EP1848506A2

EP1848506A2 - Pyrimidine-fused diazepine derivatives and indole-fused pteridines

Info

Publication number: EP1848506A2
Application number: EP06748243A
Authority: EP
Inventors: Xu Bai Bai; Qun Dang; Robert J. Ternansky Ternansky
Original assignee: Changchun Discovery Sciences Ltd; Attenuon LLC
Current assignee: Changchun Discovery Sciences Ltd; Tactic Pharma LLC
Priority date: 2005-02-18
Filing date: 2006-02-21
Publication date: 2007-10-31
Also published as: WO2006089298A3; CN101257948A; WO2006089298A2

Abstract

The present invention relates to pyrimidine-fused benzodiazepine derivative and indole-fused pteridine compounds. The invention further relates to libraries containing two or more of such compounds and methods of making such compounds. The invention also relates to methods of screening for bioactive pyrimidine-fused benzodiazepine derivative and indole-fused pteridine compounds.

Description

PYRIMIDINE-FUSED DIAZEPINE DERIVATIVES AND INDOLE-FUSED PTERD3INES

This application claims benefit of U.S. provisional patent application Serial No. 60/654,606, filed February 18, 2005, which is incorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

[0001] The present invention relates to pyrimidine-fused benzodiazepine derivative and indole-fused pteridine compounds. The invention further relates to libraries containing two or more of such compounds and methods of making such compounds. The invention also relates to methods of screening for bioactive pyrimidine-fused benzodiazepine derivative and indole-fused pteridine compounds for the treatment or prevention of diseases such as cancer.

2. BACKGROUND OF THE INVENTION

[0002] Heterocyclic compounds are often considered privileged structures in medicinal chemistry due to their various biological effects, and there are numerous reports of heterocyclic scaffolds containing the benzodiazepine moiety which show significant biological activities (Evans et al, J. Med. Chem. 1988, 31, 2235-2246; Evans et al., Bioorg. Med. Chem. Lett. 2001, 11, 1297-1300; Wyatt et al., Bioorg. Med. Chem. Lett. 2001, 11, 1301-1305; Stevens et al., J. Am. Chem. Soc. 1996, 118, 10650-10651). [0003] Benzodiazepine derivatives are probably the most explored compound class in drug discovery. These compounds have shown a variety of biological effects predominately ascribed to their actions in the central nervous system. For example, Clozapine, Olanzapine and Quetiapine are used to treat schizophrenia (Campiani et al., J. Med. Chem. 2002, 45, 344; Campiani et al., J. Med. Chem. 2004, 47, 143; Mouithys-Mickalad et al., J. Med. Chem. 2001, 44, 769); Clonazepam, Diazepam, Lorazepam, Nitrazepam and Oxazepam are used as antianxiety drugs; Midazolam is a phenobarbital-like compound typically used for anesthesia. In addition, they are cholecystokinin receptor (CCK) A and B antagonists, oxytocin antagonist and inhibitors of protein-DNA interactions (Horton et al., Chem. Rev. 2003, 103, 893; Abrous et al., Org. Lett. 2001, 3, 1089; Evans et al., J. Med. Chem. 1988, 31, 2235; Evans et al., Bioorg. Med. Chem. Lett. 2001, 11, 1297; Wyatt et al., Bioorg. Med. Chem. Lett. 2001, 11, 1301; Stevens et al., J. Am. Chem. Soc. 1996, 118, 10650; Morita et al., Tetrahedron Lett. 1970, 11, 861; Kobayashi, Chem. Lett. 1974, 967; Kobayashi, Bull. Chem. Soc. Jpn. 1975, 48, 302; Hunziker et al., HeIv. Chim. Acta 1964, 47, 1163; Eyrolles et al., J. Med. Chem. 1994, 37, 1508; Frederick and William (Bristol-Myers) Eur. Pat. Appl. DE 19742418285 19740416, 1974; Chem. Abstr. 1975, 82, 73045; Chebanov et al., J. Heterocycl. Chem, 2003, 40, 25). [0004] Tricyclic diazepine derivatives are known anti-inflammatory, anti-anginal, anti-secretory (gastric acid), anti-muscarinic, anti-arrhythmic, anti-depressant, analgesic, antipsychotic, anxiolytic, anti-convulsant, anti-neoplastic, anti-mitotic, anti-hypertensive, antiallergic, anti-asthmatic, antibiotic, anti-diabetic, anti-histamine, cardiotonic, and spasmolytic agents. In addition, they are reported to be agents for treatment of irritable bowel syndrome, urinary incontinence, renal failure, neurogenic pain and septic shock and useful as GABA A/benzodiazepine receptor complex related drugs, microtubule inhibitors, endothelin antagonists, muscarinic (M2) antagonists, vasopressin Vl antagonists, vasopressin V2 antagonists and lipoxygenase inhibitors.

[0005] Regarding tricyclic benzodiazepines, only a few studies have been directed toward their synthesis. Brodrock et al. reported a Bischler-Napieralski type cyclization of 2- benzamidodiarylsulfides in preparation of dibenzothiazepines (Brodrick et al., J. Chem. Soc. 1954, 3857). Subsequently, Hunziker extended the methodology to other dibenzothiazepines derivatives (Hunziker et al., HeIv. Chim. Acta. 1964, 47, 1163). hi 1957, Jarrett and Loudon developed a route by condensing o-aminothiophenol with reactive o-chlorophenyl-aldehydes or ketones (J. Chem. Soc. 1957, 3818). This strategy was the focus of several follow-up reports (Gait et al., J. Chem. Soc. 1958, 1588; Gait and Loudon, J. Chem. Soc. 1959, 885), and J-P. Le Roux observed dibenzothiazepine in arrangement of an azide compound (Le Roux et al. Tetrahedron Lett. 1976, 3141). Safonova et al. describe the preparation of pyrimidobenzothiazepines from the reactions of 5-amino-6-mercaptopyrimidines with derivatives of p-chloronitrobenzene containing a carbonyl group (Safonova et al., Chem. Heterocycl. Compd. (Engl. Transl.) 2001, 37, 245).

[0006] Another widely studied class of compounds are pyrimidines and pyrimidine- fused compounds because of their interesting pharmacological activities and structural similarity to the naturally-occuring nucleosides. For example, some pyrrolopyrimidines are reported to have anti-tumor activities (Wang et al., J. Med. Chem. 2004, 47, 1329), some aminopyridopyrimidines are non-nucleoside adenosine kinase inhibitors (Perner et al., J. Med. Chem. 2003, 46, 5249), certain furanopyrimidines are potent and selective inhibitors of human cytomegalovirus (HCMV) (McGuigan et al., J. Med. Chem. 2004, 47, 1847), and 5- substituted furo[2,3-d]pyrimidines exhibit potent inhibitory activity against the growth of tumor cells (Gangjee et al., J. Med. Chem. 2004, 47, 6893).

[0007] Consequently, synthetic methodologies for synthesis of novel pyrimidines or pyrimidine-fused compounds are of particular interest to organic and medicinal chemists. For example, synthetic methods are reported for the efficient syntheses of purines, pyrrolopyrimidines (Gangjee et al., Med. Chem. 2004, 47, 3689; Dang and Gomez-Galeno, J. Org. Chem. 2002, 67, 8703-8705), pyrazolopyrimidines (Bhuyan et al., J. Org. Chem. 1990, 55, 568-571; Selleri et al., J. Med. Chem. 2003, 46, 310-313), pyrimidopyrimidines (Thskur et al., Synlett. 2001, 1299-1302), imidazopyrimidines (Gudmundsson and Johns, Org. Lett. 2003, 5, 1369) and furopyrimidines (Gangjee et al., J. Med. Chem. 2004, 47, 6893-6901). [0008] A related class of compounds to the benzodiazepines are pyrimidine-fused pteridines, which have been reported to exhibit a variety of biological activities and constitute the backbones of several marketed drugs. For example, the antifolate drug methotrexate (MTX) is used as an antitumor agent and triamterene as diuretics. Other pteridines are reported to have activities against biological targets such as alkyltransferase, adenosine kinase, mycobacterial FtsZ, xanthine oxidase and neuronal nitric oxide synthase (Nelson et al., J. Med. Chem. 2004, 47, 3887; Gomtsyan et al., Bioorg. Med. Chem. Lett. 2004, 14, 4165; Reynolds et al., Bioorg. Med. Chem. Lett. 2004, 14, 3161; Oettl et al., Biochim. Biophys. Acta 1999, 1430, 387; Frohlich et al., J. Med. Chem. 1999, 42, 4108). [0009] What are needed are different classes of benzodiazepine derivatives and related compounds for screening for novel bioactive compounds that are useful for the treatment or prevention of diseases, such as cancer.

3. SUMMARY OF THE INVENTION

[0010] The present invention relates to pyrimidine-fused benzodiazepines and pyrimidine-fused pteridines, and libraries of such compounds. These compounds based on privileged structures are important for use in libraries against broad drug screening targets for new leads and may be used as the basic structures for drug discovery. In specific embodiments, a compound of the invention is purified. [0011] In one embodiment, the invention provides a compound of formula:

[0013] wherein X = O, S, or NR;

[0014] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; and

[0015] R, R₁ , R₂ = alkyl, aryl or a heterocycle.

[0016] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[0017] In certain embodiments, R, R₁, R₂ = substituted alkyl, substituted aryl or substituted heterocycle.

[0018] In another embodiment, the invention provides a compound of formula:

[0020] wherein X = O, S, or NR;

[0021] Nu = R₄S, R₄O or NR₅R₆;

[0022] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; and

[0023] R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle.

[0024] In certain embodiments, Nu = halogen.

[0025] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[0026] In certain embodiments, R, R₁, R₂, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle.

[0027] In another embodiment, the invention provides a compound of formula:

[0029] wherein X = O, S, or NR; [0030] Nu = R₄S₅ R₄O Or NR₅R₆;

[0031] Y₁ , Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; and

[0032] R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle.

[0033] In certain embodiments, Nu = halogen.

[0034] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[0035] In certain embodiments, R, R₁, R₂, R₃, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle.

[0036] In another embodiment, the invention provides a compound of formula:

[0038] wherein X = O, S, or NR; [0039] Nu = R₄S, R₄O or NR₅R₆; [0040] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; and [0041] R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [0042] In certain embodiments, Nu = halogen. [0043] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[0044] hi certain embodiments, R, R₁, R₂, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle.

[0045] hi another embodiment, the invention provides a compound of formula:

[0047] wherein X = O, S, or NR; [0048] Nu = R₄S, R₄O or NR₅R₆; [0049] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; and [0050] R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [0051] In certain embodiments, Nu = halogen.

[0052] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[0053] In certain embodiments, R, R₁, R₂, R₃, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle.

[0054] In another embodiment, the invention provides a compound of formula:

[0056] wherein:

[0057] R₇ = H, CH₃, or F; and

[0058] R₈ = 4'-F-C₆H₄, 4'-CH₃-C₆H₄, or 4'-NO₂-C₆H₄.

[0059] In another embodiment, the invention provides a compound of formula:

[0061] wherein:

[0062] NHR₉R₁₀ is n-Bu-NH₂ or pyrrolidine.

[0063] In a preferred embodiment, the compound is 4-chloro-l l-methyl-6- propylpyrimido^^-blCl^Jbenzodiazepine^-chloro-όΛl-dimethylpyrimido^^- b] [ 1 ,4]benzodiazepine; 4-chloro- 11 -methyl-6-phenyrpyrimido[4,5~&] [ 1 ,4]benzodiazepine; 4- chloro-1 l-methyl-6-(4'-fluorophenyl)-pyrimido[4,5-Z?][l,4]benzodiazepine; 4-chloro-l 1- methyl-6-(4'-methyl-phenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-l l-methyl-6-(4'- nitro-phenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6- propylpyrimido[4,5-&][l,4]benzodiazepine; 4-chloro-6,8,ll-trimethylpyrimido[4,5- b] [ 1 ,4]benzodiazepine; 4-chloro-8, 1 l-dimethyl-6-phenylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

4-chloro-8,ll-dimethyl-6-(4'-fluorophenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-

8,1 l-dimethyl-6-(4'-methyl-phenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8,l 1- dimethyl-6-(4'-nitro-phenyl)-pyrimido[4,5-b][l,4]benzodiazeρine; 4-chloro-8-fluoro-ll- methyl-6-propylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8-fluoro-6,ll- dimethylpyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-chloro-8-fluoro- 11 -methyl-6- phenylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- fluorophenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- methylphenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- nitrophenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-hydroxy -1 l-methyl-6- phenylpyrimido[4,5-b][l,4]benzodiazepine; 4-(butylamino)-8- fluoro -ll-methyl-6-(4'- methyl-phenyl)-pyrimido[4,5-b][l,4] benzodiazepine; or 4-(pyrrolidin-l-yl)-8- fluoro -11- methyl-6-(4'-methylphenyl)-pyrimido[4,5-b][l,4] benzodiazepine. [0064] The invention also provides methods for making tricyclic 4-chloro- pyrimido[4,5-&][l,4]benzodiazepines and derivatives. In one embodiment, the method comprises reacting a 6-chloro-4,5-diaminopyrimidine with an acid or acid derivative to form a 4-chloro-pyrimido[4,5-b][l,4]benzodiazepine. In other embodiments, the method further comprises conducting a nucleophilic substitution reaction on said 4-chloro-pyrimido[4,5- £>][l,4]benzodiazepine. In other embodiments, the method further comprises hydrogenating the 4-substituted-pyrimido[4,5-&][l,4]benzodiazepine under hydrogenation reaction conditions or reacting the 4-substituted-pyrimido[4,5-Z>][l,4]benzodiazepine with organometallic reagent. In yet another embodiment, the method further comprises amide formation, alkylation, or sulfonamide formation. [0065] In one embodiment, the invention provides a compound of formula:

[0066] (VIII) wherein:

Rl = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle or halogen;

R2 = alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, or substituted heterocycle.

[0067] In a preferred embodiment, the compound is 4-Chloro- 1 l-methyl-6-propyl-

5,6-dihydro-pyrimido[4,5-£] [ 1 ,4]benzodiazepine; 4-Chloro- 1 l-methyl-6-ethyl-5 ,6-dihydro- pyrimido[4,5~&] [l,4]benzodiazepine; 4-Chloro- 11 -methyl-6-ρhenyl-5,6-dihydro- pyrimido[4,5-ό][l,4]benzodiazepine; 4-Chloro-ll-methyl-6-(4'-methyl-phenyl)-5,6-dihydro- pyrimido[4,5-£] [ 1 ,4]benzodiazepine; 4-Chloro- 11 -methyl-6-(4'-fluoro-phenyl)-5,6-dihydro- pyrimido[4,5-&][l,4]benzodiazepine; 4-Chloro-ll-methyl-6-(4'-nitro-phenyl)-5,6-dihydro- pyrimido[4,5-b][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5-£][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5- b][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-phenyl-5,6-dihydro-pyrimido [4,5- Z7][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro- pyrimido[4,5-b][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-fluoro-phenyl)-5,6- dihydro-pyrimido[4,5-b][l ,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-(4'-nitro-phenyl)- 5,6-dihydro-pyrimido[4,5-£] [ 1 ,4]benzodiazepine; 4-Chloro-9, 11 -dimethyl-6-propyl-5,6- dihydro-pyrimido [4,5-&][l,4]benzodiazepine; 4-Chloro-9,l l-dimethyl-6-ethyl-5,6-dihydro- pyrimido [4,5-b][l,4]benzodiazepine; 4-Chloro~9,l l-dimethyl-6-phenyl-5,6-dihydro- pyrimido [4,5-b] [ 1 ,4]benzodiazepine; 4-Chloro-9, 11 -dimethyl-6-(4'-methyl-phenyl)-5,6- dihydro-pyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-Chloro-9, 11 -dimethyl-6-(4'-fluoro-phenyl)- 5,6-dihydro-pyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-Chloro-9, 11 -dimethyl-6-(4'-nitro- phenyl)-5,6-dihydro-pyrimido[4,5-b][l,4]benzodiazepine; or 4-Chloro-8-fluoro-l l-methyl-6- phenyl-5,6-dihydro pyrimido[4,5-b] [ 1 ,4]benzodiazepine.

[0068] The invention also provides methods for making tricyclic pyrimidine-fused

5,6-dihydrobenzodiazepines. In one embodiment, the method comprises reacting a 6-chloro- 4,5-diaminopyrimidine with an aldehyde to form a 4-chloro-dihydropyrimido[4,5- b][l ^benzodiazepine. In other embodiments, the method further comprises a nucleophilic substitution of the 4-chloro group.

[0069] In one embodiment, the invention provides a compound of formula:

wherein:

R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, halogen or alkoxy; and

R₃ = alkyl, substituted alkyl, aryl, heterocycle, substituted heterocycle, heteroaryl, or substituted aryl. [0071] In another embodiment, the invention provides a compound of formula: wherein:

R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle, or halogen. [0073] In another embodiment, the invention provides a compound of formula:

wherein:

R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle or halogen;

R₄, R₅ = H, alkyl, substituted alkyl, aryl, substitute aryl, heterocycle, substituted heterocycle, or R₄ and R₅ together form a cyclic alkyl. [0075] hi a preferred embodiment, the compound is 4-(Phenylthio)-6- phenylpyrimido[4,5-b] [1 ,4]benzothiazepine; 4-(Phenylthio)-6-(pyridin-3-yl)-pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-p-tolylpyrimido[4,5-έ] [ 1 ,4]benzothiazepine; 4- (Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(o- nitrophenyl)pyrimido[4,5-b][l,4]benzothiazepine; 4-(Phenylthio)-6-(p- mtrophenyl)pyrimido[4,5-fr] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(m- fluoroρhenyl)pyrimido[4,5-b][l,4]benzothiazepine; 4-(Phenylthio)-6-methylpyrimido[4,5- h] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-propylpyrimido[4,5 -b] [ 1 ,4]benzothiazepine; 4- (Phenylthio)-6-benzylpyrimido[4,5-b][l,4]benzothiazepine; 4-(p-Tolylthio)-6-phenyl-8- methylpyrimido[4,5-&] [1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-(pyridin-3-yl)-8- methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-p-tolyl-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-(p-fluorophenyl)-8-methylpyrimido[4,5- Z?][l,4]benzothiazepine; 4-(p-Tolylthio)-6-propyl-8-methylpyrimido[4,5- b][l,4]benzothiazepine; 4-(p-Tolylthio)-6-benzyl-8-methylpyrimido[4,5- b][l,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-phenyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-p-tolyl-8-chloropyrimido[4,5- b][l,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-(pyridin-3-yl)-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Chloro-phe nylthio)-6-Z?enzyl-8-chloropyrimido[4,5- b] [1 ,4]benzothiazepine; 4-(p-Methoxyphenylthio)-6-phenyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Methoxyphenylthio)-6-&enzyl-8-methoxypyrimido[4,5- b] [1 ,4]benzothiazepine; 4-(Phenylsulf inyl)-6-phenylpyrimido[4,5~£] [ 1 ,4]benzothiazepine; 4-

(p-Tolylsulfinyl)-6-phenyl-8-methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(p-

Chlorophenylsulfinyl)-6-phenyl-8-chloropyrimido[4,5-&][l,4]benzothiazepine; 4-(n-

Butylamino)-6-phenylpyrimido[4,5-&] [ 1 ,4]benzothiazepine; 4-(Pyrrolidin- 1 -yl)-6- phenylpyrimido[4,5-b][l,4]benzothiazeρine; 4-(n-Butylamino)-6-phenyl-8- methylpyrimido[4,5-£»] [ 1 ,4]benzothiazepine; or 4-(n-Butylamino)-6-phenyl-8- chloropyrimido[4,5-b] [ 1 ,4]benzothiazepine.

[0076] The invention also provides methods for making tricyclic pyrimido[4,5- b][l,4]benzothiazepines. In one embodiment, the method comprises reacting a 5~amino-4,6-

Msphenylthiopyrimidine with an acid or acid derivative to form a pyrimido[4,5-

Z?][l,4]benzothiadiazepine. In other embodiments, the method further comprises oxidation of the phenylthio group of the pyrimido[4,5-&][l,4]benzothiadiazepine to the corresponding sulfoxide or sulfone. In other embodiments, the method further comprises nucleophilic substitution with an amine.

[0077] In one embodiment, the invention provides a compound of:

wherein:

X = Cl, R₃R₄N, R₃O, R₃S, or aryl;

R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl or R₁ and R₂ together form a cyclic alkyl;

R₃, R₄ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle. [0079] In a preferred embodiment, the compound is 8-chloro-6-ethyl- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-propyl-l,2,6,7-tetrahydro- 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-(o-methoxyphenyl)-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-(p-nitro-phenyl)-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h] azulene; 8-chloro-6-(p-fluoro-phenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-styryl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-propenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h] azulene; 8-chloro-6,6-dimethyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene; 8-chloro~6~ethyl-6-rnethyl- l,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-propyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h] azulene; δ-chloro-ό-cyclohexylidene- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene; 8-chloro-6-methyl-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-(p-methoxyphenyl)- 1,2,6,7 -tetrahydro- 1,9, W, I lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-(p-nitro-phenyl)~ 1 ,2,6,7- tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro~6~ethyl-6-phenyl- 1 ,2,6,7- tetraliydro-7,9, 11 , 1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-ethyl- 1 ,2,6,7-tetrahydro- 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulen-6-carbonate acid; 9-chloro-7-ethyl-2,3,7,8- tetrahydro- 1H-8, 10, 12, 12b-tetraaza-benzo[4,5]cyclohepta[ 1 ,2,3-de]naphthalene; 9-chloro-7- pyproyl-2,3 ,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza-benzo[4,5]cyclohepta[ 1 ,2,3- de]naphthalene; 9-chloro-7-phenyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-(p-methylphenyl)-2,3,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza-benzo[4,5]cyclohepta[ 1 ,2,3-de]naphthalene; 9-chloro-7-(p-nitro- phenyl)-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3- dejnaphthalene; 9-chloro-7-styryl-2,3 ,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7,7-dimethyl-2,3,7,8-tetrahydro-lH- 8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; N-(n-butyl)-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulen-8-amine; N-phenyl-6-propyl- l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulen-8-amine; 8-(morpholin-l-yl)-6- phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-(pyrrolidin-l-yl)-6- propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-butoxyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-butoxy-6-phenyl- 1 ,2,6,7- tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-benzylthio-6-propyl- 1 ,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-benzylthio-6-phenyl- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-phenylthio-6-propyl- 1,2,6,7- tetrahydro-7,9, 11 , 1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-phenylthio-6-phenyl- 1 ,2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8-phenyl-6-propyl-l, 2,6,7 -tetrahydro- 7,9,11,1 lb~tetraaza-dibenzo[c,d,h]azulene; and 8-phenyl-6-phenyl- 1,2,6,7 -tetrahydro- 7,9,11 , 1 lb-tetraaza-dibenzo[c,d,h] azulene.

[0080] The invention also provides methods for making tetracyclic pyrimidine-fused benzodiazepines. In one embodiment, the method comprises reacting an indolin-1- ylpyrimidine or tetrahydroquinolin-1-ylpyrimidine with aliphatic or aromatic aldehydes or ketones to form a a tetracyclic pyrimidobenzodiazepine. La other embodiments, the method further comprises a nucleophilic substitution. [0081] In one embodiment, the invention provides a compound of formula:

wherein:

R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, or R₁ and R₂ together form a cyclic alkyl; [0083] R₃ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, or substituted heterocycle.

X = Cl or NHR; and

R = alkyl, substituted alkyl, aryl, substituted aryl.

[0084] In a preferred embodiment, the compound is 4-chloro-6-ethyl-5,6- dihydroindolo[2,l-h]ρteridine; 6-butyl-4-chloro-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro- 6-ρhenyl-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(4'-rnethylphenyl)-5,6~ dihydroindolo[2,l-h]pteridine; 4-chloro-6-(4'-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(3',4'-dichlorophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o- methoxyphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o-chlorophenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o-nitrophenyl)-5,6-diliydroindolo[2,l-h]pteridine; 4-chloro-6-(m-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-ethyl-6-methyl- 5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-propyl-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-6-cyclohexylidene-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6- methyl-ό-phenyl-S^-dihydroindolotlJ-ypteridine^-chloro-ό-methyl-ό-Cp-methylphenyl)- 5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-(p-nitrophenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-phenyl-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-methylphenyl)-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-fluorophenyl-5,6-diliydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6- cyclohexylidene-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l-h]pteridine; N-butyl-6- cyclohexylidene-indolo[2,l-h]pteridin-4-amine; and N-butyl-6-(p-methylphenyl)-indolo[2,l- h]pteridin-4-amine.

[0085] The invention also provides methods for making indole-fused pyrimidines. In one embodiment, the method comprises reacting a 5-amino-4-chloro-6-(l-indolyl)pyrimidine with an aldehyde or ketone to form a 5,6-dihydroindolo[2,l-h]pteridine. In other embodiments, the method further comprises a nucleophilic substitution reaction. [0086] The invention also provides libraries comprising a plurality of different compounds of the invention. In a specific embodiment, a plurality is two or more, three or more, four or more, five or more, 10 or more, 15 or more, 20 or more or 50 or more compounds of the invention. In specific embodiments, each compound in the library is purified.

[0087] In a specific embodiment, the invention provides a library wherein each compound in a library is of formula I. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula II. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula III. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula IV. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula V. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula VI. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula VII. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula VIII. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula IX. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula X. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula XI. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula XII. In a specific embodiment, the invention provides a library wherein each compound in a library is of formula XIH. In the foregoing embodiments, the library can be any library of the invention.

[0088] Methods of screening the libraries of the invention are also provided. Any of the libraries can screened for pharmaceutically desirable activity. In a preferred embodiment, the library to be screened contains only compounds from a specific class of compounds, e.g., compounds prepared using the same reactions. The methods of screening libraries of the invention can be applied to an entire library of compounds, a subset of a library, or a single compound in a library.

[0089] In one embodiment, the invention provides a method of screening a library for a candidate compound with a therapeutic activity, comprising:

(a) conducting a biological assay in the presence of one or more compounds of a library of the invention; and

(b) determining activity measured by said biological assay; wherein a modulation of said activity relative to the activity in the absence of said compounds indicates a therapeutic activity of said one or more compounds. In a specific embodiment, the method further comprises repeating the assay with subsets of the compounds initially tested to identify the compound with modulatory activity. [0090] In one embodiment, the libraries are tested in kinase assays. In such embodiments, kinase activity in the presence of a library is compared to kinase activity in the absence of the library. In a specific embodiment, pharmaceutically desirable actiivty in an increase or inhibition of kinase activity. In a preferred embodiment, the invention provides methods for screening a library for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library with a kinase and a kinase substrate under conditions suitable for kinase activity;

(b) determining a first ratio of unphosphorylated substrate to phosphorylated substrate to give a first measure of kinase activity; and

(c) comparing said first ratio to a second measure of kinase activity provided by a second ratio of unphosphorylated substrate to phosphorylated substrate obtained in the absence of said compounds; wherein inhibition of kinase activity is indicative of a candidate kinase inhibitor. In preferred embodiments, the kinase is FLT3, KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I

[0091] hi another embodiment, the compounds are tested for cytotoxicity with cancer cells, e.g, by a method comprising contacting one or more compounds of the invention with a cancer cell, and determining if increased cancer cell death occurs relative to that in the absence of said compounds.

4. BRIEF DESCRIPTION OF THE FIGURES

[0092] FIG. 1 shows general reaction scheme I for the synthesis of pyrimidine-fused diazepine derivative compounds.

[0093] FIG. 2 shows reaction scheme II for the synthesis of pyrimidine-fused 5,6- dihydrobenzodiazepines via a Pictet-Spengler-like cyclization.

[0094] FIG. 3 shows the reaction scheme III for the synthesis of pyrimido[4,5- b][l ,4]benzothiazepines.

[0095] FIG. 4 shows reaction scheme IV for the synthesis of tetracyclic pyrirnidobenzodiazepines .

[0096] FIG. 5 shows reaction scheme V for the synthesis of indole-fused pteridines.

5. DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention relates to pyrimidine-fused benzodiazepines and pyrimidine-fused pteridines, and libraries of a plurality of such compounds. The invention provides methods of making pyrimidine-fused benzodiazepines derivative and pyrimidine- fused pteridine compounds. The various classes of compounds encompassed by the invention are described in detail below.

5.1 Pyrimidine-fused Diazepines and Deriviatives 5.1.1 Representative Compounds

[0098] In one embodiment, the invention provides a compound of formula:

[00100] wherein X = O, S, or NR;

[00101] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; [00102] R, R₁, R₂ = alkyl, aryl or a heterocycle. [00103] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle. [00104] In certain embodiments, R, R₁, R₂ = substituted alkyl, substituted aryl or substituted heterocycle.

[00105] In another embodiment, the invention provides a compound of formula:

[00107] wherein X = O, S, or NR;

[00108] Nu = R₄S, R₄O or NR₅R₆; [00109] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; [00110] R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [00111] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[00112] In certain embodiments, R, R₁, R₂, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle.

[00113] In another embodiment, the invention provides a compound of formula:

[00115] wherein X = O, S, or NR;

[00116] Nu = R₄S, R₄O or NR₅R₆; [00117] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; [00118] R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [00119] In certain embodiments, Nu = halogen.

[00120] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[00121] In certain embodiments, R, R₁, R₂, R₃, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle. [00122] In another embodiment, the invention provides a compound of formula: [00124] wherein X = O, S, or NR;

[00125] Nu = R₄S, R₄O or NR₅R₆; [00126] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; [00127] R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [00128] In certain embodiments, Nu = halogen. [00129] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[00130] In certain embodiments, R, R₁, R₂, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle. [00131] In another embodiment, the invention provides a compound of formula:

[00133] wherein X = O, S , or NR;

[00134] Nu = R₄S, R₄O or NR₅R₆; [00135] Y₁, Y₂, Y₃, and Y₄ = H, alkyl, aryl or a heteroatom; [00136] R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle. [00137] In certain embodiments, Nu = halogen.

[00138] In certain embodiments, Y₁, Y₂, Y₃, and Y₄ = substituted alkyl, substituted aryl, heterocycle or substituted heterocycle.

[00139] In certain embodiments, R, R₁, R₂, R₃, R₄, R₅, and R₆ = substituted alkyl, substituted aryl or substituted heterocycle. [00140] In another embodiment, the invention provides a compound of formula:

[00142] wherein:

[00143] R₇ = H, CH₃, or F;

[00144] R₈ = 4'-F-C₆H₄, 4'-CH₃-C₆H₄, or 4'-NO₂-C₆H₄.

[00145] In another embodiment, the invention provides a compound of formula:

[00147] wherein:

[00148] NHR₉R₁₀ is n-Bu-NH₂ or pyrrolidine.

[00149] In preferred embodiments, the compound is:

[00150] 4-chloro-ll-methyl-6-propylpyrimido[4,5-b][l,4]benzodiazepine;

[00151] 4-chloro-6, 1 l-dimethylpyrimido[4,5-&] [ 1 ,4]benzodiazepine;

[00152] 4-chloro-ll-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine;

[00153] 4-chloro- 11 -methyl-6-(4'-fluorophenyl)-pyrimido[4,5-/7] [ 1 ,4]benzodiazepine;

[00154] 4-chloro- 1 l-methyl-6-(4'-methyl-phenyl)-pyrimido[4,5-&] [ 1 ,4]benzodiazepine;

[00155] 4-chloro-ll-methyl-6-(4'-nitro-phenyl)-pyrimido[4,5-Z?][l,4]benzodiazepine;

[00156] 4-chloro-8, 11 -dimethyl-6-propylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

[00157] 4-chloro-6,8, 1 l-trimethylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

[00158] 4-chloro-8,ll-dimethyl-6-phenylpyrimido[4,5~b][l,4]benzodiazepine;

[00159] 4-chloro-8,ll-dimethyl-6-(4'-fluorophenyl)-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00160] 4-chloro-8,ll-dimethyl-6-(4'-methyl-phenyl)-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00161] 4-chloro-8,ll-dimethyl-6-(4'-nitro-phenyl)-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00162] 4-chloro-8-fluoro- 11 -methyl-6-propylpyrimido[4,5-b] [ 1 ,4]benzodiazepine; [00163] 4-chloro-8-fluoro-6, 11 -dimethylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

[00164] 4-chloro-8-fluoro-ll-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine;

[00165] 4-chloro-8- fluoro -1 l-methyl-6-(4'-fluorophenyl)-pyrimido[4,5- b][l,4]benzodia zepine;

[00166] 4-chloro-8- fluoro -11 -methyl-6-(4'-methylphenyl)-ρyrimido[4,5- b][l,4]benzodia zepine;

[00167] 4-chloro-8- fluoro -11 -methyl-6-(4'-nitrophenyl)-pyrimido[4,5-

£][l,4]benzodia zepine;

[00168] 4-hydroxy - 1 l-methyl-6-phenylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

[00169] 4-(butylamino)-8- fluoro -1 l-methyl-6-(4'-methyl-phenyl)-pyrimido[4,5- b][l,4] benzodiazepine; or

[00170] 4-(pyrrolidin-l-yl)-8- fluoro -1 l-methyl-6-(4'-methylphenyl)-pyrimido[4,5-

Z?][l,4] benzodiazepine.

5.1.2 Synthetic Methods

[00171] In one embodiment, compounds of the invention are synthesized according to

Scheme I. See FIG. 1. Each of the pyrimidine-fused benzodiazepine analogs represented as 2-6 can be scaffolds for the production of additional compounds. See Yang et al., 2005, Org. Lett., 7 , 1541 -1543, incorporated by reference herein in its entirety. [00172] The reaction from compound 1 to compound 2 involves cyclization. The key synthetic aspect of the synthesis of 4-chloro-pyrimido[4,5-b][l,4]benzodiazepines is the intramolecular Friedel-Crafts cyclization of 5-amino-4-(N-substituted)anilino-6- chloropyrimidine with either a carboxylic acid or its derivatives to construct the 4-chloro- pyrimido[4,5-£][l,4]benzodiazepine core. An acid or Lewis acid is typically employed as a catalyst. For example, PPA could be used for this purpose, combined with or followed by treatment with POCl₃ (Norman et al., Med. Chem. 2000, 43, 4288; Caron and Vazquez, Org. Chem. 2003, 68, 4104).

[00173] Subsequent nucleophilic substitution allows the introduction of one more diversity point in the target molecules. There are four points of diversity in 4-chloro- pyrimido[4,5-b][l,4]benzodiazeρines. For instance, the 4-chloro group can be easily converted to other groups by either a substitution reaction with a nucleophile (such as an amine, alcohol and phenol) or by Suzuki-Miyaura cross-coupling reactions with boronic acids (Chorvat et al., J. Med. Chem. 1999, 42, 833; Gundersen et al., J. Med. Chem. 2002, 45, 1383; Oh et al., Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345; Havelkova et al., Synlett 1999, 1145). The reaction from compound 2 to compound 3 involves a nucleophilic subsitution.

The reaction conditions employed for reaction step b) may generally include those used in nucleophilic substitution reactions, such as the substitution of a chloro group with an amine group, using a base as a catalyst.

[00174] Compound 3 can be coverted to either compound 4 or compound 5 using hydrogenation conditions or organometallic reagents. Hydrogenation conditions include H₂ /

Pd-C. Organometallic reagents include R₃MgCl or LiR₃, wherein R₃ is an alkyl, aryl, or heterocycle.

[00175] Compounds 4 and 5 can be converted to compound 6 with reactions such as amide formation (with an acid), alkylation (with an electrophile), or sulfonamide formation

(with a sulfonyl chloride).

5.2 Tricyclic pyrimidine-fused 5,6-dihydrobenzodiazepines 5.2.1 Representative Compounds

[00176] In one embodiment, the invention provides a compound of formula:

[00177]

[00178] wherein:

[00179] R Ri₁ == HH,, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle or halogen

[00180] R₂ = alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle.

[00181] In preferred embodiments, the compound is:

[00182] 4-Chloro- 11 -methyl-6-proρyl-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00183] 4-Chloro-l l-methyl-6-ethyl-5,6-dihydro-pyrimido[4,5-b] [l,4]benzodiazepine;

[00184] 4-Chloro- 11 -methyl-6-phenyl-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00185] 4-Chloro-ll-methyl-6-(4'-methyl-phenyl)-5,6-dihydro-ρyrimido[4,5- b] [ 1 ,4]benzodiazepine; [00186] 4-Chloro-ll-methyl-6-(4'-fluoro-ρhenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00187] 4-Chloro- 11 -methyl-6-(4'-nitro-ρhenyl)-5,6-dihydro-ρyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00188] 4-Chloro-8,ll-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5- b] [ 1 ,4]benzodiazepine;

[00189] 4-Chloro-8, 1 l-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5- b\ [ 1 ,4]benzodiazepine;

[00190] 4-Chloro-8,l l-dimethyl-6-phenyl-5,6-dihydro-ρyrimido [4,5- b] [ 1 ,4]benzodiazepine;

[00191] 4-Chloro-8, 1 l-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00192] 4-Chloro-8, 1 l-dimethyl-6-(4'-fluoro-phenyl)-5,6-dihydro-ρyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00193] 4-Chloro-8,ll-dimethyl-6-(4'-nitro-phenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00194] 4-Chloro-9, 1 l-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5- b] [ 1 ,4]benzodiazepine;

[00195] 4-Chloro-9, 1 l-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5- b] [ 1 ,4]benzodiazepine;

[00196] 4-Chloro-9, 1 l-dimethyl-6-phenyl-5,6-dihydro-pyrimido [4,5- b] [ 1 ,4]benzodiazepine;

[00197] 4-Chloro-9,ll-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00198] 4-Chloro-9,ll-dimethyl-6-(4'-fluoro-phenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine;

[00199] 4-Chloro-9,ll-dimethyl-6-(4'-nitro-phenyl)-5,6-dihydro-pyrimido[4,5- b][l .^benzodiazepine; or

[00200] 4-Chloro-8-fluoro-l l-methyl-6-phenyl-5,6-dihydro pyrimido[4,5- b] [ 1 ,4]benzodiazepine.

5.2.2 Synthesis

[00201] Tricyclic pyrimidine-fused 5,6-dihydrobenzodiazepines are prepared via a

Pictet-Spengler-like cyclization with an aldehyde in place of the carboxylic acid in Friedel- Craft reaction. See Che et al., 2006, Tetrahedron, 62, 2563-2568, incorporated by reference herein in its entirety. The reaction is based on intra-molecular electrophilic substitution of the phenyl ring of 5-amino-6-chloro-4-(N-methylanilino)pyrimidine by the iminium intermediate formed with an aldehyde in one pot. The starting pyrimidines are readily prepared by a two- step process from commercially available 5-nitro-4,6-dichloro-pyrimidine and N- methylanilines (Norman et al., J. Med. Chem. 2000, 43, 4288; Caron and Vazquez, Org. Chem. 2003, 68, 4104). The resulting product may be further transformed by subsequent nucleophilic substitution of the chloro atom.

5.3 Tricyclic Pyrimido[4,5-fe][l,4]benzothiazepines 5.3.1 Representative Compounds

[00202] hi one embodiment, the invention provides a compound of formula:

[00204] wherein:

[00205] R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, halogen, heteroatom or alkoxy; and

[00206] R₃ = alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted hetercycle.

[00207] In another embodiment, the invention provides a compound of formula:

[00209] wherein:

[00210] R₂ = H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle.

[00211] hi another embodiment, the invention provides a compound of formula:

[00213] wherein:

[00214] R₂ = H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle;

[00215] R₄ , R₅ = H, alkyl substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle, or R₄ and R₅ together form a cyclic alkyl.

[00216] In preferred embodiments, the compound is:

[00217] 4-(Phenylthio)-6-phenylpyrimido[4,5-Z?][l,4]benzothiazepine;

[00218] 4-(Phenylthio)-6-(pyridin-3-yl)-pyrimido[4,5-Z?] [ 1 ,4]benzothiazepine;

[00219] 4-(Phenylthio)-6-p-tolylpyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00220] 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00221] 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5-έ][l,4]benzothiazepine;

[00222] 4-(Phenylthio)-6-(p-nitrophenyl)pyrimido[4,5-Z?][l,4]benzothiazepine;

[00223] 4-(Phenylthio)-6-(m-fluorophenyl)pyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00224] 4-(Phenylthio)-6-methylpyrimido[4,5-&] [ 1 ,4]benzothiazepine;

[00225] 4-(Phenylthio)-6-propylpyrimido[4,5-b][l,4]benzothiazepine;

[00226] 4-(Phenylthio)-6-benzylpyrimido[4,5-Z?][l,4]benzothiazepine;

[00227] 4-(p-Tolylthio)-6-phenyl-8-methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00228] 4-(p-Tolylthio)-6-(pyridin-3-yl)-8-methylpyrimido[4,5-b][l,4]benzothiazepine;

[00229] 4-(p-Tolylthio)-6-p-tolyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine;

[00230] 4-(p-Tolylthio)-6-(p-fluorophenyl)-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00231] 4-(p-Tolylthio)-6-propyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine;

[00232] 4-(p-Tolylthio)-6-benzyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine;

[00233] 4-(p-Chloro-phenylthio)-6-phenyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00234] 4-(ρ-Chloro-phenylthio)-6-ρ-tolyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00235] 4-(ρ-Chloro-ρhenylthio)-6-(ρyridin-3-yl)-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; [00236] 4-(p-Chloro-phenylthio)-6-benzyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00237] 4-(p-Methoxyphenylthio)-6-phenyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00238] 4-(p-Methoxyphenylthio)-6-benzyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00239] 4-(Phenylsulfinyl)-6-phenylpyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00240] 4-(p-Tolylsulfinyl)-6-phenyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine;

[00241] 4-(p-Chlorophenylsulfinyl)-6-phenyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine;

[00242] 4-(n-Butylamino)-6-phenylpyrimido[4,5-b][l,4]benzothiazepine;

[00243] 4-(Pyrrolidin- 1 -yl)-6-phenylpyrimido[4,5-b] [ 1 ,4]benzothiazepine;

[00244] 4-(n-Butylamino)-6-phenyl-8-methylpyrimido[4,5-^][l,4]benzothiazepine; or

[00245] 4-(n-Butylamino)-6-phenyl-8-chloropyrimido[4,5-b][l,4]benzothiazepine.

5.3.2 Synthesis

[00246] Tricyclic pyrimido[4,5-b][l,4]benzothiazepines were prepared by the procedure shown in Scheme III. See Fu et al., 2005, J. Org. Chem., 70, 10810-10816, incorporated by reference herein in its entirety. In general, these compound are prepared from 5-amino-4,6-bis-(arylthio)pyrimidines and carboxylic acids via Bischler-Napieralski- type reactions. Precursor compounds 6 in Scheme 3 were prepared by treating commercially available 5-amino-4,6-dichloropyrimidine 1 with thiophenol or its analog in the presence of base to give 5-amino-4,6-bisphenylthiopyrimidine 6 or an analogue. Additional chemical modification, such as o-methylation, was performed in some instances. The desired cyclization products 2 were prepared according to a Bischler-Napieralski-type reactions using an appropriate acid in PPA/POC1₃. Analogues of 5-amino-4,6-bisphenylthiopyrimidine 6 can be reacted with a variety of carboxylic acids or derivatives to produce structural diversity among cyclization products 2. The phenylthio group of 2 can be oxidized to produce a number of sulfoxide analogues 8. Nucleophilic substitution of the sulfoxide group generated can add additional chemical diversity, as illustrated by compounds 3.

5.4 Tetracyclic Pyrimidine-Fused Benzodiazepines 5.4.1 Representative Compounds

[00247] In one embodiment, the invention provides a compound of formula:

[00249] wherein:

[00250] X = Cl, R₃R₄N, R₃O, R₃S, or aryl;

[00251] R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl or R₁ and R₂ together form a cyclic alkyl; and

[00252] R₃, R₄ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle.

[00253] In preferred embodiments, the compound is:

[00254] 8-chloro-6-ethyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene;

[00255] 8-chloro-6-propyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo [c,d,h] azulene;

[00256] 8-chloro-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h] azulene;

[00257] 8-chloro-6-(o-methoxyphenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00258] 8-chloro-6-(p-nitro-phenyl)-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00259] 8-chloro-6-(p-fluoro-phenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00260] 8-chloro-6-styryl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene;

[00261] 8-chloro-6-propenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00262] S-chloro-β^-dimethyl-l^^J-tetrahydro^^Jl.llb-tetraaza- dibenzo[c,d,h] azulene;

[00263] 8-chloro-6-ethyl-6-methyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h] azulene;

[00264] 8-chloro-6-methyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00265] 8-chloro-6-cyclohexylidene-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h] azulene; [00266] 8-chloro-6-methyl-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00267] S-chloro-ό-methyl-δ-φ-methoxyphenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,Hb- tetraaza-dibenzo[c,d,h]azulene;

[00268] 8-chloro-6-methyl-6-(p-nitro-phenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene;

[00269] 8-chloro-6-ethyl-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo [c,d,h] azulene;

[00270] 8-chloro-6-ethyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulen-

6-carbonate acid;

[00271] 9-chloro-7-ethyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[ 1 ,2,3-de]naphthalene;

[00272] 9-chloro-7-pyproyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene;

[00273] 9-chloro-7-phenyl-2,3 ,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene;

[00274] 9-chloro-7-(p-methylphenyl)-2,3 ,7,8-tetrahydro- 1H-8,10, 12, 12b-tetraaza- benzo[4,5]cyclohepta[ 1 ,2,3-de]naphthalene;

[00275] 9-chloro-7-(p-nitro-phenyl)-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene;

[00276] 9-chloro-7-styryl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene;

[00277] 9-chloro-7,7-dimethyl-2,3 ,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene;

[00278] N-(n-butyl)-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulen-8-amine;

[00279] N-phenyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulen-8-amine;

[00280] 8-(morpholin-l-yl)-6-phenyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulene;

[00281] 8-(pyrrolidin-l-yl)-6-ρropyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00282] 8-butoxyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene; [00283] 8-butoxy-6-phenyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza~ dibenzo[c,d,h] azulene;

[00284] 8-benzylthio-6-ρroρyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h] azulene;

[00285] 8-benzylthio-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene;

[00286] 8-phenylthio-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene;

[00287] 8-phenylthio-6-ρhenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene;

[00288] 8-ρhenyl-6-ρroρyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; or

[00289] 8-phenyl-6-ρhenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene.

5.4.2 Synthesis

[00290] Tetracyclic pyrimidine-fused benzodiazepines are benzodiazepines fused with heterocycles such as pyrimidines, indolines and tetrahydroquinolines. See Zheng et al., "Design and Synthesis of a Tetracyclic Pyrimidine-fused Benzodiazepeine Library", In press. The synthetic strategy is based on an electrophilic cyclization reaction involving an iminium intermediate formed by the corresponding aminopyrimidine with a carbonyl compound. The presence of a cyclic indoline or tetrahydroquinoline ring significantly increases the reactiviy of the cyclization compared to open anilino analogs. The chloro group can be replaced with a nucleophile, such as boronic acid, amine, alcohol or thiol, or transition metal catalyzed cross coupling reactions.

[00291] A synthetic strategy starting with pyrimidine 1 in Scheme IV involves saturation of the pyrrole ring of indole moiety (indoline 1) leading to cyclization at the phenyl ring, leading to scaffolds of tetracyclic pyrimidine-fused benzodiazepines 2. Cyclization reactions occur with a wide range of adehydes, including aliphatic and aromatic aldehydes, and aliphatic ketones. The cyclization reactions are expected to follow a similar pathway as the Pictet-Spengler isoquinoline synthesis (Cox et al., Chem Rev 1995, 95, 1797). [00292] The same strategy can be applied to tetrahydroquinolinyl or similar systems, generating a library (compounds 3-6 in Scheme IV) with at least eight diversity points. The chloro group in compounds 2 presents an excellent opportunity to introduce additional diversity points and reacts well with various nucleophiles and coupling reactions. Representive nucleophiles include amines, under either acidic conditions of coned, aq. HCl (for rc-BuNH₂, aniline and morpholine; Taddei et al., Eur J Org Chem 2005, 939) or in the presence Of Et₃N (for pyrrolidine) to give the amine substituted products. Other nucleophiles with heteroatoms such alcohols and thiols are also known to react with 6-chloropurines, exemplified by n-butanol, benzylthiol and thiophenol under basic conditions to give the corresponding rc-butyloxy, benzylsulfanyl or phenylsulfanyl substituted pyrimidobenzediazopines (Lavecchia et al., Tetrahedron Lett 2005, 46, 5851). Introduction of carbon substitutents to replace the chloro further expands the scope of the current library. In one embodiment, under non-optimized Suzuki-Miyaura cross-coupling conditions (Nyerges et al., Tetrahedron 2005, 61, 8199; Havelkova et al., Synlett 1999, 7, 1145), compounds can reacted with phenylboronic acid to yield aryl substituted products.

5.5 Heterocyclic Scaffolds Consisting of Indole-Fused Pteridines 5.5.1 Representative Compounds

[00293] In one embodiment, the invention provides a compound with formula:

[00295] wherein:

[00296] R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle; or R₁ and R₂ together form a cyclic alkyl;

[00297] R₃ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle;

[00298] X = Cl or NHR; and

[00299] R = alkyl, substituted alkyl, aryl, substituted aryl.

[00300] In preferred embodiments, the compound is:

[00301] 4-chloro-6-ethyl-5,6-dihydroindolo[2, l-h]pteridine;

[00302] 6-butyl-4-chloro-5,6-dihydroindolo[2,l-h]ρteridine;

[00303] 4-chloro-6-phenyl-5,6-dihydroindolo[2, l-h]pteridine;

[00304] 4-chloro-6-(4'-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine; [00305] 4-chloro-6-(4'-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00306] 4-chloro-6-(3 \4'-dichlorophenyl)-5,6-dihydroindolo[2, 1 -h]pteridine;

[00307] 4-chloro-6-(o-methoxyphenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00308] 4-chloro-6-(o-chlorophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00309] 4-chloro-6-(o-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00310] 4-chloro-6-(m-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00311] 4-chloro-6-ethyl-6-methyl-5,6-dihydroindolo[2,l-h]pteridine;

[00312] 4-chloro-6-methyl-6-propyl-5,6-dihydroindolo[2,l-h]pteridine;

[00313] 4-chloro-6-cyclohexylidene-5,6-dihydroindolo[2,l-h]pteridine;

[00314] 4-chloro-6-methyl-6-phenyl-5,6-dihydroindolo[2,l-h]pteridine;

[00315] 4-chloro-6-methyl-6-(p-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00316] 4-chloro-6-methyl-6-(p-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

[00317] 4-chloro-7-ethoxycarbonyl-6-phenyl-5,6-dihydroindolo[2,l-h]pteridine;

[00318] 4-chloro-7-ethoxycarbonyl-6-(p-methylphenyl)-5,6-dihydroindolo[2,l- h]pteridine;

[00319] 4-chloro-7-ethoxycarbonyl-6-(p-fluorophenyl-5,6-dihydroindolo[2,l- hjpteridine;

[00320] 4-chloro-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l-h]pteridine;

[00321] 4-chloro-6-cyclohexylidene-7-ethoxycarbonyl-6-propyl-5,6- dihydroindolo[2,l-h]pteridine;

[00322] N-butyl-6-cyclohexylidene-indolo[2,l-h]pteridin-4-amine; or

[00323] N-butyl-6-(p-methylphenyl)-indolo[2,l-h]pteridin-4-amine.

5.5.2 Synthesis

[00324] Expanding on the above-mentioned cyclization reaction of pyrimidines leading to pyrimidine-fused benzodiazepines, a synthesis was designed leading to a novel heterocyclic scaffold consisting of indole-fused pteridines. See Zheng et al., 2005, J. Comb. Chem., 7, 813-815. 5-amino-4-chloro-6-(l-indolyl)pyrimidine 2 can be reacted with various aldehydes and ketones to obtain indole-fused pteridines.

[00325] The key indole-substituted pyrimidine, compound 2, is synthesized in a two- step sequence. Treatment of commercially available 4,6-dichloride-5-aminopyrimidine with indoline according to a literature procedure (Tanji et al., Chem. Pharm. Bull. 1992, 40, 227) gave pyrimidine 1. Subsequent oxidation of the indoline moiety to its corresponding indole was achieved (Yamagucm et al., Bull. Chem. Soc. Jpn. 1989, 62, 4066) to afford the key aminopyrimidine, compound 2.

[00326] Additional synthetic steps were designed such that a 5-amino-6-(l- indolinyl)pyrimidine 1 underwent an electrophilic cyclization with an aldehyde or a ketone at the phenyl ring while a 5-amino-6-(l-indolyl)pyrimidine 2 cyclized onto its electron-rich pyrrole ring.

[00327] The indole moiety of compound 3 presents five additional diversity points and can be reacted further to obtain additional compounds. The 4-Cl group also provides an additional diversity point.

5.6 Definitions

[00328] "Acid derivative" refers to a molecule, ion or functional group containing a formyl group (HCO-) or acyl group (RCO-) that is bonded to another atom or functional group, thereby forming one of a range of carbonyl-containing molecules, ions or functional groups such as aldehydes, ketones, acid halides, amides, esters, anhydrides, and thiocarboxylic acids.

[00329] "Alkyl" refers to a cyclic, branched, or straight chain chemical group containing only carbon and hydrogen, such as methyl, pentyl, adamantyl. Typically, alkyl groups will comprise 1 to 12 carbon atoms, preferably 1 to 10, and more preferably 1 to 8 carbon atoms. In some embodiments, an alkyl is at least partially unsaturated, such as an alkenyl or alkynyl.

[00330] "Substituted alkyl" refers to an alkyl substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, amino, nitro, hydroxyl, mercapto, carboxy, carboxy derivative, benzyloxy, heterocycle, substituted heterocycle, aryl, substitued aryl, heteroaryl, substituted heteroaryl, and other substituents.

[00331] "Aryl" refers to a monovalent unsaturated aromatic carbocyclic group having a single-ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).

[00332] "Substituted aryl" refers to an aryl which is substituted with amino, hydroxyl, lower alkyl, alkoxy, chloro, halo, mercapto, and other substituents.

[00333] "Heteroatom" refers to an atom other than carbon. Typically, a heteroatom is oxygen, nitrogen or fluoride.

[00334] "Heterocycle" refers to optionally substituted five-membered to eight- membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms. These five-membered to eight-membered rings may be saturated, partially unsaturated or fully unsaturated (i.e., heteroaryl). Preferred heterocyclic rings include pyridinyl, morpholino, piperidinyl, piperazinyl, 2-amino-imidazoyl, tetrahydrofurano, pyrrolo, tetrahydrothiophen-yl, hexylmethyleneimino and heptylmethyleneimino.

[00335] "Substituted heterocycle" refers to a heterocycle substituted with, for example, one or more, and preferably one or two, substituents (which are the same or different) which can be halogen, hydroxy, protected hydroxy, cyano, nitro, C₁ to C₁₂ alkyl, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy, C₁ to C₁₂ acyl, C₁ to C₁₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted)amino carboxamide, protected carboxamide, N-(C₁ to C₁₂ alkyl)carboxamide, protected N-(C₁ to C₁₂ alkyl)carboxamide, N, N-di(Q to C₁₂ alkyl)carboxamide, trifluoromethyl, N~((Ci to C₁₂ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino, heterocycle or substituted heterocycle groups.

[00336] "Nucleophile" refers to an electron-rich molecule, ion, molecular fragment or functional group, typically containing one or more heteroatoms, such as S, O or N, which may be used, for example, in a substitution reaction to displace a leaving group. Typical examples of nucleophiles include amines, alcohols, phenols and thiols. [00337] A library of the invention can contain a plurality of the (different) compounds of the invention, hi specific embodiments, a plurality is two or more, three or more, four or more, or five or more of the compounds of the invention, hi another embodiment of the invention, the plurality is ten or more of the compounds of the invention. In yet another embodiment of the invention, the plurality is fifteen or more, twenty or more, twenty-five or more, fifty or more of the above-described compounds. If desired, a plurality is 100,000 or more, or even 1,000,000 or more, of the above-described compounds.

5.7 Pharmaceutical Compositions

[00338] The compounds of the invention may be used in pharmaceutical compositions as anti-inflammatory, anti-anginal, anti-secretory (gastric acid), anti-muscarinic, antiarrhythmic, anti-depressant, analgesic, anti-psychotic, anxiolytic, anti-convulsant, antineoplastic, anti-mitotic, anti-hypertensive, anti-allergic, anti-asthmatic, antibiotic, antidiabetic, anti-histamine, cadiotonic, and spasmolytic agents, GABA A/benzodiazepine receptor complex related drugs, microtubule inhibitors, endothelin antagonists, muscarinic (M2) antagonists, vasopressin Vl antagonists, vasopressin V2 antagonists, lipoxygenase inhibitors, and treatment for septic shock, irritable bowel syndrome, urinary incontinence, renal failure and neurogenic pain.

[00339] For preparing pharmaceutical compositions containing compounds of the invention, inert, pharmaceutically acceptable carriers are used. The pharmaceutical carrier can be either solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.

[00340] A solid carrier can be one or more substances which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.

[00341] In powders, the carrier is generally a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the ^■shape and size desired.

[00342] For preparing pharmaceutical composition in the form of suppositories, a low- melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify. [00343] Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter and the like. [00344] The pharmaceutical compositions can include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it. In a similar manner, cachets are also included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. [00345] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, or suspensions, and emulsions suitable for oral administration. Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.

[00346] Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.

[00347] Aqueous solutions for oral administration can be prepared by dissolving the active compound in water and adding suitable flavorants, coloring agents, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.

[00348] Preferably, the pharmaceutical composition is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active pyrimidine-fused benzodiazepine compound. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

[00349] As pharmaceutical compositions for treating a disease or disorder as described herein, the compounds of the present invention are generally in a pharmaceutical composition so as to be administered to a subject at dosage levels of from 0.7 to 7000 mg per day, and preferably 1 to 500 mg per day, for a normal human adult of approximately 70 kg of body weight, this translates into a dosage of from 0.01 to 100 mg/kg of body weight per day. The specific dosages employed, however, can be varied depending upon the requirements of the patient, the severity of the condition being treated, and the activity of the compound being employed. The determination of optimum dosages for a particular situation is within the skill of the art.

5.8 Assays

[00350] Several methods have been developed in recent years to screen libraries of compounds to identify the compounds having the desired characteristics. Typically, where a compound exhibits a dissociation constant of 10^"6 or less when combined with the targeted enzyme or receptor, the compound is thought to demonstrate a specific interaction with the enzyme or receptor. Methods for isolating library compound species that demonstrate desirable affinity for a receptor or enzyme are well-known in the art. [00351] For example, an enzyme solution may be mixed with a solution of the compounds of a particular library under conditions favorable to enzyme-ligand binding. See Bush et al. (1993) Antimicrobial Agents and Chemotherapy 37:851-858, and Daub et al. (1989) Biochemistry 27:3701-3708. Specific binding of library compounds to the enzyme may be detected by any of the numerous enzyme inhibition assays which are well known in the art. Compounds which are bound to the enzyme may be readily separated from compounds which remain free in solution by applying the solution to a Sephadex G-25 gel filtration column. Free enzyme and enzyme-ligand complexes will pass through the column quickly, while free library compounds will be retarded in their progress through the column. The mixture of enzyme-ligand complex and free enzyme can then be treated with a powerful denaturing agent, such as guanidinium hydrochloride or urea, to cause release of the ligand from the enzyme. The solution can then be injected onto an HPLC column (for example, a Vydac C-4 reverse-phase column, eluted with a gradient of water and acetonitrile ranging from 0% acetonitrile to 80% acetonitrile). Diode array detection can provide discrimination of the compounds of the combinatorial library from the enzyme. The compound peaks can then collected and subjected to mass spectrometry for identification. [00352] Other assays suitable for testing the compounds of the invention are well known to one of skill in the art. For example, the compounds can be tested against cancer cells or cell lines in vitro for cytotoxicity, e.g, by a method comprising contacting one or more compounds of the invention with a cancer cell, and determining if increased cancer cell death occurs relative to that in the absence of said compounds.

[00353] In a preferred embodiment, the compounds are tested in kinase assays as described in greater detail below.

[00354] GENERAL PROCEDURE FOR THE IN VITRO EVALUATION OF

KINASE INHIBITORS.

[00355] The Kinase Assays will be performed using the SelectScreen™ Kinase

Profiling Service from Invitrogen Corporation (501 Charmany Drive, Madison, Wisconsin, 53719). The following general protocol will be followed to assay test compounds for inhibitory activity against a number of tyrosine, serine and threonine kinases. It would be anticipated that the compounds of this invention would initially be assayed against the following kinases: FLT3, KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) and RAF-I. Specific details for these kinases along with a number of other kinases are detailed following the general protocol described below (taken from Invitrogen Publication "Screening Protocol and Assay Conditions" rev 12/15/05): [00356] ASSAY THEORY. The Z'-LYTE™ biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores — one at each end — that make up a FRET pair. In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site- specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A ratiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 run, is used to quantitate reaction progress, as shown in the equation below: [00357] Emission Ratio = Coumarin Emission (445 nrn)/Fluorescein Emission (520 nm)

[00358] A significant benefit of this ratiometric method for quantitating reaction progress is the elimination of well-to-well variations in FRET-peptide concentration and signal intensities. As a result, the assay yields very high Z '-factor values (>0.7) at a low percent phosphorylation. Both cleaved and uncleaved FRET-peptides contribute to the fluorescence signals and therefore to the Emission Ratio. The extent of phosphorylation of the FRET-peptide can be calculated from the Emission Ratio. The Emission Ratio will remain low if the FRET-peptide is phosphorylated (i.e., no kinase inhibition) and will be high if the FRET-peptide is non-phosphorylated (i.e., kinase inhibition). [00359] ASSAY CONDITIONS. All Test Inhibitor Compounds will be initially diluted to a IOOX concentration in 100% DMSO. The IOOX concentration will then diluted to a 4X working concentration in Kinase Buffer (50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA). The Test Inhibitor Compounds will be in 1% DMSO final in the well.

[00360] Peptide/Kinase Mixtures. All Peptide/Kinase Mixtures will be diluted to a 2X working concentration in the appropriate Kinase Buffer (see Kinase Specific Assay Conditions for specific descriptions).

[00361] ATP Solution. All ATP Solutions will be diluted to a 4X working concentration in Kinase Buffer (50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA). [00362] Development Reagent Solution. The Development Reagent will be diluted in

Development Buffer (see Kinase-Specific Assay Conditions - Direct and Cascade for specific descriptions).

[00363] Assay Protocol

[00364] 1. 2.5 μL of the 4X Test Compound Solution will be added to a barcoded

Corning, low volume NBS, 384- well plate (Corning Cat. # 3676).

[00365] 2. 5 μL of the 2X Peptide/Kinase Mixture will be added to the plate.

[00366] 3. 2.5 μL of the 4X ATP Solution will be added to the plate.

[00367] 4. Assay plate will be shaken on a plate shaker for 30 seconds.

[00368] 5. Assay plate will be incubated for 60 minutes at room temperature.

[00369] 6. 5 μL of the Development Reagent Solution will be added to the plate.

[00370] 7. Assay plate will be shaken on a plate shaker for 30 seconds.

[00371] 8. Assay plate will be incubated for 60 minutes at room temperature.

[00372] 9. Assay plate will be read on fluorescence plate reader and the data is analyzed.

[00373] A ASSAY CONTROLS. The following controls will be made for each indiyidual kinase and will be located on the same plate as the kinase: [00374] 0% Phosphorylation Control (100% Inhibition Control). The maximum

Emission Ratio will be established by the 0% Phosphorylation Control (100% Inhibition Control), which will contain no ATP and therefore will exhibit no kinase activity. This control will yield 100% cleaved peptide in the Development Reaction. [00375] 100% Phosphorylation Control. The 100% Phosphorylation Control, which will consist of a synthetically phosphorylated peptide of the same sequence as the peptide substrate, will be designed to allow for the calculation of percent phosphorylation. This control will yield a very low percentage of cleaved peptide in the Development Reaction. The 0% Phosphorylation and 100% Phosphorylation Controls will allow one to calculate the percent Phosphorylation achieved in a specific reaction well. Control wells will not include any kinase inhibitors.

[00376] 0% Inhibition Control. The minimum Emission Ratio in a screen will be established by the 0% Inhibition Control, which will contain active kinase. This control will be designed to produce a 10-50% phosphorylated peptide in the Kinase Reaction and to yield 60-80% cleaved peptide in the Development Reaction.

[00377] Known Inhibitor. A known inhibitor control standard curve, 10 point titration, will be run for each individual kinase on the same plate as the kinase to ensure the kinase will be inhibited within an expected IC50 range previously determined as well as R2 and

Hillslope values within appropriate ranges.

[00378] The following controls will be prepared for each concentration of Test

Compound assayed:

[00379] Development Reaction Interference. The Development Reaction Interference will be established by comparing the Test Compound Control wells that do not contain ATP versus the 0% Phosphorylation Control (which will not contain the Test Compound).

[00380] Test Compound Fluorescence Interference. The Test Compound Fluorescence

Interference will be determined by comparing the Test Compound Control wells that do not contain the Kinase/Peptide Mixture (zero peptide control) versus the 0% Inhibition Control.

[00381] ASSAY DATA ANALYSIS

FI = Fluorescence Intensity C₁OO¹T₀ = Average Coumarin emission signal of the 100% Phos. Control Co% = Average Coumarin emission signal of the 0% Phos. Control Fioo% = Average Fluorescein emission signal of the 100% Phos. Control Fo_% = Average Fluorescein emission signal of the 0% Phos. Control

DRI = Development Reaction Interference

TCFI = Test Compound Fluorescence Intereference

[00382] Graphing Software

[00383] SelectScreen™ Kinase Profiling Service uses XLfit from IDBS. The dose response curve will be curve fit to model number 205 (sigmoidal dose-response model). If the bottom of the curve does not fit between -20% & 20% inhibition, it will be set to 0% inhibition. If the top of the curve does not fit between 70% and 130% inhibition, it will be set to 100% inhibition.

[00384] KINASE-SPECEFIC ASSAY DETAILS (Direct Format)

[00385] ABLl

[00386] The 2X ABLl / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.4 - 2.8 ng ABLl and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added.

[00387] ABLl E255K

[00388] The 2X ABLl E255K / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.8 - 5.2 ng ABLl E255K and 2 μM Tyr 02 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added.

[00389] ABLl G250E

[00390] The 2X ABLl G250E / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.8 - 7.9 ng ABLl G250E and 2 μM Tyr 02 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A is added.

[00391] ABLl T3151

[00392] The 2X ABLl T3151 / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.4 - 8.2 ng ABLl T315I and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00393] ABLl Y253F

[00394] The 2X ABLl Y253F / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.7 - 5.2 ng ABLl Y253F and 2 μM Tyr 02 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00395] ABL2 (Arg)

[00396] The 2X ABL2 (Arg) / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.4 - 5.3 ng ABL2 (Arg) and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added.

[00397] ACVRlB (ALK4)

[00398] The 2X ACVRlB (ALK4) / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnC12, 1 mM EGTA, 2 mM DTT, 0.02%

NaN3. The final 10 μL Kinase Reaction will consist of 10.0 - 62.2 ng ACVRlB (ALK4) and

2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 5 mM MgC12, 5 mM

MnC12, 1 mM EGTA, 1 mM DTT, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00399] ADRBKl (GRK2)

[00400] The 2X ADRBKl (GRK2) / Ser/Thr 16 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 11.0 - 121.2 ng ADRBKl (GRK2) and 2 μM Ser/Thr 16

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00401] ADRBK2 (GRK3)

[00402] The 2X ADRBK2 (GRK3) / Ser/Thr 16 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 9.9 - 89.0 ng ADRB K2 (GRK3) and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour

Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00403] AKTl (PKB alpha) [00404] The 2X AKTl (PKB alpha) / Ser/Thr 06 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.5 - 8.6 ng AKTl (PKB alpha) and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour

Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00405] AKT2 (PKB beta)

[00406] The 2X AKT2 (PKB beta) / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.0 - 40.0 ng AKT2 (PKB beta) and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRLF-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00407] AKT3 (PKB gamma)

[00408] The 2X AKT3 (PKB gamma) / Ser/Thr 06 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.4 - 8.3 ng AKT3 (PKB gamma) and 2 μM Ser/Thr 06

Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00409] ALK

[00410] The 2X ALK / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 20.3 - 243.5 ng ALK and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00411] AURKB (Aurora B)

[00412] The 2X AURKB (Aurora B) / Ser/Thr 01 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 11.1 - 155.4 ng AURKB (Aurora B) and 2 μM Ser/Thr 01

[00413] BLK [00414] The 2X BLK / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.5 - 4.5 ng BLK and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00415] BMX

[00416] The 2X BMX / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.8 85.0 ng BMX and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00417] BTK

[00418] The 2X BTK / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.0 - 15.0 ng BTK and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00419] CAMKlD (CaMKI delta)

[00420] The 2X CAMKlD (CaMKI delta) / Ser/Thr 10 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml Calmodulin, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 2.1 - 40.0 ng CAMKlD (CaMKI delta) and 2 μM Ser/Thr 10 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, 10 μg/ml Calmodulin, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added. [00421] CAMK2A (CaMKII alpha)

[00422] The 2X CAMK2A (CaMKII alpha) / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml Calmodulin, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.3 - 4.8 ng CAMK2A (CaMKII alpha) and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, 10 μg/ml Calmodulin, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00423] CAMK2B (CaMKII beta) [00424] The 2X CAMK2B (CaMKII beta) / Ser/Thr 10 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml

Calmodulin, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.8 - 7.0 ng

CAMK2B (CaMKII beta) and 2 μM Ser/Thr 10 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, 10 μg/ml Calmodulin, 0.01% NaN3.

After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development

Reagent A will be added.

[00425] CAMK2D (CaMKII delta)

[00426] The 2X CAMK2D (CaMKII delta) / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml

Calmodulin, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.1 - 0.8 ng

CAMK2D (CaMKII delta) and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01%

After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development

Reagent A will be added.

[00427] CAMK4 (CaMKIV)

[00428] The 2X CAMK4 (CaMKIV) / Ser/Thr 10 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml Calmodulin,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 7.5 - 67.3 ng CAMK4

(CaMKIV) and 2 μM Ser/Thr 10 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM

MgC12, 500 μM EGTA, 2 mM CaC12, 10 μg/ml Calmodulin, 0.01% NaN3. After the 1 hour

[00429] CDKl/cyclin B

[00430] The 2X CDKl/cyclin B / Ser/Thr 12 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.3 - 3.5 ng CDKl/cyclin B and 2 μM Ser/Thr 12 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:4096 dilution of Development Reagent A will be added.

[00431] CDK2/cyclin A

[00432] The 2X CDK2/cyclin A / Ser/Thr 12 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.3 - 30.5 ng CDK2/cyclin A and 2 μM Ser/Thr 12 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:4096 dilution of Development Reagent A will be added. [00433] CDK5/p35

[00434] The 2X CDK5/p35 / Ser/Thr 12 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.2 - 1.0 ng CDK5/p35 and 2 μM Ser/Thr 12 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:4096 dilution of Development Reagent A will be added. [00435] CHEKl (CHKl)

[00436] The 2X CHEKl (CHKl) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, ImM EGTA. The final 10 μL Kinase Reaction will consist of 4.0 - 115.2 ng CHEKl (CHKl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00437] CHEK2 (CHK2)

[00438] The 2X CHEK2 (CHK2) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 6.0 - 400.0 ng CHEK2 (CHK2) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00439] CLKl

[00440] The 2X CLKl / Ser/Thr 09 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 16.0 - 140.0 ng CLKl and 2 μM Ser/Thr 09 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00441] CLK2

[00442] The 2X CLK2 / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.8 - 21.6 ng CLK2 and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added. [00443] CSFlR (FMS) [00444] The 2X CSFlR (FMS) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.2 - 67.3 ng CSFlR (FMS) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00445] CSK

[00446] The 2X CSK / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.0 - 26.0 ng CSK and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added.

[00447] CSNKlAl (CKl alpha 1)

[00448] The 2X CSNKlAl (CKl alpha 1) / Ser/Thr 11 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 2 mM

DTT. The final 10 μL Kinase Reaction will consist of 16.7 - 107.0 ng CSNKlAl (CKl alpha

1) and 2 μM Ser/Thr 11 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:16 dilution of Development Reagent B will be added.

[00449] CSNKlD (CKl delta)

[00450] The 2X CSNKlD (CKl delta) / Ser/Thr 11 Peptide Mixture will be prepared in 50 mM Tris pH 8.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 17.2 - 138.2 ng CSNKlD (CKl delta) and 2 μM

Ser/Thr 11 Peptide in 50 mM Tris / HEPES pH 8.0, 0.01% BRU-35, 10 mM MgC12, 1 mM

EGTA, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:16 dilution of

Development Reagent B will be added.

[00451] CSNKlE (CKl epsilon)

[00452] The 2X CSNKlE (CKl epsilon) / Ser/Thr 11 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 6.0 - 40.0 ng CSNKlE (CKl epsilon) and 2 μM Ser/Thr 11

Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 : 16 dilution of Development Reagent B will be added.

[00453] CSNKlGl (CKl gamma 1) [00454] The 2X CSNKlGl (CKl gamma 1) / Ser/Thr 05 Peptide Mixture will be prepared in 50 niM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final

10 μL Kinase Reaction will consist of 3.7 - 19.5 ng CSNKlGl (CKl gamma 1) and 2 μM

Ser/Thr 05 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA.

After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development

Reagent B will be added.

[00455] CSNK1G2 (CKl gamma 2)

[00456] The 2X CSNK1G2 (CKl gamma 2) / Ser/Thr 05 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final

10 μL Kinase Reaction will consist of 2.0 - 16.5 ng CSNK1G2 (CKl gamma 2) and 2 μM

Ser/Thr 05 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA.

Reagent B will be added.

[00457] CSNK1G3 (CKl gamma 3)

[00458] The 2X CSNK1G3 (CKl gamma 3) / Ser/Thr 05 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final

10 μL Kinase Reaction will consist of 4.3 - 31.7 ng CSNK1G3 (CKl gamma 3) and 2 μM

Ser/Thr 05 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA.

Reagent B will be added.

[00459] CSNK2A1 (CK2 alpha 1)

[00460] The 2X CSNK2A1 (CK2 alpha 1) / Ser/Thr 11 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final

10 μL Kinase Reaction will consist of 1.4 - 22.5 ng CSNK2A1 (CK2 alpha 1) and 2 μM

Ser/Thr 11 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA.

After the 1 hour Kinase Reaction incubation, 5 μL of a 1:16 dilution of Development Reagent

B will be added.

[00461] CSNK2A2 (CK2 alpha 2)

[00462] The 2X CSNK2A2 (CK2 alpha 2) / Ser/Thr 11 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final

10 μL Kinase Reaction will consist of 1.9 - 26.3 ng CSNK2A2 (CK2 alpha 2) and 2 μM

Ser/Thr 11 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA.

B will be added. [00463] DAPK3 (ZIPK)

[00464] The 2X DAPK3 (ZIPK) / Ser/Thr 13 Peptide Mixture will be prepared in 50 niM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 11.6 - 80.0 ng DAPK3 (ZIPK) and 2 μM Ser/Thr 13 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:512 dilution of Development Reagent A will be added. [00465] DYRK3

[00466] The 2X DYRK3 / Ser/Thr 09 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.4 - 10.9 ng DYRK3 and 2 μM Ser/Thr 09 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00467] DYRK4

[00468] The 2X DYRK4 / Ser/Thr 09 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 13.6 - 103.6 ng DYRK4 and 2 μM Ser/Thr 09 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00469] EGFR (ErbBl)

[00470] The 2X EGFR (ErbB 1) / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 1.3 - 7.9 ng EGFR (ErbBl) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00471] EGFR L858R (ErbB 1 L858R)

[00472] The 2X EGFR L858R (ErbB 1 L858R) / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 0.6 - 4.5 ng EGFR L858R (ErbBl L858R) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00473] EGFR L861Q (ErbB 1 L861Q) [00474] The 2X EGFR L861Q (ErbB 1 L861Q) / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM

EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 1.2 - 8.4 ng EGFR

L861Q (ErbBl L861Q) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35,

10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added.

[00475] EPHAl

[00476] The 2X EPHAl / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 5.5 - 60.7 ng EPHAl and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00477] EPHA2

[00478] The 2X EPHA2 / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.2 - 26.2 ng EPHA2 and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00479] EPHA3

[00480] The 2X EPHA3 / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.3 - 145.5 ng EPHA3 and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00481] EPHA4

[00482] The 2X EPHA4 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 5.8 - 23.0 ng EPHA4 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00483] EPHA5

[00484] The 2X EPHA5 / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.5 - 38.1 ng EPHA5 and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00485] EPHA8

[00486] The 2X EPHA8 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.2 - 49.1 ng EPHA8 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00487] EPHBl

[00488] The 2X EPHB 1 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.4 - 63.1 ng EPHBl and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00489] EPHB2

[00490] The 2X EPHB2 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.7 - 18.0 ng EPHB2 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00491] EPHB3

[00492] The 2X EPHB3 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.0 - 43.8 ng EPHB3 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00493] EPHB4

[00494] The 2X EPHB4 / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.3 - 18.0 ng EPHB4 and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00495] ERBB2 (HER2) [00496] The 2X ERBB2 (HER2) / Tyr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MnC12, 1 mM EGTA, 2 mM DTT, 0.02% NaN3.

The final 10 μL Kinase Reaction will consist of 4.1 - 34.1 ng ERBB2 (HER2) and 2 μM Tyr

06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 5 mM MgC12, 5 mM MnC12, 1 mM

EGTA, 1 mM DTT, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a

1:64 dilution of Development Reagent A will be added.

[00497] ERBB4 (HER4)

[00498] The 2X ERBB4 (HER4) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 2.0 - 23.0 ng ERBB4 (HER4) and 2 μM Tyr

01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of

Development Reagent B will be added.

[00499] FER

[00500] The 2X FER / Tyr 05 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.6 - 21.7 ng FER and 2 μM Tyr 05 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added.

[00501] FES (FPS)

[00502] The 2X FES (FPS) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.7 - 5.2 ng FES (FPS) and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00503] FGFRl

[00504] The 2X FGFRl / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final

10 μL Kinase Reaction will consist of 0.4 - 3.7 ng FGFRl and 2 μM Tyr 04 Peptide in 50 mM

[00505] HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA,

1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of

Development Reagent B will be added.

[00506] FGFR2 [00507] The 2X FGFR2 / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 0.3 - 2.9 ng FGFR2 and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00508] FGFR3

[00509] The 2X FGFR3 / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 1.4 - 20.0 ng FGFR3 and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00510] FGFR4

[00511] The 2X FGFR4 / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 2.4 - 105.0 ng FGFR4 and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00512] FGR

[00513] The 2X FGR / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.8 - 6.0 ng FGR and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00514] FLTl (VEGFRl)

[00515] The 2X FLTl (VEGFRl) / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 5.0 - 25.0 ng FLTl (VEGFRl) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00516] FLT3 [00517] The 2X FLT3 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRϋ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.6 - 76.0 ng FLT3 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00518] FLT3 D835Y

[00519] The 2X FLT3 D835Y / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.1 - 1.3 ng FLT3 D835Y and 2 μM Tyr 02 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00520] FLT4 (VEGFR3)

[00521] The 2X FLT4 (VEGFR3) / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 1.9 - 16.0 ng FLT4 (VEGFR3) and 2 μM Tyr

04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of

Development Reagent B will be added.

[00522] FRK (PTK5)

[00523] The 2X FRK (PTK5) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.1 - 51.9 ng FRK (PTK5) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00524] FYN

[00525] The 2X FYN / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 5.0 - 34.0 ng FYN and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00526] GRK4

[00527] The 2X GRK4 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.3 - 9.6 ng GRK4 and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRϋ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00528] GRK5

[00529] The 2X GRK5 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 12.1 - 97.5 ng GRK5 and 2 μM Ser/Thr 16

Peptide in 50 mM HEPES pH 7.5, 0.01% BRLT-35, 10 mM MgC12, 2 mM MnC12, 1 mM

EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of

Development Reagent B will be added.

[00530] GRK6

[00531] The 2X GRK6 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 9.1 - 82.9 ng GRK6 and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00532] GRK7

[00533] The 2X GRK7 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 3.8 - 28.5 ng GRK7 and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00534] GSK3A (GSK3 alpha)

[00535] The 2X GSK3A (GSK3 alpha) / Ser/Thr 09 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.1 - 0.4 ng GSK3A (GSK3 alpha) and 2 μM Ser/Thr 09

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added.

[00536] GSK3B (GSK3 beta)

[00537] The 2X GSK3B (GSK3 beta) / Ser/Thr 09 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.4 - 2.5 ng GSK3B (GSK3 beta) and 2 μM Ser/Thr 09

[00538] HCK

[00539] The 2X HCK / Tyr 02 Peptide Mixture will be prepared in 50 niM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.3 - 3.5 ng HCK and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00540] IGFlR

[00541] The 2X IGFlR / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.8 - 98.8 ng IGFlR and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00542] IKBKB (IKK beta)

[00543] The 2X IKBKB (IKK beta) / Ser/Thr 05 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.2 - 10.6 ng IKBKB (IKK beta) and 2 μM Ser/Thr 05 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00544] INSR

[00545] The 2X INSR / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 2.3 - 50.0 ng INSR and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 : 128 dilution of Development Reagent B will be added. [00546] INSRR (IRR)

[00547] The 2X INSRR (IRR) / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 2.2 - 14.7 ng INSRR (IRR) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00548] IRAK4

[00549] The 2X IRAK4 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.8 - 400.0 ng IRAK4 and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00550] ITK

[00551] The 2X ITK / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.8 - 80.0 ng ITK and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00552] JAK2

[00553] The 2X JAK2 / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 0.5 - 4.0 ng JAK2 and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00554] JAK3

[00555] The 2X JAK3 / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 6.7 - 49.1 ng JAK3 and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added. [00556] KDR (VEGFR2)

[00557] The 2X KDR (VEGFR2) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.6 - 14.4 ng KDR (VEGFR2) and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00558] KIT

[00559] The 2X KIT / Tyr 06 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRϋ-35, 10 mM MnC12, 1 mM EGTA, 2 mM DTT, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 3.7 - 27.7 ng KIT and 2 μM Tyr 06 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRϋ-35, 5 mM MgC12, 5 mM MnC12, 1 mM EGTA, 1 mM DTT,

0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of

Development Reagent A will be added.

[00560] KIT T670I

[00561] The 2X KIT T670I / Tyr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnC12, 1 mM EGTA, 2 mM DTT, 0.02% NaN3.

The final 10 μL Kinase Reaction will consist of 4.8 - 127.0 ng KIT T670I and 2 μM Tyr 06

Peptide in 5OmM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgC12, 5 mM MnC12, 1 mM

1:64 dilution of Development Reagent A will be added.

[00562] LCK

[00563] The 2X LCK / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.0 - 26.5 ng LCK and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00564] LYN A

[00565] The 2X LYN A / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.1 - 12.0 ng LYN A and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00566] LYN B

[00567] The 2X LYN B / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.9 - 18.5 ng LYN B and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00568] MAP3K9 (MLKl) [00569] The 2X MAP3K9 (MLKl) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 6.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 12.0 - 55.6 ng MAP3K9 (MLKl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.0, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00570] MAP4K2 (GCK)

[00571] The 2X MAP4K2 (GCK) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.4 - 8.4 ng MAP4K2 (GCK) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00572] MAP4K4 (HGK)

[00573] The 2X MAP4K4 (HGK) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.5 - 5.0 ng MAP4K4 (HGK) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00574] MAP4K5 (KHSl)

[00575] The 2X MAP4K5 (KHS 1) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.3 - 4.0 ng MAP4K5 (KHSl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00576] MAPKl (ERK2)

[00577] The 2X MAPKl (ERK2) / Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.1 - 31.4 ng MAPKl (ERK2) and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00578] MAPKl 1 (ρ38 beta)

[00579] The 2X MAPKl 1 (p38 beta) / Ser/Thr 15 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 12.0 - 76.0 ng MAPKIl (ρ38 beta) and 2 μM Ser/Thr 15 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent B will be added.

[00580] MAPK12 (p38 gamma)

[00581] The 2X MAPK12 (p38 gamma) / Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.5 - 4.8 ng MAPK12 (p38 gamma) and 2 μM Ser/Thr 03

Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added.

[00582] MAPK13 (p38 delta)

[00583] The 2X MAPK13 (p38 delta) / Ser/Thr 03 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 3.5 - 39.6 ng MAPK13 (p38 delta) and 2 μM Ser/Thr 03

[00584] MAPK3 (ERKl)

[00585] The 2X MAPK3 (ERKl) / Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.5 - 31.5 ng MAPK3 (ERKl) and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added.

[00586] MAPKAPK2

[00587] The 2X MAPKAPK2 / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.1 - 0.3 ng MAPKAPK2 and 2 μM Ser/Thr 04 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00588] MAPKAPK3

[00589] The 2X MAPKAPK3 / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.8 - 15.8 ng MAPKAPK3 and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 niM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00590] MAPKAPK5 (PRAK)

[00591] The 2X MAPKAPK5 (PRAK) / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 2.1 - 18.0 ng MAPKAPK5 (PRAK) and 2 μM Ser/Thr 04

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 : 1024 dilution of Development Reagent A will be added.

[00592] MATK (HYL)

[00593] The 2X MATK (HYL) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.9 - 76.8 ng MATK (HYL) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00594] MERTK (cMER)

[00595] The 2X MERTK (cMER) / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 0.7 - 7.8 ng MERTK (cMER) and 2 μM Tyr

02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of

Development Reagent A will be added.

[00596] MET (cMet)

[00597] The 2X MET (cMet) / Tyr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.3 - 10.0 ng MET (cMet) and 2 μM Tyr 06 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00598] MET M1250T

[00599] The 2X MET M1250T / Tyr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 3.0 - 30.0 ng MET M1250T and 2 μM Tyr 06 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00600] MINKl

[00601] The 2X MINKl / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.2 - 35.0 ng MINKl and 2 μM Ser/Thr 07 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00602] MSTlR (RON)

[00603] The 2X MSTlR (RON) / Tyr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.1 - 1.2 ng MSTlR (RON) and 2 μM Tyr 06 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00604] MST4

[00605] The 2X MST4 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 10.5 - 89.8 ng MST4 and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added

[00606] MUSK

[00607] The 2X MUSK / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final

10 μL Kinase Reaction will consist of 16.4 - 89.5 ng MUSK and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM

DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development

Reagent B will be added.

[00608] MYLK2 (skMLCK)

[00609] The 2X MYLK2 (skMLCK) / Ser/Thr 13 Peptide Mixture will be prepared in

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 7.1 - 51.4 ng MYLK2

(skMLCK) and 2 μM Ser/Thr 13 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM

Kinase Reaction incubation, 5 μL of a 1:512 dilution of Development Reagent A will be added.

[00610] NEKl [00611] The 2X NEKl / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 1.9 - 69.1 ng NEKl and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00612] NEK2

[00613] The 2X NEK2 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.4 - 4.0 ng NEK2 and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00614] NTRKl (TRKA)

[00615] The 2X NTRKl (TRKA) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 4.8 - 77.4 ng NTRKl (TRKA) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00616] NTRK2 (TRKB)

[00617] The 2X NTRK2 (TRKB) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 0.2 - 2.0 ng NTRK2 (TRKB) and 2 μM Tyr

01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

Development Reagent B will be added.

[00618] NTRK3 (TRKC)

[00619] The 2X NTRK3 (TRKC) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 3.3 - 110.6 ng NTRK3 (TRKC) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00620] PAK3

[00621] The 2X PAK3 / Ser/Thr 14 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 3.8 - 68.0 ng PAK3 and 2 μM Ser/Thr 14 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A will be added.

[00622] PAK4

[00623] The 2X PAK4 / Ser/Thr 14 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.1 - 0.5 ng PAK4 and 2 μM Ser/Thr 14 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A will be added.

[00624] PAK6

[00625] The 2X PAK6 / Ser/Thr 14 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.0 - 12.6 ng PAK6 and 2 μM Ser/Thr 14 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A will be added.

[00626] PASK

[00627] The 2X PASK / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 10.5 - 59.8 ng PASK and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00628] PDGFRA (PDGFR alpha)

[00629] The 2X PDGFRA (PDGFR alpha) / Tyr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction will consist of 4.0 - 132.8 ng PDGFRA (PDGFR alpha) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32 dilution of Development Reagent B will be added.

[00630] PDGFRA D842V

[00631] The 2X PDGFRA D842V / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 4.4 - 39.6 ng PDGFRA D842V and 2 μM Tyr

04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

Development Reagent B will be added. [00632] PDGFRA T674I

[00633] The 2X PDGFRA T674I / Tyr 04 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 14.6 - 58.4 ng PDGFRA T674I and 2 μM Tyr

04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM MnC12, 1 mM

Development Reagent B will be added.

[00634] PDGFRB (PDGFR beta)

[00635] The 2X PDGFRB (PDGFR beta) / Tyr 04 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM

DTT. The final 10 μL Kinase Reaction will consist of 4.8 - 33.0 ng PDGFRB (PDGFR beta) and 2 μM Tyr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 2 mM

MnC12, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a

1:32 dilution of Development Reagent B will be added.

[00636] PHKGl

[00637] The 2X PHKGl / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 20 μg/ml Calmodulin, 0.02%

NaN3. The final 10 μL Kinase Reaction will consist of 6.3 - 117.8 ng PHKGl and 2 μM

Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM

EGTA, 2 mM CaC12, 10 μg/ml Calmodulin, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00638] PHKG2

[00639] The 2X PHKG2 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 0.02% NaN3. The final 10 μL

Kinase Reaction will consist of 5.2 - 26.0 ng PHKG2 and 2 μM Ser/Thr 07 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, 0.01% NaN3.

After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development

Reagent A will be added.

[00640] PMl

[00641] The 2X PIMl / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.8 - 39.4 ng PIMl and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00642] PIM2

[00643] The 2X PIM2 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.5 - 25.0 ng PIM2 and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01%

BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00644] PKNl (PRKl)

[00645] The 2X PKNl (PRKl) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM Tris pH 8.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 2.9 - 29.8 ng PKNl (PRKl) and 2 μM Ser/Thr 07 Peptide in 50 mM Tris / HEPES pH 8.0, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.01% NaN3.

Reagent A will be added.

[00646] PLKl

[00647] The 2X PLKl / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.8 - 25.3 ng PLKl and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 :8 dilution of Development Reagent B will be added.

[00648] PLK2

[00649] The 2X PLK2 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 13.0 - 186.2 ng PLK2 and 2 μM Ser/Thr 16 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

Reaction incubation, 5 μL of a 1 :8 dilution of Development Reagent B will be added.

[00650] PLK3

[00651] The 2X PLK3 / Ser/Thr 16 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.8 - 4.5 ng PLK3 and 2 μM Ser/Thr 16 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:8 dilution of Development Reagent B will be added.

[00652] PRKACA (PKA)

[00653] The 2X PRKACA (PKA) / Ser/Thr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.0 - 0.1 ng PRKACA (PKA) and 2 μM Ser/Thr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRD-35, 10 mM MgC12, 1 niM EGTA. After the 1 hour Kinase

[00654] PRKCA (PKC alpha)

[00655] The 2X PRKCA (PKC alpha) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.0 - 0.3 ng PRKCA (PKC alpha) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM

MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour

Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00656] PRKCB 1 (PKC beta I)

[00657] The 2X PRKCB 1 (PKC beta I) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid

Mix, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.0 - 0.3 ng PRKCBl

(PKC beta I) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00658] PRKCB2 (PKC beta II)

[00659] The 2X PRKCB2 (PKC beta II) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid

Mix, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.0 - 0.6 ng PRKCB2

(PKC beta II) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00660] PRKCD (PKC delta)

[00661] The 2X PRKCD (PKC delta) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.1 - 1.3 ng PRKCD (PKC delta) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM

MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00662] PRKCE (PKC epsilon)

[00663] The 2X PRKCE (PKC epsilon) / Ser/Thr 07 Peptide Mixture will be prepared in 50 niM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid

Mix, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.1 - 0.7 ng PRKCE

(PKC epsilon) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00664] PRKCG (PKC gamma)

[00665] The 2X PRKCG (PKC gamma) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid

Mix, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.0 - 0.4 ng PRKCG

(PKC gamma) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00666] PRKCH (PKC eta)

[00667] The 2X PRKCH (PKC eta) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.1 - 1.1 ng PRKCH (PKC eta) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase

[00668] PRKCI (PKC iota)

[00669] The 2X PRKCI (PKC iota) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.8 - 5.0 ng PRKCI (PKC iota) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase

[00670] PRKCN (PKD3) [00671] The 2X PRKCN (PKD3) / Ser/Thr 10 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.3 - 5.8 ng PRKCN (PKD3) and 2 μM Ser/Thr 10 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00672] PRKCQ (PKC theta)

[00673] The 2X PRKCQ (PKC theta) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.0 - 0.4 ng PRKCQ (PKC theta) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM

[00674] PRKCZ (PKC zeta)

[00675] The 2X PRKCZ (PKC zeta) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM CaC12, 2X Novel Lipid Mix,

0.02% NaN3. The final 10 μL Kinase Reaction will consist of 0.9 - 8.0 ng PRKCZ (PKC zeta) and 2μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 500 μM EGTA, 2 mM CaC12, IX Novel Lipid Mix, 0.01% NaN3. After the 1 hour Kinase

[00676] PRKDl (PKC mu)

[00677] The 2X PRKD 1 (PKC mu) / Ser/Thr 10 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.1 - 1.7 ng PRKDl (PKC mu) and 2 μM Ser/Thr 10 Peptide in 50 mM HEPES pH 7.5, 0.01% BRJJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour

[00678] PRKD2 (PKD2)

[00679] The 2X PRKD2 (PKD2) / Ser/Thr 10 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.2 - 2.4 ng PRKD2 (PKD2) and 2 μM Ser/Thr 10 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00680] PRKGl [00681] The 2X PRKGl / Ser/Thr 14 Peptide Mixture will be prepared in 50 rnM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 20 μM cGMP. The final 10 μL Kinase Reaction will consist of 0.1 - 0.8 ng PRKGl and 2 μM Ser/Thr 14 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA, 10 μM cGMP. After the

1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A will be added.

[00682] PRKG2 (PKG2)

[00683] The 2X PRKG2 (PKG2) / Ser/Thr 14 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA, 20 μM cGMP. The final

10 μL Kinase Reaction will consist of 0.1 - 2.0 ng PRKG2 (PKG2) and 2 μM Ser/Thr 14

Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 10 μM cGMP. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development

Reagent A will be added.

[00684] PRKX

[00685] The 2X PRKX / Ser/Thr 14 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.3 - 2.8 ng PRKX and 2 μM Ser/Thr 14 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 :256 dilution of Development Reagent A will be added.

[00686] PTK2 (FAK)

[00687] The 2X PTK2 (FAK) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.7 - 40.1 ng PTK2 (FAK) and 2 μM Tyr 01 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00688] PTK6 (Brk)

[00689] The 2X PTK6 (Brk) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 17.5 - 400.0 ng PTK6 (Brk) and 2 μM Tyr 01

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

Development Reagent B will be added.

[00690] RET [00691] The 2X RET / Tyr 02 Peptide Mixture will be prepared in 50 rnM HEPES pH

7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.5 - 4.6 ng RET and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00692] ROCKl

[00693] The 2X ROCKl / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.8 - 12.3 ng ROCKl and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00694] ROCK2

[00695] The 2X ROCK2 / Ser/Thr 13 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.9 - 6.1 ng ROCK2 and 2 μM Ser/Thr 13 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:512 dilution of Development Reagent A will be added. [00696] ROSl

[00697] The 2X ROS 1 / Tyr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.6 - 23.9 ng ROSl and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00698] RPS6KA1 (RSKl)

[00699] The 2X RPS6KA1 (RSKl) / Ser/Thr 06 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.8 - 7.1 ng RPS6KA1 (RSKl) and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00700] RPS6KA2 (RSK3)

[00701] The 2X RPS6KA2 (RSK3) / Ser/Thr 06 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 2.3 - 15.6 ng RPS6KA2 (RSK3) and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour

[00702] RPS6KA3 (RSK2)

[00703] The 2X RPS6KA3 (RSK2) / Ser/Thr 06 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRLT-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 0.3 - 2.7 ng RPS6KA3 (RSK2) and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRLT-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour

[00704] RPS6KA4 (MSK2)

[00705] The 2X RPS6KA4 (MSK2) / Ser/Thr 01 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 12.4 - 126.1 ng RPS6KA4 (MSK2) and 2 μM Ser/Thr 01

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00706] RPS6KA5 (MSKl)

[00707] The 2X RPS6KA5 (MSKl) / Ser/Thr 01 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 1.0 - 11.0 ng RPS6KA5 (MSKl) and 2 μM Ser/Thr 01

[00708] RPS6KB1 (p70S6K)

[00709] The 2X RPS6KB 1 (p70S6K) / Ser/Thr 07 Peptide Mixture will be prepared in

50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL

Kinase Reaction will consist of 6.3 - 51.3 ng RPS6KB1 (p70S6K) and 2 μM Ser/Thr 07

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added.

[00710] SGK (SGKl)

[00711] The 2X SGK (SGKl) / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.1 - 1.0 ng SGK (SGKl) and 2 μM Ser/Thr 06 Peptide in 50 niM

HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00712] SGK2

[00713] The 2X SGK2 / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.2 - 3.0 ng SGK2 and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added.

[00714] SGKL (SGK3)

[00715] The 2X SGKL (SGK3) / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.1 - 1.8 ng SGKL (SGK3) and 2 μM Ser/Thr 06 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00716] SRC

[00717] The 2X SRC / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.4 - 36.0 ng SRC and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00718] SRC Nl

[00719] The 2X SRC Nl / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.9 - 15.5 ng SRC Nl and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00720] SRC N2

[00721] The 2X SRC N2 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.8 - 31.5 ng SRC N2 and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00722] SRMS (Srm) [00723] The 2X SRMS (Srm) / Tyr 01 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 13.3 - 67.7 ng SRMS (Srm) and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added. [00724] SRPK2

[00725] The 2X SRPK2 / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 10.6 - 123.1 ng SRPK2 and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00726] STK22B (TSSK2)

[00727] The 2X STK22B (TSSK2) / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 5.6 - 37.5 ng STK22B (TSSK2) and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00728] STK22D (TSSKl)

[00729] The 2X STK22D (TSSKl) / Ser/Thr 04 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.5 - 11.4 ng STK22D (TSSKl) and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00730] STK24 (MST3)

[00731] The 2X STK24 (MST3) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 14.7 - 84.0 ng STK24 (MST3) and 2 μM Ser/Thr 07 Peptide in 50 mM Tris / HEPES pH 8.0, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00732] STK25 (YSKl)

[00733] The 2X STK25 (YSKl) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.1 - 56.8 ng STK25 (YSKl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00734] STK3 (MST2)

[00735] The 2X STK3 (MST2) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 6.5, 0.01% BRLT-35, 10 mM MgC12, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction will consist of 5.7 - 150.0 ng STK3 (MST2) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.0, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA, 0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00736] STK4 (MSTl)

[00737] The 2X STK4 (MSTl) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 8.0 - 200.0 ng STK4 (MSTl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00738] STK6 (Aurora A)

[00739] The 2X STK6 (Aurora A) / Ser/Thr 01 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.9 - 6.2 ng STK6 (Aurora A) and 2 μM Ser/Thr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added. [00740] SYK

[00741] The 2X SYK / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRJJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.4 - 6.4 ng SYK and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent A will be added. [00742] TAOK2 (TAOl)

[00743] The 2X TAOK2 (TAOl) / Ser/Thr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 10.8 - 70.4 ng TAOK2 (TAOl) and 2 μM Ser/Thr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:32768 dilution of Development Reagent A will be added. [00744] TBKl [00745] The 2X TBKl / Ser/Thr 05 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRϋ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 0.6 - 9.2 ng TBKl and 2 μM Ser/Thr 05 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:128 dilution of Development Reagent B will be added.

[00746] TEK (Tie2)

[00747] The 2X TEK (Tie2) / Tyr 05 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 4 mM MnC12, 1 mM EGTA, 2 mM DTT.

The final 10 μL Kinase Reaction will consist of 0.5 - 5.8 ng TEK (Tie2) and 2 μM Tyr 05

Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 2 mM MnC12, 1 mM

EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added.

[00748] TYRO3 (RSE)

[00749] The 2X TYRO3 (RSE) / Tyr 02 Peptide Mixture will be prepared in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL Kinase

Reaction will consist of 2.8 - 40.8 ng TYRO3 (RSE) and 2 μM Tyr 02 Peptide in 50 mM

HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase

[00750] YESl

[00751] The 2X YES 1 / Tyr 02 Peptide Mixture will be prepared in 50 mM HEPES pH

7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.5 - 18.0 ng YESl and 2 μM Tyr 02 Peptide in 50 mM HEPES pH 7.5, 0.01%

[00752] ZAP70

[00753] The 2X ZAP70 / Tyr 07 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnC12, 1 mM EGTA, 2 mM DTT, 0.02% NaN3. The final

10 μL Kinase Reaction will consist of 20.0 - 113.0 ng ZAP70 and 2 μM Tyr 07 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgC12, 5 mM MnC12, 1 mM EGTA, 1 mM DTT,

0.01% NaN3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:16 dilution of

Development Reagent B will be added.

[00754] KINASE-SPECIFIC ASSAY DETAILS (Cascade Format)

[00755] BRAF [00756] The 2X BRAF / inactive MAP2K1 (MEKl) / inactive MAPKl (ERK2) /

Ser/Thr 03 Peptide Mixture will be prepared in 50 niM HEPES pH 7.5, 0.01% BRU-35, 10 niM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.03 - 0.13 ng BRAF, 10 ng inactive MAP2K1 (MEKl), 100 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU- 35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00757] BRAF V599E

[00758] The 2X BRAF V599E / inactive MAP2K1 (MEKl) / inactive MAPKl (ERK2)

/ Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.004 - 0.014 ng BRAF V599E, 10 ng inactive MAP2K1 (MEKl), 100 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00759] MAP2K1 (MEKl)

[00760] The 2X MAP2K1 (MEKl) / inactive MAPKl (ERK2) / Ser/Thr 03 Peptide

Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.00 - 4.00 ng MAP2K1 (MEKl), 105 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1 : 1024 dilution of Development Reagent A will be added. [00761] MAP2K2 (MEK2)

[00762] The 2X MAP2K2 (MEK2) / inactive MAPKl (ERK2) / Ser/Thr 03 Peptide

Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 1.00 - 4.00 ng MAP2K2 (MEK2), 105 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00763] MAP2K6 (MKK6)

[00764] The 2X MAP2K6 (MKK6) / inactive MAPK12 (p38 gamma) / Ser/Thr 03

Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 9.80 - 39.00 ng MAP2K6 (MKK6), 100 ng inactive MAPKlK (p38 gamma), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00765] MAP3K8 (COT)

[00766] The 2X MAP3K8 (COT) / inactive MAP2K1 (MEKl) / inactive MAPKl

(ERK2) / Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.5 - 2.0 ng MAP3K8 (COT), 10 ng inactive MAP2K1 (MEKl), 100 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRD-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00767] MAPK14 (p38 alpha)

[00768] The 2X MAPK14 (p38 alpha) / inactive MAPKAPK2 / Ser/Thr 04 Peptide

Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.005 - 0.020 ng MAPK14 (p38 alpha), 5 ng inactive MAPKAPK2, and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. [00769] PDKl

[00770] The 2X PDKl / inactive AKT2 (PKB beta) / Ser/Thr 06 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 3.20 - 12.60 ng PDKl, 150 ng inactive AKT2 (PKB beta), and 2 μM Ser/Thr 06 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:2048 dilution of Development Reagent A will be added. [00771] RAFl (cRAF)

[00772] The 2X RAFl (cRAF) / inactive MAP2K1 (MEKl) / inactive MAPKl (ERK2)

/ Ser/Thr 03 Peptide Mixture will be prepared in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. The final 10 μL Kinase Reaction will consist of 0.002 - 0.008 ng RAFl (cRAF), 10 ng inactive MAP2K1 (MEKl), 100 ng inactive MAPKl (ERK2), and 2 μM Ser/Thr 03 Peptide in 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgC12, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A will be added. 6. EXAMPLES

6.1 Tricyclic 4-chloro-pyrimido[4,5-£][l,4]benzodiazepines

[00773] A general synthetic procedure may be described for instances of this invention in which the desired product is a 4-chloro-pyrimido[4,5-b][l,4]benzodiazepine, such that compound 2 in Scheme I has Rl = H, X = N-CH₃, Y3 = R, Yl = H, Y2 = H and Y3 = H. See FIG. 1. In such instances, reaction a) may be performed as follows. To a solution of 4,6-dichloro-5-nitro-pyrimidine and triethylamine in THF was added a solution of the appropriate amine (e.g., N-methylbenzenamines) in THF. The reaction mixture was stirred at room temperature over night. The reaction mixture was concentrated in vacuo, diluted with water, and extracted with EtOAc. The organic phase was washed with HCl, brine, dried over anhydrous MgSO₄, and concentrated in vacuo to yield the crude product as a solid. Purification by recrystallization was additionally used in some instances to obtain purified 6- chloro-5-nitropyrimidin-4-amine product (compound 1, scheme I, where X = NR, Y3 = R and Yl, Y2 and Y4 = H). The 6-chloro-5-nitropyrimidin-4-amine was dissolved in a mixture of ethanol and water. Iron powder and NH₄Cl was added to it. The mixture was then stirred in reflux for 5 h, cooled to room temperature, and filtered through a pad of celite. The filtrate was concentrated in vacuo. The residue was extracted with EtOAc, and the organic extract was washed with saturated NaHCO₃, water, and brine and dried over anhydrous MgSO₄. It was then filtered and concentrated in vacuo to the crude product which was purified by flash chromatography to give the corresponding 6-chloro-N4-pyrimidine-4,5-diamine (compound 1, Scheme I).

[00774] Preparation of the 4-chloropyrimido[4,5-b][l,4] benzodiazepine (compound 2,

Scheme I), was carried out by the following general procedure. Two cyclization conditions were employed for the formation of tricyclic 4-chloro-pyrimido[4,5-&][l,4]benzodiazepines. 6-Chloro-N4-pyrimidinyl-4,5-diamine, the appropriate acid or its derivatives and PPA were dissolved in POCl₃, and stirred under refluxing xylene for 0.5-12 h. The reaction mixture was concentrated in vacuo, diluted with water, and extracted with EtOAc. The water layer was treated with 5N NaOH to pH 10 and extracted with EtOAc. The combined EtOAc layer was washed with saturated NaHCO₃, brine, dried over anhydrous MgSO₄, concentrated in vacuo and purified by flash chromatography. R₁=H, R₂=CH₃; 2.1,

2.2, R₃=CH₃, 84%; 3.2, R₃=CH₃, 80%

2.3, R₃=F; 3.3, R₃=F, 77%(2 steps) method i) 4.19, R₃=H, X=O, 61% method ii) 4.3, R₃=H, X=Cl, 74% a) P-R₃-Ph-NH-R₂, Et₃NZTHF, r.t.; b) FeZNH₄Cl, C₂H₅OH/H₂O, reflux 7h; c) benzoic acid, methold i) PPA/xylene, method ii) POCI₃/PPA.

[00775] When pyrimidine 3.1 and benzoic acid were treated with pPA in refluxing xylene for 5 h 4-hydroxy-ll-methyl-6-phenyl-pyrimido[4,5-b][l,4]benzodiazepine (4.19) was resulted in 61% isolated yield. Compound 4.19 could be readily converted to 4-chloro- ll-methyl-6-phenyl-pyrimido[4,5-b][l,4] benzodiazepine (4.3) by treatment with POCl₃. The two-step transformations could be achieved using pPA / POCl₃ in a one-pot procedure to give 4.3 in 74% yield.

[00776] Other examples of compounds synthesized according to these procedure are shown in Table 1. Under POC13/PPA conditions, 6-chloro-4,5-diamino pyrimidines 3 were reacted with various acids or derivatives to yield the desired 4-chloro-pyrimido[4,5-b][l,4] benzodiazepine ring system.

Table 1. Formation of tricyclic 4-Chloro-pyrimido[4,5-b][l,4] benzodiazepines

comp no. R₃ R₄ X Yield (%) time

4.1 H CH3C-JH.2CH2 Cl 60 45min

4.2 H CH₃ OAc 52 30min

4.3 H Ph OH 74 5h

4.4 H 4'-F-C₆H₄ OH 79 5h

4.5 H 4'-CH₃-C₆H₄ OH 90 5h

4.6 H 4'-NO₂-C₆H₄ OH 38 12h 4.9 CH₃ Ph OH 85 5h

4.10 CH₃ 4'-F-C₆H₄ OH 88 5h

4.11 CH₃ 4'-CH₃-C₆H₄ OH 97 5h

4.12 CH₃ 4'-NO₂-C₆H₄ OH 49 12h

4.13 F CH₃CH₂CH₂ Cl 50 Ih

4.14 F CH₃ OAc 45 30min

4.15 F Ph OH 88 5h

4.16 F 4'-F-C₆H₄ OH 81 5h

4.17 F 4'-CH₃-C₆H₄ OH 90 5h

4.18 F 4'-NO₂-C₆H₄ OH 37 12h

[00777] In order to generate additional library diversity, nuclephilic substitution reactions were conducted on the 4-Cl group in tricyclic 4-chloro-pyrimido[4,5- £][l,4]benzodiazepines with amines.

[00778] C-4 substituted compound 5.1, 5.2 were prepared by nuclephilic substitution of the 4-Cl group with an primary amine or secondary amine in refluxing n-BuOH/Et₃N in good chemical yields.

5.1, R₅R₆NH= n-Bu-NH₂, 84%, 5.2, R₅R₆NH= pyrrolidine, 89%

[00779] Commercial reagents were used as received without additional purification.

Melting point was uncorrected. Mass spectra and HPLC (ELSD) data was recorded on an 1100 LC/MS system (Agilent Technology Corporation) with Alltech ELSD 2000 using a YMC ODS-A, 5-μm, 120-A, 4.6 x 50 mm (Waters, Inc.), and the retention time (Rt) for the expected product was recorded. IH NMR data was obtained using a 300-MHz Varian VXR- 300S NMR spectrometer with TMS as the internal standard and CDCl₃ as solvent. Multiplicities are indicated as the following: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doubled doublet; br, broad. Coupling constants (J values) where noted are quoted in hertz. Starting compounds for the cyclization (compounds 3 in Table 1) were prepared according to the literature method (Tanji et al., Chem. Pharm. Bull. 1992, 40, 227).

6.1.1 General synthetic procedure and characterization details for the Preparation of 6-chloro-5-nitropyrimidin-4-amine

[00780] To a solution of 4,6-dichloro-5-nitro-pyrimidine (8.00 g, 41.5 mmol) and triethylamine (6.9 mL, 49.2 mmol) in anhydrous THF (40 mL) was added a solution of the appropriate amine (41.5 mmol) in THF (40 niL) slowly. The reaction mixture was stirred at room temperature over night. The reaction mixture was concentrated in vacuo, diluted with water, and extracted with EtOAc. The organic phase was washed with IN HCl, brine, dried over anhydrous MgSO₄, and concentrated in vacuo to yield the crude product as a solid.

Purification by recrystallization from methanol provided the desired pure product 2.1 and 2.2

(Product 2.3 was used directly in the next step without further purification).

[00781] (6-Chloro-5-nitro-pyrimidine-4-yl)-methylphenylamine (2.1). Yellow solid,

81%, mp: 133.8-135.5 ⁰C. ES-MS: 265 ((M+l)+). IH NMR δ 8.52 (s, IH), 7.37-7.39 (m, 3H),

7.15-7.18 (m, 2H), 3.58 (s, 3H).

[00782] (6-Chloro-5-nitropyrimidin-4-yl)-methyl-p-tolylamine (2.2). Yellow solid, 84

%, mp: 133.3-134.3 ⁰C. ES-MS: 279 ((M+l)+). IH NMR δ 8.49 (s, IH), 7.16-7.19 (d, J = 9.0

Hz, 2H), 7.03-7.06 (d, J = 9.0 Hz, 2H), 3.55 (s, 3H), 2.38 (s, 3H).

[00783] (6-Chloro-5-nitropyrimidine-4-yl)-(4-fluorophenyl)-methylamine (2.3).

Brown solid, ES-MS: 283 ((M+l)+).

6.1.2 General synthetic procedure and characterization details for the Preparation of 6-Chloro-N4-pyrimidine-4,5-diamines

[00784] Compound 2 (33.0 mmol) was dissolved in a mixture of ethanol (118.0 niL) and water (33.0 mL). Iron powder (5.54 g, 98.9 mmol) and NH₄Cl (1.06 g, 19.8 mmol) was added to it. The mixture was then stirred in reflux for 5 h, cooled to room temperature, and filtered through a pad of celite. The filtrate was concentrated in vacuo. The residue was extracted with EtOAc, and the organic extract was washed with saturated NaHCO₃, water, and brine and dried over anhydrous MgSO₄. It was then filtered and concentrated in vacuo to the crude product which was purified by flash chromatography (elution with 9% EtOAc in petroleum ether followed by 20% EtOAc in petroleum ether) to give 3.1, 3.2, 3.3. [00785] 6-chloro-N4-methyl-N4-phenylpyrimidine-4,5-diamine (3.1). White solid, 80

%, mp: 81.0-82.6 ⁰C. ES-MS: 235 ((M+l)+). IH NMR δ 8.22 (s, IH), 7.35-7.40 (t, J = 7.5 Hz, 2H), 7.17-7.22 (t, J = 7.5 Hz, IH), 7.05-7.08 (d, J = 9.0 Hz, 2H), 3.51 (s, 3H). [00786] 6-chloro-N4-methyl- N4-p-tolylpyrimidine-4,5-diamine (3.2). White solid,

80%, mp: 114.9-116.1 ⁰C. ES-MS: 249 ((M+l)+). IH NMR δ 8.19 (s, IH), 7.16-7.19 (d, J = 9.0 Hz, 2H), 6.97-7.00 (d, J = 9.0 Hz, 2H), 4.82 (br, 2H), 3.47 (s, 3H), 2.35(s, 3H). [00787] 6-chloro-N4-(4-fluorophenyl) -N4-methylpyrimidine-4,5-diamine (3.3).

Yellow solid, 77 % (two steps), mp: 106-108 °C. ES-MS: 253 ((M+l)+). IH NMR δ 8.21 (s, IH), 7.05-7.08 (m, 4H), 3.47 (s, 3H). 6.1.3 General synthetic procedure and Characterization Details for the Preparation of 4-chIoropyrimido[4,5-£][l,4]benzodiazepine

[00788] 6-Chloro-N4-pyrimidinyl-4,5-diamines (1.3 mmol), the appropriate acid or its derivatives (1.9 mmol) and PPA (0.63 g, 1.9 mmol) were dissolved in POCl₃ (6.0 mL), and stirred under reflux for 0.5-12 h. The reaction mixture was concentrated in vacuo, diluted with water (15 mL), and extracted with EtOAc (3 x 10 mL). The water layer was treated with 5N NaOH to pH 10 and extracted with EtOAc (2 x 10 mL). The combined EtOAc layer was washed with saturated NaHCO₃, brine, dried over anhydrous MgSO₄, concentrated in vacuo and purified by flash chromatography (elution with 20 % EtOAc in petroleum ether for the compound 4.6, 4.12, 4.18, elution with 2.5 % EtOAc in petroleum ether followed by 9 % EtOAc in petroleum ether for the others).

[00789] 4-chloro-ll-methyl-6-propylpyrimido[4,5-έ»][l,4]benzodiazepine (4.1).

Yellow solid, 60%, mp: 125.3-126.9 °C. ES-MS: 287 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.66 min). IH NMR (300 Hz, CDCl₃) δ 8.35 (s, IH), 7.38-7.44 (t, J = 9.0 Hz, IH), 7.32-7.35 (d, J = 9.0 Hz, IH), 7.08-7.14 (t, J = 9.0 Hz, IH), 6.98-7.01 (d, J = 9.0 Hz, IH), 3.28 (s, 3H), 2.84-2.89 (t, J = 7.5 Hz, 2H), 1.70-1.83 (m, 2H), 1.02-1.07 (t, J = 7.5 Hz, 3H). 13C NMR (300 Hz, CDCl₃) δ 177.5, 162.9, 156.0, 153.3, 153.0, 132.1, 131.0, 130.4, 128.3, 124.2, 119.3, 42.2, 35.9, 20.4, 13.7.

[00790] 4-chloro-6,l l-dimethylpyrimido[4,5-b][l,4]benzodiazepine (4.2). Yellow solid, 52%, mp: 121.1-122.6°C. ES-MS: 259 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.10min). IH NMR (300 Hz, CDCl₃) δ 8.36 (s, IH), 7.40-7.45 (t, J = 7.8 Hz, IH), 7.34-7.36 (d, J = 6.9 Hz, IH), 7.10-7.15 (t, J = 7.5Hz, IH), 6.98-7.00 (d, J = 8.1 Hz, IH), 3.30 (s, 3H), 2.64(s, 3H). [00791] 4-chloro-l l-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine (4.3)

Yellow solid, 74%, mp: 127.1-128.9 °C. ES-MS: 321 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.89 min). IH NMR (300 Hz, CDCl₃) δ 8.39 (s, IH), 7.80-7.83 (d, J = 9.0 Hz, 2H), 7.42-7.55 (m, 4H), 7.09-7.14 (t, J = 7.5 Hz, 3H), 3.35 (s, 3H). 13C NMR (300 Hz, CDCl₃) δ 173.0, 162.8, 156.7, 154.0, 153.9, 139.6, 133.0, 132.0, 131.6, 130.2, 129.1, 128.5, 124.1, 120.1, 36.2. [00792] 4-chloro- 11 -methyl-6-(4'-fluorophenyl)-pyrimido[4,5-b] [ 1 ,4]benzodiazepine

(4.4). Orange solid, 79%, mp: 196.1-197.1 °C. ES-MS: 339 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.76 min). IH NMR (300 Hz, CDCl₃) δ 8.40 (s, IH), 7.81-7.86 (m, 2H), 7.48-7.52 (m, IH), 7.10-7.19 (m, 5H), 3.35 (s, 3H).

[00793] 4-chloro- 11 -memyl-6-(4'-methyl-phenyl)-pyrimido[4,5-Z?] [ 1 ,4]benzodiazepine

(4.5) Orange solid, 90%, mp: 200.9-201.8 °C. ES-MS: 335 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.93 min). IH NMR (300 Hz, CDCl₃) δ 8.38 (s, IH), 7.70-7.73 (d, J = 8.1 Hz, 2H), 7.46-7.52 (m, IH), 7.23-7.26(d, J = 8.4 Hz, 2H), 7.06-7.15(m, 3H), 3.34 (s, 3H), 2.43(s, 3H). 13C NMR (300 Hz, CDCl₃) δ 173.0, 162.9, 156.4, 153.6, 142.2, 136.7, 133.0, 132.1, 131.4, 130.4, 130.2, 129.4, 129.2, 129.1, 124.0, 120.0, 36.2, 21.8.

[00794] 4-chloro-ll-methyl-6-(4'-nitro-phenyl)-pyrimido[4,5-b][l,4]benzodiazepine

(4f). Orange solid, 38%, mp: 213.4-214.6 °C. ES-MS: 366 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.83 min). IH NMR (300 Hz, CDCl₃) δ 8.44(s, IH), 8.29-8.31 (d, J = 8.4 Hz, 2H), 7.99-8.02 (d, J=8.4 Hz, 2H), 7.52-7.57 (t, J = 7.5 Hz, IH), 7.12-7.14 (m, 2H), 7.01-7.04 (m, IH), 3.37(s, 3H).

[00795] 4-chloro-8,l l-dimethyl-6-propylpyrimido[4,5-b][l,4]benzodiazepine (4.7).

Yellow solid, 65%, mp: 114.8-116.4 ⁰C. ES-MS: 301 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.88 min). IH NMR (300 Hz, CDCl₃) δ 8.33 (s, IH), 7.19-7.23 (d, J = 6.0 Hz, IH), 7.12 (s, IH), 6.87-6.90 (d, J = 9.0 Hz, IH), 3.26 (s, 3H), 2.83-2.88 (t, J = 7.5 Hz, 2H), 2.31 (s, 3H), 1.70-1.83 (m, 2H), 1.02-1.07 (t, J = 7.5 Hz, 3H). 13C NMR (300 Hz, CDCl₃) δ 177.5, 163.1, 155.8, 153.2, 150.4, 134.0, 132.7, 130.9, 130.2, 128.6, 119.1, 42.1, 35.8, 20.6, 20.4, 13.7 [00796] 4-chloro-6,8,ll-trimethylpyrimido[4,5-&][l,4]benzodiazepine (4.8). Yellow solid, 57%, mp: 117.9-119.6 ⁰C. ES-MS: 273 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.35 min). IH NMR (300 Hz, CDCl₃) δ 8.34 (s, IH), 7.20-7.23 (m, IH), 7.14 (s, IH), 6.86-6.89 (d, J = 8.1 Hz, IH), 3.27 (s, 3H), 2.63 (s, 3H), 2.31 (s, 3H).

[00797] 4-chloro-8,l l-dimetfiyl-6-phenylpyrimido[4,5-&][l,4]benzodiazepine (4.9).

Orange solid, 85%, mp: 170.4-171.5 ⁰C. ES-MS: 335 ((M+l)+), HPLC (ELSD): 100% (Rt = 4.08 min). IH NMR (300 Hz, CDCl₃) δ 8.37 (s, IH), 7.82-7.85 (m, 2H), 7.43-7.53 (m, 3H), 7.28 (s, IH), 6.98-7.00 (d, J=8.4Hz, IH), 6.92 (s, IH), 3.32 (s, 3H), 2.25 (s, 3H). [00798] 4-chloro-8, 1 l-dimethyl-6-(4'-fluorophenyl)-pyrimido[4,5-

Z7][l,4]benzodiazepine (4.10). Yellow solid, 88%, mp: 166.0-167.6 °C. ES-MS: 353 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.92 min). IH NMR (300 Hz, CDCl₃) δ 8.38 (s, IH), 7.82-7.87 (m, 2H), 7.27-7.32 (m, IH), 7.12-7.18 (m, 2H), 6.98-7.00 (d, J=8.4 Hz, IH), 6.90 (s, IH), 3.31 (s, 3H), 2.25 (s, 3H).

[00799] 4-chloro-8,ll-dimethyl-6-(4'-methyl-phenyl)-pyrimido[4,5-

£][l,4]benzodiazepine (4.11). Yellow solid, 97%, mp: 231.0-232.1 ⁰C. ES-MS: 349 ((M+l)+), HPLC (ELSD): 100% (Rt = 4.08 min). IH NMR (300 Hz, CDCl₃) δ 8.36 (s, IH), 7.71-7.74 (d, J = 8.1 Hz, 2H), 7.24-7.30 (m, 3H), 6.97-7.00 (d, J = 8.4 Hz, IH), 6.93 (s, IH), 3.32 (s, 3H), 2.44 (s, 3H), 2.24 (s, 3H). 13C NMR (300 Hz, CDCl₃) δ 172.9, 163.0, 156.3, 153.5, 151.3, 142.0, 136.7, 133.7, 133.5, 132.2, 130.4, 130.2, 129.3, 129.2, 128.9, 119.8, 36.1,

21.7, 20.7.

[00800] 4-chloro-8, 1 l-dimethyl-6-(4'-nitro-phenyl)-pyrimido[4,5- b][l,4]benzodiazepine (4.12). Orange solid, 49%, nip: 229.6-230.8 °C. ES-MS: 380

((M+l)+), HPLC (ELSD): 100% (Rt = 4.00 min). IH NMR (300 Hz, CDCl₃) δ 8.42 (s, IH),

8.29-8.32 (d, J = 8.7 Hz, 2H), 7.99-8.02 (d, J = 8.7 Hz, 2H), 7.33-7.35 (d, J = 7.2 Hz, IH),

7.00-7.03 (d, J = 8.4 Hz, IH), 6.80 (s, IH), 3.34 (s, 3H), 2.26 (s, 3H).

[00801] 4-chloro-8-fluoro- 11 -methyl-6-propylpyrimido[4,5-b] [ 1 ,4]benzodiazepine

(4.13). Yellow solid, 50%, mp: 119.4-120.5 °C. ES-MS: 305 ((M+l)+), HPLC (ELSD):

100% (Rt = 3.49 min). IH NMR (300 Hz, CDCl₃) δ 8.37 (s, IH), 7.11-7.15 (m, IH), 7.02-

7.06 (m, IH), 6.94-6.98 (m, IH), 3.26 (s, 3H), 2.82-2.86 (t, J = 7.2 Hz, 2H), 1.72-1.84 (m,

2H), 1.03-1.08 (t, J = 7.2 Hz, 3H).

[00802] 4-chloro-8-fluoro-6,l l-dimethylpyrimido[4,5-£][l,4]benzodiazepine (4.14).

Yellow solid, 45%, mp: 140.5-141.5 °C. ES-MS: 277 ((M+l)+), HPLC (ELSD): 100% (Rt =

3.18 min). IH NMR (300 Hz, CDCl₃) δ 8.37 (s, IH), 7.10-7.15 (m, IH), 7.03-7.07 (m, IH),

6.93-6.98 (m, IH), 3.27 (s, 3H), 2.63 (s, 3H).

[00803] 4-chloro-8-fluoro- 1 l-methyl-6-phenylpyrimido[4,5-b] [ 1 ,4]benzodiazepine

(4.15). Orange solid, 88%, mp: 207.7-208.7 °C. ES-MS: 339 ((M+l)+), HPLC (ELSD):

100% (Rt = 3.90 min). IH NMR (300 Hz, CDCl₃) δ 8.40 (s, IH), 7.82-7.84 (d, J = 8.1 Hz,

2H), 7.44-7.57 (m, 3H), 7.18-7.26 (m, IH), 7.04-7.09 (m, IH), 6.83-6.86 (m, IH), 3.20 (s,

3H).

[00804] 4-chloro-8- fluoro -1 l-methyl-6-(4'-fluoroρhenyl)-pyrimido[4,5-

Z?][l,4]benzodia zepine (4.16). Yellow solid, 81%, mp: 204.9-205.7 °C. ES-MS: 357

((M+l)+), HPLC (ELSD): 100% (Rt = 3.91 min). IH NMR (300 Hz, CDCl₃) δ 8.40 (s, IH),

7.83-7.88 (m, 2H), 7.05-7.22 (m, 4H), 6.82-6.85 (m, IH), 3.32 (s, 3H).

[00805] 4-chloro-8- fluoro -1 l-methyl-6-(4'-methylphenyl)-pyrimido[4,5-

£][l,4]benzodia zepine (4.17).Yellow solid, 90%, mp: 190.8-19.1.9 ⁰C. ES-MS: 353

((M+l)+), HPLC (ELSD): 100% (Rt = 4.12 min). IH NMR (300 Hz, CDCl₃) δ 8.40 (s, IH),

7.71-7.74 (d, J = 8.1 Hz, 2H), 7.17-7.28 (m, 3H), 7.03-7.08 (m, IH), 6.84-6.87 (m, IH), 3.31

(s, 3H), 2.44 (s, 3H).

[00806] 4-chloro-8- fluoro -1 l-methyl-6-(4'-nitrophenyl)-pyrimido[4,5-

£][l,4]benzodia zepine (4.18).Orange solid, 37%, mp: 226.7-229.0°C. ES-MS: 384 ((M+l)+),

HPLC (ELSD): 100% (Rt = 3.82 min). IH NMR (300 Hz, CDCl₃) δ 8.45 (s, IH), 8.31-8.34 (d, J = 8.7 Hz, 2H), 8.00-8.03 (d, J = 8.4 Hz, 2H), 7.24 (s, IH), 7.08-7.12 (m, IH), 6.73-6.76 (m, IH), 3.34 (s, 3H).

[00807] 4-hydroxy -1 l-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine (4.19).

Orange oil, 61%. ES-MS: 303 ((M+l)+). IH NMR (300 Hz, CDCl₃) δ 7.92 (s, IH), 7.82-7.84 (d, J = 6.6 Hz, 2H), 7.37-7.46 (m, 5H), 7.04-7.07 (m, 2H), 3.29 (s, 3H).

6.1.4 General synthetic procedure and characterization details for the Preparation of 4-aminopyrimido[4,5-Z»][l,4] benzodiazepine

[00808] 4-chlomo-8- fluoro -11 -methyl-6-(4'-methyl-phenyl)-pyrimido[4,5-

Z?][l,4]benzodiazepine (4.17) (0.14 mmol), the appropriate amine (0.28 mmol) and Et3N (0.04mL, 0.28 mmol) were dissolved in n-BuOH (2.0 mL), and stirred under reflux for 5 h. The reaction mixture was concentrated in vacuo, dissolved with EtOAc (15 mL), and washed with water (2 x 15 mL), brine, dried over anhydrous MgSO₄, concentrated in vacuo and purified by flash chromatography (CH₂Cl₂/ EtOAc, 10:1 for compound 5.1 and CH₂Cl₂/ EtOAc,5: 1 for compound 5.2).

[00809] 4-(butylamino)-8- fluoro -1 l-methyl-6-(4'-methyl-phenyl)-pyrimido[4,5- b][l,4] benzodiazepine^). Orange oil, 84%. ES-MS: 390 ((M+l)+), HPLC (ELSD): 100% (Rt = 3.80 min).

[00810] 4-(pyrrolidin-l-yl)-8- fluoro -1 l-methyl-6-(4'-methylphenyl)-pyrimido[4,5- b][l,4] benzodiazepine^). Orange oil, 89%. ES-MS: 388 ((M+l)+), HPLC (ELSD): 100% (Rt = 2.58 min).

6.2 Tricyclic pyrimidine-fused 5,6-dihydrobenzodiazepines

[00811] A variety of compounds were synthesized according to Scheme II (see FIG. 2) and are shown in Table 2.

Table 2: Synthesis of 4-chloro-5,6-dmydro-pyrimido[4,5-Z?][l,4]benzodiazepines

1 3

Pyrimidines R¹ R¹ Products Yield Time

1.1 H CH₃CH₂CH₂ 3.1 65% 27h

1.1 H CH₃CH₂ 3.2 47% 34h

1.1 H Ph 3.3 65% 48h

1.1 H 4'-CH₃-C₆H₄ 3.4 44% 24h

1.1 H 4'-F-C₆H₄ 3.5 88% 29h 1.1 H 4'-NO₂-C₆H₄ 3.6 97% 16h

1.2 P-CH₃ CH₃CH₂CH₂ 3.7 72% 24h

1.2 P-CH₃ CH₃CH₂ 3.8 57% 17h

1.2 P-CH₃ Ph 3.9 72% 26h

1.2 P-CH₃ 4'-CH₃-C₆H₄ 3.10 46% 17h

1.2 P-CH₃ 4'-F-C₆H₄ 3.11 75% 35h

1.2 P-CH₃ 4'-NO₂-C₆H₄ 3.12 97% 18h

1.3 m-CH₃ CH₃CH₂CH₂ 3.13 81% 23h

1.3 m-CH₃ CH₃CH₂ 3.14 74% 21h

1.3 m-CH₃ Ph 3.15 71% 24h

1.3 m-CH₃ 4'-CH₃-C₆H₄ 3.16 45% 17h

1.3 m-CH₃ 4'-F-C₆H₄ 3.17 91% 21h

1.3 m-CH₃ 4'-NO₂-C₆H₄ 3.18 99% 22h

1.4 p-F Ph 3.19 19% 6ά

[00812] The starting compound 1 were prepared by a modified procedure similar to that described for the tricyclic 4-chloro-pyrimido[4.5-/?][l,4]benzodiazepines, above. Phosphoryl oxychloride was freshly distilled. All other commercial reagents were used as received without additional purification. Melting point was uncorrected. Mass spectra and HPLC (ELSD) data was recorded on an 1100 LC/MS system (Agilent Technology Corporation) with Alltech ELSD 2000, using a 4.6x50 mm Column (CenturySIL C-18 AQ+, 5μ) with a linear gradient 30-90% (v/v) acetonitrile - water with 0.035% trifluoroacetic acid over 8 minutes with a flow rate of 3.5 mL/min. Analytical TLC was performed using 2.5 x 5 cm plated coated with a 0.25 mm thickness of silica gel 60 F254. Column chromatography was performed using silica gel G (200-300 mesh). All IH NMR spectra (300 MHz) are reported as follows: chemical shifts in ppm downfield from TMS as internal standard (δ scale ) and CDCl₃ or DMSO-d6 as the solvent. Multiplicities are indicated as the following: multiplicity [s=singlet, d=doublet, t=triplet, m=multiplet, integration and coupling constant (Hz)]. AU 13C NMR spectra (75 MHz) were determined with complete proton decoupling and reported in ppm.

[00813] 5-amino-6-chloro-4-N-metliyl-anilino-pyrimidine (1) (0.85 mmol), the appropriate aldehyde or its derivatives (1.275 mmol) and TFA (0.8 mL) were dissolved in CH₃CN (10.0 mL), and stirred under reflux for 16-48 h. The reaction mixture was concentrated in vacuo, diluted with EtOAc (15 mL), and washed with saturated NaHCO₃ (3 x 15 mL). The water layer was extracted with EtOAc (3 x 10 mL). The combined EtOAc layer was washed with brine, dried over anhydrous Na₂SO₄, concentrated in vacuo and purified by flash chromatography (elution with EtOAc : petroleum ether =1:10 for the compound 3.6, 3.12, 3.18, elution with EtOAc : petroleum ether =1:30 for the others). [00814] 4-Chloro- 1 l-methyl-6-propyl-5,6~dihydro-pyrimido[4,5~

Z?][l,4]benzodiazepine(3.1) Yellow oil, yield: 65 %, ES-MS: 289.2 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.01 (s, 1 H), 7.29-7.34 (m, 1 H), 7.03-7.17 (m, 3 H), 4.54-4.59 (m, 1 H), 4.48 (d, J = 4.2 Hz, 1 H), 3.49 (s, 3 H), 1.97-2.07 (m, 2 H), 1.39-1.54 (m, 2 H), 1.02 (t, J = 7.5 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 150.9, 145.8, 145.6, 142.5, 135.5, 128.4, 128.1, 126.0, 123.8, 122.3, 57.3, 40.0, 35.1, 20.1, 13.9. Anal. Calcd for C₁₅H₁₇ClN₄: C, 62.39; H, 5.93; N, 19.40. Found C, 62.37; H, 5.69; N, 19.40. [00815] 4-Chloro- 11 -methyl-6-ethyl-5,6-dihydro-pyrimido[4,5-6]

[l,4]benzodiazepin(3.2) Yellow oil, yield: 47 %, ES-MS: 275.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.01 (s, 1 H), 7.29-7.34 (m, 1 H), 7.03-7.16 (m, 3 H), 4.52 (d, J = 3.9 Hz, 1 H), 4.41-4.48 (m, 1 H), 3.49 (s, 3 H), 1.98-2.14 (m, 2 H), 1.05 (t, J = 7.2 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.0, 145.9, 145.6, 142.6, 135.4, 128.5, 128.1, 126.1, 123.9, 122.4, 59.4, 40.1, 26.1, 11.5.

[00816] 4-Chloro-l l-methyl-6-phenyl-5,6-dihydro-pyrimido[4,5-

Z>][l,4]benzodiazepine(3.3) Yellow oil, yield: 65 %, ES-MS: 323.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.99 (s, 1 H), 7.31-7.40 (m, 4 H), 7.24-7.27 (m, 2 H), 7.16 (d, J = 7.8 Hz, 1 H), 7.06 (td, J = 7.5 Hz, 0.9Hz, 1 H), 6.95 (dd, J = 7.5 Hz, 1 H), 5.82 (d, J = 4.2 Hz, 1 H), 4.97 (d, J = 3.9 Hz, 1 H), 3.26 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.3, 146.4, 146.0, 143.0, 140.1, 135.7, 129.0, 128.8, 128.0, 127.8, 127.7, 126.3, 124.0, 122.8, 60.8, 39.7. Anal. Calcd for C₁₈H₁₅ClN₄: C, 66.98; H, 4.68; N, 17.36. Found C, 67.02; H, 4.95; N, 17.07. [00817] 4-Chloro-ll-methyl-6-(4'-methyl-ρhenyl)-5,6-diliydro-pyrimido[4,5-

£][l,4]benzodiazepine(3.4) Yellow oil, yield: 44 %, ES-MS: 337.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.99 (s, 1 H), 7.32-7.38 (m, 1 H), 7.14-7.20 (m, 5 H), 7.04 (td, J = 7.2 Hz, 1.2Hz, 2 H), 6.92 (dd, J = 7.5 Hz, 1.5 Hz, 1 H), 5.84 (d, J = 3.9 Hz, 1 H), 4.91 (d, J = 3.3 Hz, 1 H), 3.32 (s, 3 H), 2.36 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.3, 146.4, 146.0, 142.9, 137.5, 136.9, 135.9, 129.4, 128.9, 128.2, 127.6, 126.4, 123.9, 122.7, 60.5, 39.9, 21.1. [00818] 4-Chloro-ll-methyl-6-(4'-fluoro-phenyl)-5,6-dihydro-pyrimido[4,5-

Z>][l,4]benzodiazepine(3.5) Yellow oil, yield: 88 %, ES-MS: 341.1[(M+1)+]. IH NMR (300 MHz, CDCl₃) δ: 8.00 (s, 1 H), 7.35-7.40 (m, 1 H), 7.16-7.23 (m, 3 H), 6.97-7.10 (m, 4 H), 5.74 (d, J = 3.0 Hz, 1 H), 4.96 (d, J = 3.3 Hz, 1 H), 3.26 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 162.0 (d, J = 245.0 Hz, 1 C), 151.2, 146.5, 145.8, 143.1, 136.1, 136.1, 135.4, 129.2, 127.9 (d, J = 5.7 Hz, 2 C), 127.7, 124.1, 122.8, 115.5 (d, J = 21.8 Hz, 2 C), 60.3, 39.7. [00819] 4-Chloro- 11 -methyl-6-(4 ' -nitro~ρhenyl)-5,6-dihydro-pyrimido[4,5- b][l,4]benzodiazepine(3.6) Orange solid, yield: 97 %, ES-MS: 368.1 [(M+l)+]. mp 174.5- 175.8 ⁰C. IH NMR (300 MHz, CDCl₃) δ : 8.16 (d, J = 8.7 Hz, 2 H), 8.00 (s, 1 H), 7.41-7.46 (m, 1 H), 7.35 (d, J = 8.4 Hz, 2 H), 7.20-7.16(m, 3 H), 5.60 (d, J = 5.7 Hz, 1 H), 5.19 (d, J = 5.7 Hz, 1 H), 3.11 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.1, 148.3, 147.2, 146.8, 145.7, 143.5, 134.5, 129.8, 128.4, 126.8, 126.6, 124.5, 123.8, 123.1, 60.8, 39.1. Anal. Calcd for C₁₈H₁₄ClN₅O₂: C, 58.78; H, 3.84; N, 19.04. Found C, 58.86; H, 3.74; N, 18.93. [00820] 4-Chloro-8, 1 l-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5-

Z>][l,4]benzodiazepine (3.7) Yellow oil, yield: 72 %, ES-MS: 303.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.00 (s, 1 H), 7.12 (d, J = 8.4 Hz, 1 H), 7.04 (d, J = 8.4 Hz, 1 H), 6.94 (d, J = 1.8Hz, 1 H), 4.45-4.54 (m, 2 H), 3.47 (s, 3 H), 2.32 (s, 3 H), 1.96-2.04 (m, 2 H), 1.36-1.53 (m, 2 H), 1.00 (t, J = 7.2 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.1, 145.9, 143.0, 142.5, 135.2, 133.5, 128.9, 128.0, 126.7, 122.2, 57.5, 40.0, 35.2, 20.7, 20.2, 13.9. [00821] 4-Chloro-8,ll-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5- b][l,4]benzodiazepine (3.8) Yellow oil, yield: 57 %, ES-MS: 289.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.00 (s, 1 H), 7.11 (d, J = 8.1 Hz, 1 H), 7.04 (d, J = 8.1 Hz, 1 H), 6.93 (s, 1 H), 4.49 (s, 1 H), 4.37-4.41 (m, 1 H), 3.47 (s, 3 H), 2.32 (s, 3 H), 1.98-2.12 (m, 2 H), 1.05 (t, J = 7.2 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.0, 145.8, 142.8, 142.4, 134.9, 133.4, 128.8, 127.8, 126.7, 122.1, 59.4, 39.9, 26.0, 20.6, 11.4. [00822] 4-Chloro-8, 1 l-dimethyl-6-phenyl-5,6-dihydro-pyrimido [4,5-

£][l,4]benzodiazepine (3.9) Yellow oil, yield: 72 %, ES-MS: 337.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.97 (s, 1 H), 7.27-7.39 (m, 3 H), 7.23-7.26 (m, 2 H), 7.16 (dd, J = 8.7 Hz, 1.5 Hz, 1 H), 7.05 (d, J = 8.7 Hz, 1 H), 6.79 (d, J = 1.5 Hz, 1 H), 5.73 (d, J = 3.9 Hz, 1 H), 4.98 (d, J = 3.9 Hz, 1 H), 3.23 (s, 3 H), 2.29 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.5, 146.5, 143.3, 142.9, 140.2, 135.3, 133.8, 129.5, 128.6, 128.5, 127.8, 127.6, 126.3, 122.7, 60.9, 39.7, 20.8.

[00823] 4-Chloro-8, 1 l-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro-pyrimido[4,5-

£][l,4]benzodiazepine (3.10) Yellow oil, yield: 46 %, ES-MS: 351.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.97 (s, 1 H), 7.12-7.19 (m, 4 H), 7.06 (d, J = 8.1 Hz, 2 H), 6.75 (s, 1 H), 5.76 (d, J = 3.6 Hz, 1 H), 4.91 (d, J = 3.6 Hz, 1 H), 3.28 (s, 3 H), 2.36 (s, 3 H), 2.28 (s, 3H). 13C NMR (75 MHz, CDCl₃) δ: 151.5, 146.4, 143.3, 142.9, 137.3, 137.0, 135.5, 133.7, 129.4, 129.3, 128.2, 127.9, 126.3, 122.6, 60.6, 39.8, 21.1, 20.8. [00824] 4-Chloro-8, 1 l-dimethyl-6-(4 ' -fluoro-phenyl)-5,6-dihydro-ρyrimido[4,5- b][l,4]benzodiazepine (3.11) Yellow solid, yield: 75 %, ES-MS: 355.1 [(M+l)+]. mp 153.7- 155.6 ^°C. IH NMR (300 MHz, CDCl₃) δ _: 7.98 (s, 1 H), 7.16-7.22 (m, 3 H), 7.00-7.07 (m, 3 H), 6.81 (d, J = 1.5 Hz, 1 H), 5.66 (d, J = 4.8 Hz, 1 H), 4.96(d, J = 4.8 Hz, 1 H), 3.22 (s, 3 H), 2.31 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 162.0 (d, J = 245.0 Hz, 1 C), 151.4, 146.6, 143.2, 143.1, 136.1, 135.0, 133.9, 129.6, 128.5, 127.8 (d, J = 8.0 Hz, 2 C), 127.4, 122.8, 115.5 (d, J = 21.8 Hz, 2 C), 60.4, 39.6, 20.7. Anal. Calcd for C₁₉H₁₆ClFN₄: C, 64.32; H, 4.55; N, 15.79. Found C, 64.14; H, 4.49; N, 15.52.

[00825] 4-Chloro-8,l l-dimethyl-6-(4 ' -nitro-phenyl)-5,6-dihydro-pyrimido[4,5-

£][l,4]benzodiazepine (3.12) Orange solid, yield: 97 %, ES-MS: 382.1 [(M+l)+]. mp 225.6- 227.3 ⁰C. IH NMR (300 MHz, CDCl₃) δ _: 8.16 (d, J = 8.7 Hz, 2 H), 7.98 (s, 1 H), 7.34 (d, J = 8.1 Hz, 2 H), 7.23 (d, J = 8.1 Hz, 1 H), 7.06 (d, J = 8.1 Hz, 1 H), 7.00 (s, 1 H), , 5.53 (d, J = 5.7 Hz, 1 H), 5.18 (d, J = 6.0 Hz, 1 H), 3.07 (s, 3 H), 2.37 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.3, 148.4, 147.1, 146.8, 143.4, 142.9, 134.4, 134.2, 130.2, 129.0, 126.6, 123.7, 123.0, 60.8, 39.0, 20.7.

[00826] 4-Chloro-9, 1 l-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5-

6][l,4]benzodiazepine(3.13) Yellow oil, yield: 81 %, ES-MS: 303.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.00 (s, 1 H), 7.01 (d, J = 7.8 Hz, 1 H), 6.96 (s, 1 H), 6.88 (dd, J = 7.8 Hz, 1.2Hz, 1 H), 4.51-4.56 (m, 1 H), 4.45-4.46 (m, 1 H), 3.48 (s, 3 H), 2.34 (s, 3 H), 1.94- 2.06 (m, 2 H), 1.33-1.55 (m, 2 H), 0.99 (t, J = 7.2 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.0, 145.8, 145.5, 142.5, 138.2, 132.6, 128.2, 125.9, 124.5, 122.9, 57.0, 40.0, 35.2, 21.2, 20.1, 13.9.

[00827] 4-Chloro-9,ll-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5-

£][l,4]benzodiazepine(3.14) Yellow oil, yield: 74 %, ES-MS: 289.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.00 (s, 1 H), 7.01 (d, J = 7.5 Hz, 1 H), 6.96 (s, 1 H), 6.88 (d, J = 7.5 Hz, 1 H), 4.49-4.50 (m, 1 H), 4.38-4.45 (m, 1 H), 3.48 (s, 3 H), 2.34 (s, 3 H), 1.98-2.14 (m, 2 H), 1.05 (t, J = 7.5 Hz, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.0, 145.8, 145.4, 142.5, 138.2, 132.4, 128.1, 126.1, 124.5, 122.9, 59.1, 40.0, 26.2, 21.2, 11.5. Anal. Calcd for C₁₅H₁₇ClN₄: C, 62.39; H, 5.93; N, 19.40. Found C, 62.56; H, 6.12; N, 19.22. [00828] 4-Chloro-9,l l-dimethyl-6-phenyl-5,6-dihydro-pyrimido [4,5-

£][l,4]benzodiazepine(3.15) Yellow oil, yield: 71 %, ES-MS: 337.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.98 (s, 1 H), 7.30-7.38 (m, 3 H), 7.24-7.27 (m, 2 H), 6.98 (s, 1 H), 6.86 (d, J = 7.8 Hz, 1 H), 6.82 (d, J = 7.8 Hz, 1 H), 5.78 (d, J = 3.9 Hz, 1 H), 4.95 (d, J = 3.9 Hz, 1 H), 3.27 (s, 3 H), 2.35 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.3, 146.3, 145.8, 142.9, 140.4, 139.0, 132.8, 128.7, 128.1, 127.7, 127.6, 126.3, 124.7, 123.4, 60.5, 39.7, 21.2 [00829] 4-Chloro-9,ll-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro-ρyrimido[4,5- b][l,4]benzodiazepine(3.16) Yellow oil, yield: 45 %, ES-MS: 351.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.98 (s, 1 H), 7.11-7.16 (m, 4 H), 6.98 (s, 1 H), 6.85 (d, J = 8.4 Hz, 1 H), 6.79 (d, J = 8.4 Hz, 1 H), 5.80 (d, J = 3.3 Hz, 1 H), 4.89 (d, J = 3.3 Hz, 1 H), 3.30 (s, 3 H), 2.36 (s, 3 H), 2.35 (s, 3H). 13C NMR (75 MHz, CDCl₃) δ: 151.3, 146.3, 145.8, 142.9, 138.8, 137.4, 137.1, 133.0, 129.4, 128.2, 127.6, 126.4, 124.6, 123.3, 60.2, 39.9, 21.2, 21.1. [00830] 4-Chloro-9,ll-dimethyl-6-(4'-fluoro-phenyl)-5,6-dihydro-pyrimido[4,5- b][l,4]benzodiazepine(3.17) Yellow oil, yield: 91 %, ES-MS: 355.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 7.99 (s, 1 H), 7.19-7.23 (m, 2 H), 7.98-7.06 (m, 3 H), 6.87 (s, 2 H), 5.70 (d, J = 4.2 Hz, 1 H), 4.94 (d, J = 4.2 Hz, 1 H), 3.25 (s, 3 H), 2.36 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 162.0 (d, J = 245.0 Hz, 1 C), 151.3, 146.4, 145.6, 143.1, 139.1, 136.3, 132.4, 127.9, 127.8 (d, J = 3.4 Hz, 2 C), 127.7, 124.7, 123.5, 115.5 (d, J = 21.8 Hz, 2 C), 60.0, 39.6, 21.2.

[00831] 4-Chloro-9,l l-dimethyl-6-(4 ' -nitro-phenyl)-5,6-dihydro-pyrimido[4,5-

Z?][l,4]benzodiazepine(3.18) Orange solid, yield: 99 %, ES-MS: 382.1 [(M+l)+], nip 82.4- 88.7 °C (no clear melting point observed). IH NMR (300 MHz, CDCl₃) δ : 8.16 (d, J = 8.4 Hz, 2 H), 7.99 (s, 1 H), 7.34 (d, J = 8.4 Hz, 2 H), 7.07 (d, J = 7.2 Hz, 1 H), 6.98 (s, 1 H), 6.96 (d, J = 7.5 Hz, 1 H), 5.56 (d, J = 5.4 Hz, 1 H), 5.17 (d, J = 5.4 Hz, 1 H), 3.09 (s, 3 H), 2.38 (s, 3 H). 13C NMR (75 MHz, CDCl₃) δ: 151.2, 148.6, 147.1, 146.7, 145.5, 143.4, 139.9, 131.6, 128.2, 126.9, 126.1, 125.1, 123.7, 123.6, 60.5, 39.0, 21.3

[00832] 4-Chloro-8-fluoro-l l-methyl-6-phenyl-5,6-dihydro pyrimido[4,5- b][l,4]benzodiazepine(3.19) 5-amino-6-chloro-4-N-methyl anilinopyrimidine (1.4) (200 mg, 0.85 mmol), the benzaldehyde (0.14 mL, 1.275 mmol) and TFA (0.8 mL) were dissolved in CH₃CN (10.0 mL), and stirred under reflux for 6 d. The reaction mixture was concentrated in vacuo, diluted with EtOAc (15 mL), and washed with saturated NaHCO₃ (3 x 15 mL). The water layer was extracted with EtOAc (3 x 10 mL). The combined EtOAc layer was washed with brine, dried over anhydrous Na₂SO₄, concentrated in vacuo and purified by flash chromatography to give 3.19 (55 mg, 19%) as a yellow oil (elution with EtOAc : petroleum ether =1:30). Yield: 19 %, ES-MS: 341.1 [(M+l)+]. IH NMR (300 MHz, CDCl₃) δ: 8.01 (s, 1 H), 7.36-7.43 (m, 3 H), 7.28-7.33 (m, 2 H), 7.11-7.16 (m, 1 H), 7.02 (td, J = 8.7 Hz, 3.0 Hz, 1 H), 6.60-6.63 (dd, J = 8.7 Hz, 3.0 Hz, 1 H), 5.87 (d, J = 3.3 Hz, 1 H), 4.87 (d, J = 3.0 Hz, 1 H), 3.28 (s, 3 H).

6.3 Tricyclic Pyrimido[4,5-ft][l,4]benzothiazepines

[00833] Tricyclic Pyrimido[4,5-b][l,4]benzothiazepines were synthesized according to

Scheme III (FIG. 3). Various cyclization conditions of 5-amino-4,6-bisphenylthiopyrimidine with benzoic acid in PPA/POCI₃ was studied. While no cyclization product was obtained below the temperature of refluxing POCl₃, 95% of desired cyclization product 4 was isolated in the refluxing temperature of POCl₃ after 30 hours. The reactions of several analogues of 5- amino-4,6-bisphenylthiopyrimidine with a variety of carboxylic acids or derivatives were investigated under the above condition and the results are shown in Table 3.

Table 3. Cyclization of 5-amino-4,6-bis(phenylthio)pyrimidine

Entry R^z R^J Time Yield (%)

2.1 H Ph 3Oh 95

2.2 H Pyridin-3-yl 8d 93

2.3 H 4'-CH₃-C₆H₄ 35h 97

2.4 H 2'-NO₂-C₆H₄ 3Oh 70

2.5 H 4'-NO₂-C₆H₄ —

2.6 H 3'-NO₂-C₆H₄ —

2.7 H 4'-F-C₆H₄ 35h 52

2.8 H CH₃ 14h 45

2.9 H CH₃CH₂CH₂ ⁰ 14h 47

2.10 H PhCH₂ 12h 65

2.11 CH₃ Ph 2Oh 97

2.12 CH₃ Pyridin-3-yl 3d 97 2.13 CH₃ 4'-CH₃-C₆H₄ 15h 96

2.14 CH₃ 4'-F-C₆H₄ 3Oh 80

2.15 CH₃ CH₃CH₂CH₂ ⁰ 1Oh 67

2.16 CH₃ PhCH₂ 1Oh 60

2.17 Cl Ph 5d 98

2.18 Cl 4'-CH₃-C₆H₄ 12d 95

2.19 Cl Pyridin-3-yl 16d 12^b

2.20 Cl PhCH₂ 32h 30

2.21 MeO Ph 18h 80

2.22 MeO PhCH₂ 7h 30 ^aUnder the condition of PPA/POC1₃ 1.5 equiv of aromatic acid was added and 1.0 equiv of aliphatic acid was added, (a) trace product was obtained; (b) 80% intermediate was recovered; (c) CH₃CH₂CH₂COCl was utilized.

[00834] The 4-phenylthio group was put in by design to provide an entrance to an additional diversity point. The phenylthio compound 2 could be oxidized to its corresponding sulfoxide or sulphone. Although there are two sulfur atoms present in compound 2, it was anticipated that the sulfur atom that is part of the pyrimidobenzothiazepine ring system should be less prone to oxidation compared to the A- phenylthio group. Therefore, treatment of compound 2 with /w-CPB A readily provided the desired sulfoxides 8, which was achieved by dropwise addition of 1.2 equivalent of m-CPBA in CH₂Cl₂ at O⁰C (Table 4). Elevated temperature or increase in amount of oxidant resulted in increased amount of byproducts from overoxidation.

Table 4. Selective Oxidation

Entry IC Time Yield (%)

8.1 H ^~4h 77 8.2 CH₃ 3h 75

8.3 Cl 4h 75

[00835] The sulfoxide group in compound 8 could be readily replaced by a nucleophilie. To test its versatility, the desired amine-substituted products were obtained with high yields in dry CH₃CN at room temperature in 15 minutes (Table 5).

Table 5. Substitution with Amines

Entry R^z R" R^: 5 Yield (%)

3.1 H H n-Bu 90 3.2 H -(CHz)₄- 90 3.3 CH₃ H n-Bu 88 3.4 Cl H n-Bu 90

[00836] AU reactions were carried out under the nitrogen atmosphere. Phosphoryl oxychloride was freshly distilled. Acetonitrile was dried over anhydrous K₂CO₃. Dichloromethane was dried over anhydrous CaCl₂. All other commercial reagents were used as received without additional purification. Melting point was uncorrected. Mass spectra and HPLC (ELSD) data was recorded on an 1100 LC/MS system (Agilent Technology Corporation), using a 4.6x50 mm column (CenturySIL C- 18 AQ+, 5μ) with a linear gradient 30-90% (v/v) acetonitrile - water with 0.035% trifluoroacetic acid over 8 minutes with a flow rate of 3.5 mL/min. Analytical TLC was performed using 2.5 x 5 cm plates coated with a 0.25 mm thickness of silica gel 60 F254. Column chromatography was performed using silica gel G (200-300 mesh). AU IH NMR spectra (300 MHz) are reported as follows: chemical shifts in ppm downfield from TMS as internal standard (δscale ) and CDCl₃ or DMSO-d6 as the solvent. Multiplicities are indicated as the following: multiplicity [br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, integration and coupling constant (Hz)]. AU 13C NMR spectra (75 MHz) were determined with complete proton decoupling and reported in ppm. Combustion elemental analysis was performed on a Flash EA 1112 instrument.

6.3.1 Procedures and characterization details for 5-amino-4,6- bis(phenylthio)pyrimidine

[00837] 5-Amino-4,6-dichloroρyrimidine (0.652 g, 4.00 mmol) and thiophenol (0.97 g,

0.9 mL, 8.80 mmol) were added to a solution of triethylamine (1.62 g, 2.24 mL, 16.00 mmol) in n-BuOH (20 mL). The reaction mixture was stirred and refluxed over night, then concentrated in vacuo. CH₂Cl₂ (150 mL) was added to the residue. The organic phase was washed twice with brine (60 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo to yield the crude product as a solid. Purification by recrystallization from petroleum ether/EtOAc (10:1, v/v) provided the desired product 5-amino-4,6-bis(phenylthio)pyrimidine 6.1 (1.182 g, 95%) as a yellow solid. Mp: 80-82°C; IH NMR (CDCl₃)δ: 8.24 (s, 1 H), 7.52- 7.47 (m, 4 H), 7.42-7.37 (m, 6 H); 13C NMR (CDCl₃)δ: 149.6, 148.5, 136.3, 133.8, 129.4, 129.0, 128.9; ES-MS: 312.1 [M+H+].

[00838] 5-Amino-4,6-bis(p-tolylthio)pyrimidine 95%; white plates, mp: 185-186⁰C;

IH NMR (CDCl₃)δ: 8.22 (s, 1 H), 7.40 (d, J = 8.1 Hz, 4 H), 7.21 (d, J = 8.1 Hz, 4 H), 4.17 (s, 2 H), 2.37 (s, 6 H); 13C NMR (CDCl₃)δ: 150.1, 148.7, 139.2, 135.6, 134.2, 130.2, 125.1, 21.3; ES-MS: 340.1 [M+H+].

[00839] 5-Amino-4,6-bis(p-chlorophenylthio)pyrimidine 96%; white plates, mp: 205-

206⁰C; IH NMR (DMSO-d6)δ: 7.98 (s, 1 H), 7.51 (s, 8 H), 5.52 (s, 2 H); 13C NMR (DMSO- d6)δ: 153.1, 151.5, 141.2, 141.0, 139.1, 134.5, 133.0; ES-MS: 380.0 [M+H+]. [00840] 5-Amino-4,6-bis(p-hydroxy)phenylthiopyrimidine 98%; white powder, mp:

226-228°C; IH NMR (DMSO-d6)δ: 9.86 (s, 2 H), 7.90 (s, 1 H), 7.30 (d, J = 8.7 Hz, 4 H), 6.82 (d, J = 8.4 Hz, 4 H), 5.17 (s, 2 H); 13C NMR (DMSO-d6)δ: 164.1, 154.9, 151.9, 142.4, 139.8, 121.8, 121.7; ES-MS: 344.0 [M+H+].

[00841] 5-Amino-4,6-bis(p-methoxyphenylthio)pyrimidine was prepared by methylation of compound 6.4 with iodomethane. The procedure was as follows. To a suspension of anhydrous K₂CO₃ (0.415 g, 3.00 mmol) in acetone (5 mL) 4,6~bis(p- hydroxy)phenylthio-5-aminopyrimidine (0.343 g, 1.00 mmol) and iodomethane (0.596 g, 4.20 mmol) was added and the mixture was refluxed with stirring over night. After cooled to room temperature, the solvent was removed in vacuo. EtOAc (20 mL) was added and washed twice with water (10 mL), dried over anhydrous Na₂SO₄. After evaporation of the solvent, the crude product was purified by recrystallization from EtOAc to provide the desired product 0.334 g (90%) as white plates.

[00842] 5-Amino-4,6-bis(p-methoxy)ρhenylthiopyrimidine 90%; white plates, mp:

189-191°C; IH NMR (CDCl₃)B: 8.20 (s, 1 H), 7.46 (d, J = 9.0 Hz, 4 H), 6.94 (d, J = 8.7 Hz, 4 H), 4.13 (s, 2 H), 3.83 (s, 6 H); 13C NMR (CDCl₃)δ: 160.5, 150.6, 148.7, 136.3, 134.7, 118.7, 115.0, 55.3; ES-MS: 372.1 [M+H+].

6.3.2 General Procedure for synthesis of 4-(phenyIthio)-6- phenylpyrimido[4,5-Z»][l,4]benzothiazepine

[00843] 5-Amino-4,6-bis(phenylthio)pyrimidine (0.156 g, 0.50 mmol), benzoic acid

(0.092 g, 0.75 mmol) and PPA (0.253g, 0.75 mmol) were dissolved in POCl₃ (5.0 mL), and stirred under reflux for 30h. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate (15 mL), and water (15 mL) was added slowly. The water layer was treated with 5N aqueous NaOH to pH 10 and extracted with EtOAc (2x15 mL). The combined EtOAc layer was washed with saturated Na₂CO₃, brine, dried over anhydrous Na₂SO₄, concentrated in vacuo and purified by flash chromatography with petroleum ether/EtOAc (15:1, v/v) as eluent afforded 0.189 g (95%) of 2.1 as a yellow solid. [00844] 4-(Phenylthio)-6-phenylpyrimido[4,5-&] [1 ,4]benzothiazepine (2.1) 95%; yellow solid, mp: 171-173°C; IH NMR (CDCl₃)δ: 8.45 (s, 1 H), 7.94-7.91 (m, 2 H), 7.65- 7.31 (m, 12 H); 13C NMR (CDCl₃)δ: 171.2, 166.7, 154.5, 153.0, 139.7, 137.9, 137.5, 137.3, 135.8, 134.0, 132.6, 131.9, 131.4, 130.5, 129.8, 129.6, 128.7, 128.6, 128.5; ES-MS: 398.1 [M+H+]. Anal. Calcd for C₂₃H₁₅N₃S₂: C, 69.49; H, 3.80; N, 10.57. Found: C, 69.48; H, 3.72; N, 10.62.

[00845] 4-(Phenylthio)-6-(pyridin-3-yl)-pyrimido[4,5-Z?] [ 1 ,4]benzothiazepine (2.2)

93%; yellow solid, mp: 241-243°C; IH NMR (CDCl₃)δ: 9.05 (br s, 1 H), 8.78 (br s, 1 H), 8.48 (s, 1 H), 8.34 (dt, J = 8.4, 1.8 Hz, 1 H), 7.67 (dd, J = 7.2, 0.9 Hz, 1 H), 7.61-7.56 (m, 3 H), 7.49-7.41 (m, 5 H), 7.34 (dd, J = 6.6, 1.5 Hz, 1 H); 13C NMR (CDCl₃)δ: 168.9, 166.9, 154.9, 153.1, 152.4, 151.5, 137.6, 137.3, 136.4, 135.8, 135.4, 134.4, 133.1, 131.0, 129.9, 129.6, 129.0, 128.2, 123.6; ES-MS: 399.1 [M+H+].

[00846] 4-(Phenylthio)-6-(pyridin-3-yl)-pyrimido[4,5-b][l,4]benzothiazepine (2.2)

93%; yellow solid, mp: 241-243⁰C; IH NMR (CDCl₃)δ: 9.05 (br s, 1 H), 8.78 (br s, 1 H), 8.48 (s, 1 H), 8.34 (dt, J = 8.4, 1.8 Hz, IH), 7.67 (dd, J = 7.2, 0.9 Hz, IH), 7.61-7.56 (m, 3H), 7.49-7.41 (m, 5H), 7.34 (dd, J = 6.6, 1.5 Hz, IH); 13C NMR (CDCl₃)δ: 168.9, 166.9, 154.9, 153.1, 152.4, 151.5, 137.6, 137.3, 136.4, 135.8, 135.4, 134.4, 133.1, 131.0, 129.9, 129.6,

129.0, 128.2, 123.6; ES-MS: 399.1 [M+H+].

[00847] 4-(Phenylihio)-6-p-tolylpyrimido[4,5-b][l,4]benzothiazepine (2.3) 97%; yellow solid, mp: 164-167⁰C; IH NMR (CDCl₃)δ: 8.44 (s, 1 H), 7.82 (d, J = 8.1 Hz, 2 H),

7.65-7.25 (m, 11 H), 2.45( s, 3 H); 13C NMR (CDCl₃)δ: 170.8, 166.4, 154.0, 152.7, 142.3,

137.8, 137.2, 137.1, 136.7, 135.5, 133.7, 132.3, 131.2, 130.2, 129.5, 129.3, 129.2, 128.3, 21.6;

ES-MS: 412.1 [M+H+].

[00848] 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5-£] [1 ,4]benzothiazepine (2.4)

70%; yellow solid, mp: 190-192⁰C; IH NMR (CDCl₃)δ: 8.49 (s, 1 H), 8.07 (dd, J = 8.1, 1.2

Hz, 1 H), 7.97 (dd, J = 7.8, 1.2 Hz, 1 H), 7.81 (td, J = 7.8, 1.2 Hz, 1 H), 7.68 (td, J = 8.1, 1.5

Hz, 1 H), 7.62-7.54 (m, 3 H), 7.52 (dd, J = 7.5, 1.5 Hz, 1 H), 7.49-7.43 (m, 3 H), 7.29 (td, J =

8.1, 1.4 Hz, 1 H), 7.09 (dd, J = 7.5, 1.2 Hz, 1 H); 13C NMR (CDCl₃)δ: 170.1, 167.6, 155.5,

153.7, 149.1, 137.6, 137.5, 137.1, 136.7, 135.9, 134.0, 133.7, 133.4, 132.7, 131.3, 129.9,

129.6, 129.5, 129.0, 128.1, 125.0; ES-MS: 443.1 [M+H+].

[00849] 4-(Phenylthio)-6-(p-fluorophenyl)pyrimido[4,5-/?][l,4]benzothiazepine (2.7)

52%; yellow solid, mp: 197-198⁰C; IH NMR (CDCl₃)δ: 8.46 (s, 1 H), 7.97-7.92 (m, 2 H),

7.65 (dd, J = 8.0, 1.4 Hz, 1 H), 7.61-7.57 (m, 2 H), 7.54 (dd, J = 7.5, 1.5 Hz, 1 H), 7.48-7.43

(m, 3 H), 7.40 (dd, J = 7.2, 1.2 Hz, 1 H), 7.33 (dd, J = 7.7, 1.7 Hz, 1 H), 7.22-7.14 (m, 2 H);

13C NMR (CDCl₃)δ: 169.8, 166.9, 165.1 (J = 235.8 Hz), 154.5, 152.9, 137.5, 137.0, 135.8,

135.5, 134.2, 132.8, 132.7 (J = 9.2 Hz), 131.2, 129.9, 129.6, 129.5, 128.7, 128.3, 115.8 (J =

20.6 Hz); ES-MS: 416.1 [M+H+].

[00850] 4-(Phenylthio)-6-methylpyrimido[4,5-b][l,4]benzothiazepine (2.8) 45%; yellow oil; IH NMR (CDCl₃)δ: 8.42 (s, 1 H), 7.55-7.52 (m, 4 H), 7.46-7.42 (m, 5 H), 2.77 (s,

3 H); 13C NMR (CDCl₃)δ: 173.5, 165.9, 153.9, 153.2, 138.9, 137.5, 136.2, 135.5, 133.5,

132.0, 129.5, 129.3, 129.0, 128.2, 29.1; ES-MS: 336.0 [M+H+].

[00851] 4-(Phenylthio)~6-propylρyrimido[4,5-£][l,4]benzothiazepine (2.9) 47%; yellow solid, mp: 166-168⁰C; IH NMR (CDCl₃)δ: 8.41 (s, 1 H), 7.55-7.46 (m, 4 H), 7.45-

7.39 (m, 4 H), 3.01 (t, J = 7.5 Hz, 2 H), 1.90-1.78 (m, 2 H), 1.10 (t, J = 7.5 Hz, 3 H); 13C

NMR (CDCl₃)δ: 176.8, 165.9, 153.8, 153.2, 138.5, 137.6, 136.7, 135.5, 133.5, 131.7, 129.5,

129.2, 129.0, 128.3, 128.1, 43.7, 20.4, 13.9; ES-MS: 364.1 [M+H+].

[00852] 4-(Phenylthio)-6-benzylpyrimido[4,5-b][l,4]benzothiazepine (2.10) 65%; yellow solid, mp: 149-151°C; IH NMR (CDCl₃)δ: 8.42 (s, 1 H), 7.57-7.52 (m, 3 H), 7.48-

7.36 (m, 8 H), 7.35-7.20 (m, 3 H), 4.38 (s, 2 H); 13C NMR (CDCl₃)δ: 175.1, 166.2, 154.4, 153.7, 138.3, 137.7, 137.3, 136.4, 135.8, 133.8, 132.1, 129.8, 129.6, 129.5, 129.2, 128.9, 128.5, 128.3, 127.2, 48.6; ES-MS: 412.1 [M+H+].

[00853] 4-(p-Tolylthio)-6-phenyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine (2.11)

97%; yellow solid, mp: 210-211⁰C; IH NMR (CDCl₃)δ: 8.44 (s, 1 H), 7.96-7.93 (m, 2 H), 7.55-7.45 (m, 6 H), 7.33 (dd, J = 7.8, 1.7 Hz, 1 H), 7.25 (d, J = 7.8 Hz, 2 H), 7.13 (d, J = 1.5 Hz, 1 H), 2.40 (s, 3 H), 2.32 (s, 3 H); 13C NMR (CDCl₃)δ: 170.9, 166.6, 154.1, 152.9, 139.7, 139.5, 138.6, 137.6, 136.8, 135.4, 133.9, 133.5, 133.2, 131.5, 131.4, 130.1, 128.4, 124.6, 21.4, 21.0; ES-MS: 426.1 [M+H+].

[00854] 4-(p-Tolylthio)-6-(pyridin-3-yl)-8-methylpyrimido[4,5-b][l,4]benzothiazepine

(2.12) 97%; yellow solid, mp: 266-267°C; IH NMR (CDCl₃)δ: 9.04 (d, J = 1.8 Hz, 1 H), 8.78 (dd, J = 4.8, 1.5 Hz, 1 H), 8.47 (s, 1 H), 8.38 (dt, J = 8.1, 1.8 Hz, 1 H), 7.55-7.36 (m, 5 H), 7.26 (d, J = 8.1 Hz, 2 H), 7.12 (d, J = 1.8 Hz, 1 H), 2.41 (s, 3 H), 2.34 (s, 3 H); 13C NMR (CDCl₃)δ: 168.9, 167.1, 154.9, 153.3, 152.3, 151.6, 140.2, 139.4, 137.7, 137.3, 136.2, 135.7, 135.5, 134.3, 134.2, 134.0, 131.4, 130.5, 124.5, 123.7, 21.7, 21.4; ES-MS: 427.1 [M+H+]. [00855] 4-(p-Tolylthio)-6-p-tolyl-8-methylpyrimido[4,5-Z?][l,4]benzothiazepine (2.13)

96%; yellow solid, mp: 194-196°C; IH NMR (CDCl₃)δ: 8.43 (s, 1 H), 7.83 (d, J = 8.1 Hz, 2 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.46 (d, J = 8.1 Hz, 2 H), 7.34-7.24 (m, 5 H), 7.13 (d, J = 1.5 Hz, 1 H), 2.45 (s, 3 H), 2.40 (s, 3 H), 2.31 (s, 3 H); 13C NMR (CDCl₃)δ: 170.7, 166.5, 153.9, 152.8, 142.1, 139.7, 138.6, 137.7, 136.8, 136.7, 135.4, 133.8, 133.5, 133.1, 131.5, 130.1, 129.1, 124.6, 21.5, 21.4, 21.0; ES-MS: 440.3 [M+H+]. [00856] 4-(ρ-Tolylthio)-6-(p-fluorophenyl)-8-methylpyrimido[4,5-

Z>][l,4]benzothiazepine (2.14) 80%; yellow solid, mp: 152-153°C; IH NMR (CDCl₃)δ: 8.45 (s, 1 H), 7.98-7.93 (m, 2 H), 7.51 (d, J = 7.8 Hz, 1 H), 7.45 (d, J = 8.1 Hz, 2 H), 7.34 (dd, J = 8.1, 1.8 Hz, 1 H), 7.25 (d, J = 8.1 Hz, 2 H), 7.21-7.15 (m, 2 H), 7.11 (d, J = 1.5 Hz, 1 H), 2.40 (s, 3 H), 2.33 (s, 3 H); 13C NMR (CDCl₃)δ: 169.8, 166.9, 165.2 (J = 251.9 Hz), 154.5, 153.1, 140.1, 139.1, 137.8, 136.8, 136.0, 135.9, 135.7, 134.2, 134.0, 133.7, 132.6 (J = 8.6 Hz), 131.6, 130.5, 124.7, 115.8 (J = 21.8 Hz), 21.7, 21.4; ES-MS: 444.1 [M+H+]. [00857] 4-(p-Tolylthio)-6-propyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine (2.15)

67%; yellow solid, mp: 164-166°C; IH NMR (CDC13)δ: 8.40 (s, 1 H), 7.43-7.39 (m, 3 H), 7.30-7.22 (m, 4 H), 3.01 (t, J = 7.2 Hz, 2 H), 2.39 (s, 3 H), 2.38 (s, 3 H), 1.90-1.78 (m, 2 H), 1.10 (t, J = 7.8 Hz, 3 H); 13C NMR (CDC13)δ: 176.7, 166.1, 153.7, 153.3, 139.6, 139.1, 138.3, 137.5, 135.4, 133.3, 133.2, 132.4, 130.0, 128.4, 124.6, 43.5, 21.3, 21.1, 20.4, 13.8;ES- MS: 392.1 [M+H+]. Anal. Calcd for C₂₂H₂iN₃S₂: C, 67.48; H, 5.41; N, 10.73. Found: C, 69.39; H, 5.43; N, 10.66. [00858] 4-(p-Tolylthio)-6-benzyl-8-nieihylpyrimido[4,5-b][l,4]benzothiazeρine (2.16)

60%; yellow solid, mp: 179-181⁰C; IH NMR (CDCl₃)δ: 8.40 (s, 1 H), 7.42 (d, J = 8.1 Hz, 4 H), 7.35-7.18 (m, 8 H), 4.37 (s, 2 H), 2.39 (s, 3 H), 2.34 (s, 3 H); 13C NMR (CDCl₃)δ: 174.7, 166.2, 154.0, 153.6, 139.7, 139.1, 137.8, 137.4, 136.2, 135.4, 133.7, 133.3, 132.6, 130.1, 129.4, 128.5, 128.4, 126.8, 124.5, 48.1, 21.4, 21.2; ES-MS: 440.1 [M+H+]. [00859] 4-(p-Chloro-phenylthio)-6-phenyl-8-chloropyrimido[4,5- b][l,4]benzothiazepine (2.17) 98%; yellow solid, mp: 203-205°C; IH NMR (CDCl₃)δ: 8.46 (s, 1 H), 7.94-7.90 (m, 2 H), 7.61-7.49 (m, 7 H), 7.44-7.40 (m, 2 H), 7.31 (d, J = 2.4 Hz, 1 H); 13C NMR (CDCl₃)δ: 169.9, 166.3, 154.6, 152.7, 139.0, 138.3, 137.8, 137.1, 136.3, 125.7,

135.3. 135.2, 132.7, 132.3, 131.0, 130.3, 129.9, 128.9, 126.7; ES-MS: 466.0 [M+H+]. [00860] 4-(p-Chloro-phenylthio)-6-p-tolyl-8-chloropyrimido[4,5- 6][l,4]benzotbiazepine (2.18) 95%; yellow solid, mp: 192-193⁰C; IH NMR (CDCl₃)δ: 8.44 (s, 1 H), 7.81 (d, J = 8.1 Hz, 2 H), 7.58-7.47 (m, 4 H), 7.43-7.39 (m, 2 H), 7.33-7.30 (m, 3 H), 2.46 (s, 3 H); 13C NMR (CDCl₃)δ: 169.4, 165.9, 154.1, 152.4, 138.1, 137.6, 137.0, 136.8, 136.0, 135.3, 134.9, 134.8, 132.3, 130.7, 130.1, 129.6, 129.5, 129.4, 126.6, 21.6; ES-MS: 480.1 [M+H+].

[00861] 4-(p-Chloro-phenylthio)-6-(pyridin-3-yl)-8-chloropyrimido[4,5-

6][l,4]benzothiazepine (2.19) 12%; yellow solid, mp: 264-266°C; IH NMR (CDCl₃)δ: 9.07 (s, 1 H), 8.82 (d, J = 3.6 Hz, 1 H), 8.49 (s, 1 H ), 8.32 (dt, J = 8.1, 1.8 Hz, 1 H), 7.61 (d, J = 8.4 Hz, 1 H), 7.56 (d, J = 2.1 Hz, 1 H), 7.54-7.48 (m, 3 H), 7.45-7.41 (m, 2 H), 7.31 (d, J = 2.1 Hz, 1 H); 13C NMR (CDCl₃)δ: 167.3, 166.2, 154.7, 152.5, 152.4, 151.0, 137.1, 136.9, 136.7, 136.1, 135.5, 135.2, 135.1, 134.4, 132.9, 130.2, 129.6, 126.1, 123.5; ES-MS: 467.0 [M+H+].

[00862] 4-(p-Chloro-phenylthio)-6-benzyl-8-chloropyrimido[4,5- b][l,4]benzothiazepine (2.20) 30%; yellow solid, mp: 177-179°C; IH NMR (CDCl₃)δ: 8.42 (s, 1 H), 7.51-7.25 (m, 12 H), 4.35 (s, 2 H); 13C NMR (CDCl₃)δ: 173.9, 166.0, 154.5, 153.4,

139.3. 137.1, 136.8, 136.3, 135.8, 135.5, 135.0, 132.2, 129.8, 129.6, 129.0, 128.6, 128.2, 127.5, 126.7, 48.4; ES-MS: 480.0 [M+H+].

[00863] 4-(p-Methoxyphenylthio)-6-phenyl-8-methoxypyrimido[4,5-

£][l,4]benzothiazepine (2.21) 80%; yellow solid, mp: 206-209⁰C; IH NMR (CDCl₃)δ: 8.45 (s, 1 H), 7.99-7.96 (m, 2 H), 7.58-7.48 (m, 6 H), 7.06 (dd, J = 8.4, 3.0 Hz, 1 H), 7.00-6.95 (m, 2 H), 6.83 (d, J = 2.7 Hz, 1 H), 3.85 (s, 3 H), 3.74(s, 3 H); 13C NMR (CDCl₃)δ: 170.6, 167.2, 161.0, 159.8, 154.4, 153.4, 139.5, 138.2, 137.9, 137.4, 135.2, 131.9, 130.4, 128.7, 128.3, 118.8, 118.4, 116.5, 115.3, 55.9, 55.6;ES-MS: 458.1 [M+H+]. [00864] 4-(p-Methoxyphenylthio)-6-benzyl-8-methoxypyrimido[4,5-

£][l,4]benzothiazepine (2.22) 30%; yellow solid, mp: 175-177°C; IH NMR (CDCl₃)δ: 8.42 (s, 1 H), 7.47-7.43 (m, 4 H), 7.37-7.24 (m, 4 H), 7.00-6.97 (m, 2 H), 6.96-6.94 (m, 1 H), 6.91 (dd, J = 8.9, 2.9 Hz, 1 H), 4.36 (s, 2 H), 3.84 (s, 3 H), 3.74 (s, 3 H); 13C NMR (CDCl₃)δ: 174.4, 166.7, 161.0, 160.1, 154.3, 154.2, 139.3, 137.6, 137.4, 136.5, 134.9, 129.6, 128.9, 128.0, 127.3, 118.8, 117.6, 115.2, 113.8, 55.8, 55.6, 48.5; ES-MS: 472.1 [M+H+].

6.3.3 General Procedure for synthesis of 4-(phenyIsuIfinyl)-6- phenylpyrimido[4,5-2>] [l,4]benzothiazepine 8.1.

[00865] 4-(Phenylthio)-6-phenylpyrimido[4,5-£] [ 1 ,4]benzothiazepine 2.1 (0.397 g,

1.00 mmol) was dissolved in CH₂Cl₂ (10 mL) and cooled to 0-5°C in an ice bath. A solution of m-CPBA (0.206 g, 1.20 mmol) in CH₂Cl₂ (15 mL) was added dropwise over 30min. After stirring 3h, the reaction mixture was treated with saturated NaHSO₃, saturated Na₂CO₃, brine, dried over anhydrous Na₂SO₄, concentrated in vacuo and purified by flash chromatography with petroleum ether/EtOAc (5:1, v/v) as eluent afforded 0.318 g (77%) of 8.1 as a yellow solid.

[00866] 4-(Phenylsulfinyl)-6-phenylpyrimido[4,5-Z?][l,4]benzothiazepine (8.1) 77%; yellow solid, mp: 200-202°C; IH NMR (CDCl₃)δ: 8.96 (s, 1 H), 7.92 (br d, J = 6.9 Hz, 2 H), 7.69 (br s, 1 H), 7.66-7 '.47 (m, 7 H), 7.33 (br s, 3 H), 7.06 (br s, 1 H); 13C NMR (CDCl₃)δ: 166.0, 157.8, 154.7, 142.9, 136.4, 136.2, 133.9, 132.6, 132.4, 131.6, 130.3, 129.3, 128.7, 128.6, 125.4;ES-MS: 414.0 [M+H+].

6.3.4 General Procedure for synthesis of 4-(n-butylamino)-6- phenylpyrimido[4,5-Z>][l,4]benzothiazepine 3.1.

[00867] 4-(Phenylsulfinyl)-6-phenylpyrimido[4,5-b][l,4]benzothiazepine 8.1 (0.413 g,

1.00 mmol) was dissolved in dry CH₃CN (10 mL). n-Butylamine (0.219 g, 0.30 mL, 3.00 mmol) was added at room temperature. After stirring 15min, the reaction mixture was concentrated in vacuo and purified by flash chromatography with petroleum ether/EtOAc (8:1, v/v) as eluent afforded 0.324 g (77%) of 3.1 as a yellow solid. [00868] 4-(n-Butylammo)-6-phenylpyrimido[4,5-&][l,4]benzothiazepine (3.1) 90%; yellow oil; IH NMR (CDCl₃)δ: 8.30 (s, 1 H), 7.80-7.77 (m, 2 H), 7.63 (dd, J = 7.8, 0.9 Hz, 1 H), 7.55-7.44 (m, 4 H), 7.34 (td, J = 7.2, 0.9 Hz, 1 H), 7.24 (dd, J = 7.8, 1.4 Hz, 1 H), 5.81 (t, J = 5.4 Hz, 1 H), 3.54 (q, J = 6.7 Hz, 2 H), 1.74-1.64 (m, J = 7.4 Hz, 2 H), 1.53-1.41 (m, J = 7.5 Hz, 2 H), 0.99 (t, J = 7.4 Hz, 3 H) ; 13C NMR (CDCl₃)δ: 170.9, 158.1, 155.7, 150.1, 139.7, 138.2, 137.4, 133.8, 132.3, 131.5, 130.8, 129.8, 128.7, 128.2, 127.2, 41.3, 31.9, 20.4, 14.1 ; ES-MS: 361.1 [M+H+]. Anal. Calcd for C₂₁H₂₀N₄S: C, 69.97; H, 5.59; N, 15.54. Found: C, 70.00; H, 5.76; N, 15.30.

[00869] 4-(Pyrrolidin-l-yl)-6-ρhenylpyrimido[4,5-b][l,4]benzothiazepine (3.2) 90%; yellow solid, mp: 231-232⁰C; IH NMR (CDCl₃)δ: 8.17 (s, 1 H), 7.81-7.77 (m, 2 H), 7.63 (dd, J = 7.8, 1.5 Hz, 1 H), 7.51-7.34 (m, 5 H), 7.29 (dd, J = 7.8, 1.7 Hz, 1 H), 3.88 (br s, 2 H), 3.63 (br s, 2 H), 2.00 (br s, 2 H), 1.85 (br s, 2 H); 13C NMR (CDCl₃)δ: 168.0, 156.5, 153.8, 153.7, 139.0, 138.8, 138.2, 133.5, 131.6, 131.3, 129.3, 129.1, 128.7, 128.3, 127.7, 50.0, 29.9; ES- MS: 359.1 [M+H+]. Anal. Calcd for C₂₁H₁₈N₄S: C, 70.36; H, 5.06; N, 15.63. Found: C, 70.36; H, 5.15; N, 15.82.

[00870] 4-(n-Butylamino)-6-phenyl-8-methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine

(3.3) 88%; yellow solid, mp: 173-175°C; IH NMR (CDCl₃)δ: 8.28 (s, 1 H), 7.81-7.77 (m, 2 H), 7.56-7.44 (m, 4 H), 7.30 (dd, J = 7.8, 1.5 Hz, 1 H), 7.03 (d, J = 1.5 Hz, 1 H), 5.76 (t, J = 5.6 Hz, 1 H), 3.53 (q, J = 6.7 Hz, 2 H), 2.29 (s, 3 H), 1.73-1.64 (m, 2 H), 1.53-1.41 (m, 2 H), 0.99 (t, J = 7.4 Hz, 3 H) ; 13C NMR (CDCl₃)δ: 170.6, 157.7, 155.4, 150.4, 139.5, 138.1, 137.0, 134.7, 133.2, 132.9, 131.1, 130.8, 129.5, 128.4, 126.9, 40.9, 31.6, 21.0, 20.1, 13.8; ES- MS: 375.2 [M+H+].

[00871] 4-(n-Butylamino)-6-phenyl-8-chloropyrimido[4,5~&] [ 1 ,4]benzothiazepine (3.4)

90%; yellow solid, mp: 155-157⁰C; IH NMR (CDCl₃)δ: 8.30 (s, 1 H), 7.79-7.76 (m, 2 H), 1.51-1 A5 (m, 5 H), 7.22 (d, J = 2.1 Hz, 1 H), 5.75 (t, J = 5.6 Hz, 1 H), 3.55 (q, J = 6.7 Hz, 2 H), 1.74-1.64 (m, 2 H), 1.53-1.41 (m, 2 H), 0.99 (t, J = 7.5 Hz, 3 H) ; 13C NMR (CDCl₃)δ: 169.3, 158.0, 156.1, 150.0, 139.0, 138.6, 136.6, 134.9, 134.6, 132.3, 131.7, 130.3, 129.6, 128.9, 127.0, 41.2, 31.9, 20.4, 14.1; ES-MS: 395.1 [M+H+].

6.4 Tetracyclic pyrimidine-fused benzodiazepines

6.4.1 General Synthetic and Characterization Details

[00872] Tetracyclic pyrimidine-fused benzodiazepines were synthesized according to

Scheme IV (FIG. 4). The scope of the cyclization was explored with a variety of aldehydes and ketones and the results are summarized in Table 6.

Table 6. Preparation of Tetracyclic Pyrimidobenzodiazepines _R1COR2 TFAZCH₃CN reflux

Entry R¹ R^z time yield products mp (⁰C) (h) (%)

1 Et H 3 92 2.1 140-142

2 nPr H 3 90 2.2 151-152

3 Ph H 4 92 2.3 142-144

4 o-Me- Ph H 4 97 2.4 201-203

5 P-NO₂- Ph H 5 96 2.5 195-196

6 P-F- Ph H 3 90 2.6 147-148

7 PhCH=CH H 2 81 2.7 162-163

8 CH₃CH=CH H 0.5 83 2.8 132-135

9 Me Me 5 95 2.9 117-118

10 Et Me 15 92 2.10 86-88

11 nPr Me 10 84 2.11 99-101

12 R¹ R²= -(CH₂)_S- 5 92 2.12 182-183

13 Ph Me 12 87 2.13 188-189

14 /7-MeO-Ph Me 60 62 2.14 168-169

15 P-NO₂- Ph Me 44 82 2.15 182-183

16 Ph Et 65 57 2.16 145-147

17 Me COOH 6.5 59 2.17 273(dec)

Table 7. Preparation of Tetracyclic Pyrimidobenzodiazepines

Entry R¹ time (h) yield (%) products mp (°C)

1 Et H 3.5 97 2.18 syrup

2 nPr H 4 95 2.19 108-110

3 Ph H 5 96 2.20 140-142

4 p-Me-Ph H 7 91 2.21 syrup

5 P-NO₂-Ph H 4 85 2.22 245-247

6 PhCH=CH H 10 40 2.23 114-116

7 Me Me 56 37 2.24 130-132

[00873] Other variants of the tetracyclic pyrimidine-fused benzodiazepines were obtained with additional chemical modification to illustrate the possible library diversity. The chloro group in compounds 2 (n = 1, 2) presents an excellent opportunity to introduce additional diversity points. Two compounds (2.2 and 2.3) were selected as representative examples to examine their reactivity towards various nucleophiles and coupling reactions. Compounds 2.2 or 2.3 reacted readily with an amine under either acidic conditions of coned, aq. HCl (for n-BuNH₂, aniline and morpholine) or in the presence of Et₃N (for pyrrolidine) to give the desired amine substituted products in moderate to high yields (Entry 1-4, Table 8). Other nucleophiles with heteroatoms such alcohols and thiols were also known to react with 6-chloropurines, therefore their reactions were exemplified with n-BuOH, BnSH and PhSH. Compounds 2.2 or 2.3 reacted with n-butanol, benzylthiol and thiophenol smoothly under basic conditions to give the corresponding n-butyloxy, benzylsulfanyl or phenylsulfanyl substituted pyrimidobenzediazopines in high yields (Entry 5-10, Table 8). Introduction of carbon substitutents to replace the chloro further expands the scope of the current library, therefore under non-optimized Suzuki-Miyaura cross-coupling condition (Nyerges et al., Tetrahedron. 2005, 61, 8199; Havelkova et al., Synlett. 1999, 7, 1145) compound 2.2 or 2.3 reacted with phenylboronic acid to yield aryl substituted products (Entry 11-12, Table 8) in good yields. [00874] Table 8. Introduction of final diversity points to compounds 2

Entry ArCl Nucleophiles time Product Yield (%) mp (°C)

1 2.3 R³R⁴NH = 11Bu-NH₂ 3 d 3a 74 105-107

2 2.2 R³R⁴NH = Ph-NH₂ 4.5 d 3b 60 149-151 3 2.3 R³R⁴NH = morpholine 3 d 3c 90 158-160

4 2.2 R³R⁴NH = pyrrolidine 3 d 3d 93 116-118

5 2.2 R³OH = nBu-OH 3O h 4a 94 syrup

6 2.3 R³OH = nBu-OH 33 h 4b 94 89-91

7 2.2 R³SH = Bn-SH 7 h 5a 94 syrup

8 2.3 R³SH = Bn-SH 7 h 5b 96 113-115

9 2.2 R³SH = Ph-SH 9 h 5c 95 syrup

10 2.3 R³SH = Ph-SH 7 h 5d 94 syrup

11 2.2 NA 1O h 6a 53 syrup

12 2.3 NA 5 h 6b 53 syrup

[00875] Acetonitrile was treated with calcium hydride and distilled after refluxing for

3 h in a nitrogen atmosphere. AU other commercial reagents were used as received without additional purification. Melting point was uncorrected. Mass spectra and HPLC (ELSD) data was recorded on an 1100 LC/MS system (Agilent Technology Corporation). The IH and 13C NMR data were obtained on a 300-MHz Varian spectrometer with TMS as the internal standard and CDCl₃ or DMSO-d6 as solvent. Multiplicities are indicated as the following: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doubled doublet; br, broad. Coupling constants (J values) where noted are quoted in hertz.

6.4.2 Synthesis and characterization of precursors

[00876] The indole-derivative of benzodiazepine precusor was prepared according to the procedure described for the indole-fused pteridines, below.

[00877] The quinoline derivative of benzodiazepine precusor was prepared as described below.

[00878] l-(6-Chloro-5-nitro-pyrimidin-4-yl)-l,2,3,4-tetrahydroquinoline.

[00879] To a solution of 4,6-dichloro-5-nitro-pyrimidine (3.00 g, 15.5 mmol) in anhydrous THF (40 mL) was added dropwise a solution of 1,2,3,4-tetrahydroquinoline 10

(2.07 g, 15.5 mmol) and triethylamine (3.3 mL, 23.3 mmol) in anhydrous THF (20 mL) in ice bath. After warmed to room temperature, the reaction mixture was stirred over night then concentrated in vacuo. The residue was dissoved in CH₂Cl₂ (50 mL) and washed with IN

HCl (30 mLx3), brine (30 mLx3), dried over anhydrous MgSO₄. Concentration in vacuo and purification by flash chromatography on a silica gel column (Petroleum/ EtOAc 10:1, v/v) provided the desired product 3.46 g (77%), brown solid, mp 130-131 ⁰C. IH NMR (CDCl₃):δ8.06 (s, IH), 7.21 (d, J = 7.5 Hz, IH), 7.14 (td, J = 7.2 Hz, 1.5 Hz, IH), 7.07 (td, J = 8.1 Hz, 1.8 Hz, IH), 6.91 (dd, J = 7.5 Hz, 0.9 Hz, IH), 4.02 (t, J = 6.6 Hz, 2H), 2.79 (t, J = 6.6 Hz, 2H), 2.13-2.04 (m, 2 H); 13C NMR (CDC1₃):6157.1, 154.4, 153.2, 137.5, 132.6, 128.8, 126.4, 126.2, 119.3, 47.2, 26.3, 23.8; ES-MS: 291.0 [M+H] +. [00880] 4-Chloro-6-(3,4-dihydro-2H-quinolin-l-yl)-ρyrimidin-5-ylamine.

[00881] l-(6-Chloro-5-nitro-pyrimidin-4-yl)-l,2,3,4-tetrahydroquinoline (3.14 g, 10.8 mmol) was dissolved in a mixture of ethanol (40 niL) and water (10 mL). Iron powder (1.82 g, 32.5 mmol) and NH₄Cl (0.34 g, 6.26 mmol) was added and the mixture was stirred under reflux for 1 h, cooled, and filtered through a pad of celite. The filtrate was concentrated in vacuo. The residue was extracted with EtOAc, and the organics were washed with saturated NaHCO3 and brine in sequence and dried over anhydrous MgSO₄. Concentration in vacuo gave the crude product which was purified by recrystallization in EtOH to give Ib 2.33 g (83%), white solid, mp 115-116 ⁰C. IH NMR (CDC1₃):6 8.26 (s, IH), 7.16 (d, J = 7.2 Hz, IH), 7.07 (td, J = 8.1 Hz, 1.5 Hz, IH), 6.94 (td, J = 7.5 Hz, 1.2 Hz, IH), 6.39 (d, J = 7.8 Hz, IH), 3.85 (t, J = 6.0 Hz, 2H), 3.82 (s, br, 2H), 2.89 (t, J = 6.6 Hz, 2H), 2.13-2.05 (m, 2H); 13C NMR (CDCl₃):δl50.4, 146.9, 144.1, 138.7, 129.5, 129.3, 127.7, 126.3, 122.0, 117.1, 47.5, 26.9, 23.5; ES-MS: 261.0 [M+H] +.

6.4.3 General procedure for the synthesis of tetracyclic benzodiazepines

[00882] To a solution of indolin-1-ylpyrimidine Ia or tetrahydroquinolinylpyrimidine

(0.5 mmol) and an aldehyde (0.6 mmol) or ketone (0.6 mmol) in 3 mL acetonitrile was added 3 drops of TFA. The mixture was refluxed with stirring until the disappearance of the starting material. After cooling to room temperature, the solvent was removed in vacuo to give the crude product. Purification by recrystallization or flash chromatography on a silica gel column provided the desired products.

6.4.4 Specific synthetic procedures and characterization details

[00883] Compound 2.1 (8-chloro-6-ethyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene): 92%, mp 140-142 ⁰C. IH NMR (CDCl₃): δ 8.14 (s, IH), 7.12-7.10 (m, IH), 6.91-6.84 (m, 2H), 4.88 (d, J = 5.4 Hz, IH), 4.50-4.41 (m, IH), 4.32-4.13 (m, 2H), 3.22-3.11 (m, 2H), 1.63-1.53 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl₃): δ 152.0, 149.5, 146.3, 140.6, 133.7, 127.7, 126.5, 125.0, 124.3, 121.8, 62.3, 51.1, 30.3, 26.8, 11.3; ES-

MS: 287.1 [M+H+].

[00884] Compound 2.2 (δ-chloro-ό-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 90%, mpl51-152 ⁰C. IH NMR (CDCl₃): δ 8.15 (s, IH), 7.11 (dd, J =

6.6 Hz, 0.9 Hz, IH), 6.90-6.83 (m, 2H), 4.84 (s, br, IH), 4.50-4.41 (m, IH), 4.32-4.22 (m,

2H), 3.27-3.06 (m, 2H), 1.63-1.23 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl₃): δ

152.1, 149.5, 146.3, 140.7, 133.7, 127.8, 126.4, 125.1, 124.3, 121.8, 60.3, 51.1, 39.4, 26.9,

19.7, 13.9; ES-MS: 301.1 [M+H+].

[00885] Compound 2.3 (8-chloro-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 92%, mpl42-144 ⁰C. IH NMR (CDCl₃): δ 8.10 (s, IH), 7.31-7.21 (m,

3H), 7.17 (dd, J = 7.2 Hz, 1.2 Hz, IH), 7.11-7.08 (m, 2H), 6.85 (t, J = 7.8 Hz, IH), 6.74 (d, J

= 7.5 Hz, IH), 5.47 (s, IH), 5.11 (s, br, IH), 4.43-4.32 (m, 2H), 3.22 (t, J = 8.7 Hz, 2H); 13C

NMR (CDCl₃): δ 152.2, 149.7, 146.9, 141.4, 141.2, 133.3, 128.8, 127.9, 127.4, 127.3, 125.6,

125.4, 124.5, 121.5, 63.7, 50.9, 26.7; ES-MS: 335.1 [M+H+].

[00886] Compound 2.4 (8-chloro-6-(o-methoxyphenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene): 97%, mp 201-203 ⁰C. IH NMR (CDCl₃): δ 8.01 (s, IH),

7.20-7.12 (m, 2H), 6.92-6.84 (m, 3H), 6.62 (t, J = 7.5 Hz, IH), 6.38 (dd, J = 7.2 Hz, 1.5 Hz,

IH), 5.89 (d, J = 3.6 Hz, IH), 5.63 (s, br, IH), 4.46-4.31 (m, 2H), 3.96 (s, 3H), 3.28-3.20 (m,

2H); 13C NMR (CDCl₃): δl56.8, 152.3, 149.7, 147.2, 142.6, 133.4, 129.5, 129.4, 127.9,

127.6, 126.0, 124.7, 124.5, 122.0, 120.3, 110.4, 59.0, 55.4, 51.2, 27.0; ES-MS: 365.2

[M+H+].

[00887] Compound 2.5 (8-chloro-6-(p-nitro-phenyl)~ 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene): 96%, mpl95-196 ⁰C. IH NMR (CDCl₃): δ 8.11 (d, J = 8.7

Hz, 2H), 8.10 (s, IH), 7.25-7.22 (m. 3H), 6.91 (t, J = 7.5 Hz, IH), 6.81 (d, J = 7.8 Hz, IH),

5.62 (d, J = 2.7 Hz, IH), 5.22 (s, br, IH), 4.48-4.27 (m, 2H), 3.28-3.21 (m, 2H); 13C NMR

(CDCl₃): δ 152.2, 150.2, 148.5, 147.4, 147.3, 141.3, 133.7, 128.3, 127.2, 125.1, 124.4, 124.0,

123.7, 121.9, 62.9, 50.9, 26.6; ES-MS: 380.0 [M+H+].

[00888] Compound 2.6 (8-chloro-6-(p-fluoro-phenyl)-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene): 90%, mpl47-148 ⁰C. IH NMR (CDCl₃): δ 8.12 (s, IH), 7.19

(d, J = 7.2 Hz, IH), 7.08-7.04 (m, 2H), 6.99-6.94 (m, 2H), 6.87 (t, J = 7.2 Hz, IH), 6.75 (d, J

= 7.5 Hz, IH), 5.47 (s, IH), 5.12 (s, br, IH), 4.46-4.28 (m, 2H), 3.23 (t, J = 8.1 Hz, 2H); ES-

MS: 353.1 [M+H+].

[00889] Compound 2.7 (8-chloro-6-styryl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 81%, mρl62-163 ⁰C. IH NMR (CDCl₃): δ 8.16 (s, IH), 7.25-7.21 (m, 5H), 7.16 (d, J = 7.2 Hz, IH), 6.99 (d, J = 7.5 Hz, IH), 6.90 (t, J = 7.5 Hz, IH), 6.33-6.18 (m, 2H), 5.06 (t, J = 4.8 Hz, IH), 4.96 (d, J = 4.2 Hz, IH), 4.43-4.30 (m, 2H), 3.22-3.16 (m, 2H); 13C NMR (CDCl₃): δ 152.0, 149.8, 147.1, 140.8, 135.9, 133.5, 133.1, 129.6, 128.5, 128.0, 126.8, 126.6, 125.3, 125.0, 124.5, 121.7, 61.8, 50.9, 26.7; ES-MS: 361.1 [M+H+]. [00890] Compound 2.8 (δ-chloro-ό-propenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 83%, mpl32-135 ⁰C. IH NMR (CDCl₃): δ 8.18 (s, IH), 7.13 (dd, J = 7.2 Hz, 1.2 Hz, IH), 6.93 (dd, J = 7.8 Hz, 1.2 Hz, IH), 6.88 (t, J = 6.9 Hz, IH), 5.56-5.37 (m, 2H), 4.83 (d, J = 6.3 Hz, IH), 4.62 (s, br, IH), 4.44-4.25 (m, 2H), 3.21-3.14 (m, 2H), 1.62 (dd, J = 6.0 Hz, 1.5 Hz, 3H); 13C NMR (CDCl₃): δ 151.9, 149.4, 146.4, 140.5, 133.2, 131.2, 129.5, 126.4, 125.5, 124.1, 123.9, 121.5, 61.5, 50.7, 26.5, 17.6; ES-MS: 299.1 [M+H+]. [00891] Compound 2.9 (8-chloro-6,6-dimethyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 95%, mpll7-118 ⁰C. IH NMR (CDCl₃): δ 8.18 (s, IH), 7.13-7.10 (m, 2H), 6.89 (t, J = 7.2 Hz, IH), 4.36 (t, J = 8.7 Hz, 2H), 3.17 (t, J = 8.7 Hz, 2H), 1.57 (s, 6H); 13C NMR (CDCl₃): δ 152.3, 149.7, 146.7, 140.3, 133.5, 131.7, 125.0, 124.0, 123.1, 121.5, 56.8, 50.7, 30.4, 26.6; ES-MS: 287.1 [M+H+].

[00892] Compound 2.10 (8-chloro-6-ethyl-6-methyl- 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene): 92%, 86-88 ⁰C. IH NMR (CDCl₃): δ 8.16 (s, IH), 7.11 (d, J = 7.2 Hz, IH), 7.04 (d, J = 7.8 Hz, IH), 6.88 (t, J = 7.5 Hz, IH), 4.48-4.22 (m, 3H), 3.19-3.12 (m, 2H), 1.85-1.75 (m, IH), 1.67-1.55 (m, IH), 1.60 (s, 3H), 0.86 (t, J = 7.8 Hz, 3H); 13C NMR (CDCl₃): δ 152.0, 149.4, 146.1, 140.4, 133.6, 130.7, 124.9, 124.0, 123.9, 121.3, 59.8, 50.7, 34.5, 27.1, 26.6, 8.1; ES-MS: 301.1 [M+H+].

[00893] Compound 2.11 (8-chloro-6-methyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene): 84%, mp 99-101 ⁰C. IH NMR (CDCl₃): δ 8.17 (s, IH), 7.11 (dd, J = 7.2 Hz, 1.2 Hz, IH), 7.04 (d, J = 8.1 Hz, IH), 6.88 (t, J = 7.5 Hz, IH), 4.48-4.23 (m, 3H), 3.20-3.09 (m, 2H), 1.78-1.48 (m, 2H), 1.61(s, 3H), 1.41-1.18 (m, 2H), 0.82 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl₃): δ 152.1, 149.5, 146.2, 140.4, 133.7, 130.8, 124.9, 124.0, 123.9, 121.3, 59.7, 50.7, 44.4, 27.7, 26.6, 17.0, 14.2; ES-MS: 315.1 [M+H+]. [00894] Compound 2.12 (8-chloro-6-cyclohexylidene-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene): 92%, mp 182-183 °C. IH NMR (CDCl₃): δ 8.12 (s, IH), 7.10 (t, J = 8.1 Hz, 2H), 6.88 (d, J = 7.8 Hz, IH), 4.59 (s, br, IH), 4.34 (td, J = 8.4 Hz, 1.8 Hz, 2H), 3.12 (t, J = 9.0 Hz, 2H), 2.00-1.76 (m, 5H), 1.69-1.49 (m, 4H), 1.33-1.25 (m, IH); 13C NMR (CDCl₃): δ 152.3, 149.1, 145.5, 140.9, 133.6, 133.3, 124.5, 124.0, 121.9, 121.7, 58.2, 50.7, 34.9, 26.6, 25.1, 20.8; ES-MS: 327.1 [M+H+]. [00895] Compound 2.13 (8-chloro-6-methyl-6-ρhenyl-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene): 87%, mp 188-189 ⁰C. IH NMR (CDCl₃): δ 8.06 (s, IH), 7.30-7.20 (m, 3H), 7.17-7.14 (m, 3H), 6.88-6.80 (m, 2H), 4.76 (s, br, IH), 4.45-4.36 (m, IH), 4.23-4.14 (m, IH), 3.21-3.14 (m, IH), 1.90 (s, 3H); 13C NMR (CDCl₃): δ 152.0, 149.6, 146.5, 145.8, 141.1, 133.4, 130.8, 128.6, 127.6, 126.5, 125.9, 125.5, 124.3, 121.3, 63.6, 50.7, 30.6, 26.7; ES-MS: 349.1 [M+H+].

[00896] Compound 2.14 (8-chloro-6-methyl-6-(p-methoxyphenyl)- 1 ,2,6,7-tetrahydro-

7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene): 62%, mp 168-169 ⁰C. IH NMR (CDCl₃): δ 8.07 (s, IH), 7.14 (d, J = 6.6 Hz, IH), 7.07 (d, J = 9.0 Hz, 2H), 6.86-6.77 (m, 4H), 4.71 (s, IH), 4.41-4.34 (m, IH), 4.25-4.15 (m, IH), 3.76 (s, 3H), 3.16 (t, J = 8.7 Hz, 2H), 1.86 (s, 3H); 13C NMR (CDCl₃): δ 158.8, 152.1, 149.6, 146.3, 141.0, 138.0, 133.4, 131.1, 127.8, 125.9, 125.5, 124.2, 121.2, 113.8, 63.1, 55.1, 50.7, 30.5, 26.7; ES-MS: 379.1 [M+H+]. [00897] Compound 2.15 (8-chloro-6-methyl-6-(p-nitro-phenyl)-l,2,6,7-tetrahydro-

7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene): 82%, mp 182-183 ⁰C. IH NMR (CDCl₃): δ 8.10 (d, J = 8.7 Hz, 2H), 8.05 (s, IH), 7.28-7.21 (m, 3H), 6.95-6.89 (m, 2H), 4.80 (s, IH), 4.48- 4.39 (m, IH), 4.18-4.07 (m, IH), 3.23-3.14 (m, IH), 1.98 (s, 3H); 13C NMR (CDCl₃): δ 153.2, 151.7, 149.9, 147.1, 146.5, 141.1, 133.9, 129.1, 127.5, 125.3, 125.0, 124.8, 123.8, 121.7, 63.5, 50.7, 30.9, 26.6; ES-MS: 394.2 [M+H+].

[00898] Compound 2.16 (8-chloro-6~ethyl-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,11b- tetraaza-dibenzo[c,d,h]azulene): 57%, mp 145-147 ⁰C. IH NMR (CDCl₃): δ 8.06 (s, IH), 7.31-7.21 (m, 3H), 7.16-7.14 (m, 3H), 6.83 (t, J = 7.5 Hz, IH), 6.72 (d, J = 8.1 Hz, IH), 4.86 (s, br, IH), 4.37-4.17 (m, 2H), 3.15 (t, J = 8.7 Hz, 2H), 2.32-2.12 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl₃): δ 151.4, 149.1, 145.7, 144.5, 141.2, 133.7, 129.3, 128.5, 127.5, 127.1, 126.6, 125.5, 124.2, 120.9, 66.9, 50.7, 34.3, 26.5, 8.5; ES-MS: 363.2 [M+H+], [00899] Compound 2.17 (8-chloro-6-ethyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulen-6-carbonate acid): 59%, mp 273 ⁰C (dec). IH NMR (DMSO-d6): δ 8.14 (s, IH), 7.24-7.21 (m, 2H), 6.91 (t, J = 7.2 Hz, IH), 5.03 (s, IH), 4.38-4.29 (m, IH), 4.21- 4.11 (m, IH), 3.21-3.09 (m, 2H), 1.88 (s, 3H); ES-MS: 317.2 [M+H+]. [00900] Compound 2.18 (9-chloro-7-ethyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 97%, colorless syrup. IH NMR (CDCl₃): δ 7.97 (s, IH), 7.09 (dd, J = 6.9 Hz, 1.2 Hz, IH), 7.01-6.94 (m, 2H), 4.50 (s, br, IH), 4.46-4.39 (m, IH), 4.15-4.09 (m, IH), 3.85-3.77 (m, IH), 2.89 (t, J = 6.9 Hz, 2H), 2.13-1.99 (m, 4H), 1.05 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl₃): δ 150.4, 145.8, 142.6, 141.9, 135.3, 133.0, 131.4, 129.5, 128.3, 123.5, 59.2, 46.1, 26.7, 26.2, 21.4, 11.5; ES-MS: 301.1 [M+H+]. [00901] Compound 2.19 (9-chloro-7-pyproyl-2,3,7,8-tetrahydro-lH-8,10,12,12b- tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 95%, mρl08-110 ⁰C. IH NMR (CDCl₃): δ 7.97 (s, IH), 7.09 (dd, J = 6.9 Hz, 2.1 Hz, IH), 7.01-6.94 (m, 2H), 4.55 (t, J = 8.1 Hz, IH), 4.12-4.06 (m, IH), 3.88-3.80 (m, IH), 2.89 (t, J = 7.5 Hz, 2H), 2.13-1.95 (m, 4H), 1.52-1.35 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl₃): δ 150.5, 145.8, 142.5, 141.9, 135.4, 131.4, 129.5, 128.4, 123.5, 123.4; ES-MS: 315.1 [M+H+]. [00902] Compound 2.20 (9-chloro-7-ρhenyl-2,3,7,8-tetrahydro-lH-8,10,12,12b- tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 96%, mρl40-142°C. IH NMR (CDCl₃): δ 7.96 (s, IH), 7.39-7.26 (m, 5H), 7.13 (d, J = 7.2 Hz, IH), 6.97 (t, J = 7.2 Hz, IH), 6.76 (d, J = 7.5 Hz, IH), 5.81 (s, IH), 3.90-3.82 (m, IH), 3.66-3.59 (m, IH), 2.89 (t, J = 7.2 Hz, 2H), 2.11-2.02 (m, 2H); 13C NMR (CDCl₃): δ 150.7, 146.3, 142.9, 142.2, 140.3, 135.3, 133.1,

131.8, 130.0, 128.7, 128.3, 127.6, 126.3, 125.3, 123.5, 60.6, 45.8, 26.5, 21.4; ES-MS: 349.2 [M+H+].

[00903] Compound 2.21 (9-chloro-7-(p-methylphenyl)-2,3,7,8-tetrahydro-lH-

8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 91%, colorless syrup. IH NMR (CDCl₃): δ 7.95 (s, IH), 7.15 (m, 5H), 7.11 (dd, J = 7.8 Hz, 1.2 Hz, IH), 6.94 (t, J = 7.8 Hz, IH), 6.72 (d, J = 7.2 Hz, IH), 5.80 (s, IH), 3.80-3.74 (m, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.35 (s, 3H), 2.08-2.04 (m, 2H); 13C NMR (CDCl₃): δ 150.7, 146.3, 142.9, 142.3, 137.3, 137.2, 135.5, 131.7, 129.9, 129.3, 128.4, 126.4, 125.2, 123.5, 60.3, 45.8, 26.5, 21.4, 21.0; ES- MS: 363.2 [M+H+].

[00904] Compound 2.22 (9-chloro-7-(p-nitro-phenyl)-2,3 ,7,8-tetrahydro- IH-

8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 85%, mp 245-247°C. IH NMR (CDCl₃): δ 8.16 (d, J = 8.7 Hz, 2H), 7.96(s, IH), 7.34 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 7.2 Hz, IH), 7.07 (t, J = 7.2 Hz, IH), 7.00 (d, J = 6.3 Hz, IH), 5.56 (d, J = 5.7 Hz, IH), 5.19 (d, J = 5.7 Hz, IH), 4.26-4.19 (m, IH), 2.90-2.79 (m, 3H), 2.13-1.92 (m, 2H); 13C NMR (CDCl₃): δ 150.5, 148.6, 147.1, 146.6, 143.5, 141.9, 134.1, 132.3, 130.7, 127.0, 126.6, 125.8,

123.9, 123.7, 60.7, 45.5, 26.3, 21.0; ES-MS: 394.1 [M+H+].

[00905] Compound 2.23 (9-chloro~7-styryl-2,3,7,8-tetrahydro-lH-8,10,12,12b- tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 40%, mp 114-116 ⁰C. IH NMR (CDCl₃): δ 8.00 (s, IH), 7.40-7.23 (m, 5H), 7.13 (dd, J = 7.2 Hz, 1.2 Hz, IH), 7.05 (d, J = 7.2 Hz, IH), 7.00 (t, J = 7.2 Hz, IH), 6.63 (d, J = 2.7 Hz, 2H), 5.34 (t, J = 2.7 Hz, IH), 4.13-4.05 (m, IH), 3.88-3.80 (m, IH), 2.91 (t, J = 6.9 Hz, 2H), 2.15-2.05 (m, 2H); 13C NMR (CDCl₃): δ 150.6, 146.2, 136.2, 134.8, 132.4, 131.6, 129.9, 128.6, 128.1, 128.0, 127.9, 126.5, 124.1, 123.7, 59.3, 46.1, 26.6, 21.4; ES-MS: 375.1 [M+H+]. [00906] Compound 2.24 (9-chloro-7,7-dime%l-2,3,7,8-tetrahydro-lH-8,10,12,12b- tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene): 37%, mpl30-132 ⁰C. IH NMR (CDCl₃): δ 7.98 (s, IH), 7.14 (d, J = 7.8 Hz, IH), 7.10 (d, J = 7.5 Hz, IH), 6.97 (t, J = 7.5 Hz, IH), 4.31 (s, br, IH), 3.99-3.96 (m, 2H), 2.90 (t, J = 7.2 Hz, 2H), 2.18-2.10 (m, 2H), 1.79 (s, 6H); 13C NMR (CDCl₃): δ 149.7, 145.9, 141.6, 138.1, 132.0, 129.8, 127.4, 123.3, 121.1, 55.6, 46.1, 29.4, 26.7, 21.6; ES-MS: 301.1 [M+H+].

6.4.5 General procedure for displacement of the chloro group in 2.2 or 2.3 with H-BuNH₂, aniline and morpholine, preparation of compounds 3a, 3b and 3c.

[00907] To a solution of compound 2.2 or 2.3 (0.3 mmol) and excess amine (3.0 mmol) in n-BuOH (3.0 mL) was added coned, aq. HCl (2-3 drops). The mixture was stirred under a nitrogen atmosphere in refluxing until completion (monitored by TLC). After cooling of the resulting mixture and concentration in vacuo to dryness, the residue was purified by flash chromatography on silica gel to give the desire product.

[00908] Compound 3a (N-(n-butyl)-6-phenyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulen-8-arnine): 74%, brown solid, mp 105-107 ⁰C. IH NMR (CDCl₃): δ 8.09 (s, IH), 7.20-7.15 (m, 3H), 7.12 (t, J = 4.2 Hz, IH), 7.08-7.05 (m, 2H), 7.76 (d, J = 4.2 Hz, 2H), 5.85 (t, J = 2.4 Hz, IH), 5.51 (s, IH), 4.35-4.29 (m, 2H), 3.30-3.13 (m, 4H), 1.48-1.40 (m, 2H), 1.37-1.23 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl₃): δ 161.4, 157.2, 154.7, 154.6, 142.4, 141.2, 133.1, 128.5, 128.2, 128.0, 127.4, 127.3, 124.0, 120.3, 65.1, 49.6, 40.4, 32.0, 27.1, 20.0, 13.8.

[00909] Compound 3b (N-phenyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulen-8-amine): 60%, pale yellow solid, mp 149-151 °C. IH NMR (CDCl₃): δ 8.40 (s, IH), 8.32 (s, IH), 7.68 (dd, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.09 (d, J = 7.5 Hz, IH), 7.03 (t, J = 7.5 Hz, IH), 6.89 (d, J = 6.9 Hz, IH), 6.82 (t, J = 7.5 Hz, IH), 4.42-4.33 (m, IH), 4.30-4.27 (m, 2H), 3.17-3.12 (m, 2H), 2.69 (s, br, IH), 1.62-1.56 (m, 2H), 1.45-1.38 (m, 2H), 0.88 (t, J = 6.9 Hz, 3H).

[00910] Compound 3c (8-(morpholin-l-yl)-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 Ib- tetraaza-dibenzo[c,d,h]azulene): 90%, white solid, mp 158-162 °C. IH NMR (CDCl₃): δ 8.22 (s, IH), 7.35-7.25 (m, 3H), 7.17 (d, J = 7.2 Hz, 2H), 7.11 (d, J = 7.2 Hz, IH), 6.74 (t, J = 7.5 Hz, IH), 6.58 (d, J = 7.5 Hz, IH), 5.28 (s, IH), 4.93 (s, br, IH), 4.46-4.30 (m, 2H), 3.75-3.65 (m, 4H), 3.19 (t, J = 8.7 Hz, 2H), 3.00-2.93 (m, 2H), 2.87-2.80 (m, 2H); 13C NMR (CDCl₃): 5157.2, 151.7, 150.0, 149.9, 141.8, 132.9, 128.8, 127.8, 127.5, 127.1, 125.8, 124.1, 121.2, 120.5, 66.8, 64.1, 64.0, 50.3, 49.6, 26.8.

[00911] Preparation of compound 3d. (8-(pyrroridin-l-yl)-6-propyl~l,2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene): To a solution of compound 2.2 (90 mg, 0.3 mmol) and pyrrolidine (213 mg, 3.0 mmol) in n-BuOH (3.0 mL) was added Et₃N (1 mL). The mixture was refluxed with stirrring under a nitrogen atmosphere for 3 days. After cooling to room temperature, the solvent was evaporated in vacuo and the residue was purified by flash chromatography on silica gel (petroleum/EtOAc 5:1, v/v) to give the desired product (93.5 mg) as a pale yellow solid, yield 93%, mp 116-118⁰C. IH NMR (CDCl₃): δ 8.21 (s, IH), 7.06 (dd, J = 7.5 Hz, 1.5 Hz, IH), 6.85 (d, J = 7.5 Hz, IH), 6.78 (t, J = 7.5 Hz, IH), 4.43-4.22 (m, 2H), 4.17 (t, J = 6.9 Hz, IH), 3.81-3.73 (m, 2H), 3.64-3.53 (m, 2H), 3.19- 3.08 (m, 2H), 1.99-1.84 (m, 4H), 1.41-1.19 (m, 4H), 0.86 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl₃): δ 155.8, 150.8, 150.7, 141.5, 133.1, 129.7, 125.9, 123.5, 120.0, 112.0, 60.5, 50.6, 50.3, 37.6, 26.8, 25.3, 19.3, 14.0.

6.4.6 General procedure for displacement of the chloro group in 2.2 or 2.3 with n-BuOH or PI1CH2SH, preparation of compounds 4a, 4b, 5a, 5b.

[00912] To a solution of compounds 2.2 or 2.3 (0.3 mmol) in THF (2 mL) was added n-BuOH (222 mg, 0.27 mL, 3 mmol) or PhCH₂SH (74.4 mg, 0.07 mL, 0.6 mmol) and then sodium hydride (48 mg, 1.2 mmol). The mixture was warmed to 70 °C with stiring and the reaction was monitored by TLC until completion. Cold water was poured to quench the reaction then extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous MgSO₄. After concentration in vacuo, the crude residue was purified by flash chromatography on silica gel to give the desired product. [00913] Compound 4a (8-butoxyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 94%, yellow syrup. IH NMR (CDCl₃): δ 8.10 (s, IH), 7.05 (d, J = 7.5 Hz, IH), 6.86 (d, J = 7.5 Hz, IH), 6.77 (t, J = 7.5 Hz, IH), 4.63 (s, IH), 4.45-4.38 (m, 3H), 4.37-4.13 (m, 2H), 3.21-3.05 (m, 2H), 1.82-1.73 (m, 2H), 1.57-1.43 (m, 6H), 1.39-1.22 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl₃): δ 158.9, 150.3, 148.4, 148.3, 141.2, 132.9, 127.4, 126.3, 123.5, 120.1, 113.1, 66.4, 60.1, 50.0, 39.3, 31.1, 26.7, 19.3, 19.1, 13.7, 13.6.

[00914] Compound 4b (8-butoxy-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 94%, yellow solid, 89-91 ⁰C. IH NMR (CDCl₃): δ 8.03 (s, IH), 7.26- 7.19 (m, 3H), 7.13-7.06 (m, 3H), 6.79-6.71 (_m, 2H), 5.40 (s, IH), 4.88 (s, br, IH), 4.38-4.31 (m, 2H), 4.29-4.23 (m, 2H), 3.19 (t, J = 8.1 Hz, 2H), 1.70-1.65 (m, 2H), 1.42-1.34 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl₃): δ 159.1, 150.9, 149.0, 148.9, 142.5, 142.1, 132.7, 128.4, 127.6, 127.4, 127.3, 124.9, 124.0, 120.1, 114.2, 66.4, 64.1, 50.1, 30.9, 26.8, 19.0, 13.7. [00915] Compound Sa (8-benzylthio-6-ρropyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 94%, yellow syrup. IH NMR (CDCl₃): δ 8.33 (s, IH), 7.38-7.34 (m, 2H), 7.31-7.19 (m, 3H), 7.04 (dt, J = 6.6 Hz, 0.9 Hz, IH), 6.84-6.76 (m, 2H), 4.54 (d, J = 13.2 Hz, IH), 4.44 (d, J = 13.5 Hz, IH), 4.43-4.37 (m, IH), 430-4.10 (m, 3H), 3.19-3.07 (m, 2H), 1.52-1.15 (m, 4H), 0.80 (t, J = 7.2 Hz, 3H); 13C NMR(CDCl₃): δ 153.1, 150.7, 150.0, 149.9, 140.8, 137.7, 133.1, 128.9, 128.4, 127.9, 127.1, 126.0, 124.8, 123.7, 120.7, 60.1, 50.3, 38.7, 35.4, 26.6, 19.6, 13.7.

[00916] Compound 5b (8-benzylthio-6-ρhenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 96%, yellow solid, mp 113-115 °C. IH NMR (CDCl₃): δ 8.27 (s, IH), 7.30-7.19 (m, 8H), 7.14-7.07 (m, 3H), 6.78 (t, J = 7.5 Hz, IH), 6.67 (d, J = 7.2 Hz, IH), 5.36 (s, IH), 4.51 (s, br, IH), 4.41-4.31 (m, 4H), 3.20 (t, J = 9.0 Hz, 2H); 13C NMR(CDCl₃): δ 154.2, 151.2, 150.6, 150.4, 141.9, 141.5, 137.6, 133.0, 128.9, 128.5, 127.7, 127.3, 127.2, 125.9, 125.7, 124.2, 120.9, 109.7, 64.0, 50.4, 35.3, 26.8.

6.4.7 General procedure for displacement of the chloro group in 2.2 or 2.3 with thiophenol, preparation of compounds 5c and 5d.

[00917] To a solution of compound 2.2 or 2.3 (0.3 mmol) and thiophenol (330 mg, 3.0 mmol) in n-BuOH (3.0 niL) was added Et₃N (1 mL). The mixture was refluxed with stirrring under a nitrogen atmosphere until completion. After cooling to room temperature, the solvent was evaporated in vacuo and the residue was purified by flash chromatography on silica gel (petroleum/EtOAc 5:1, v/v) to give the desire product.

[00918] Compound 5c (8-phenylthio-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 95%, yellow syrup. IH NMR (CDCl₃): δ 8.27 (s, IH), 7.43-7.38 (m, 2H), 7.36-7.28 (m, 3H), 7.09 (d, J = 7.2 Hz, IH), 6.88-6.81 (m, 2H), 5.03 (s, br, IH), 4.49- 4.41 (m, IH), 4.33-4.22 (m, 2H), 3.23-3.07 (m, 2H), 1.61-1.51 (m, 2H), 1.49-1.26 (m, 2H), 0.87 (t, J == 6.9 Hz, 3H).

[00919] Compound 5d (8-ρhenylthio-6-phenyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulene): 94%, yellow syrup. IH NMR (CDCl₃): δ 8.22 (s, IH), 7.26-7.05 (m, HH), 6.80 (t, J = 7.5 Hz, IH), 6.71(d, J = 7.5 Hz, IH), 5.41 (s, IH), 5.31 (s, br, IH), 4.41- 4.28 (m, 2H), 3.19 (t, J = 8.1 Hz, 2H). 6.4.8 General procedure for displacement of the chloro group in 2.2 and 2.3 with PhB(OH)₂, preparation of compounds 6a and 6b.

[00920] Compound 3.2 or 3.3 (0.33 mmol) and phenylboronic acid (61 mg, 0.5 mmol) was dissolved in DME (4 mL) under nitrogen atmosphere. Potassium carbonate (138 mg, 1.0 mmol) dissolved in water (4 mL) was added, and followed by palladium (II) acetate (0.9 mg, 0.004 mmol) and triphenylphosphine (4.2 mg, 0.016 mmol). The reaction mixture was refluxed with stirring for 6 h. After cooling, the solid was filtered off through a pad of Celite and washed with ethyl acetate. The organic phase was separated and the aqueous phase was extracted with ethyl acetate (5 mL x 3). The combined organic extracts were washed with saturated NaHCO₃, water and brine in sequence, dried with anhydrous MgSO₄, concentrated in vacuo and purified by flash chromatography on silica gel to give the desired product. [00921] Compound 6a (8-phenyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 53%, yellow syrup. IH NMR (CDCl₃): δ 8.46 (s, IH), 7.55-7.42 (m, 5H), 7.09 (t, J = 4.5 Hz, IH), 6.80 (d, J = 4.2 Hz, 2H), 4.81 (d, J = 5.4 Hz, IH), 4.54-4.45 (m, IH), 4.38-4.28 (m, IH), 4.05-3.99 (m, IH), 3.24-3.12 (m, 2H), 1.46 (q, J = 7.8Hz, 2H), 1.12- 0.86 (m, 2H), 0.74 (t, J = 7.2 Hz, 3H); 13C NMR(CDCl₃): δ 152.0, 151.6, 150.0, 149.9, 141.0, 136.7, 133.3, 128.9, 128.5, 127.3, 125.9, 125.8, 123.9, 120.9, 59.8, 50.8, 38.2, 26.6, 19.2, 13.5.

[00922] Compound 6b (8-phenyl-6-phenyl- 1 ,2,6,7-tetrahydro~7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene): 53%, yellow syrup. IH NMR (CDCl₃): δ 8.43 (s, IH), 7.39-7.35 (m, 3H), 7.27-7.19 (m, 5H), 7.14 (d, J = 7.2 Hz, IH), 6.97-6.94 (m, 2H), 6.78 (t, J = 7.5 Hz, IH), 6.65 (d, J = 7.8 Hz, IH), 5.29 (d, J = 3.9 Hz, IH), 4.78 (d, J = 3.9 Hz, IH), 4.45 (t, J = 8.7 Hz, 2H), 3.24 (t, J = 8.7 Hz, 2H); 13C NMR (CDCl₃): δ 153.4, 153.1, 151.0, 150.9, 141.7, 141.4, 136.5, 133.1, 128.8, 128.5, 127.8, 127.2, 126.6, 125.7, 124.2, 121.0, 64.6, 50.8, 26.8.

6.5 Heterocyclic Scaffolds Consisting of Indole-Fused Pteridines 6.5.1 General Synthetic and Characterization Details

[00923] Indole-fused Pteridines were synthesized according to Scheme V (FIG. 5).

The scope of this reaction was further studied with a wide variety of aldehydes and ketones, and results are summarized in Table 9.

Table 9. Synthesis of 5,6-dihydroindolo[2,l-h]pteridines

no

R¹ R time product yield mp (⁰C)

(h) (%)

CH₃CH₂ H 0.5 3.1 93 139-140

CH₃CH₂CH₂ H 0.5 3.2 94 83-85

Ph H 3 3.3 91 176-177

^-Me-C₆H₄ H 3 3.4 96 155-157

P-NO₂-C₆H₄ H 6 3.5 83 202-204

3',4'-di-Cl-C₆H₃ H 5 3.6 82 225-227

0-MeO-C₆H₄ H 2.5 3.7 93 148-150

0-Cl-C₆H₄ H 8.5 3.8 98 142-144

0-NO₂-C₆H₄ H 28 3.9 72 154-156 ra-NO₂-C₆H₄ H 18 3.10 87 214-216

CH₃CH₂ CH₃ 6 3.11 93 135-136

CH₃CH₂CH₂ CH₃ 15 3.12 90 110-112

R¹, R² = -(CH₂)_S- 3 3.13 89 153-154

Ph CH₃ 24 3.14 64 149-151

P-Me-C₆H₄ CH₃ 45 3.15 70 134-136

P-NO₂-C₆H₄ CH₃ 53 3.16 47 201-202

[00924] The indole moiety of compounds 3 presents ample opportunity for additional diversity points. To illustrate this diversity, 3-ethoxycarbonylindolylpyrimidine (Compound 7 in Table 10) was prepared via ethyl l-(6-chloro-5-nitropyrimidin-4-yl)indole-3-carboxylate and subjected to a number of cyclizations (Table 10).

Table 10. Synthesis of 7-ethoxycarbonyl substitued 4-chloro-5,6-dihydroindolo[2,l-h]pteridines

R ,r R time compd. yield mp (°C)

(h) (%)

Ph H 20 8.1 86 210-213 p-Me-Ph H 20 8.2 82 179-181 p-F-Ph H 20 8.3 58 204-206

CH₃(CH₂)₂ H 20 8.4 82^α 168-170

[00925] The 4-Cl group was put in by design to provide an entry to introduce an additional diversity point. For this purpose, two compounds were selected to illustrate the propensity of the 4-Cl group towards nucleophilic substitutions.

= H

6.5.2 Preparation and Characterization of 4-chloro-6-(2,3-dihydro- indol-l-yl)-pyrimidin-5-ylamine 1:

[00926] 5-Amino-4,6-dichloro-pyrimidine (16.3 g, 0.1 mol) was added to a solution of

70 mL ethanol, 250 mL water, 4 mL of concentrated hydrochloric acid and (11.9 g, 0.1 mmol) of indoline. The solution was stirred and refluxed for 9.5 h, then allowed to cool at room temperature overnight. The precipitate was filtered and washed with ethanol to give the desired product 1 (16 g) which was used without further purification. From the filtrate, 3 g of the starting material 5 was recovered. Compound 1: 79%, white solid, mp 135-137°C. ES-MS: 247.1 [M+H+]; IH NMR (CDCl₃)δ: 8.21 (s, 1 H), 7.25 (d, J = 7.2, 1 H), 7.14 (t, J = 7.5 H, IH), 6.91 (t, J = 7.5, 1 H), 6.57 (t, J = 7.5, 1 H), 4.16 (t, J = 8.1, 2 H), 3.96 (s, br., 2 H), 3.15 (t, J = 8.1, 2 H). 13C NMR (CDCl₃)δ: 149.6, 146.8, 144.3, 143.9, 132.6, 129.0, 127.1, 125.4, 121.5, 112.5, 52.8, 28.7.

6.5.3 Preparation and Characterization of 4-chloro-6-indol-l-yl- pyrimidin-5-ylaniine 2.

[00927] A mixture of compound 1 in Scheme V (6.73 g, 27.4 mmol) and DDQ (7.45 g,

32.8 mmol) was stirred under reflux in 150 mL benzene for 2 h. The mixture was then cooled to room temperature, filtered and the solid washed with benzene. The combined filtrate was washed with saturated NaHCO₃, brine, and dried over anhydrous MgSO₄. After concentration in vacuo and purification by recrystallization in EtOH the desired product 2 (4.52 g) was obtained. Concentration of the mother liquid and purification by column chromatography give another fraction (0.41 g) of 2 to total yield 74%. Mp 149-151°C. ES- MS: 245.1 [M+H+]; IH NMR (CDCl₃)δ: 8.39(s, 1 H), 7.69 (dd, J = 6.9, 1.2, 1 H), 7.52 (d, J = 3.3, 1 H), 7.46 (dd, J = 7.2, 1.2, 1 H), 7.30 (td, J = 6.9, 1.5, 1 H), 7.24 (td, J = 7.2, 1.2, 1 H), 6.78 (dd, J = 3.6, 0.9, 1 H), 4.27 (s, 2 H); 13C NMR (CDCl₃)δ: 146.6, 144.1, 140.3, 134.6, 131.5, 129.5, 126.1, 123.3, 121.7, 121.4, 112.2, 106.5.

6.5.4 General procedure and specific characterization details for the cyclization preparation of compounds 3.

[00928] To a solution of 4-chloro-6-indol-l-yl-pyrimidin-5-ylamine (0.5 mmol), an aldehyde (0.6 mmol) or ketone (0.6 mmol) in 3 mL dry acetonitrile was added 3 drops TFA. The solution was refluxed with stirring under a nitrogen atmosphere, monitored by TLC until the disappearance of starting material 2. After cooling to room temperature, the solvent was removed by evaporation in vacuo and the crude product was purified by recrystallization or flash column chromatography.

[00929] Compound 3.1 (4-chloro-6-ethyl-5,6-dihydroindolo[2,l-h]pteridine): 93%, mpl39-140 °C, ES-MS: 285.1 [M+H+]; IH NMR (CDCl₃)δ: 8.80 (d, J = 8.1, 1 H), 8.32 (s, 1 H), 7.57 (d, J = 7.5, 1 H), 7.35 (td, J = 7.2, 1.5, 1 H), 7.26 (td, J = 7.2, 1.2, 1 H), 6.46 (s, 1 H), 4.72 (t, J = 5.7, 1 H), 4.50 (s, br, 1 H), 2.02-1.86 (m, 2 H), 1.09 (t, J = 7.2, 3 H); 13C NMR (CDCl₃)δ: 146.6, 144.9, 141.2, 134.6, 130.0, 125.4, 123.9, 123.0, 120.5, 116.1, 103.0, 51.8,

29.3, 9.4.

[00930] Compound 3.2 (6-butyl-4-chloro-5,6-dihydroindolo[2,l-h]pteridine): 94%, mp

83-85 ⁰CES-MS: 299.1 [M+H+]; IH NMR (CDCl₃)δ: 8.80 (d, J = 8.4, 1 H), 8.32 (s, 1 H), 7.57 (d, J = 6.9, 1 H), 7.37-7.32 (m, 1 H), 7.29-7.23 (m, 1 H), 6.46 (s, 1 H), 4.76 (t, J = 6.0, 1 H), 4.50 (s, br, 1 H), 1.94-1.84 (m, 2 H), 1.59-1.47 (m, 2 H), 1.01 (t, J = 7.5, 3 H); 13C NMR (CDCl₃)δ: 146.6, 144.9, 141.3, 134.9, 134.6, 130.0, 125.3, 123.9, 123.0, 120.5, 116.0, 102.9,

50.4, 38.4, 18.5, 13.8.

[00931] Compound 3.3 (4-chloro-6-ρhenyl-5,6-dihydroindolo[2,l-h]pteridine): 91%, mp 176-177 ⁰C. ES-MS: 333.1 [M+H+]; IH NMR (CDCl₃)δ: 8.81 (d, J = 8.4, 1 H), 8.36 (s, 1

H), 7.51-7.42 (m, 6 H), 7.35 (t, J = 7.5, 1 H), 7.22 (t, J = 7.5, 1 H), 6.03 (s, 1 H), 5.74 (s, 1 H),

4.70 (s, 1 H); 13C NMR (CDCl₃)δ: 147.0, 145.1, 141.5, 139.3, 135.3, 134.8, 129.8, 129.0,

128.9, 127.8, 125.8, 124.2, 123.1, 120.7, 116.0, 105.2, 55.3.

[00932] Compound 3.4 (4-chloro-6-(4'-methylphenyl)-5,6-dihydroindolo[2,l- h]pteridine): 96%, mp 155-157 °C. ES-MS: 347.1 [M+H+]; IH NMR (CDCl₃)δ: 8.82 (d, J =

7.8, 1 H), 8.37 (s, 1 H), 7.47 (d, J = 7.5, 1 H), 7.40-7.33 (m, 3 H), 7.27-7.23 (m, 3 H), 6.05 (s,

1 H), 5.74 (s, 1 H), 4.67 (s, 1 H), 2.41 (s, 3 H); 13C NMR (CDCl₃)δ: 147.0, 145.2, 141.5,

139.0, 136.3, 135.6, 134.9, 130.0, 129.7, 127.8, 125.9, 124.2, 123.1, 120.7, 116.0, 105.2, 55.1,

21.2.

[00933] Compound 3.5 (4-chloro-6-(4'-nitrophenyl)-5,6-dihydroindolo[2,l- h]pteridine): 83%, mp 202-204 °C. ES-MS: 378.1 [M+H+]; IH NMR (DMSO-d6)δ: 8.73 (d,

J = 8.4, 1 H), 8.33 (s, 1 H), 8.24 (d, J = 8.7, 2 H), 7.68 (d, J = 8.7, 1 H), 7.61 (d, J = 7.8, 1 H),

7.37-7.31 (m, 2 H), 7.24 (t, J = 7.2, 1 H), 6.52 (d, J = 0.6, 1 H), 6.25 (s, 1 H).

[00934] Compound 3.6 (4-chloro-6-(3 ',4'-dichloroρhenyl)-5,6-dihydroindolo[2, 1 - h]ρteridine): 82%, mp 225-227 °C. ES-MS: 401.0 [M+1+]; IH NMR (CDCl₃)δ: 8.82 (d, J =

8.7, 1 H), 8.41 (s, 1 H), 7.63 (d, J = 2.1, 1 H), 7.52 (t, J = 8.1, 2 H), 7.41-7.34 (m, 2 H), 7.26

(t, J = 7.5, 2 H), 6.07 (s, 1 H), 5.75 (s, 1 H), 4.68 (s, 1 H); 13C NMR (CDCl₃)δ: 147.8, 145.4,

142.3, 139.7, 135.2, 134.4, 133.7, 133.6, 131.4, 130.2, 129.9, 127.5, 125.6, 124.9, 123.6,

121.1, 116.4, 105.9, 54.8.

[00935] Compound 3.7 (4-chloro-6-(o-methoxyphenyl)-5,6-dihydroindolo[2,l- h]pteridine): 93%, mp 148-150 °C. ES-MS: 363.1 [M+1+]; IH NMR (CDCl₃)δ: 8.85 (d, J =

8.1, 1 H), 8.32 (s, 1 H), 7.57 (d, J = 7.5, 1 H), 7.38 (td, J = 7.2, 1.5, 1 H), 7.32-7.25 (m, 2 H),

6.96 (d, J = 8.1, 1 H), 6.88-6.79 (m, 2 H), 6.41 (s, 1 H), 6.27 (s, 1 H), 5.22 (s, br, 1 H), 3.96 (s, 3 H); 13C NMR (CDCl₃)δ: 156.7, 147.0, 142.1, 135.0, 133.6, 130.2, 130.0, 129.2, 127.9,

126.0, 124.3, 123.3, 121.2, 121.0, 116.4, 110.9, 110.0, 105.0, 55.8, 49.7.

[00936] Compound 3.8 (4-chloro-6-(o-chlorophenyl)-5,6-dihydroindolo[2,l- bjpteridine): 98%, mp 142-144 °C. ES-MS: 367.0 [M+1+]; IH NMR (CDCl₃)δ: 8.85 (dd, J =

8.4, 0.9, 1 H), 8.37 (s, 1 H), 7.55-7.65 (m, 2 H), 7.41-7.22 (m, 6 H), 6.37 (t, J = 1.2, 1 H),

6.31 (s, 1 H), 4.95 (s, 1 H); 13C NMR (CDCl₃)δ: 147.1, 145.0, 141.9, 137.7, 134.7, 132.9,

132.8, 129.9, 129.8, 128.8, 127.7, 125.2, 124.3, 123.2, 120.8, 116.1, 105.2, 51.5.

[00937] Compound 3.9 (4-chloro-6-(o-nitxophenyl)-5,6-dihydroindolo[2,l-h]pteridine):

72%, mp 154-156 °C. ES-MS: 378.1 [M+H+]; IH NMR (CDCl₃)δ: 8.90 (dd, J = 8.1, 0.9, 1

H) , 8.39 (s, 1 H), 8.07-8.04 (m, 1 H), 7.59-7.25 (m, 5 H), 7.15-7.12 (m, 1 H), 6.49 (s, 1 H),

6.41 (d, J = 0.6, 1 H), 5.44 (s, br, 1 H).

[00938] Compound 3.10 (4-chloro-6-(m-nitrophenyl)-5,6-dihydroindolo[2,l- hjpteridine): 87%, mp 214-216 °C. ES-MS: 378.0 [M+H+]; IH NMR (CDCl₃)δ: 8.83 (d, J =

8.4, 1 H), 8.43 (s, 1 H), 8.42 (s, 1 H), 8.33-8.29 (m, 1 H), 7.86 (d, J = 8.1, 1 H), 7.66 (t, J =

8.1, 1 H), 7.49 (d, J = 7.8, 1 H), 7.39 (td, J = 8.4, 1.2, 1 H), 7.26 (td, J = 8.4, 0.9, 1 H), 6.03 (s,

1 H), 5.92 (s, 1 H), 4.77 (s, 1 H); 13C NMR (CDCl₃)δ: 148.6, 147.7, 142.1, 141.5, 134.9, 134.0, 133.8, 130.2, 129.6, 125.2, 124.7, 124.1, 123.4, 123.1, 120.9, 116.1, 109.7, 105.8, 54.8. [00939] Compound 3.11 (4-chloro-6-ethyl-6-methyl-5,6-dihydroindolo[2,l- h]pteridine): 93%, mp 135-136 °C. ES-MS: 299.1 [M+H+]; IH NMR (CDCl₃)δ: 8.82 (d, J = 8.1, 1 H), 8.32 (s, 1 H), 7.59 (dd, J = 7.2, 2.1, 1 H), 7.35 (td, J = 7.2, 1.5, 1 H), 7.26 (td, J = 7.5, 1.2, 1 H), 6.45 (s, 1 H), 4.26 (s, br, 1 H), 1.92-1.76 (m, 2 H), 1.67 (s, 3 H), 0.94 (t, J = 7.2, 3 H); 13C NMR (CDCl₃)δ: 146.5, 144.9, 141.3, 139.0, 134.6, 129.9, 125.1, 123.9, 122.9, 120.5, 116.1, 102.1, 55.0, 35.7, 27.2, 8.5.

[00940] Compound 3.12 (4-chloro-6-methyl-6-propyl-5,6-dihydroindolo[2,l- h]pteridine): 90%, mp 110-112 °C. ES-MS: 313.1 [M+H+]; IH NMR (CDCl₃) δ: 8.83 (d, J = 8.1, 1 H), 8.33 (s, 1 H), 7.58 (dd, J = 7.2, 0.6, 1 H), 7.35 (td, J = 7.5, 1.5, 1 H), 7.26 (td, J = 7.8, 1.2, 1 H), 6.45 (s, 1 H), 4.25 (s, br, 1 H), 1.85-1.68 (m, 2 H), 1.70 (s, 3 H), 1.46-1.32 (m,

2 H), 0.88 (t, J = 7.2, 3 H); 13C NMR (CDCl₃)δ: 146.5, 144.9, 141.2, 139.2, 134.6, 129.9, 125.1, 123.9, 123.0, 120.5, 116.1, 102.1, 54.8, 45.5, 27.8, 17.5, 14.1.

[00941] Compound 3.13 (4-chloro-6-cyclohexylidene-5,6-dihydroindolo[2,l- hjpteridine): 89%, mp 153-154 ⁰C. ES-MS: 325.2 [M+H+]; IH NMR (CDCl₃)δ: 8.79 (dd, J = 8.1, 1.2, 1 H), 8.34 (s, 1 H), 7.58 (d, J = 7.8, 1 H), 7.34 (td, J = 7.2, 1.5, 1 H), 7.26 (td, J = 7.5, 1.2, 1 H), 6.49 (s, 1 H), 4.85 (s, 1 H), 2.01-1.36 (m, 10 H); 13C NMR (CDCl₃)δ: 146.9, 145.5, 142.1, 141.1, 134.6, 130.0, 124.6, 123.9, 123.0, 120.6, 116.0, 101.2, 53.2, 36.5, 24.8, 21.3. [00942] Compound 3.14 (4-chloro-6-methyl-6-phenyl-5,6-dihydroindolo[2,l- h]pteridine): 64%, nip 149-151 °C. ES-MS: 347.1 [M+H+]; IH NMR (CDCl₃)δ: 8.78 (d, J = 8.1, 1 H), 8.33 (s, 1 H), 7.58 (dd, J = 7.2, 0.6, 1 H), 7.39-7.21 (m, 7 H), 6.55 (s, 1 H), 4.91 (s, br, 1 H), 2.04 (s, 3 H); 13C NMR (CDC1₃)6: 147.2, 145.8, 144.1, 142.0, 138.7, 134.8, 129.6, 128.7, 127.8, 125.2, 124.2, 123.1, 120.8, 115.9, 109.6, 103.6, 57.5, 29.4. [00943] Compound 3.15 (4-chloro-6-methyl-6-(p-methylphenyl)-5,6- dihydroindolo[2,l-h]pteridine): 70%, mp 134-136 ⁰C. ES-MS: 361.1 [M+H+]; IH NMR (CDCl₃)δ: 8.78 (d, J = 8.1, 1 H), 8.33 (s, 1 H), 7.61 (d, J = 7.5, 1 H), 7.36 (t, J = 7.2, 1 H), 7.28 (t, J = 7.5, 1 H), 7.20 (d, J = 7.2, 2 H), 7.09 (d, J = 7.8, 2 H), 6.54 (s, 1 H), 4.88 (s, br, 1 H), 2.29 (s, 3 H), 2.02 (s, 3 H); 13C NMR (CDCl₃)δ: 147.2, 145.8, 142.1, 141.2, 139.0, 137.7, 134.8, 129.7, 129.4, 125.2, 124.2, 123.1, 120.8, 116.0, 103.5, 57.4, 29.5, 20.9. [00944] Compound 3.16 (4-chloro-6-methyl-6-(p-nitrophenyl)-5,6-dihydroindolo[2,l- hjpteridine): 47%, mp 201-202 °C. ES-MS: 392.1 [M+H+]; IH NMR (CDCl₃)δ: 8.79 (d, J = 7.5, 1 H), 8.37 (s, 1 H), 8.14 (d, J = 8.4, 2 H), 7.65 (d, J = 7.5, 1 H), 7.49 (d, J = 8.4, 2 H), 7.40 (t, J = 7.2, 1 H), 7.32 (t, J = 7.2, 1 H), 6.64 (s, 1 H), 4.96 (s, br, 1 H), 2.10 (s, 3 H); 13C NMR (CDCl₃)δ: 151.3, 147.9, 147.3, 145.8, 142.5, 137.1, 134.9, 129.4, 126.5, 124.7, 124.4, 124.0, 123.4, 121.0, 116.1, 104.0, 57.5, 29.0.

6.5.5 Preparation and characterization of ethyl l-(5-amino-6-chIoro- pyrimidin-4-yl)-lH-indole«3-carboxylate:

[00945] To a solution of ethyl l-(6-chloro-5-nitropyrimidin-4-yl)indole-3-carboxylate

(100 mg, 0.29 mmol) in EtOH (5 mL) was added SnCl₂ ^«2H₂O (326 mg, 1.45 mmol). The mixture was refluxed with stirring for 2 h. After cooling to room temperature, the solution was treated with saturated NaHCO₃ to PH 8, and extracted with EtOAc (3 x 15 mL). The combined organics were washed with brine, dried over anhydrous MgSO₄ and concentrated in vacuo. Purification by flash chromatography on silica gel (petroleum/EtOAc, 5:1, v/v) gave ethyl l-(5-amino -6-chloropyrimidin-4-yl)indole-3-carboxylate 7 (80 mg, 88%). mp 166-168 ⁰C; ES-MS: 317.1 [M+H+]; IH NMR (CDCl₃)δ: 8.44 (t , 1 H), 8.31-8.20 (m, 1 H), 8.18 (s , 1 H), 7.43-7.26 (m, 3 H), 4.44-4.33 (m, 4 H), 1.43 (t, J = 7.2, 1 H); 13C NMR (CDCl₃)δ: 164.5, 147.5, 146.7, 142.8, 135.3, 132.7, 132.6, 127.1, 124.5, 123.6, 122.4, 112.5, 112.0, 60.4, 14.7 6.5.6 Preparation and characterization of indole-substituted pteridines

[00946] Compounds 8 were synthesized from compound 7 and aldehydes or ketones according to a procedure similar to compounds 3:

[00947] Compound 8 (1 4-chloro-7-ethoxycarbonyl-6-phenyl-5,6-dihydroindolo[2,l- hjpteridine): 86%, mp 210-213 ⁰C. ES-MS: 405.3 [M+H+]; IH NMR (CDCl₃)δ: 8.95 (d, J = 7.8, 1 H), 8.39 (s, 1 H), 8.11 (d, J = 6.9, 1 H), 7.45-7.40 (m, 2 H), 7.26-7.16 (m, 5 H), 6.77 (d, J = 1.8, 1 H), 4.99 (s, 1 H), 4.42-4.32 (m, 2 H), 1.40 (t, J = 7.2 , 3 H); 13C NMR (CDCl₃)δ: 164.5, 146.9, 144.8, 142.9, 141.4, 140.0, 134.7, 129.4, 128.6, 127.9, 126.2, 125.8, 125.4, 124.7, 122.3, 116.6, 108.0, 60.7, 53.3, 14.6

[00948] Compound 8.2 (4-chloro-7-ethoxycarbonyl-6-(p-methylphenyl)-5,6- dihydroindolo[2,l-h]pteridine): 82%, mp 179-181 ⁰C. ES-MS: 419.2 [M+H+]; IH NMR (CDCl₃)δ: 8.93 (dd, J = 5.4, 1.8, 1 H), 8.38 (s, 1 H), 8.214-8.182 (m, 1 H), 7.46-7.36 (m, 2 H), 7.05-7.01 (m, 4 H), 6.72 (d, J = 2.1 , 1 H), 4.95 (s, 1 H), 4.42-4.34 (m, 2 H), 2.26 (s, 1 H), 1.40 (t, J = 7.2 , 3 H); 13C NMR (CDCl₃)δ: 164.5, 146.8, 144.8, 142.9, 140.3, 138.6, 138.5, 134.7, 130.0, 127.9, 126.1, 125.9, 125.3, 124.7, 122.2, 116.5, 107.8, 60.7, 53.1, 21.3, 14.6 [00949] Compound 8.3 (4-chloro-7-ethoxycarbonyl-6-(p-fluorophenyl-5,6- dihydroindolo[2,l-h]pteridine): 58%, mp 204-206 °C. ES-MS: 423.1[M+H+]; IH NMR (CDCl₃)δ: 8.95 (dd, J = 5.7, 1.5, 1 H), 8.41 (s, 1 H), 8.214-8.184 (m, 1 H), 7.48-7.38 (m, 2 H), 7.19-7.13 (m, 2 H), 6.98-7.13 (m, 2 H), 6.76 (d, J = 2.4, 1 H), 4.97 (d, J = 2.1 ,1 H), 4.44-4.34 (m, 2 H), 2.26 (s, 1 H), 1.42 (t, J = 7.2, 3 H).

[00950] Compound 8.4 (4-chloro-7-etfioxycarbonyl-6-propyl-5,6-dihydroindolo[2,l- h]pteridine): 82%, mp 168-170 ⁰CES-MS: 371.2 [M+H+]; IH NMR (CDCl₃)δ: 8.86 (d, J = 6.3, 1 H), 8.36 (s, 1 H), 8.19 (d, J = 7.2, 1 H), 7.40-7.35 (m, 2 H), 5.58 (s, br, 1 H), 4.71 (s, 1 H), 4.45(q, J = 7.5, 2 H), 1.76-1.74 (m, 2 H), 1.51-1.42 (m, 5 H), 0.96 (t, J = 7.2, 3 H); 13C NMR (CDCl₃)δ: 164.4, 146.3, 144.5, 142.4, 134.2, 127.5, 125.7, 124.7, 124.2, 121.7, 116.1, 109.7, 106.3, 60.3, 50.0, 38.5, 18.6, 14.4, 13.5.

[00951] Compound 8.5 (4-chloro-6-cyclohexylidene-7-ethoxycarbonyl-6-propyl-5,6- dihydroindolo[2,l-h]pteridine): 53%, mp 187-189 ⁰C. ES-MS: 397.2 [M+H+]; IH NMR (CDCl₃)δ: 8.93 (d, J = 7.2, 1 H), 8.36 (s, 1 H), 8.02 (d, J = 7.2, 1.5, 1 H), 7.43-7.32 (m, 2 H), 5.08 (s, 1 H), 4.49(q, J = 6.9, 2 H), 2.74-2.65 (m, 2 H), 1.89-1.81 (m, 6 H), 1.59-1.49 (m, 2 H), 1.51 (t, J = 6.9, 3 H); 13C NMR (CDCl₃)δ: 165.1, 146.3, 144.6, 144.4, 142.0, 133.4, 128.2, 124.8, 124.7, 123.9, 121.3, 116.4, 108.3, 61.0, 56.0, 33.6, 24.1, 21.1, 14.4. 6.5.7 General procedure and characterization details for the preparation of amino-substituted pteridines 9.

[00952] A mixture of compound 3 (0.154 mmol), n-BuNH2 (1.232 mmol) and Et₃N

(0.231 mmol) was refluxed with stirring in n-BuOH (2.0 mL) until the disappearance of compound 3 monitored by TLC. Concentration in vacuo and purification by flash chromatography (petroleum/EtOAc, 5:1, v/v) gave the desired product 9. [00953] Compound 9.1 (N-butyl-6~cyclohexylidene-indolo[2,l-h]pteridin-4- amine):88%, mp 137-138 °C; ES-MS: 362.3 [M+H+]; IH NMR (CDCl₃) δ: 8.77 (d, J = 8.4, 1 H), 8.40 (s, 1 H), 7.56 (d, J = 6.6, 1 H), 7.29 (t, J = 6.9, 1 H), 7.19 (t, J = 7.2, 1 H), 6.36 (s, 1 H), 5.42 (s, br, 1 H), 3.53 (q, J = 6.6, 2 H), 2.16 (s, br, 1 H), 1.84-0.73 (m, 17 H); 13C NMR (CDCl₃)δ: 153.0, 148.4, 143.1, 134.7, 129.7, 123.0, 121.7, 120.2, 115.5, 109.7, 106.9, 98.6, 52.0, 40.7, 35.2, 32.0, 29.6, 25.7, 21.3, 20.1, 13.8.

[00954] Compound 9.2 (N-butyl-6-(p-methylphenyl)-indolo[2,l-h]pteridin-4-amhie):

91%, mp 139-140 °C; ES-MS: 382.2 [M+H+]; IH NMR (CDCl₃) δ: 9.34 (d, J = 8.4, 1 H), 8.60 (s, 1 H), 7.92 (d, J = 7.8, 2 H), 7.86 (d, J = 7.5, 1 H), 7.55 (t, J = 7.5, 1 H), 7.46 (t, J = 6.9, 1 H), 7.39 (d, J = 7.2, 2 H), 7.28 (s, 1 H), 6.79(s, br, 1 H), 3.65 (q, J = 6.3, 2 H), 2.49 (s, 1 H), 1.75-1.67 (m, 2 H), 1.52-1.45 (m, 2 H), 0.99 (t, J = 6.9, 3 H); 13C NMR (CDCl₃) δ: 158.9, 155.9, 153.0, 144.3, 140.3, 135.1, 132.6, 130.3, 129.2, 129.0, 128.5, 124.3, 123.7, 121.4, 118.1, 113.0, 102.9, 40.7, 31.8, 21.5, 20.2, 13.9.

[00955] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. [00956] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

[00957] Citation of a reference herein shall not be deemed an admission that such is prior art to the invention.

Claims

WHAT IS CLAIMED IS:

1. A library of a plurality of different compounds selected from the group consisting of:

wherein X = O, S, or NR;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom; R, R₁, R₂ = alkyl, aryl or a heterocycle;

wherein X = O, S, or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein X = O, S, or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein X = O, S, or NR; Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein X = O, S , or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein:

R₇ = H, CH₃, or F;

R₈ = 4'-F-C₆H₄, 4'-CH₃-C₆H₄, or 4'-NO₂-C₆H₄; and

wherein:

NHR₉R₁₀ is n-Bu-NH2 or pyrrolidine.

2. The library of claim 1, wherein the plurality of compounds are selected from the group consisting of:

4-chloro-l l-methyl-6-propylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-6,l 1- dimethylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-ll-methyl-6-phenylpyrimido[4,5-

&][l,4]benzodiazepine; 4-chloro-ll-methyl-6-(4'-fluorophenyl)-pyrimido[4,5- b][l,4]benzodiazepine; 4-chloro-ll-methyl-6-(4'-methyl-phenyl)-pyrimido[4,5- &][l,4]benzodiazepine; 4-chloro-ll-methyl-6-(4'-nitro-phenyl)-pyrimido[4,5- b) [ 1 ,4]benzodiazepine; 4-chloro-8, 1 l-dimethyl-6-propylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

4-chloro-6,8,ll-trimethylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6- phenylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6-(4'-fluorophenyl)- pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6-(4'-methyl-phenyl)~ pyrimido[4,5-Z?][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6-(4'-nitro-phenyl)- pyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-chloro-8-fluoro- 11 -methyl-6-propylpyrimido[4,5- b] [ 1 ,4]benzodiazepine; 4-chloro-8-fluoro-6, 1 l-dimethylpyrimido[4,5-b] [ 1 ,4]benzodiazepine;

4-chloro-8-fluoro-ll-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8- fluoro -1 l-methyl-6-(4'-fluorophenyl)-pyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-8- fluoro -1 l-methyl-6-(4'-methylphenyl)-pyrimido[4,5-Z>][l,4]benzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'-nitrophenyl)-pyrimido[4,5-&][l,4]benzodiazepine; 4-hydroxy -11- methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine; 4-(butylamino)-8- fluoro-1 l-methyl-6-

(4'-methyl-phenyl)-pyrimido[4,5-&][l,4] benzodiazepine; or 4-(pyrrolidin-l-yl)-8- fluoro -

11 -methyl-6-(4'-methylphenyl)-pyrimido[4,5-&] [ 1 ,4] benzodiazepine.

3. A compound selected from the group consisting of

wherein X = O, S, or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle; wherein X = O, S, or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein X = O, S, or NR; Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom; R, R₁, R₂, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein X = O, S, or NR;

Nu = R₄S, R₄O or NR₅R₆;

Y₁, Y₂, Y₃, and Y₄ = H, alkyl or a heteroatom;

R, R₁, R₂, R₃, R₄, R₅, and R₆ = alkyl, aryl or a heterocycle;

wherein:

R₇ = H, CH₃, or F; R₈ = 4'-F-C₆H₄, 4'-CH₃-C₆H₄, or 4'-NO₂-C₆H₄;

wherein:

NHR₉R_1O is n-Bu-NH₂ or pyrrolidine.

4. The compound of claim 3 selected from the group consisting of 4-chloro-l 1-methyl- 6-propylpyrimido[4,5-έ»][l,4]benzodiazepine; 4-chloro-6,ll-dimethylpyrimido[4,5- &][l,4]benzodiazepine; 4-chloro-l l-methyl-6-phenylpyrimido[4,5-b][l,4]benzodiazepine; 4- chloro-1 l-methyl-6-(4'-fluorophenyl)-pyrimido[4,5-&][l,4]benzodiazepine; 4-chloro-l 1- methyl-6-(4'-methyl-phenyl)-pyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-chloro- 11 -methyl-6-(4'- nitro-phenyl)-pyrimido[4,5-&][l,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6- propylpyrimido[4,5-b][l,4]benzodiazepine; 4-chloro-6,8,ll-trimethylpyrimido[4,5- b] [ 1 ,4]benzodiazepine; 4-chloro-8, 11 -dimethyl-6-phenylpyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-chloro-8,ll-dimethyl-6-(4'-fluorophenyl)-pyrimido[4,5-&][l,4]benzodiazepine; 4-chloro- 8,1 l-dimethyl-6-(4'-methyl-phenyl)-pyrimido[4,5-^][l,4]benzodiazepine; 4-chloro-8,l 1- dimethyl-6-(4'-nitro-ρhenyl)-pyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-chloro- 8-fluoro- 11- methyl-6-propylpyrimido[4,5-Z?][ 1 ,4]benzodiazepine; 4-chloro-8-fluoro-6, 11- dimethylpyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-chloro-8-fluoro- 11 -methyl-6- phenylpyrimido[4,5-^][l,4]benzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- fluorophenyl)-pyrimido[4,5-ft][l,4]benzodia zepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- methylphenyl)-pyrimido[4,5-Z?][l,4Jbenzodiazepine; 4-chloro-8- fluoro -ll-methyl-6-(4'- nitroρhenyl)-ρyrimido[4,5-b][l,4]benzodiazeρine; 4-hydroxy -1 l-methyl-6- phenylpyrimido[4,5-i»][l,4]benzodiazepine; 4-(butylamino)-8- fluoro-ll-methyl-6-(4'- methyl-phenyl)-pyrimido[4,5-b][l,4] benzodiazepine; and 4-(pyrrolidin-l-yl)-8- fluoro -11- methyl-6-(4'-methylphenyl)-pyrimido[4,5-b] [ 1 ,4] benzodiazepine.

5. A method of preparing the compound of claim 3 comprising reacting a 6-chloro-4,5- diaminopyrimidine with an acid or acid derivative to form a 4-chloro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine.

6. The method of claim 5, which further comprises conducting a nucleophilic substitution reaction on said 4-chloro-pyrimido[4,5-b][l, ^benzodiazepine to produce a 4- substituted pyrimido[4,5-b] [1 ,4]benzodiazepine.

7. The method of claim 6, which further comprises hydrogenating the 4-substituted- pyrimido[4,5-£][l,4]benzodiazepine under hydrogenation reaction conditions or reacting the 4-substituted-pyrimido[4,5-Z?][l,4]benzodiazepine with an organometallic reagent.

8. The method of claim 7, which further comprises forming an amide, alkylating, or forming a sulfonamide at the 5 position of the cyclic secondary amine of the 4-substituted- pyrimido [4,5 -b] [ 1 ,4]benzodiazepine.

9. A method of screening the library of claim 1 for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library of claim 1 with a kinase and a kinase substrate under conditions suitable for kinase activity;

(c) comparing said first ratio to a second measure of kinase activity provided by a second ratio of unphosphorylated substrate to phosphorylated substrate obtained in the absence of said compounds; wherein inhibition of kinase activity in step(b) is indicative of the presence of a candidate kinase inhibitor among said compounds.

10. The method of claim 9, where the kinase is FLT3, KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I.

11. A library of a plurality of different compounds selected from the group consisting of:

wherein:

Rl = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle or halogen; R2 = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, or substituted heterocycle.

12. The library of claim 11 , wherein the plurality of compounds are selected from the group consisting of:

4-Chloro-l l-methyl-6-propyl-5,6-dihydro-pyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-Chloro- 11- methyl-6-ethyl-5,6-dihydro-pyrimido[4,5-b] [l,4]benzodiazepine; 4-Chloro-l l-methyl-6- phenyl-5,6-dihydro-ρyrimido[4,5-b][l,4]benzodiazepine; 4-Chloro-ll-methyl-6-(4'-methyl- phenyl)-5,6-dihydro-pyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-Chloro- 11 -methyl-6-(4'-fluoro- phenyl)-5 ,6-dihydro-pyrimido [4,5 -b] [ 1 ,4]benzodiazepine; 4-Chloro- 11 -methyl-6-(4'-nitro- phenyl)-5,6-dihydro-pyrimido[4,5-b][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-propyl- 5,6-dihydro-pyrimido [4,5-&][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-ethyl-5,6- dihydro-pyrimido [4,5-£][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-phenyl-5,6-dihydro- pyrimido [4,5~b][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-(4'-methyl-phenyl)-5,6- dihydro-pyrimido[4,5-&] [ 1 ,4]benzodiazepine; 4-Chloro-8, 11 -dimethyl-6-(4'-fluoro-phenyl)- 5,6-dihydro-pyrimido[4,5-&][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-nitro- phenyl)-5,6-dihydro-pyrimido[4,5-b][l,4]benzodiazepine; 4-Chloro-9,ll-dimethyl-6-propyl- 5,6-dihydro-pyrimido [4,5-b][l,4]benzodiazepine; 4-Chloro-9,ll-dimethyl-6-ethyl-5,6- dihydro-pyrimido [4,5-Z?][l,4]benzodiazepine; 4-Chloro-9,l l-dimethyl-6-phenyl-5,6-dihydro- pyrimido [4,5-b][l,4]benzodiazepine; 4-Chloro-9,l l-dimethyl-6-(4'-methyl-phenyl)-5,6- dihydro-pyrimido[4,5-Z?][l,4]benzodiazepine; 4-Chloro-9,ll-dimethyl-6-(4'-fluoro-phenyl)- 5,6-dihydro-pyrimido[4,5-£] [ 1 ,4]benzodiazepine; 4-Chloro-9, 1 l-dimethyl-6-(4'-nitro- phenyl)-5,6-dihydro-pyrimido[4,5-b][l,4]benzodiazepine; and 4-Chloro-8-fluoro-l 1-methyl- 6-phenyl-5,6-dihydro pyrimido[4,5-&] [ 1 ,4]benzodiazepine.

13. A compound selected from the group consisting of:

wherein:

14. The compound of claim 13, wherein the compound is selected from the group consisting of: 4-Chloro-l l-methyl-6-propyl-5,6-dihydro-pyrimido[4,5- b][l,4]benzodiazepine; 4-Chloro-ll-methyl-6-ethyl-5,6-dihydro-pyrimido[4,5-&] [l,4]benzodiazepine; 4-Chloro-ll-methyl-6-phenyl-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine; 4-Chloro- 1 l-methyl-6-(4'-methyl-phenyl)-5,6-dihydro-pyrimido[4,5- b] [ 1 ,4]benzodiazepine; 4-Chloro- 11 -methyl-6-(4'-fluoro-phenyl)-5,6-dihydro-pyrimido[4,5- b][l,4]benzodiazepine; 4-Chloro-ll-methyl-6-(4'-nitro-phenyl)-5,6-dihydro-pyrimido[4,5- b][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-propyl-5,6-dihydro-pyrimido [4,5- &][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-ethyl-5,6-dihydro-pyrimido [4,5- b][l,4]benzodiazepine; 4-Chloro-8,l l-dimethyl-6-phenyl-5,6-dihydro-pyrimido [4,5- b][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-methyl-phenyl)-5,6-dihydro- pyrimido[4,5~Z?][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-fluoro-phenyl)-5,6- dihydro-pyrimido[4,5-έ][l,4]benzodiazepine; 4-Chloro-8,ll-dimethyl-6-(4'-nitro-phenyl)- 5 ,6-dihydro-pyrimido[4,5-/?] [ 1 ,4]benzodiazepine; 4-Chloro-9, 11 ~dimethyl-6-propyl-5,6~ dihydro-pyrimido [4,5-&][l,4]benzodiazepine; 4-Chloro-9,ll-dimethyl-6-ethyl-5,6-dihydro- pyrimido [4,5-ό][l,4]benzodiazepine; 4-Chloro-9,l l-dimethyl-6-phenyl-5,6-dihydro- pyrimido [4,5-&][l,4]benzodiazepine; 4-Chloro-9,l l-dimethyl-6-(4'-methyl-phenyl)-5,6- dihydro-pyrimido[4,5-Z?][l,4]benzodiazepine; 4-Chloro-9,ll-dimethyl-6-(4'-fluoro-phenyl)- 5,6-dihydro-pyrimido[4,5-b] [ 1 ,4]benzodiazepine; 4-Chloro-9, 1 l-dimethyl-6-(4'-nitro- phenyl)-5,6-dihydro-pyrimido[4,5-b][l,4]benzodiazepine; and 4-Chloro-8-fluoro-l 1-methyl- 6-phenyl-5,6-dihydro pyrimido[4,5-b][l,4]benzodiazepine.

15. A metliod of preparing the compound of claim 13 comprising reacting a 6-chloro-4,5- diaminopyrimidine with an acid or acid derivative to form a 4-chloro-dihydropyrimido[4,5~ b] [ 1 ,4]benzodiazepine.

16. The method of claim 15, which further comprises conducting a nucleophilic substitution reaction on said 4-chloro-dihydropyrimido[4,5-b][l,4]benzodiazepine.

17. A method of screening the library of claim 11 for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library of claim 11 with a kinase and a kinase substrate under conditions suitable for kinase activity;

18. The method of claim 17, where the kinase is FLT3, KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I.

19. A library of a plurality of different compounds selected from the group consisting of:

wherein:

R₃ = H, alkyl, substituted alkyl, aryl, heterocycle, substituted heterocycle, heteroaryl, or substituted aryl;

wherein:

R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle, alkoxy, or halogen;

wherein:

R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted . heterocycle or halogen;

R₄, R₅ = H, alkyl, substituted alkyl, aryl, substitute aryl, heterocycle, substituted heterocycle, or R₄ and R₅ together form a cyclic alkyl.

20. The library of claim 19, wherein the two or more compounds are selected from the group consisting of: 4-(Phenylthio)-6-phenylpyrimido[4,5-b][l,4]benzothiazepine; 4-

(Phenylthio)-6-(pyridin-3-yl)-pyrimido[4,5-έ] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-p- tolylpyrimido[4,5-&][l,4]benzothiazepine; 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5- b\ [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(p-nitrophenyl)pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(m-fluorophenyl)pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-

(Phenylthio)-6-propylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6- benzylpyrimido[4,5-b][l,4]benzothiazepine; 4-(p-Tolylthio)-6-phenyl-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-(pyridin-3-yl)-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-p-tolyl-8-methylpyrimido[4,5-

&][l,4]benzothiazepine; 4-(p-Tolylthio)-6-(p-fluorophenyl)-8-methylpyrimido[4,5- b][l_>4]benzothiazepine; 4-(p-Tolylthio)-6-propyl-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-benzyl-8-methylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-phenyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-p-tolyl-8-chloropyrimido[4,5-

Z7][l,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-(pyridin-3-yl)-8-chloropyrimido[4,5-

Z?][l,4]benzothiazepine; 4-(p-Chloro-phe nylthio)-6-benzyl-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Methoxyphenylthio)-6-phenyl-8-methoxypyrimido[4,5- b][l,4]benzothiazepine; 4-(p-Methoxyphenylthio)-6-benzyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylsulf inyl)-6-phenylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-

(p-Tolylsulfinyl)-6-phenyl-8-methylpyrimido[4,5-6] [ 1 ,4]benzothiazepine; 4-(p-

Chlorophenylsulfinyl)-6-ρhenyl-8-chloropyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(n-

Butylamino)-6-phenylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; 4-(Pyrrolidin- 1 -yl)-6- phenylpyrimido[4,5-b][l,4]benzothiazepine; 4-(n-Butylamino)-6-phenyl-8- methylpyrimido[4,5-&] [ 1 ,4]benzothiazepine; and 4-(n-Butylamino)-6-phenyl-8- chloropyrimido[4,5-b] [ 1 ,4]benzothiazepine.

21. A compound selected from the group consisting of:

wherein:

R₄, R₅ = H, alkyl, substituted alkyl, aryl, substitute aryl, heterocycle, substituted heterocycle, or R₄ and R₅ together form a cyclic alkyl..

22. The compound of claim 21, wherein the compound is selected from the group consisting of: 4-(Phenylthio)-6-phenylpyrimido[4,5-b][l,4]benzothiazepine; 4-(Phenylthio)- 6-(ρyridin-3-yl)-pyrimido[4,5-b][l,4]benzothiazepine; 4-(Phenylthio)-6-p-tolylpyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(o-nitrophenyl)pyrimido[4,5-

&][l,4]benzothiazepine; 4-(Phenylthio)-6-(p-nitrophenyl)pyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-(m-fluorophenyl)pyrimido[4,5~ b] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6-methylpyrimido [4,5-&] [ 1 ,4]benzothiazepine; 4-

(Phenylthio)-6-propylpyrimido[4,5-£»] [ 1 ,4]benzothiazepine; 4-(Phenylthio)-6- benzylpyrimido[4,5-b][l,4]benzothiazeρine; 4-(ρ-Tolylthio)-6-phenyl-8-niethylpyrimido[4,5- fo][l,4]benzothiazepine; 4-(p-Tolylthio)-6-(pyridin-3-yl)-8-methylpyrimido[4,5-

&][l,4]benzothiazepine; 4-(p-Tolylthio)-6-p-tolyl-8-methylpyrimido[4,5-

^][l,4]benzothiazepine; 4-(p-Tolylthio)-6-(p-fluorophenyl)-8-methylpyrimido[4,5- fe][l,4]benzothiazepine; 4-(p-Tolylthio)-6-propyl-8-methylpyrimido[4,5- b] [1 ,4]benzothiazepine; 4-(p-Tolylthio)-6-benzyl-8-methylpyrimido[4,5-

Z7][l,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-phenyl-8-chloropyrimido[4,5-

&][l,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-p-tolyl-8-chloropyrimido[4,5- b] [1 ,4]benzothiazepine; 4-(p-Chloro-phenylthio)-6-(pyridin-3-yl)-8-chloropyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Chloro-phe nylthio)-6-benzyl-8-chloropyrimido[4,5-

&][l,4]benzothiazepine; 4-(p-Methoxyplienylthio)-6-phenyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(p-Methoxyphenylthio)-6-benzyl-8-methoxypyrimido[4,5- b] [ 1 ,4]benzothiazepine; 4-(Phenylsulfinyl)-6-phenylpyrimido[4,5-Z?] [ 1 ,4]benzothiazepine; 4-

(p-Tolylsulfinyl)-6-phenyl-8-methylpyrimido[4,5-b][l,4]benzothiazepine; 4-(p-

Chloroph.enylsulfinyl)-6-phenyl-8-chloropyrimido[4,5-b][l,4]benzothiazepine; 4-(n-

Butylamino)-6-phenylpyrimido[4,5-b][l,4]benzothiazepine; 4-(Pyrrolidin-l-yl)-6- phenylpyrimido[4,5-&] [ 1 ,4]benzothiazepine; 4-(n-Butylamino)-6-phenyl-8- methylpyrimido[4,5-b] [ 1 ,4]benzothiazepine; and 4-(n-Butylamino)-6-phenyl-8- chloropyrimido[4,5-&][l,4]benzothiazepine.

23. A method of preparing the compound of claim 21 comprising reacting a 5-amino-4,6- bisphenylthiopyrimidine with an acid or acid derivative to form a pyrimido[4,5- b] [ 1 ,4]benzothiadiazeρine.

24. The method of claim 23, which further comprises oxidizing the phenylthio group of said pyrimido[4,5-b][l,4]benzothiadiazepine to the corresponding sulfoxide or sulfone.

25. The method of claim 24, which further comprises conducting a nucleophilic substitution reaction on said pyrimido[4,5-&][l,4]benzothiadiazepine with an amine.

26. A method of screening the library of claim 19 for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library of claim 19 with a kinase and a kinase substrate under conditions suitable for kinase activity;

27. The method of claim 26, where the kinase is FLT3, KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I.

28. A library of a plurality of different compounds selected from the group consisting of:

wherein:

X = Cl, R₃R₄N, R₃O, R₃S, or aryl;

R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, carboxylic acid group or R₁ and R₂ together form a cyclic alkyl;

R₃, R₄ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle or substituted heterocycle.

29. The library of claim 28, wherein the plurality of compounds are selected from the group consisting of: 8-chloro-6-emyl-l,2,6,7-tetrahyάϊo-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene; S-chloro-ό-phenyl-l^βJ-tetrahydro^^ll_jllb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-(o-methoxyphenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-(p-nitro-phenyl)-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-(ρ-fluoro-phenyl)-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulene; S-chloro-ό-styryl-l^θJ-tetrahydro^^llJlb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-propenyl- 1 ,2,6,7-tetrahydro-7,9,l 1 , 1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6,6-dimethyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-ethyl-6-methyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-propyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h]azulene; 8-chloro-6-cyclohexylidene-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza- dibenzo[c,d,h] azulene; 8-chloro-6-methyl-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza- dibenzo[c,d,h] azulene; 8-chloro-6-methyl-6-(p-methoxyphenyl)- 1 ,2,6,7-tetrahydro- 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-(p-nitro-phenyl)- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-ethyl-6-phenyl-l,2,6,7- tetrahydro-7 ,9,11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-chloro-6-ethyl- 1 ,2,6,7-tetrahydro- 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulen-6-carbonate acid; 9-chloro-7-ethyl~2,3,7,8- tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7- pyproyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3- de]naphthalene; 9-chloro-7-phenyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-(p-methylphenyl)-2,3,7,8-tetrahydro- lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-(p-nitro- phenyl)-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3- de]naphthalene; 9-chloro-7-styryl-2,3,7,8-tetrahydro- 1H-8, 10, 12, 12b-tetraaza- benzo[4,5]cyclohepta[ 1 ,2,3-de]naphthalene; 9-chloro-7,7-dimethyl-2,3,7,8-tetrahydro- IH- SJOj^j^b-tetraaza-benzo^^Jcycloheptatl^^-delnaphthalene^-Cn-butyO-θ-phenyl- 1,2,6,7 -tetrahydro-7 ,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulen-8-amine; N-phenyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulen-8-amine; 8-(morpholin- l-yl)-6- phenyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8-(pyrrolidin-l-yl)-6- propyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-butoxyl-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-butoxy-6-phenyl- 1 ,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; δ-benzylthio-ό-propyl- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-benzylthio-6-phenyl- 1, 2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8-phenylthio-6-propyl- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-phenylthio-6-phenyl- 1,2,6,7- tetrahydro-7,9,11 , 1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-phenyl-6-propyl- 1 ,2,6,7-tetrahydro- 7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; and 8-phenyl-6-phenyl-l , 2,6,7 -tetrahydro- 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene.

30. A compound of formula:

wherein:

X = Cl, R₃R₄N, R₃O, R₃S, or aryl;

31. The compound of claim 30, wherein the compound is selected from the group consisting of: 8-chloro-6-ethyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-propyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6- phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-(o- methoxyphenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-(p- nitro-phenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-(p- fluoro-phenyl)- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6- styryl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-propenyl- l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6,6-dimethyl- 1,2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-ethyl-6-methyl- 1,2,6,7- tetrahydro-7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-propyl-l, 2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; δ-chloro-ό-cyclohexylidene- 1,2,6,7- tetrahydro-7 ,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-phenyl- 1 ,2,6,7- tetrahydro-7,9, 11 , 1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-chloro-6-methyl-6-(p- methoxyphenyl)- 1 ,2,6,7-tetrahydro-7 ,9,11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-chloro-6- methyl-6-(p-nitro-phenyl)-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8- chloro-6-ethyl-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8- chloro-6-ethyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulen-6-carbonate acid; 9-chloro-7-ethyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3- de]naphthalene; 9-chloro-7-pyproyl-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-phenyl-2,3,7,8-tetrahydro-lH- 8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-(p- methylphenyl)-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3- de]naρhthalene; 9-chloro-7-(ρ-nitro-phenyl)-2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza- benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7-styryl-2,3,7,8-tetrahydro-lH- 8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; 9-chloro-7,7-dimethyl- 2,3,7,8-tetrahydro-lH-8,10,12,12b-tetraaza-benzo[4,5]cyclohepta[l,2,3-de]naphthalene; N- (n-butyl)-6-phenyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulen-8-amine; N- phenyl-6-propyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulen-8-amine; 8- (morpholin- 1 -yl)-6-phenyl- 1 ,2,6,7-tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8- (pyrrolidin-l-yl)-6-propyl-l,2,6,7-tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8- butoxyl-6-propyl-l,2,6,7-tetrahydro-7,9,l 1,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-butoxy-6- phenyl- 1 ,2,6,7-tetrahydro-7 ,9,11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-benzylthio-6-propyl- l,2,6,7-tetrahydro~7,9,l 1,1 lb~tetraaza-dibenzo[c,d,h]azulene; 8-benzylthio-6-phenyl- 1,2,6,7- tetrahydro-7, 9,11,1 lb-tetraaza-dibenzo[c,d,h] azulene; 8-phenylthio-6-propyl- 1,2,6,7- tetrahydro-7,9, 11,1 lb-tetraaza-dibenzo[c,d,h]azulene; 8-phenylthio-6-phenyl- 1 ,2,6,7- tetrahydro-7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; 8-phenyl-6-propyl-l, 2,6,7 -tetrahydro- 7,9,ll,llb-tetraaza-dibenzo[c,d,h]azulene; and 8-phenyl-6-phenyl-l,2,6,7-tetrahydro~ 7,9,11,1 lb-tetraaza-dibenzo[c,d,h]azulene.

32. A method of preparing the single compound of claim 30 comprising reacting an indolin-1-ylpyrimidine or tetrahydroquinolin-1-ylpyrimidine with an aldehyde or ketone to form a tetracyclic pyrimidobenzodiazepine.

33. The method of claim 32, which further comprises conducting a nucleophilic substitution reaction on said tetracyclic pyrimidobenzodiazepine.

34. A method of screening the library of claim 28 for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library of claim 28 with a kinase and a kinase substrate under conditions suitable for kinase activity;

(c) comparing said first ratio to a second measure of kinase activity provided by a second ratio of unphosphorylated substrate to phosphorylated substrate obtained in the absence of said compounds; wherein inhibition of kinase activity is indicative of a candidate kinase inhibitor.

35. The method of claim 34, where the kinase is FLT3 , KIT, PDGFR-B, RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I.

36. A library of a plurality of different compounds selected from the group consisting of:

wherein:

R₁, R₂ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, or R₁ and R₂ together form a cyclic alkyl;

R₃ = H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, or substituted heterocycle.

X = Cl or NHR; and

R = alkyl, substituted alkyl, aryl, substituted aryl.

37. The library of claim 36, wherein the plurality of compounds are selected from the group consisting of: 4-chloro-6-ethyl-5,6-dihydroindolo[2,l-h]pteridine; 6-butyl-4-chloro- 5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-phenyl-5,6-dihydroindolo[2,l-h]pteridine; 4- chloro-6-(4'-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(4'-nitrophenyl)- 5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(3',4'-dichlorophenyl)-5,6-dihydroindolo[2,l- hjpteridine; 4-chloro-6-(o-methoxyphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o- chlorophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o-nitrophenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-6-(m-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-ethyl-6-methyl-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-propyl- 5,6-dihydroindolo[2,l-h]ρteridine; 4-chloro-6-cyclohexylidene-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-6-methyl-6-phenyl-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6- methyl-6-(p-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-(p- nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-phenyl-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-methylphenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-fluorophenyl-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-6-cyclohexylidene-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l- hjpteridine; N-butyl-β-cyclohexylidene-mdolo^l-hlpteridm^-amine; and N-butyl-6-(p- methylphenyl)-indolo[2, 1 -h]pteridin-4-amine.

38. A compound of formula:

wherein:

X = Cl or NHR; and

R = alkyl, substituted alkyl, aryl, substituted aryl.

39. The compound of claim 38, wherein the compound is selected from the group consisting of: 4-chloro-6-ethyl-5,6-dihydroindolo[2,l-h]pteridine; 6-butyl-4-chloro-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-6-phenyl-5,6-dihydroindolo[2,l-h]pteridine;.4- chloro-6-(4'-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(4'-nitrophenyl)-

5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(3',4'-dichlorophenyl)-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-6-(o-methoxyphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o- chlorophenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-(o-nitrophenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-6-(m-nitrophenyl)-5,6-dihydroindolo[2,l-h]pteridine;

4-chloro-6-ethyl-6-methyl-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-propyl-

5,6-dihydroindolo[2,l-h]ρteridine; 4-chloro-6-cyclohexylidene-5,6-dihydroindolo[2,l- h]pteridine; 4-chloro-6-methyl-6-phenyl-5,6-dihydroindolo[2, l-hjpteridine; 4-chloro-6- methyl-6-(p-methylphenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-6-methyl-6-(p- nitroρhenyl)-5,6-dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-phenyl-5,6- diliydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-methylphenyl)-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-(p-fluorophenyl-5,6- dihydroindolo[2,l-h]pteridine; 4-chloro-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2,l- hjpteridine; 4-chloro-6-cyclohexylidene-7-ethoxycarbonyl-6-propyl-5,6-dihydroindolo[2, 1 - h]pteridine; N-butyl-6-cyclohexylidene-indolo[2,l-h]pteridin-4-amine; and N-butyl-6-(ρ- meihylphenyl)-indolo[2, 1 -h]pteridin-4-amine.

40. A method of preparing the compound of claim 38 comprising reacting a 5-amino-4- chloro-6-(l-mdolyl)pyrimidine with an aldehyde or ketone to form a 5,6-dihydroindolo[2,l- bjpteridine.

41. The method of claim 40, which further comprises conducting a nucleophilic substitution reaction on said 5,6-dihydroindolo[2,l-h]pteridine.

42. A method of screening the library of claim 36 for a candidate kinase inhibitor, comprising:

(a) contacting one or more compounds in the library of claim 36 with a kinase and a kinase substrate under conditions suitable for kinase activity;

43. The method of claim 42, where the kinase is FLT3 , KIT, PDGFR-B , RET, SRC, FLTl(VEGFRl), FLT4(VEGFR3), KDR(VEGFR2) or RAF-I.

44. A method of screening a library for a candidate compound with a therapeutic activity, comprising:

(b) determining activity measured by said biological assay; wherein a modulation of said activity relative to the activity in the absence of said compounds indicates a therapeutic activity of said one or more compounds.