated disorder, disease or condition, such as thrombosis, cancer and/or inflammation. In another embodiment, the subject is a cell. The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents should preferably not interfere with the biological activity of the solute. Solvents may be, by way of example, water, acetone, ethanol or acetic acid. Methods of solvation are generally known within the art.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. For example, the terminal -NR⁵R⁶ amine group, the tertiary amine group of the morpholino group and/or the tertiary amine group of the piperazinyl group of the compounds of the invention may undergo reaction with an acid to form the acid addition salt.

Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases. Corresponding counterions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts. Organic bases include primary, secondary and tertiary amines, substituted amines including naturally- occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine. For example, where the compound of the invention possesses a carboxylic acid group (in R³ for example) the compound may undergo reaction with a base to form the base addition salt.

Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms.

The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

While the compound as hereinbefore described, or pharmaceutically acceptable salt thereof, may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention. For example, the compound could be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the compound as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition.

As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the compound care should be taken to ensure that the activity of the compound is not destroyed in the process and that the compound is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the compound by means known in the art, such as, for example, micro encapsulation or coating (such as the use of enteric coating). Similarly the route of administration chosen should be such that the compound reaches its site of action. Those skilled in the art may readily determine appropriate formulations for the compounds of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.

The compounds as hereinbefore described, or pharmaceutically acceptable salt thereof, may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.

The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the compound, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolarity, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active compound may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may^be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active compound to specific regions of the gut.

Liquid formulations may also be administered enterally via a stomach or oesophageal tube.

Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the compounds of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like.

The present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.

The compounds of the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The compounds may also be administered using a nebuliser.

Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

As mentioned above the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 μg to about 200 mg. Expressed in proportions, the active compound may be present in from about 0.25 μg to about 200 mg/mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The terms "therapeutically effective amount" and "effective amount" refer to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, preferably a mammal, more preferably a human in need of such treatment. The therapeutically effective amount or effective amount will vary depending on the subject and nature of symptom, disease or condition being treated, the severity of the symptom, disease or condition and the manner of administration, and may be determined routinely by one of ordinary skill in the art. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The invention will now be described with reference to some specific examples and drawings. However, it is to be understood that the particularity of the following description is not to supercede the generality of the invention as hereinbefore described.

EXAMPLES

Chemistry

All chemical reagents acquired from Sigma-Aldrich, Fluka, Merck, BDH laboratories, CSL, Ajax Finechem, Merck Schuhardt, ChemSupply, Auspep, Prolabo, Lancaster, TCI, Matrix Scientific, Boron Molecular, Alfa Aesar, Chem-Impex and May and Baker were used without further purification. 1H-NMR spectra were recorded with either a 300 MHz Varian widebore NMR spectrometer or a 400 MHz Bruker Ultrashield-Avance NMR spectrometer. ^I3C-NMR spectra were recorded with a 400 MHz Bruker Ultrashield-Avance NMR spectrometer.

Results were recorded as follows: chemical shift values are expressed units acquired in either CDC1₃ (7.26 ppm), (CD₃)₂SO (2.50 ppm) or CD₃OD (3.31 ppm) as references, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration and coupling constants (J) in Hertz. Mass spectra were acquired in the positive and negative mode using an atmospheric pressure (ESI/APCI) ion source on a Micromass Platform ESI/APCI single quadrupole mass spectrometer with sample management facilitated by an Agilent 1 100 series HPLC system using MassLynx version 3.5 software. High Resolution Mass Spectrometry analyses were collected on a Waters Micromass LCT Premier XE Orthogonal Acceleration Time-of-Flight Mass Spectrometer coupled to an Alliance 2795 Separation Module using MassLynx version 4.1 software. LCMS analysis was performed using an Agilent 6100 series Single Quad (Series 1200 HPLC, Column: Luna C8(2), 50 x 4.6 mm; solvent system: 5-100% acetonitrile in water with 0.1% Formic acid over 10 minutes; flow rate 0.5 ml/min; molecular weight range 100-1000; cone Voltage ; column temperature 30 °C). All masses were reported as the protonated parent ions. Analytical RJP- HPLC was obtained on a Waters Millenium 2690 system, with UV detection at 254 nM. 28Method used gradient elution through a Supelco C8 column (150 x 2.1 mm ID) 20- 100% Buffer B (Buffer A: H₂0, 0.1% TFA; Buffer B: 80% CH₃CN, 0.1% TFA, 19.9% H₂0 or Buffer B: 80% CH₃OH, 0.1% TFA, 19.9% H₂0) over 10 minutes at 1.0 ml/min. Preparative RP-HPLC was obtained on a Waters 600 HP LC system and Waters 486 tunable absorbance detector, with UV detection at 254 nM.. Gradient elution through a Phenomonex Luna C8 (2) 10 μ column (50 x 21.2 mm ID), 20-100% Buffer B (Buffer A: H₂0, Buffer B: 80% CH₃CN, 20% H₂0) over 30 minutes at 5 ml/min. Melting point determination was performed uncorrected using a Mettler Toledo MP50 melting point apparatus. Microwave chemistry was performed using a Biotage Initiator Microwave Reactor.

1. Precursors

N-Boc-piperazine A 1

To a stirred solution of piperazine (5.0 g, 58 mmol, 2 equiv) in DCM (145 ml) at 0 °C, was added dropwise a solution of di-tert-butyldicarbonate (6.3 g, 1 equiv) in DCM (58 ml) over 20 minutes, then stirred a further 1 hour at 0 °C. The reaction mixture was filtered and remaining filtrate was evaporated under reduced pressure, with remaining oil diluted with H₂0, refiltered, saturated with K₂C0₃, extracted with ether, evaporated under reduced pressure to yield the product Al as white crystals; yield: 3.5 g, 65.2%; IH-NMR (300 MHz, CD₃OD), 3.40 (t, J = 4.8 Hz, 4H), 2.77 (t, J = 5.4 Hz, 4H), 1.46 (s, 9H); ESI-MS, m/z 187.3 [M+H]+.

2-Difluoromethylbenzimidazole A2

A mixture of 1 ,2-phenylenediamine (0.50 g, 4.6 mmol, 1 equiv) and difluoroacetic acid (2.2 g, 5 equiv) in H₂0 (10 ml) in a sealed tube was exposed to MW irradiation (90 W, 130 °C) for 10-15 minutes. The reaction mixture was diluted with H₂0 (50 ml), neutralized with 50% NaOH with resulting precipitate filtered, washed with H₂0, then dried to yield the product as orange powder. Remaining product was extracted into EtOAc, evaporated under reduced pressure and dried to yield the product (A2) as brown crystals; yield: 0.70 g, 90.3%; 1H-NMR (300 MHz, CD₃OD), 7.65 (s, 2H), 7.35 (dd, J = 6.1, 3.1 Hz, 2H), 7.02 (t, J = 53.1 Hz, 1H); ESI-MS, m/z 169.2 [M+H]+.

N-Boc-L-phenylalanyl-piperazine Bl

Boc-Phenylalanine (264mg, 1.0 mmol) dissolved in DMF was treated with HCTU (414 mg, 1 mmol) and diisopropylethylamine (200 μί,) followed by piperazine (860 mg, 10 mmol) and the mixture was allowed to stir at room temperature overnight. The mixture was then poured into ice water (100 ml) and extracted with ethyl acetate (3 x 30 ml). The combined organic extracts were dried, and the solvent removed to yield a colourless oil (240 mg), m/z 334.2 [M + H]⁺.

2. Key intermediates

Intermediate 1: Synthesis of 4-(4-(2-(Difluoromethyl)-lH-benzoidHmidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)morpholine (A3)

A6 A3 -(4,6-dichloro-l,3,5-triazin-2-yl)morpholine - (A4)

o a stirred solution of cyanuric chloride (10.0 g, 54 mmol, 1.4 equiv) in acetone (100 ml) was added dropwise a solution of morpholine (3.4 g, 1 equiv) and triethylamine (3.9 g, 1 equiv) in acetone (100 ml) at -20 °C, then quenched with H₂0, stirred for a few minutes, filtered, washed with MeOH and dried to yield the product (A4) as white powder, yield:

8.4 g, 93.0%; IH-NMR (300 MHz, CD₃OD), 3.87 (t, J = 4.8 Hz, 4H, OCH₂), 3.73 (t, J = 4.8 Hz, 4H, NCH₂); LCMS (ESI): (m/z) = 236.1 [M+H]+; HPLC (MeCN): 5.99 min.

4-(4-Chloro-6-(2-(difluoromethyl)-lH-benzo[d/imidazol-l-yl)-l,3,S-trw

yl)morpholine- (AS)

A mixture of 4-(4,6^ϊοη1θΓθ-1,3,5^π3ζίη-2^1^ο ηο1ίηε (A4) (0.74 g, 3.2 mmol, 1 equiv), 2-difluoromethylbenzimidazole (A2) (0.53 g, 1 equiv) and K₂C0₃ (0.44 g, 1 equiv) in DMF (10 ml) was stirred at room temperature for 4 hours. The reaction mixture was poured onto H₂0, filtered, washed with H₂0 and small amount of MeOH, then dried to yield the product AS as off-white powder, yield: 0.49 g, 42.6%; IH-NMR (300 MHz, CDC1₃), 8.44 (dd, J = 7.4, 1.4 Hz, 1H), 7.91 (dd, J = 7.1, 1.4 Hz, 1H), 7.58 (t, J = 53.4 Hz, 1H), 7.47 (m, 2H), 3.97 (m, 4H), 3.83 (m, 4H); LCMS (ESI): (m/z) = 367.2 [M+H]+; ESI- MS, m/z 367.8 [M+H]+; HPLC (MeCN): 8.30 min.

Tert-butyl-4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morp

triazin-2-yl)piperazine-l-carboxylate (A6)

A mixture of 4-(4-chloro-6-(2-(difluoromethyl)-lH-benzo[ijT|imidazol-l-yl)-l,3,5-triazin-2- yl)morpholine (A5) (0.48 g, 1.3 mmol, 1 equiv), K₂C0₃ (0.36 g, 2 equiv) and boc- piperazine (Al) (0.49 g, 2 equiv) in DMF (15 ml) in a sealed tube was exposed to microwave irradiation (90 W, 140 °C) for 30 - 40 min. The reaction mixture was cooled, taken up into H₂0, extracted into EtOAc, dried over Na₂S0₄, evaporated under reduced pressure with product crystallising overnight as off-white crystals which were filtered, washed with ether and CHCI3, yielding A6: 0.27 g, 39.5%; IH-NMR (300 MHz, CDC1₃), 8.33 (dd, J = 7.1, 1.7 Hz, 1H), 7.89 (dd, J = 6.7, 1.5 Hz, 1H), 7.56 (t, J = 53.0 Hz, 1H), 7.42 (m, 2H), 3.87 (t, J= 4.5 Hz, 8H), 3.79 (t, J = 4.2 Hz, 4H), 3.54 (br s, 4H), 3.35 (t, J =

4.5 Hz, 4H), 1.50 (s, 9H); LCMS (ESI): (m/z) = 517.1 [M+H]+; ESI-MS, m/z 517.4 (100%), 417.4 (85%) [M+H]+; HPLC (MeCN): 9.75 min. 4-(4-(2- ifluoromethyl)-lH-benzofd/imidazol-l-yl)-6-(pipera^

yl)morpholine (A3)

A solution of tert-butyl 4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6- mo holino-l,3,5-triazin-2-yl) iperazine-l-carboxylate (A6) (0.13 g, 0.25 mmol) in DCM'.TFA (1 :1 , 2 ml) was stirred at room temperature for 20 minutes. The mixture was evaporated under reduced pressure, diluted with H₂0, neutralized with 2 N NaOH, filtered, washed with H₂0, then dried to yield product (A3) as off-white crystals, yield: 0.13 g, 100%, Mp: 250-252 °C; 1H-NMR (300 MHz, CD₃OD), 8.44 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.4 Hz, 1H), 7.69 (t, J = 53.0 Hz, 1H), 7.47 (m, 2H), 4.14 (t, J = 6.1 Hz, 4H), 3.91 (t, J =7.8 Hz, 4H), 3.76 (br s, 4H), 3.35 (t, J = 13.1 Hz, 4H); ¹³C-NMR (101 MHz, CD3OD), 166.30, 166.22, 163.28, 147.72, 142.43, 134.48, 127.25, 125.85, 121.31, 1 17.42, 109.82 (t, J = 240 Hz), 67.58, 45.42, 45.34, 44.27; LCMS (ESI): (m/z) = 417.1 [M+H]+; ESI-MS, m/z 417.6 [M+H]+; HR-MS calculated for Ci₉H₂₂N₈OF₂ [M+H]+: 417.1957; found 417.1975; HPLC (MeCN): 5.57 min.

Intermediate 2: Synthesis of 3-(4-Morpholino-6-(piperazin-l-yl)-l,3,5-triazin-2- yl)phenol (A 7)

H

tert-Butyl 4-(4-chloro-6-morpholino-l,3,5-triazin-2-yl)piperazine-l-carboxylate (A 8) A mixture of 4-(4,6-dichloro-l,3,5-triazin-2-yl)morpholine (A4) (0.50 g, 2.1 mmol, 1 equiv), K₂C0₃ (0.29 g, 1 equiv) and boc-piperazine (Al) (0.40 g, 1 equiv) in DMF (8 ml) was strirred at room temperature for 4 - 8 hr. The reaction mixture was poured onto H₂0, stirred several minutes, left overnight, precipitate filtered, washed with H₂0 and small amount MeOH then dried to yield product A8 as a white powder, yield: 0.75 g, 91.7%; 1 H- NMR (300 MHz, CDC1₃), 3.77 (br s, 8H), 3.71 (m, 4H), 3.46 (t, J = 5.1 Hz, 4H), 1.48 (s, 9H); LCMS (ESI): (m/z) = 385.2 [M+H]+; ESI-MS, m/z 385.3 [M+H]+; HPLC (MeCN): 8.68 min. tert-Butyl 4-(4-(3-hydroxyphenyl)-6-morpholino-l,3,5-triazin-2-yl)piperazine-l- carboxylate (Λ9)

To a solution of ert-butyl 4-(4-chloro-6-mo holino-l,3,5-triazin-2-yl)piperazine-l- carboxylate (A8) (0.10 g, 0.35 mmol, 1 equiv), CsC0₃ (0.23 g, 2 equiv) and 3- hydroxyphenylboronic acid (0.047 g, 1.3 equiv) in DMF (3.0 ml) degassed for 10 minutes, was added Pd(OAc)₂ (0.005 g, 0.01 equiv), P(Cy)₃ (0.007 g, 0.01 equiv), TBAB (0.008 g, 0.01 equiv) and H₂0 (0.5 ml) in a sealed tube was heated under microwave conditions of 150 °C for 30 minutes. The reaction mixture was taken up into EtOAc, filtered through celite, evaporated under reduced pressure, addition of H₂0 with resulting precipitate filtered, washed with MeOH and H₂0, then dried to yield product (A9) as an off- white powder. Purification via column chromatography may be required using hexane:EtOAc (l :l),yield: θ!θ85 g, 73.7%; 1H-NMR (400 MHz, CDC1₃), 7.95 (d, J = 7.8 Hz, 1H, ArH), 7.86 (dd, J = 2.5, 1.4 Hz, 1H, ArH), 7.30 (t, J = 7.9 Hz, 1H, ArH), 6.97 (dd, J = 8.4, 3.0 Hz, 1H, ArH), 5.57 (br s, 1H, NH), 3.91 (br s, 8H, OCH₂, NCH2), 3.76 (t, J = 5.1 Hz, 4H, NCH₂), 3.49 (t, J = 5.1 Hz, 4H, CH₂), 1.49 (s, 9H, CH₃); LCMS (ESI): (m/z) = 443.1 [M+H]+; ESI-MS, m/z 443.3 [M+H]+; HPLC (MeCN): 7.36 min.

3-(4-Morpholino-6-(piperazin-l-yl)-l,3,5-triazin-2-yl)phenol (A 7)

terf-butyl 4-(4-(3-hydroxyphenyl)-6-morpholino- 1 ,3 ,5-triazin-2-yl)piperazine- 1 - carboxylate (A9) (0.085 g, 0.2 mmol) in DCM:TFA (1 :1, 2 ml) was stirred at room temperature for 20 minutes. Mixture was diluted with H₂0, neutralized with 2 N NaOH, filtered, washed with H₂0, then dried to yield product (A7) as off-white crystals, yield: 0.035 g, 50.3%; Mp: 255-258 °C; 1H-NMR (400 MHz, CD₃OD), 7.85 (m, 2H), 7.26 (t, J = 7.9 Hz, 1H), 6.94 (dd, J= 8.1, 2.6 Hz, 1H), 4.62 (br s, 4H), 4.16 (br s, 4H), 3.92 (br s, 4H), 3.76 (t, J = 5.1 Hz, 4H); LCMS (ESI): (m/z) = 343.1 [M+H]+; ESI -MS, m/z 343.5 [M+H]+; HR-MS calculated for C₁₇H₂₂N₆0₂ [M+H]+: 343.1877; found 343.1895; HPLC (MeCN): 4.27 min.

Synthesis of Final compounds

3a. General methods

General method for amino acid coupling - Method I

To a solution of a Boc-protected or Fmoc protected amino acid (0.24 mmol, 2 equiv) in DMF (3 ml) was added HBTU or HCTU (0.091 g, 2 equiv) followed by DIPEA (0.062 g, 4 equiv). This solution was added to 4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6- (ρίρεΓ3ζίη-1^1)-1,3,5-ίπ3ζϊη-2^1^θ ^1ϊη6 (A3) (0.050 g, 1 equiv) in DMF (1 ml) then stirred at room temperature for 2 hr. The reaction mixture was diluted with MeCN:H₂0 (1 :1, 50 ml) then placed on freezedrier overnight resulting in a solid residue. The product was purified by elution through silica (100% EtOAc) or crystallization from MeOH and H₂0. General procedure for Boc-deprotection - Method H

A solution of the Boc-protected aminoacylpiperazinyltriazine () (1.3 mmol) in DCM.TFA (1 :1, 2 ml) was stirred at room temperature for 20 minutes. The mixture was evaporated under reduced pressure, diluted with H₂0, neutralized with 1 or 2 N NaOH, filtered, washed with H₂0, then dried to yield the product which was purified as described.

General procedure for Fmoc deprotection- Method J

A solution of the Fmoc-protected aminoacylpiperazinyltriazine (0.062 mmol) in THF (4 ml) was added 1 -octanethiol (0.091 g, 10 equiv) followed by DBU (0.003 g, 0.03 equiv) under N₂, then stirred at room temperature for 4 hr. The reaction mixture was evaporated under reduced pressure then triturated with ether. This was left to stand for approximately 1 hr, filtered, washed with ether then dried to yield the product.

3b. Final Benzimidazoles

Example 1 (AA = Gly)

Tert-butyl-(2-(4-(4-(2-(difluoromethyl)-lH-benzo[d/imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)-2-oxoethyl)carbamate (A 10) was prepared via Method I from 4-(4-(2-(difluoromethyl)- 1 H-benzo[</]imidazol- 1 -yl)-6-(piperazin- 1 -y 1)- 1 ,3 ,5-triazin-2- yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-Gly-OH (0.042 g, 2 equiv) then purified using silica plug (100% EtOAc) to yield product as yellow powder, yield: 0.01 1 g, 16.4%; LCMS (ESI): (m/z) = 574.2 [M+HJ+; ESI-MS, m/z 574.4 [M+H]+; HPLC (MeCN): 8.67 min.

2-Amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morphoUno triazin-2-yl)piperazin-l-yl)ethanone (1) was prepared via Method H from tert-butyl (2-(4- (4-(2-(difluoromethyl)-lH-benzo[i^imidazol-l-yl)-6-morpholino-l ,3,5-triazin-2- yl)piperazin-l-yl)-2-oxoethyl)carbamate (A10) (0.01 1 g, 0.02 mmol) to yield product as yellow powder, yield: 0.010 g, 100%; Mp: 154-157 °C; 1H-NMR (300 MHz, CD₃OD), 8.44 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 53.7 Hz, 1H), 7.50 (dd, J = 18.1 , 8.5 Hz, 2H), 4.62 (br s, 2H), 3.98 (br s, 4H), 3.92 (br s, 4H), 3.78 (br s, 6H), 3.59 (br s, 2H); ¹³C-NMR (101 MHz, CD₃OD), 166.56, 166.28, 163.58, 142.64, 141.41 , 134.78, 127.37, 125.93, 121.45, 1 17.49, 109.91 (t, J = 248 Hz), 68.72, 67.65, 45.36, 42.87, 41.1 1 , 27.12; LCMS (ESI): (m/z) = 474.1 [M+H]+; ESI-MS, m/z 474.4 [M+H]+; HR-MS calculated for C₂,H₂₅N₉0₂F₂ [M+H]+: 474.2172; found 474.2179; HPLC (MeCN): 6.29 min. = L-Phe)

(S)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-niorpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-l-oxo-3-phenylpropan-2-yl)carbamate (All) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lHbenzo[<flimidazol-l-yl)-6-

(A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Phe-OH (0.064 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.070 g, 87.9%; LCMS (ESI): (m/z) = 664.2 [M+H]+; HPLC (MeCN): 9.79 min.

(S)~2-amino-l-(4-(4-(2-(difluoromethyl)-l -benzofd/imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)-3-phenylpropan-l-one (2) was prepared via Method H from (S)-tert-butyl ( 1 -(4-(4-(2-(difluoromethyl)- 1 Hbenzo[d]imidazol- 1 -yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin-l-yl)-l-oxo-3-phenylpropan-2-yl)carbamate (All) (0.087 g, 0.13 mmol) to yield product as yellow crystals, yield: 0.024 g, 35.8%, yellow crystals; 1 H- NMR (400 MHz, CD₃OD), 8.21 (m, 2H), 7.85 (d, 2H), 7.39 (m, 6H), 4.70 (br s, 1H), 3.80 (s, 8H), 3.67 (s, 2H), 3.54 (m, 2H), 3.36 (m, 2H), 3.06 (m, 3H), 2.80 (t, J = 8.0 Hz, 1 H);

LCMS (ESI): (m/z) = 564.2 [M+H]+; HR-MS calculated for C₂₈H3iN₉0₂F₂ [M+H]+:

564.2642; found 564.2647; HPLC (MeCN): 6.94 min. (S)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-mo

od]

A mixture of 4-(4-chloro-6-(2-(difluoromethyl)-lH-benzo[i/limidazol-l-yl)-l,3,5-triazin-2- yl)morpholine (A5) (0.26 g, 0.7 mmol, 1 equiv), K₂C0₃ (0.36 g, 2 equiv) and boc-L- phenylalaninyl-piperazine (Bl) (0.24 g, 0.72 mmol) in DMF (3 ml) in a sealed tube was exposed to microwave irradiation (90 W, 140 °C) for 30 min. The reaction mixture was cooled and diluted in 50% acetonitrile then evaporated to dryness. The residue was chromatographed by semi-preparative RP-HPLC eluting with acetonitrile in 0.1% aq. TFA. The purified fractions were then dissolved in 0.2% aq. HCl and freeze-dryed again to provide the 2.HC1 salt, yield: 0.032 g.

Example 3 (AA = D-Ala)

(R)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzoldjimidazol-l-yl)-6-mo l,3,5-triazin-2-yl)piperazin-l-yl)-l-oxopropan-2-yl)carbamate (A12) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i ]imidazol-l-yl)-6-(piperazin-l-yl)- l ,3,5-triazin-2-yl)morpholine (A8) (0.050 g, 0.12 mmol, 1 equiv) and Boc-D-Ala-OH (0.045 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.066 g, 93.6% LCMS (ESI): (m/z) = 588.2 [M+H]+; HPLC (MeCN): 8.78 min.

(R)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-m

triazin-2-yl)pip razin-l-yl)propan-l-one (3) was prepared from (R)-tert-butyl (l-(4-(4-(2- (difluoromethyl)- 1 H-benzo[d] imidazole -yl)-6-morpholino- 1 ,3 ,5-triazin-2-yl)piperazin- 1 - yl)-l-oxopropan-2-yl)carbamate via Method H from (A12) (0.066 g, 0.11 mmol) to yield product as off-white powder, yield: 0.007 g, 12.8%; IH-NMR (400 MHz, CD₃OD), 8.43 (d, J= 8.2 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.70 (t, J= 53.2 Hz, 1H), 7.46 (m, 2H), 4.22 (dd, J = 13.5, 6.5 Hz, 1H), 4.02 (m, 2H), 3.98 (s, 2H), 3.88 (dd, J = 18.8, 13.7 Hz, 6H), 3.75 (s, 4H), 3.69 (s, 2H), 1.39 (d, J = 6.9 Hz, 3H); LCMS (ESI): (m/z) = 488.1 [M+H]+; HR-MS calculated for C₂₂H2₇N₉0₂F₂ [M+H]+: 488.2329; found 488.2329; HPLC (MeCN): 5.96 min.

Example 4 (AA = L-Ile)

tert-butyl ((2S,3S)-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-3-methyl-l-oxopentan-2-yl)carbamate (A13) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i ]imidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)mo holine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Ile-OH (0.056 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.066 g, 95.2%; LCMS (ESI): (m/z) = 630.2 [M+H]+; HPLC (MeCN): 9.97 min.

l,3,5-triazin-2-yl)piperazin-l-yl)-3-methylpentan-l-one (4) was prepared from tertbutyl ((2S,3S)- 1 -(4-(4-(2-(difluoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3,5- triazin-2-yl)piperazin-l-yl)-3-methyl-l-oxopentan-2-yl)carbamate via Method H from (A13) (0.068 g, 0.1 1 mmol) to yield product as off-white crystals, yield: 0.017 g, 29.0%; 1H-NMR (400 MHz, CD₃OD), 8.44 (d, J= 8.1 Hz, IH), 7.81 (d, J= 7.5 Hz, IH), 7.72 (t, J = 53.2 Hz, IH), 7.46 (m, 2H), 4.39 (d, J= 5.0 Hz, IH), 4.10 (m, 2H), 3.90 (m, 8H), 3.78 (s, 4H), 3.67 (s, 2H), 1.95 (m, IH), 1.60 (m, IH), 1.26 (m, 2H), 1.12 (m, 3H), 0.97 (m, 3H); LCMS (ESI): (m/z) = 530.2 [M+H]+; HR-MS calculated for C₂₅H₃₃N₉0₂F₂ [M+H]+: 530.2798; found 530.2813; HPLC (MeCN): 6.73 min. = L-Ala)

(S)-tert-butyl (l-(4-(4-(2_'(difluoromethyl)-lH-benzo[djimidazol-l-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-l-oxopropan-2-yl)carbamate (A14) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i ]imidazol-l-yl)-6-(piperazin-l-yl)- l,3,5-triazin-2-yl)mdrpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Ala-OH (0.045 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.066 g, 92.9%; LCMS (ESI): (m/z) = 588.2 [M+H]+; HPLC (MeCN): 8.83 min. (S)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzofdJimidazol-l-yl)-6-morph

triazin-2-yl)piperazin-l-yl)propan-l-one (5) was prepared from (S)-tert-butyl (l -(4-(4-(2- (difluoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3,5-triazin-2-yl)piperazin-l - yl)-l -oxopropan-2-yl)carbamate via Method H from (A14) (0.066 g, 0.1 1 mmol) to yield product as white crystals, yield: 0.004 g, 7.7%; 1 H-NMR (400 MHz, CD₃OD), 8.42 (t, J = 8.0 Hz, 1 H), 7.80 (d, J = 7.7 Hz, 1 H), 7.71 (t, J = 53.2 Hz, 1H), 7.46 (m, 2H), 4.23 (m, 1H), 4.03 (m, 2H), 3.98 (m, 2H), 3.90 (t, 6H), 3.78 (s, 4H), 3.69 (s, 2H), 1.40 (d, J = 6.9 Hz, 3H); LCMS (ESI): (m/z) = 488.1 [M+H]+; HR-MS calculated for C₂₂H₂7N₉0₂F₂ [M+H]+: 488.2329; found 488.2320; HPLC (MeCN): 6.15 min.

Example 6 (AA = I Tyr)

(S)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morphoU l,3,5-triazin-2-yl)piperazin-l-yl)-3-(4-hydroxyphenyl)-l-oxopropan^

(A15) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i/]imidazol- l - yl)-6-(piperazin-l-yl)-l ,3,5-triazin-2-yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Tyr-OH (0.068 g, 2 equiv) then purified by column chromatography using

100% EtOAc to yield product as an oil, yield: 0.091 g, > 100% ; LCMS (ESI): (m/z) =

680.2 [M+H]+; HPLC (MeCN): 8.82 min.

(S)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol -yl)-6-mo

triazin-2-yl)piperazin-l-yl)-3-(4-hydroxyphenyl)propan-l-one (6) was prepared from (S)- tert-butyl ( 1 -(4-(4-(2-(difIuoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3 ,^'5- triazin-2-yl)piperazin-l -yl)-3-(4-hydroxyphenyl)-l -oxopropan-2-yl)carbamate via Method H from (A15) (0.091 g, 0.13 mmol) to yield product as off-white powder yield: 0.020 g, 25.4%; 1H-NMR (400 MHz, CD₃OD), 8.36 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 10.4 Hz, 1H), 7.43 (m, 2H), 7.07 (d, J = 8.4 Hz, 1 H), 6.71 (m, 2H), 4.70 (s, 1 H), 4.41 (s, 1H), 3.86 (s, 4H), 3.71 (s, , 6H), 3.57 (m, 1H), 3.52 (m, l H), 3.46 (m, 2H), 3.05 (m, 2H), 2.85 (m, 2H); LCMS (ESI): (m/z) = 580.1 [M+H]+; HR-MS calculated for C₂₈H₃₁N₉0₃F₂ [M+H]+: 580.2591 ; found 580.2590; HPLC (MeCN): 6.31 min.

Example 7 (AA = L-Met)

(S)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[dJimidazol-l-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-4-(methylthio)-l-oxobutan-2-yl)carbamate (A16) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[if|imidazol- l-yl)-6- (piperazin-l-yl)-l ,3,5-triazin-2-yl)moφholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Met-OH (0.060 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.092 g, > 100% ; LCMS (ESI): (m/z) = 648.2 [M+H]+; HPLC (MeCN): 9.44 min.

(S)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)-4-(methylthio)butan-l-one (7) was prepared from (S)-tert- butyl (1 -(4-(4-(2-(difluoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3,5-triazin- 2-yl)piperazin-l-yl)-4-(methylthio)-l-oxobutan-2-yl)carbamate via Method H from (A16) (0.092 g, 0.14 mmol) then purified by semi preparative RP-HPLC to yield product as white powder, yield: 0.007 g, 9.0%; 1 H-NMR (400 MHz, CD₃OD), 8.42 (t, J= 8.3 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 53.2 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1 H), 7.43 (m, 1 H), 4.44 (m, 1H), 4.10 (m, 1H), 3.90 (t, J = 7.5 Hz, 4H), 3.77 (br s, 4H), 3.65 (s, 4H), 3.18 (td, J = 7.4, 3.6 Hz, 2H), 2.96 (dd, J = 13.6, 5.7 Hz, 2H), 2.08 (m, 2H), 1.88 (m, 2H), 1.85 (s, 3H); LCMS (ESI): (m/z) = 545.2 [M+H]+; HR-MS calculated for C₂₄H₃iN₉0₂F₂S [M+H]+: 548.2362; found 545.2917; HPLC (MeCN): 6.78 = L-Pro)

(S)-tert-butyl 2-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino-l,3,5- triazin-2-yl)piperazine-l-carbonyl)pyrrolidine-l-carboxylate (A 17) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[£/]imidazol-l-yl)-6-(piperazin-l-yl)- l,3,5-triazin-2-yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Pro-OH (0.048 g, 2 equiv) then purified by column chromatography using EtOAc.MeOH (95:5) to yield product as an oil, yield: 0.075 g, 100%; LCMS (ESI): (m/z) = 614.2 [M+H]+; HPLC (MeCN): 9.63 min.

(S)-(4-(4-(2-(difluoromethyl)-lH-benzo(dJimidazol-l-yl)-6-morpholin^

yl)piperazin-l-yl)(pyrrolidin-2-yl)methanone (8) was prepared from (S)-tert-butyl 2-(4-(4- (2-(difluoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3,5-triazin-2- yl)piperazine-l-carbonyl)pyrrolidine-l-carboxylate via Method H from (A17) (0.059 g, 0.1 mmol) then purified by semi preparative RP-HPLC to yield product as white powder, yield: 0.004 g, 8.2%; 1H-NMR (400 MHz, CD₃OD), 8.41 (d, J = 8.0 Hz, 1 H), 7.80 (m, 1H), 7.71 (t, J = 52.8 Hz, 1H), 7.42 (m, 3H), 4.76 (dd, J = 8.8, 7.0 Hz, 1H), 4.00 (m, 2H), 3.90 (m, 6H), 3.77 (s, 4H), 3.65 (s, 2H), 3.45 (m, 2H), 2.59 (m, 1H), 2.10 (m, 2H), 2.00 (m, 1H); LCMS (ESI):

[M+H]+: 514.2485; found 514.2502; HPLC (MeCN): 6.59 min. Example 9 (AA = Lys)

(S)-di-tert-butyl (6-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-6-oxohexane-l,5-diyl)dicarbantate (A18) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[isf]imidazol-l -yl)-6-(piperazin- l-yl)- l ,3,5-triazin-2-yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-Lys(Boc)- OH.DCHA (0.13 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.080 g, 89.0%; LCMS (ESI): (m/z) = 745.1 [M+H]+; HPLC (MeCN): 9.67 min.

(S)-2,6-diamino-l-(4-(4-(2-(difluoromethyl)-lH-benzofdJimidaz l-l-yl)-6-m

l,3,5-triazin-2-yl)pipe'razin-l-yl)hexan-l-one (9) was prepared from (S)-di-tert-butyl(6- (4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-mo holίno- l ,3,5-triazin-2- yl)piperazin-l-yl)-6-oxohexane-l ,5-diyl)dicarbamate via Method H from (A18) (0.089 g, 0.12 mmol) then purified by semi preparative RP-HPLC to yield product as a white powder, yield: 0.006 g, 9.2%; 1H-NMR (400 MHz, CD₃OD), 8.44 (d, J = 8.1 Hz, 1 H), 7.80 (d, J = 7.9 Hz, 1 H), 7.71 (t, J = 52.8 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1 H), 7.42 (m, 1 H), 4.46 (t, J = 6.1 Hz, 1 H), 4.12 (m, 2H), 3.91 (m, 6H), 3.78 (s, 6H), 3.64 (s, 2H), 2.96 (m, 2H), 1.90 (m, 2H), 1.71 (m, 2H), 1.52 (m, 2H); LCMS (ESI): (m/z) = 545.2 [M+H]+; HR- MS calculated for C₂₅H₃₄Nio0₂F₂ [M+H]+: 545.2907; found 545.2921 ; HPLC (MeCN):

5.17 min. = β-AIa)

(9H-fluoren-9-yl)methyl (3-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazoI-l-yl)-6- morpholino-l,3,5-triazin'2-yl)piperazin-l-yl)-3-oxopropyl)carbamate (A19) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i/|imidazol-l-yl)-6-(piperazin-l-yl)- l,3,5-triazin-2-yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Fmoc-P-Ala-OH (0.075 g, 2 equiv) then purified by column chromatography using EtOAc:MeOH (95:5) to yield product as an oil, yield: 0.044 g, 51.6%; LCMS (ESI): (m/z) = 710.1 [M+H]+; HPLC (MeCN): 8.93 min.

3-amino-l-(4-(4-(2-(difluoromethyl)-IH-benzo/d/imidazol-l-yl)-6-morpItolin^ triazin-2-yl)piperazin-l-yl)propan-l-one (10) was prepared via Method J from (9Hfluoren-9-yl)methyl (3-(4-(4-(2-(difluoromethyl)-l H-benzo[d]imidazol-l -yl)-6- mo holino-l,3,5-triazin-2-yl)piperazin-l-yl)-3-oxopropyl)carbamate (A19) (0.044 g, 0.1 mmol) to yield product as off-white powder, yield: 0.010 g, 33.1%; IH-NMR (400 MHz, CD₃OD) 8.45 (d, J= 8.0 Hz, 1H), 7.81 (d, J= 7.8 Hz, 1H), 7.72 (t, J = 52.8 Hz, lH), 7.45 (m, , 2H), 3.96 (t, J= 5.2 Hz, 2H), 3.89 (t, J = 4.0 Hz, 4H), 3.76 (s, 4H), 3.71 (s, 2H), 3.61 (t, J = 5.2 Hz, 2H), 3.55 (t, J = 6.0 Hz, 2H), 3.35 (t, J = 6.0 Hz, 2H), 2.96 (t, J = 6.4 Hz, 1H), 2.65 (t, J = 6.4 Hz, 2H), 1.75 (br s, 2H); LCMS (ESI): (m/z) = 488.1 [M+H]+; HR- MS calculated for C₂₂H₂7N₉0₂F2 [M+H]+: 488.2329; found 488.2343; HPLC (MeCN): 6.07 min. = D-Phe)

(R)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino- l,3_f5-triazin-2-yl)piperazin-l-yl)-l-oxo-3-phenylpropan-2-yl)carbamate (Λ20) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[^imidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and

Boc-D-Phe-OH (0.064 g, 2 e quiv) then purified by column chromatography using 100%

EtOAc to yield product as an oil, yield: 0.1 1 g, > 100% ; LCMS (ESI): (m/z) = 664.2

[M+H]+; HPLC (MeCN): 9.62 min.

J

(R)-2-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)-3-phenylpropan-l-one (11) was prepared via Method H from (R)-tert-butyl ( 1 -(4-(4-(2-(difluoromethy 1)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-l-oxo-3-phenylpropan-2-yl)carbamate A20) (0.080 g, 0.12 mmol) then purified by semi preparative RP-HPLC to yield product as off- white powder, yield: 0.042 g, 62.4%; 1H-NMR (400 MHz, CD₃OD), 8.38 (s, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 9.6 Hz, 1H), 7.40 (m, 7H), 4.72 (m, 1H), 3.90 (s, 4H), 3.77 (s, 4H), 3.70 (dd, J = 8.9, 4.8 Hz, 4H), 3.45 (br s, 2H), 3.10 (m, 4H); LCMS (ESI): (m/z) = 564.1 [M+H]+; HR-MS calculated for C^H^N?^ [M+H]+: 564.2642; found 564.2632; HPLC (MeCN): 7.09 min. Example 12 (AA = N-Me-L-Ala)

-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[dJimidazol-l-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazin-l-yl)-l-oxopropan-2-yl)(methyl)carbamate (Λ21) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[i0imidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)moφholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-N-Me-Ala-OH (0.049 g, 2 equiv) then purified by column chromatography using 100% EtOAc to yield product as an oil, yield: 0.066 g, 92.0%; LCMS (ESI): (m/z) = 602. 2 [M+H]+; HPLC ( eCN): 9.29 min.

(S)-l~(4-(4-(2-(difluoromethyl)-lH-benzold]imidazol-l-yl)-6-morpho

yl)piperazin-l-yl)-2-(methylamino)propan-l-one (12) was prepared via Method H from (S)-tert-butyl (l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino-l ,3,5- triazin-2-yl)piperazin-l-yl)-l-oxo-3-phenylpropan-2-yl)carbamate A21) (0.066 g, 0.1 1 mmol) then purified by semi preparative RP-HPLC to yield product as off-white powder, yield: 0.010 g, 18.2%; 1H-NMR (400 MHz, CD₃OD), 8.43 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.71 (t, J = 53.2 Hz, 1H), 7.46 (m, 2H), 4.57 (s, 1H), 4.39 (q, J= 6.9 Hz, lH), 4.03 (m, 2H), 3.89 (t, J= 4.8 Hz, 6H), 3.78 (s, 4H), 3.67 (s, 4H), 2.69 (s, 3H), 1.51 (d, J = 6.8 Hz, 3H); LCMS (ESI): (m/z) = 502.1 [M+H]+; HR-MS calculated for C₂₃H₂₉N₉0₂F₂ [M+H]+: 502.2485; found 502.2494; HPLC (MeCN): 6.14 min. Example 13 (AA = GABA)

tert-butyl (4-(4-(4-(2-(difl oromethyl)-lH-benzofdJimidazol-l-yl)-6-morpholino-l,3 triazin-2÷yl)piperazin-l-yl)-4-oxobutyl)carbamate (Λ22) was prepared via Method I from 4-(4-(2-(difluoromethyl)- lH-benzo[i/]imidazol- 1 -yl)-6-(piperazin- 1 -yl)- 1 ,3 ,5-triazin-2- yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-y-Abu-OH (0.049 g, 2 equiv) then purified by semi-preparative RP-HPLC to yield product as white powder, yield: 0.007 g, 9.7%; LCMS (ESI): (m/z) = 602. 1 [M+H]+; HPLC (MeCN): 8.67 min.

4-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)butan-l-one (13) was prepared via Method H from 4-amino-l- (4-(4-(2-(difIuoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-morpholino- 1 ,3 ,5-triazin-2- yl)piperazin-l-yl)butan-l-one (A22) (0.050 g, 0.1 mmol) then purified by semi preparative RP-HPLC to yield product as white powder, yield: 0.010 g, 16.8%; IH-NMR (400 MHz, CD₃OD), 8.42 (d, J= 7.8 Hz, 1H), 7.78 (d, J= 7.6 Hz, 1H), 7.70 (t, J= 53.2 Hz, 1H), 7.46 (m, 2H), 3.97 (t, J= 4.8 Hz, 2H), 3.88 (t, J= 4.8 Hz, 6H), 3.77 (s, 4H), 3.73 (s, 2H), 3.68 (s, 2H), 3.01 (t, J = 7.6 Hz, 2H), 2.64 (t, J= 6.9 Hz, 2H), 1.98 (m, 2H); LCMS (ESI): (m/z) = 502.1 [M+H]+; HR-MS calculated for C₂₃H₂₉N₉0₂F₂ [M+H]+: 502.2485; found 502.2482; HPLC (MeCN): 6.21 min. Example 14 (AA = L- ³hPhe)

(S)-(9H-fluoren-9-yl)methyl (4-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6- morpholino-l,3,5-triazin-2-yl)piperazin-l-yl)-4-oxo-l-phenylbuto^ (423) was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[if]imidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)mo holine (A3) (0.050 g, 5.12 mmol, 1 equiv) and Fmoc-L-P-HoPhe-OH (0.096 g, 2 equiv) then purified by semi-preparative RP-HPLC to yield product as white powder, yield: 0.010 g, 10.4%; LCMS (ESI): (m/z) = 800. 2 [M+H]+; HPLC (MeCN): 10.04 min.

(S)-3-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-m

triazin-2-yl)piperazin-l-yl)-4-phenylbutan-l-one (14) was prepared via Method J from (S)-(9H-fluoren-9-yl)methyl (4-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6- mo holino-l,3,5-triazin-2-yl)piperazin-l-yl)-4-oxo-l-phenylbutan-2-yl)carbamate (A23) (0.010 g, 0.012 mmol) to yield product as white powder, yield: 0.070 g, 97.2%; 1H-NMR (400 MHz, CD₃OD), 8.44 (d, J - 8.4 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.70 (t, J = 53.2 Hz, lH), 7.48 (m, 2H), 7.36 (s, 1H), 7.30 (m, 3H), 4.59 (s, 1H), 3.92 (dd, J = 9.5, 4.9 Hz, 8H), 3.75 (m, 6H), 3.56 (s, 2H), 2.93 (m, 2H), 2.77 (dd, J = 16.7, 3.2 Hz, 1H), 2.60 (dd, J = 17, 8.8 Hz, 2H); LCMS (ESI): (m/z) = 578.1 [M+H]+; HR-MS calculated for C29H₃3N₉0₂F₂ [M+H]+: 578.2798; found 578.2809; HPLC (MeCN): 7.07 min. Example 15 (AA = Ac-Gly)

N-(2_'(4-(4-(2-(difluoromethyl)-lH-benzofdJimidazol-l-yl)-6-morpholino

yl)piperazin-l-yl)-2-oxoethyl)acetamide (15) was prepared via Method I from 4-(4-(2- (difluoromethyl)- 1 H-benzo[d]imidazol- 1 -y l)-6-(piperazin- 1 -y 1)- 1 ,3 ,5 -triazin-2- yl)morpholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and N-acetylglycine (0.028 g, 2 equiv) then purified by semi preparative RP-HPLC to yield product as white powder, yield: 0.005 g, 10.0%; IH-NMR (400 MHz, CD₃OD), 8.46 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 52.8 Hz, 1H), 7.48 (m, 2H), 4.13 (d, J = 1 1.2 Hz, 2H), 3.99 (t, J = 4.8 Hz, 2H), 3.93 (t, J = 4.8 Hz, 6H), 3.78 (m, 4H), 3.71 (t, J = 4.8 Hz, 2H), 3.67 (s, 2H), 2.03 (s, 3H); LCMS (ESI): (m/z) = 516.1 [M+H]+; HR-MS calculated for C^^^C^ [M+H]+: 516.2278; found 516.2291 ; HPLC (MeCN): 7.26 min.

Example 16 (AA = N,N-dimethylgIycine)

l-(4-(4-(2-(difluoromethyl)-l -benzo[d]i idazol-l-yl)-6-morpholino-l,3

yl)piperazin-l-yl)-2-(dimethylamino)ethanone (16) was prepared via Method I from 4-(4- (2-(difluoromethyl)- 1 H-benzo[d]imidazol- 1 -yl)-6-(piperazin- 1 -y I)- 1 ,3 ,5-triazin-2- yl)mo holine (A3) (0.050 g, 0.12 mmol, 1 equiv) and dimethylglycine.HCl (0.034 g, 2 equiv) to yield product as white powder, yield: 0.026 g, 17.2%; IH-NMR (400 MHz, CD3OD), 8.44 (d, J= 7.8 Hz, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.72 (t, J= 52.8 Hz, 1H), 7.48 (m, 2H), 4.28 (s, 2H), 4.00 (dt, J= 15.6, 1.6 Hz, 4H), 3.92 (t, J= 4.8 Hz, 4H), 3.74 (s, 6H), 3.54 (s, 2H), 2.96 (s, 6H); LCMS (ESI): (m/z) = 502.3 [M+H]+; HR-MS calculated for C₂3H₂₉N₉0₂F₂ [M+H]+: 502.2485; found '502.2473; HPLC (MeCN): 6.20 min. = L-p³-hAla)

(S)-tert-butyl (4-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-morpholino- 1,3, 5 riazin-2-yl)piperazin-l-yl)-4-oxobutan-2-yl) carbamate (A24)

was prepared via Method I from 4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6- (piperazin-l-yl)-l,3,5-triazin-2-yl)moφholine (A3) (0.050 g, 0.12 mmol, 1 equiv) and Boc-L-β³ -hAla-OH (0.049 g, 2 equiv) to yield product as an oil, yield: 0.007 g, 10.0%; LCMS (ESI): (m/z) = 602.3 [M+H]+; HPLC (MeCN): 8.60 min.

(S)-3-amino-l-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-mo

triazin-2-yl)piperazin-l-yl)butan-l-one (17) was prepared via Method H from (S)-tert- butyl (4-(4-(4-(2-(difluoromethyl)-lH-benzo[d]imidazol-l-yl)-6-moφholino-l ,3,,5-triazin- 2-yl)piperazin-l-yl)-4-oxobutan-2-yl)carbamate (A24) (0.007 g) and purified by semi preparative RP-HPLC to yield compound as white powder, yield: 0.003 g, 51.7%, white powder; LCMS (ESI): (m/z) = 502.3 [M+H]+; HR-MS calculated for C₂₃H₂9N₉0₂F2 [M+H]+: 502.2485; found 502.2504; HPLC (MeCN): 6.03 min.

In a similar fashion compounds 19-23 were prepared. 3c. Phenol compound

Example 18 (AA = L-Phe)

(S)-tert-butyl (l-(4-(4-(3-hydroxyphenyl)-6-morpholino-l,3,5-triazin-2-yl)piperazin-l~ yl)-l-oxo-3-phenylpropan-2-yl)carbamate (A25) was prepared via Method I from 3-(4- mo holino-6-(piperazin-l-yl)-l ,3,5-triazin-2-yl)phenol (A7) (0.022 g, 0.064 mmol, 1 equiv) and Boc-L-Phe-OH (0.034 g, 2 equiv) to yield product as off-white crystals, yield: 0.012 g, 31.8%; LCMS (ESI): (m/z) = 590.2 [M+H]+; HPLC (MeCN):8.03 min.

(S)-2-amino-l-(4-(4-(3-hydroxyphenyl)-6-morpholino-l,3,5-triazin-2-yl)pi^

3-phenylpropan-l-one (18) was prepared from (S)-tert-butyl (l-(4-(4-(3-hydroxyphenyl)- 6-mo holino-l ,3,5-triazin-2-yl)piperazin-l-yl)-l-o o-3-phenylpropan-2-yl)carbamate via Method H from (A25) (0.012 g, 0.01 mmol) then purified by semi preparative RP-HPLC to yield product as white powder yield: 0.006 g, 23.5%; IH-NMR (400 MHz, CD₃OD), 7.82 (d, J = 7.8 Hz, 1H), 7.79 (s, 1H), 7.36 (m, 6H), 7.25 (td, J = 7.9, 3.9 Hz, 1H), 6.93 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 4.72 (dd, J = 8.7, 6.5 Hz, 1H), 3.89 (s, 4H), 3.75 (m, 4H), 3.61 (m, 2H), 3.36 (m, 1H), 3.16 (m, 4H), 3.03 (m, lH); ¹³C-NMR (100 MHz, CD₃OD), 171.91, 169.26, 168.63, 166.69, 158.57, 139.80, 135.27, 130.75, 130.32, 130.1Q, 129.13, 120.72, 119.46, 116.16, 67.76, 52.12, 44.92, 43.68, 43.27, 38.94; LCMS (ESI): (m/z) = 490.1 [M+H]+; HR-MS calculated for C₂₆H3iN₇03 [M+H]+: 490.2561 ; found 490.2574; HPLC (MeCN): 5.63 min.

Materials and Methods

Generation of recombinant baculovirus containing pi 10 DNA

Known techniques using the pFastBac™ system (Invitrogen, U.S.A.) were used to generate recombinant baculovirus. Recombinant bacmid DNA was then transfected, using lipofectin (Invitrogen, U.S.A.), into SOI cells and supernatant containing recombinant virus was collected after 3-5 days at 27 °C. High titre virus stock was then produced by amplification through two cycles of infection. Production of pi 10 protein was confirmed by western blotting of cell extracts separated by SDS-PAGE using a pi 10 isoform specific antibody.

Protein expression and purification

pi 10 virus (20 ml) and p85 virus (5 ml) were added to each 200 ml of SF21 cells (2 x 10⁶ cells/ml) and incubated shaking at 140 rpm for 48 h at 27 °C, after which time the cells were collected by centrifugation and stored at -80 °C until ready for extraction. The pi 10/p85 PI3K protein complex was extracted from the cells and purified using Ni-agarose chromatography. Fractions containing the PI3 protein were pooled and dialysed against 50 mM TrisHCl pH 7.5, 300 mM NaCl at 4 °C. PI3 protein was then made 20% (v/v) glycerol and 2 mM dithiothreitol and stored at -80 °C.

Inhibition assays

The PI3K inhibitors were dissolved at 10 mM in dimenthyl sulphoxide (DMSO) and stored at -20 °C until use. PI3K enzyme activity was determined using a luminescence assay measuring ATP consumption. PI3K enzyme activity was determined in 50 μΐ of 20 mM HEPES pH 7.5, 5 mM MgCl₂ with PI and ATP at the indicated concentrations. After a 60 min incubation at room temperature the reaction was stopped by the addition of 50 μΐ of Kinase-Glo (Promega) followed by a further 15 min incubation. Luminescence was then read using a Fluostar plate reader (BMG Labtech). Inhibitors were diluted in 20% (v/v) DMSO at the indicated concentrations in order to generate a concentration versus inhibition of enzyme activity curve which was then analysed using GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego California USA) in order to calculate the IC50. The results are shown in Table 1 : IC50 data for PI3K inhibition

PI3 isoform inhibition (IC50 - nM) Isoform

Compound AA residue

a P Ί δ selectivity

ZST -474 6 6 38 ^" 3 Non-selective

TGX-221 , 5070 52 376 δ:β=7 fold

1 Gly 1500 35 9900 1 10 δ:β=3 fold

2 L-Phe 4700 63 >100 μΜ 2200 δ:β=35 fold

3 ) -Ala 3700 1000 2000 70 β:δ=14 fold

4 L-Ile 6300 67 > 10 μΜ 1000 δ:β=15 fold

5 L-Ala 3500 31 3600 490 δ:β=16 fold

6 L-Tyr 3300 220 > 10 μΜ 1200 δ:β=5 fold

7 L-Met 13040 483 2947 δ:β=6 fold

8 L-Pro 2200 26 > 10 μΜ 390 δ:β=15 fold

9 L-Lys 2300 50 > 10 μΜ 670 δ:β=13 fold

10 β-Ala 720 98 3900 58 δ:β=2 fold

1 1 D-Phe 34000 78000 58000 3600 β:δ=16 fold

12 N-Me-L-Ala 1500 260 8000 290 δ:β=2 fold

13 γ-Abu 850 81 7500 77 β:δ =1 fold

14 L-p³hPhe 26000 7100 >100 μΜ 2800 β:δ ^'=2 fold

15 Ac-Gly 42 54 380 26 β:δ =2 fold

16 N,N-diMeGly 1600 2400 > 10 μΜ 330 β.δ =8 fold

17 L-p³hAla 3000 1300 7500 1 10 β:δ =12 fold

18 L-Phe 19000 190 >100 μΜ 8900 δ:β=47 fold

19 L-p-Phe 62000 1800 >100μΜ 17000 δ:β=9 fold

20 L-p³hLeu 21000 19000 >100 μΜ 9100 δ:β=2 fold

21 Isonipecotoyl 1500 3200 6500 330 δ:β=10 fold

22 D-p³hAla 2200 1700 16000 1300 δ:β=1.5 fold

23 D-p³hPro 4400 1600 5500 26 δ:β=62 fold Cell based assays

Assay of Akt phosphorylation in MB-MDA-468 cells

Cells were seeded in 6-well plates at a density of 5 x 10⁵ cells per well and allowed to grow for 32 h. Cells were then serum-starved overnight before exposing to various concentrations of compounds for 2 h, followed by IGF-1 (50ng/ml) stimulation for 15 min. The cell lysates were subsequently subjected to western blot analysis. Blots were detected by Odyssey infrared imaging system (LI-COR) and bands were quantified by Image-J. The results are shown in Figure 2.

Assay of cell growth in MB-MDA-468 cells

Cell viability was assessed using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay according to Gozgit JM, Wong MJ, Wardwell S, Tyner JW, Loriaux MM, et al. 2011. Mol Cancer Ther 10: 1028-35. Exponentially growing cell lines were plated into 96-well plates and incubated for twenty-four hours at 37 °C. Wells were treated with compound or vehicle (dimethyl sulfoxide) for 48 hours. Absorbance was measured using a VERSA max microplate reader (Molecular, Devices), data were analysed by GraphPadPrism ^"5 and are the results of three independent experiments, each tested in duplicate, and shown in Table 2. , ^!

TABLE 2 Inhibition of cell growth in breast cancer cell line by ZSTK474 analogues

MDA-MB-468

growth

ΕΟ₅₀ (μΜ)

ZSTK474 3.2±1.4

CPD 1 15±5.3

CPD 2 4.6±1.1

CPD 4 l lil .l

CPD 5 13±1.2 Preparation of human washed platelets and platelet-rich plasma (PRP) - Blood was collected into anticoagulant ACD from healthy volunteers. Preparation of human washed platelets and platelet-rich plasma (PRP) was performed as previously described (Cazenave, Megakaryocytes and platelets functional assays 2004).

Platelet aggregation assay - Platelet aggregation assays were performed according to Schoenwaelder et al. [J. Biol. Chem. (2007) 282, 28648-28658]. Briefly, 400 washed human platelets (3X10⁸/ml) or PRP (adjuested with platelet-poor plasma to a final platelet count of 3X10⁸/ml) were preincubated with vehicle (0.25% DMSO), or different concentrations of test compound for rnin with stirring before stimulating with agonist ADP or TRAP. Platelet aggregation was monitored for 10 min using light transmission aggregometry. All assays were performed with constant stirring at 800 rpm, 37 °C. The results are shown in Figure 3.

In vitro whole blood flow studies - Human blood anticoagulated with hirudin was preincubated with vehicle (0.1% DMSO), TGX-221 (2 μΜ) or various concentrations of test compound (2) for 5 min before perfusion through vWF-coated microcapillary tubes at 1800 s^"1 for 4 min, as previously described by Yap et al. Blood (2002) 99, 151-158. Images were captured in real time and analysed using Image J. The results are shown in Figure 4.

In vivo thrombosis model - Electrolytic preparation in mice

The method described here was adapted from the electrolytic preparation in the rat described by Guarini [J. Pharmacol. Toxicol. Methods, 35 (1996), pp. 101-105]. Platinum wire (diameter 1 mm) twin circular hook electrodes (shaped to 0.75 mm diameter, 0.25 mm apart) were placed distal to the flow probe on the left carotid artery to induce subsequent vascular injury. A piece of Parafilm (2 * 5 mm; American National Can, Chicago, IL, USA) was inserted under the vessel for electrical isolation. Test drugs were administered p.o. at both 24 and 2 h prior to electrolytic injury. The artery was then clamped distally to the electrode to occlude blood flow (blood stasis), and a current of 4 raA was delivered for 1.25 min via the electrode using a constant current unit (Model r

CCUl, Grass, Quincy, MA, USA) connected to a Grass SD9 stimulator. The artery clamp was released immediately after this 1.25 min period. Blood flow was monitored for 30 min after the end of stimulation; thrombus formation was defined by a decrease to zero kHz. The total blood flow through the injured artery over the 30 min following electrolytic injury was calculated as the area under the blood flow curve. The results are shown in Figure 5.

/

Claims

1. Compound of formula:

wherein:

R¹ is selected from optionally substituted Ce-naryl, optionally substituted Ci-siheteroaryl, optionally . substituted .₆alkylene)-C_6-i₄aryl, optionally substituted

-(Ci.₆alkylene)-Ci.9heteroaryl, optionally substituted -(C_2-6alkyenylene)-C6-i aryl and optionally substituted -(Chalky enylene)-Ci-9heteroaryl;

q is 0, 1 , 2, 3 or 4;

where present, each R² is independently selected from optionally substituted Ci_-8alkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^! ') wherein R^{1 1} is hydrogen or optionally substituted C|_-6alkyl; and R⁹ is optionally substituted C|_-6alkyl or where present two R² groups together form a methylene, 1 ,2-ethylene or 1 ,3- propylene group;

r is 0, 1 , 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted C_\. salkyl, optionally substituted C_2-8alkenyl, optionally substituted C_2-8alkynyl, optionally substituted Ci^alkoxy, optionally substituted Ci.₆alkylthio, optionally substituted C|. ₆alkylamino, optionally substituted di-[Ci-6alkyl]amino, optionally substituted Ci. ₆alkoxycarbonyl, optionally substituted N-Ci-6alkylcarbamoyl, optionally substituted N J- di-[Ci-₆alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C_2- ₆alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1,3 -propylene group;

m, n and p are each independently selected from 0 or 1 with the proviso that at least one of m, n or p is 1 ; R , R and R ^a are each independently selected from: hydrogen, optionally substituted C|. ₆alkyl, optionally substituted Ci.₆alkenyl, optionally substituted C₆-i₄aryl, optionally substituted Cj.gheterocycloalkyl, optionally substituted Q.gheteroaryl, optionally substituted Ci.|₄cycloaIkyl, optionally substituted Ci.₆alkyloxy, optionally substituted C₆. i aryloxy, optionally substituted Ci-6alkenyloxy, optionally substituted Ci.gheteroaryloxy, optionally substituted Ci-gheterocycloalkyloxy, optionally substituted Ci.i₄cycloalkyloxy, amino, optionally substituted Ci^alkylamino, optionally substituted Ci^alkenylamino, optionally substituted C6-i₄arylamino, optionally substituted Ci.Qheteroarylamino, optionally substituted Ci-ncycloalkylamino and optionally substituted d_ gheterocycloalkylamino;

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R^4a forms 1 ,2-ethylene or 1 ,3 -propylene;

wherein, where present, the or each optional substituent is independently selected from: optionally substituted Q^alkyl; optionally substituted C6-io ryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(Ci.₆alkyl); -NHCOO(C,_-6alkyl); -NH-COR³ wherein R^a is H or C_1-6alkyl; -NR^aR^b wherein R^a is H or Ci.₆alkyl and R^b is H or C^alkyi; -C(0)NR^aR^b, wherein R^a is H or Ci_-6alkyl and R^b is H, Ci_-6alkyl; -C(0)R^a wherein R^a is H or Ci_-6alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(Ci_-6alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(Ci_-6alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C₆-ioaryl; optionally substituted 5-10 membered Ci-gheteroaryl; optionally substituted 3-10 membered C . 9heterocyclyl; optionally substituted C_3-iocycloalkyl; optionally substituted Ci_-6alkyl; optionally substituted C_2-6alkenyl; optionally substituted C₂. 6alkynyl; and hydrogen;

with the proviso that when the moiety:

NH₂ then R¹ is selected from:

wherein: R⁷ is .selected from hydrogen, halo and hydroxy and R⁸ is selected from CH_ZF₃._Z wherein z is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

2. Compound according to claim 1 of formula:

wherein R³ is not hydrogen.

a:

wherein R is not hydrogen.

4. Compound according to any one of claims 1 to 3 wherein R is selected from :

wherein: R is selected from hydrogen, halo and hydroxy and R is selected from CH_ZF3__; wherein z is 0, 1 or 2.

. Compound of formula:

wherein:

Xi, X₂ and X₃ are each independently selected from C(Ci-8alkyl), CH and N with the proviso that at least one of Xj, X₂ and X₃ is N, and wherein R¹, R², R⁵, R⁶, R¹⁰, q, r, m and p are as defined in claim 1

or R⁵ together with any one of R³, R⁴ or R^4a forms 1 ,2-ethylene or 1,3-propylene;

with the proviso that when the moiety:

then X² and X³ do not represent CH;

2 3

H then X and X do not represent CH;

7 ίϊ

wherein: R is selected from hydrogen, halo and hydroxy and R is selected from CH_zF_3-; wherein z is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

6. Compound according to claim 5 wherein at least two of X , X and X

7. Compound according to claim 5 wherein X² and X³ are N.

8. Compound of formula:

wherein:

X, is selected from C(d.₈alkyl), CH and N, and wherein R², R³, R⁴, R^4a, R⁵, R⁶, R¹⁰, q, r, m and p are as defined in claim 1 ;

1 is selected fro

wherein: R⁷ is selected from hydrogen, halo, N¾ NHR³⁰, NR³⁰R³¹, hydroxy and hydrOxymethyl, wherein R³⁰ and R³¹ are each independently selected from Ci_-6alkyl and R⁸ is selected from CH_ZF_3-Z wherein z is 0, 1 or 2, hydroxy C_)-6alkyl, NHR¹² wherein R¹² represents hydrogen, Ci_-6alkyl or Ci-ealkoxy;

and pharmaceutically acceptable salts thereof.

9. Compound according to claim 8 wherein X¹ is N.

10. Compound according to any one of claims 5 to 9 wherein the moiety:

and R³ is not hydrogen.

1 1. Compound according to any one of claims 5 to 9 wherein the moiety:

is and R is not hydrogen.

12. Pharmaceutical composition comprising a compound according to any one of claims 1 to 1 1 or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

13. Use of a compound of any one of claims 1 to 1 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting PI3K.

14. Use of a compound of formula:

wherein:

Xi, X₂ and X₃ are each independently selected from C(Ci_-8alkyl), CH and N with the proviso that at least one of Xi, X₂ and X₃ is N;

R¹ is selected from optionally substituted Ce-uaryl, optionally substituted Ci.gheteroaryl, optionally substituted -(Ci-₆alkylene)-C6-i4aryl, optionally substituted -(Ci-6alkylene)-Ci-9heteroaryl, optionally substituted -(C₂.6alkyenylene)-C₆.i4aryl and optionally substituted -(C_2-6alkyenylene)-Ci-9heteroaryl;

q is 0, 1 , 2, 3 or 4;

where present, each R² is independently selected from optionally substituted C).₈alkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^U) wherein Rⁿ is hydrogen or optionally substituted

and R⁹ is optionally substituted C|.6alkyl or where present two R² groups together form a methylene, 1 ,2-ethyIene or 1 ,3- propylene group;

r is 0, 1 , 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifluoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted C|. ₈alkyl, optionally substituted C₂.₈alkenyl, optionally substituted C_2-8alkynyl, optionally substituted optionally substituted C|.

6alkylamino, optionally substituted di-[Ci-6alkyl]amino, optionally substituted C|. ₆alkoxycarbonyl, optionally substituted N-Ci.6alkylcarbamoyl, optionally substituted N,N- di-[Ci.₆alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C_2- ₆alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1,3-propylene group;

R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted Ci. ₆alkyl, optionally substituted Ci-ealkenyl, optionally substituted C^-naryl, optionally substituted Ci-9heterocycloalkyl, optionally substituted Ci^heteroaryl, optionally substituted C|.i₄cycloalkyl, optionally substituted Ci_-6alkyloxy, optionally substituted C₆. i₄aryloxy, optionally substituted Ci^alkenyloxy, optionally substituted Cj.gheteroaryloxy, optionally substituted C|-9heterocycloalkyIoxy, optionally substituted Ci.ncycloalkyloxy, amino, optionally substituted

optionally substituted Ci_-6alkenylamino, optionally substituted C6-i₄arylamino, optionally substituted ^'Ci-gheteroarylamino, optionally substituted Ci.ncycloalkylamino and optionally substituted C). ₉heterocycloalkylamino;

or any two of R³, R⁴ and R^4a together with the carbon atoms to which they are attached form a 3 to 7 membered optionally substituted ring that is aromatic or non-aromatic and may contain one or more.heteroatoms,

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

wherein, where present, the or each optional substituent is independently selected from: optionally substituted

optionally substituted C_6-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(C_1-6alkyl); -NHCOO(C,.₆alkyl); -NH-COR⁸ wherein R^a is H or C^alkyl; -NR^aR^b wherein R^a is H or C,_-6alkyl and R^b is H or C_1-6alkyl; -C(0)NR^aR^b, wherein R^a is H or C_{] -6}alkyl and R^b is H, C].₆alkyl; -C(0)R^a wherein R^a is H or C|.₆alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(C_1-6alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C_1-6alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted Ce-ioaryl; optionally substituted 5-10 membered Ci.9heteroaryl; optionally substituted 3-10 membered C|. 9heterocyclyl; optionally substituted C_3-iocycloalkyl; optionally substituted Ci-ealkyl; optionally substituted C_2-6alkenyl; optionally substituted C₂.

₆alkynyl; and hydrogen;

15. Use according to claim 14 wherein the compound is of formula:

wherein R is not hydrogen.

16. Use according to claim 14 wherein the compound is of formula:

wherein R³ is not hydrogen.

17. Method of inhibiting PI3K comprising administering an effective amount of a compound of any one of claims 1 to 1 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

18. Method of selectively inhibiting one or more isoforms of PI3 comprising of formula:

wherein:

Xi, X₂ and X₃ are each independently selected from C(C|_-8alkyl), CH and N with the proviso that at least one of Xi, X₂ and X₃ is N; R is selected from optionally substituted C6-i4aryl, optionally substituted Ci-gheteroaryl, optionally substituted -(C_1-6alkylene)-C6-i₄aryl, optionally substituted -(C|.6alkylene)-Ci.₉heteroaryl, optionally substituted -(C2-6alkyenylene)-C6-i4aryl and optionally substituted -(Chalky enylene)-C i .9heteroary 1;

q is 0, 1, 2, 3 or 4;

where present, each R² is independently selected from optionally substituted Ci_-8alkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(R^N) wherein R^{1 1} is hydrogen or optionally substituted Ci_-6alkyl; and R⁹ is optionally substituted Ci^alkyl or where present two R² groups together form a methylene, 1 ,2-ethylene or 1 ,3- propylene group;

r is 0, 1 , 2, 3 or 4;

optionally substituted Ci.6alkylthio, optionally substituted Ci. ₆alkylamino, optionally substituted di-[C|.₆alkyl]amino, optionally substituted C i. ealkoxycarbonyl, optionally substituted N-Ci^alkylcarbamoyl, optionally substituted N,N- di-[Ci.₆alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C₂. ₆alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1 ,3-propylene group;

R³, R⁴ and R^4A are each independently selected from: hydrogen, optionally substituted C i. ₆alkyl, optionally substituted C|.6alkenyl, optionally substituted C6-i aryl, optionally substituted Ci-gheterocycloalkyl, optionally substituted Ci-gheteroaryl, optionally substituted Ci.i₄cycloalkyl, optionally substituted Ci^alkyloxy, optionally substituted C_6- ₁₄aryloxy, optionally substituted Ci^alkenyloxy, optionally substituted C|.9heteroaryloxy, optionally substituted Ci.gheterocycloalkyloxy, optionally substituted Ci-ncycloalkyloxy, amino, optionally substituted Ci-ealkylamino, optionally substituted Ci-6alkenylamino, optionally substituted C6-i₄arylamino, optionally substituted Ci.₉heteroarylamino, optionally substituted Ci.Mcycloalkylamino and optionally substituted C]_ gheterocycloalkylamino;

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

or R⁵ together with any one of R³, R⁴ or R^4a forms 1,2 -ethylene or 1,3 -propylene;

wherein, where present, the or each optional substituent is independently selected from: optionally substituted C|.₆alkyl; optionally substituted C₆-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(C,.₆alkyl); -NHCOO(C,.₆alkyl); -NH-COR³ wherein R^a is H or C|.₆alkyl; -NR^aR^b wherein R^a is H or Ci_-5alkyl and R^b is H or C alkyl; -C(0)NR^aR^b, wherein R^a is H or Ci_-6alkyl and R^b is H, C|.₆alkyl; -C(0)R^a wherein R^a is H or C_1-6alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(Ci_-6alkyl , -NHS0₂-, -S0₂NH-, -NHCONH-, -NHCON(C|_-6alkyl)-, -S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C₆-io ryl; optionally substituted 5-10 membered Ci^heteroaryl; optionally substituted 3-10 membered C|. 9heterocyclyl; optionally substituted C₃.|ocycloalkyl; optionally substituted Ci.₆alkyl; optionally substituted C_2-6alkenyl; optionally substituted C_2- ₆alkynyl; and hydrogen;

or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

19. Method according to claim 18 wherein the compound is of formula:

wherein R³ is not hydrogen. Method according to claim 18 wherein the compound is of formula:

wherein R is not hydrogen.

21. Compound of any one of claims 1 to 1 1 or a pharmaceutically acceptable salt thereof for use as a selective inhibitor of PI3K.

22. Compound of formula:

wherein:

Xi, X₂ and X₃ are each independently selected from C(Ci-galkyl), CH and N with the proviso that at least one of Xi, X₂ and X₃ is N;

R¹ is selected from optionally substituted C_6-i₄aryl, optionally substituted Ci- iheteroaryl, optionally substituted -(Ci_6alkylene)-C₆-i₄aryl, optionally substituted -(Ci_-6alkylene)-Ci-9heteroaryl, optionally substituted -(C_2-6alkyenylene)-C6-i₄aryl and optionally substituted -(C₂.6alkyenylene)-Ci.₉heteroaryl;

q is 0, 1, 2, 3 or 4; ^!

where present, each R² is independently selected from optionally substituted Ci.₈alkyl or a group of the formula -A-R⁹ wherein A is a direct bond or is selected from O and N(Rⁿ) wherein R^{1 1} is hydrogen or optionally substituted C].6alkyl; and R⁹ is optionally substituted C|_-6alkyl or where present two R² groups together form a methylene, 1 ,2-ethylene or 1 ,3- propylene group;

r is 0, 1 , 2, 3 or 4;

where present, each R¹⁰ is independently selected from halogeno, trifiuoromethyl, cyano, nitro, hydroxy, mercapto, amino, carboxy, carbamoyl, ureido, optionally substituted C_\. salkyl, optionally substituted C₂.galkenyl, optionally substituted C_2-8alkynyl, optionally substituted C^alkoxy, optionally substituted Ci^alkylthio, optionally substituted Ci. 6alkylamino, optionally substituted di-[C|^alkyl]amino, optionally substituted C|. 6alkoxycarbonyl, optionally substituted N-Ci.₆alkylcarbamoyl, optionally substituted N,N- di-[Ci,6alkyl]carbamoyl, optionally substituted C_2-6alkanoyl, optionally substituted C_2- 6alkanoyloxy, optionally substituted C_2-6alkanoylamino, or two R¹⁰ groups together form a methylene, 1 ,2-ethylene or 1,3-propylene group;

R³, R⁴ and R^4a are each independently selected from: hydrogen, optionally substituted C|. ₆alkyl, optionally substituted Ci^alkenyl, optionally substituted C₆.i₄aryl, Optionally substituted Ci.gheterocycloalkyl, optionally substituted Ci^heteroaryl, optionally substituted Ci-ncycloalkyl, optionally substituted

optionally substituted C6- naryloxy, optionally substituted Ci^alkenyloxy, optionally substituted Ci-gheteroaryloxy, optionally substituted Ci.gheterocycloalkyloxy, optionally substituted Ci-ncycloalkyloxy, amino, optionally substituted C].₆alkylamino, optionally substituted Ci.6alkenylamino, optionally substituted Ce-Marylamino, optionally substituted Ci.9heteroarylamino, optionally substituted C^ncycloalkylamino and optionally substituted Ci_ ₉heterocycloalkylamino ;

R⁵ and R⁶ are each independently selected from hydrogen, methyl and ethyl

wherein, where present, the or each optional substituent is independently selected from: optionally substituted Chalky!; optionally substituted C₆-ioaryl; halogen; -OH; -NH₂; -N0₂; -S0₂NH₂; -COOH; -COO(Ci_-6alkyl); -NHCOO(C,_-6alkyl); -NH-COR³ wherein R^a is H or C^alkyl; -NR^aR^b wherein R^a is H or C,_-6alkyl and R^b is H or d_-6alkyl; -C(0)NR^aR^b, wherein R^a is H or d_-6alkyl and R^b is H, Ci_-6alkyl; -C(0)R^a wherein R^a is H or C|.₆alkyl; or -Y-Q wherein:

Y is selected from: -0-, -NH-, -N(C,.₆alkyl)-, -NHS0₂-, -S0₂NH-, -NHCONH-,

-S(0)_q- wherein q is 0, 1 or 2, -C(0)NH-, -C(0)N(CH₃)-, -NHC(O)-, -C(O)-, -NHC(NH)NH-, or absent, and

Q is selected from: optionally substituted C6-i₀aryl; optionally substituted 5-10 membered d.gheteroaryl; optionally substituted 3-10 membered C|. 9heterocyclyl; optionally substituted C3-iocycloalkyl; optionally substituted Ci.₆alkyl; optionally substituted C_2-6alkenyl; optionally substituted C₂. 6alkynyl; and hydrogen;

or a pharmaceutically acceptable salt thereof, for use as a selective inhibitor of one or more isoforms of PI3K.