Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/48—Two nitrogen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/14—Decongestants or antiallergics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to compounds, prodrugs, and methods of using these compounds and prodrugs thereof in the treatment of conditions in which modulation of the JAK pathway or inhibition of JAK kinases, particularly JAK3, are therapeutically useful.
• Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within cells (see, e.g., Hardie and Hanks, The Protein Kinase Facts Book, I and II, Acad 6 mic Press, San Diego, CA, 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases can be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein- serine/threonine, lipids, etc.).
• phosphorylate e.g., protein-tyrosine, protein- serine/threonine, lipids, etc.
• JAK kinases are a family of cytoplasmic protein tyrosine kinases including JAKl, JAK2, JAK3 and TYK2. Each of the JAK kinases is selective for the receptors of certain cytokines, though multiple JAK kinases can be affected by particular cytokine or signaling pathways. Studies suggest that JAK3 associates with the common gamma ( ⁇ c) chain of the various cytokine receptors. JAK3 in particular selectively binds to receptors and is part of the cytokine signaling pathway for IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21.
• ⁇ c common gamma
• JAKl interacts with, among others, the receptors for cytokines IL-2, IL-4, IL- 7, IL-9 and IL-21, while JAK2 interacts with, among others, the receptors for IL-9 and TNF - ⁇ .
• cytokines e.g., IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21
• receptor oligomerization occurs, resulting in the cytoplasmic tails of associated JAK kinases being brought into proximity and facilitating the trans- phosphorylation of tyrosine residues on the JAK kinase. This trans-phosphorylation results in the activation of the JAK kinase.
• STAT proteins which are DNA binding proteins activated by phosphorylation of tyrosine residues, function both as signaling molecules and transcription factors and ultimately bind to specific DNA sequences present in the promoters of cytokine-responsive genes (Leonard et al., (2000), J. Allergy Clin. Immunol.105:877-888).
• JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant (allograft) rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as well as in solid and hematologic malignancies such as leukemia and lymphomas.
• autoimmune diseases such as transplant (allograft) rejection
• rheumatoid arthritis amyotrophic lateral sclerosis and multiple sclerosis
• solid and hematologic malignancies such as leukemia and lymphomas.
• JAK3 in particular has been implicated in a variety of biological processes. For example, the proliferation and survival of murine mast cells induced by IL-4 and IL-9 have been shown to be d 6 pend 6 nt on JAK3- and gamma chain- signaling (Suzuki et al., (2000), Blood 96:2172-2180). JAK3 also plays a crucial role in IgE receptor- mediated mast cell d 6 granulation responses (Malaviya et al., (1999), Biochem. Biophys. Res.
• JAK3 kinases have also been implicated in the mechanism involved in early and late stages of rheumatoid arthritis (Muller-Ladner et al, (2000), J. Immunol.
• JAK kinases are abundantly expressed in primary leukemic cells from children with acute lymphoblastic leukemia, the most common form of childhood cancer, and studies have correlated STAT activation in certain cells with signals regulating apoptosis (Demoulin et al, (1996), MoI Cell. Biol. 16:4710-6; Jurland 6 r et al, (1997), Blood. 89:4146-52; Kaneko et al, (1997), Clin. Exp. Immun. 109:185-193; and Nakamura et a/.,(1996), J. Biol. Chem. 271:19483-8). They are also known to be important to lymphocyte differentiation, function and survival.
• JAK-3 in particular plays an essential role in the function of lymphocytes, macrophages, and mast cells.
• compounds which modulate the JAK pathway including those selective for JAK3, can be useful for treating diseases or conditions where the function of lymphocytes, macrophages, or mast cells is involved (Kudlacz et al, (2004) Am. J. Transplant 4:51-57; Changelian (2003) Science 302:875- 878).
• Conditions in which targeting of the JAK pathway or modulation of the JAK kinases, particularly JAK3, are contemplated to be therapeutically useful included 6 , leukemia, lymphoma, transplant rejection (e.g., pancreas islet transplant rejection, bone marrow transplant applications (e.g., graft- versus -host disease), autoimmune diseases (e.g., diabetes), and inflammation (e.g., asthma, allergic reactions). Conditions which can benefit for inhibition of JAK3 are discussed in greater d 6 tail below.
• Provid 6 d herein are novel 2,4-pyrimidinediamine compounds for use in the treatment of conditions in which targeting of the JAK pathway or inhibition of JAK kinases, particularly JAK3, are therapeutically useful.
• the invention is directed to compounds, prodrugs, pharmaceutical compositions and methods of using them in the treatment of conditions in which modulation of the JAK pathway or inhibition of JAK kinases, particularly JAK3, will be therapeutically useful.
• this invention provid 6 s a compound of formula I:
• X is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl;
• Y is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene;
• R 1 is hydrogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, aminoacyl, aminoacyloxy, carboxyl, carboxyl ester, carbonate ester, -C(O)N(R 4 )R 5 , nitro or halo; each R 3 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted
• R 6 is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene, linking the nitrogen bearing R 6 and the ring bearing Y R 7 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl;
• R 8 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; optionally, R 7 and R 8 , together with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group;
• R 7 and R 8 are non-hydrogen
• each of R 6 , R 7 , and R 8 optionally are M + , wherein M + , a counter-ion, is K + , Na + , Li + or +N(R 9 ) ⁇ wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• the invention provid 6 s compounds of formula II: wherein
• R 10 is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene, and the remaining variables are as d 6 fined as for formula I except that included 6 d are compounds where R and R are simultaneously hydrogen.
• X is alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; and the remaining variables are as d 6 fined for compounds of formula I.
• Another implementation of the invention provid 6 s compounds of formula IV:
• X is alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; and the remaining variables are as d 6 fined for compounds of formula I.
• Another embodiment is a method of inhibiting an activity of a JAK kinase, including contacting the JAK kinase with an amount of a compound effective to inhibit an activity of the JAK kinase where the compound is according to formula I as d 6 scribed herein.
• the contact is mad 6 in vitro, in another embodiment the contact is mad 6 in vivo.
• Another embodiment is a method of treating a T-cell mediated autoimmune disease, including administering to a patient suffering from such an autoimmune disease an amount of a compound effective to treat the autoimmune disease where the compound is according to formula I as d 6 scribed herein.
• Another embodiment is a method of treating or preventing allograft transplant rejection in a transplant recipient, including administering to the transplant recipient an amount of a compound effective to treat or prevent the rejection where the compound is according to formula I as d 6 scribed herein.
• Administration in this context may included 6 contacting a transplant organ with a compound or pharmaceutical composition d 6 scribed herein prior to transplant and/or concurrent with administration to the transplant recipient.
• Yet another embodiment is a method of treating or preventing a Type IV hypersensitivity reaction, including administering to a subject an amount of a compound of effective to treat or prevent the hypersensitivity reaction where the compound is according to formula I as d 6 scribed herein.
• Another embodiment is a method of treating or preventing an ocular disease or disord 6 r, including administering to a subject an amount of a compound of effective to treat or prevent the ocular disease or disord 6 r where the compound is according to formula I as d 6 scribed herein.
• Another embodiment is a method of inhibiting a signal transduction cascad 6 in which JAK3 kinase plays a role, including contacting a cell expressing a receptor involved in such a signaling cascad 6 with a compound where the compound is according to formula I as d 6 scribed herein.
• Another embodiment is a method of treating or preventing a JAK kinase-mediated disease, including administering to a subject an amount of compound effective to treat or prevent the JAK kinase-mediated disease where the compound is according to formula I as d 6 scribed herein.
• Another embodiment is a pharmaceutical formulation including a compound of formula I as d 6 scribed herein. Therapy using the 2,4-pyrimidinediamine compounds and pharmaceutical formulations d 6 scribed herein can be applied alone, or it can be applied in combination with or adjunctive to other common immunosuppressive therapies
• the invention encompasses compounds having formula I and the compositions and methods using these compounds in the treatment of conditions in which modulation of the JAK pathway or inhibition of JAK kinases, particularly JAK3 , are therapeutically useful.
• Alky refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includ 6 s, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 -).
• Alkyjene refers to divalent saturated aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight- chained or branched. This term is exemplified by groups such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), iso-propylene (-CH 2 CH(CH 3 )-) or (-CH(CH 3 )CH 2 -), and the like.
• Substituted alkylene refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as d 6 scribed for carbons in the d 6 friendship of "substituted” below.
• Alkoxy refers to the group -O-alkyl, wherein alkyl is as d 6 fined herein. Alkoxy includ 6 s, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t- butoxy, sec-butoxy, n-pentoxy, and the like.
• Acyl refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substitute
• Acylamino refers to the groups -NR 20 C(O)alkyl, -NR 20 C(O)substituted alkyl, N R 20 C(O)cycloalkyl, -NR 20 C(O)substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O)substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O)substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O)substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O)substituted heteroaryl, -NR 20 C(O)he
• Acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, alkenyl- C(O)O-, substituted alkenyl-C(O)O-, alkynyl-C(O)O-, substituted alkynyl-C(O)O-, aryl- C(O)O-, substituted aryl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, cycloalkenyl-C(O)O-, substituted cycloalkenyl-C(O)O-, heteroaryl-C(O)O-, substituted heteroaryl-C(O)O-, heterocyclyl-C(O)O-, and substituted heterocyclyl-C(O)O-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
• Amino refers to the group -NH 2 .
• Aminocarbonyi refers to the group -C(O)NR 21 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted substituted
• Aminothiocarbonyl refers to the group -C(S)NR 21 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitute
• Aminocarbonylamino refers to the group -NR 20 C(O)NR 21 R 22 , wherein R 20 is hydrogen or alkyl and R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl
• Aminothiocarbonylamino refers to the group -NR 20 C(S)NR 21 R 22 , wherein R 20 is hydrogen or alkyl and R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cyclo
• Aminocarbonyloxy refers to the group -0-C(O)NR 21 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitute
• Aminosulfonyl refers to the group -SO 2 NR 21 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkenyl, substituted cyclo
• Aminosulfonyloxy refers to the group -0-SO 2 NR 21 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl; R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group; and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted substitute
• Aminosulfonylamino refers to the group - NR 20 -SO 2 NR 21 R 22 , wherein R 20 is hydrogen or alkyl and R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
• “Sulfonylamino” refers to the group -NR 21 SO 2 R 22 , wherein R 21 and R 22 ind 6 pend 6 ntly are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and where R 21 and R 22 are optionally joined together with the atoms bound thereto to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
• “Aryi” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple cond 6 nsed rings (e.g., naphthyl or anthryl) which cond 6 nsed rings may or may not be aromatic (e.g., 2- benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like), provid 6 d that the point of attachment is through an atom of the aromatic aryl group.
• Preferred aryl groups included 6 phenyl and naphthyl.
• Aryloxy refers to the group -O-aryl, wherein aryl is as d 6 fined herein, including, by way of example, phenoxy, naphthoxy, and the like.
• Arylthio refers to the group -S -aryl, wherein aryl is as d 6 fined herein.
• sulfur may be oxidized to -S(O)- or -SO 2 - moieties.
• the sulfoxid 6 may impart chirality to the molecule.
• Alkenyl refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. Such groups are exemplified, for example, bi-vinyl, allyl, and but-3-en-l-yl. Includ 6 d within this term are the cis and trans isomers or mixtures of these isomers.
• Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups included 6 acetylenyl (-C ⁇ CH), and propargyl (-CH 2 C ⁇ CH).
• Alkynyloxy refers to the group -O-alkynyl, wherein alkynyl is as d 6 fined herein. Alkynyloxy includ 6 s, by way of example, ethynyloxy, propynyloxy, and the like.
• Carboxyl refers to -CO 2 H or salts thereof.
• Carboxyl ester or “carboxy ester” refers to the groups -C(O)O-alkyl,
• (Carboxyl esteriamino) refers to the groups -NR-C(O)O-alkyl, -NR-C(O)O- substituted alkyl, -NR-C(O)O-alkenyl, -NR-C(O)O-substituted alkenyl, -NR-C(O)O- alkynyl, -NR-C(O)O-substituted alkynyl, -NR-C(O)O-aryl, -NR-C(O)O-substituted aryl, -NR-C(O)O-cycloalkyl, -NR-C(O)O-substituted cycloalkyl, -NR-C(O)O-cycloalkenyl, -NR-C(O)O-substituted cycloalkenyl, -NR-C(O)O-heteroaryl, -NR-C(O)O-sub
• (Carboxyl ester)oxy or “carbonate” refers to the groups -O-C(O)O-alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O-
• Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
• suitable cycloalkyl groups include 6 , for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
• Cycloalkenyl refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.
• Cycloalkynyl refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
• Cycloalkylene refers to divalent cycloalkyl groups, wherein cycloalkyl is as d 6 fined herein.
• Cycloalkoxy refers to -O-cycloalkyl.
• Cycloalkylthio refers to -S-cycloalkyl.
• sulfur may be oxidized to -S(O)- or -SO2- moieties.
• the sulfoxid 6 may impart chirality to the molecule.
• Cycloalkenyloxy refers to -O-cycloalkenyl.
• Cycloalkenylthio refers to -S-cycloalkenyl.
• sulfur may be oxidized to sulfinyl or sulfonyl moieties. Depending on the pendant substitution, the sulfoxid 6 may impart chirality to the molecule.
• Halo or halogen refers to fluoro, chloro, bromo, and iodo and is preferably fluoro or chloro.
• Hydroxy or “hydroxyl” refers to the group -OH.
• Heteroaryloxy refers to -O-heteroaryl.
• Heteroarylthio refers to the group -S-heteroaryl.
• sulfur may be oxidized to -S(O)- or -SO 2 - moieties.
• the sulfoxid 6 may exist as one or more stereoisomers.
• Heterocyclyl in the broad 6 st sense included 6 s aromatic and non-aromatic ring systems and more specifically refers to a stable three- to fifteen-membered ring radical that consists of carbon atoms and from one to five heteroatoms.
• the heterocyclyl radical can be a monocyclic, bicyclic or tricyclic ring system, which can includ 6 fused or bridged ring systems as well as spirocyclic systems; and the nitrogen, phosphorus, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized to various oxidation states, hi a specific example, the group -S(O) 0-2 -, refers to - S- (sulfid 6 ), -S(O)- (sulfoxid 6 ), and -SO 2 - (sulfone) linkages.
• nitrogens particularly but not exclusively, those d 6 fined as annular aromatic nitrogens, are meant to included 6 their corresponding N-oxid 6 form, although not explicitly d 6 fined as such in a particular example.
• the corresponding pyridyl-N-oxid 6 is meant to be includ 6 d as another compound of the invention, hi addition, annular nitrogen atoms can be optionally quaternized.
• Heterocycle includ 6 s heteroaryl and heteroalicyclyl, that is a heterocyclyl ring can be partially or fully saturated or aromatic.
• heterocyclylalkyl included 6 s heteroalicyclylalkyls and heteroarylalkyls.
• heterocyclyl radicals include 6 , but are not limited to, azetidinyl, acridinyl, benzodioxolyl, benzodioxanyl, benzofuranyl, carbazoyl, cinnolinyl, dioxolanyl, indolizinyl, naphthyridinyl, perhydroazepinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrazoyl, tetrahydroisoquinolyl, piperidinyl, piperazinyl, 2-o
• Heteroaryl refers to an aromatic group of having from 1 to 10 annular carbon atoms and 1 to 4 annular heteroatoms within the ring. Heteroaryl groups have at least one aromatic ring component, but heteroaryls can be fully unsaturated or partially unsaturated. If any aromatic ring in the group has a heteroatom, then the group is a heteroaryl, even, for example, if other aromatic rings in the group have no heteroatoms.
• heteroaryls 2H-pyrido[3,2-b][l,4]oxazin-3(4H)-one-7-yl, indolyl and benzimidazolyl are "heteroaryls.”
• Heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl or furyl) or multiple cond 6 nsed rings (e.g., indolizinyl, quinolinyl, benzimidazolyl or benzothienyl), where the cond 6 nsed rings may or may not be aromatic and/or contain a heteroatom, provid 6 d that the point of attachment to the parent molecule is through an atom of the aromatic portion of the heteroaryl group.
• the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provid 6 for the N-oxid 6 (N ⁇ O), sulfinyl, or sulfonyl moieties.
• N ⁇ O N-oxid 6
• Compounds d 6 scribed herein containing phosphorous, in a heterocyclyl ring or not, included 6 the oxidized forms of phosphorous.
• Heteroaryl groups are monocyclic, bicyclic, , tricyclic or tetracyclic, and the like.
• Heteroaryloxy refers to -O-heteroaryl.
• Heteroarylene generically refers to any heteroaryl that has at least two groups attached thereto.
• pyridylene refers to a divalent pyridyl ring radical.
• a pyridylene thus can have more than two groups attached, but is d 6 fined by a minimum of two non-hydrogen groups attached thereto.
• Heteroalicyclic refers specifically to a non-aromatic heterocyclyl radical. A heteroalicyclic may contain unsaturation, but is not aromatic. As mentioned, aryls and heteroaryls are attached to the parent structure via an aromatic ring.
• 2H-1,4- benzoxazin-3(4H)-one-4-yl is a heteroalicyclic, while 2H-l,4-benzoxazin-3(4H)-one-7-yl is an aryl.
• 2H-pyrido[3,2-b][l,4]oxazin-3(4H)-one-4-yl is a heteroalicyclic, while 2H-pyrido[3,2-b][l,4]oxazin-3(4H)-one-7-yl is a heteroaryl.
• Heterocyclylalkyl refers to a heterocyclyl group linked to the parent structure via e.g an alkylene linker, for example (tetrahydrofuran-3-yl)methyl- or (pyridin-4- yl)methyl
• Heterocyclyloxy refers to the group -O-heterocycyl.
• Heterocyclylthio refers to the group -S-heterocycyl.
• sulfur may be oxidized to -S(O)- or -SO 2 - moieties.
• the sulfoxid 6 may exist as one or more stereoisomers.
• Niro refers to the group -NO 2 .
• Niroso refers to the group -NO.
• “Sulfonyl” refers to the group SO 2 -alkyl, SO 2 -substituted alkyl, SO 2 -alkenyl, SO 2 -substituted alkenyl, SO 2 -cycloalkyl, SO 2 -substituted cylcoalkyl, SO 2 -cycloalkenyl, SO 2 -substituted cylcoalkenyl, SO 2 -aryl, SO 2 -substituted aryl, SO 2 -heteroaryl, SO 2 - substituted heteroaryl, SO 2 -heterocyclyl, and SO 2 -substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
• “Sulfonyloxy” refers to the group -OSO 2 -alkyl, OSO 2 -substituted alkyl, OSO 2 - alkenyl, OSO 2 -substituted alkenyl, OSO 2 -cycloalkyl, OSO 2 -substituted cylcoalkyl, OSO 2 -cycloalkenyl, OSO 2 -substituted cylcoalkenyl, OSO 2 -aryl, OSO 2 -substituted aryl, OSO 2 -heteroaryl, OSO 2 -substituted heteroaryl, OSO 2 -heterocyclyl, and OSO 2 substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloal
• Thioacyl refers to the groups H-C(S)-, alkyl-C(S)-, substituted alkyl-C(S)- , alkenyl-C(S)-, substituted alkenyl-C(S)-, alkynyl-C(S)-, substituted alkynyl-C(S)-, cycloalkyl-C(S)-, substituted cycloalkyl-C(S)-, cycloalkenyl-C(S)-, substituted cycloalkenyl-C(S)-, aryl-C(S)-, substituted aryl-C(S)-, heteroaryl-C(S)-, substituted heteroaryl-C(S)-, heterocyclyl-C(S)-, and substituted heterocyclyl-C(S)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl
• Alkylthio refers to the group -S-alkyl, wherein alkyl is as d 6 fined herein.
• sulfur may be oxidized to -S(O)-.
• the sulfoxid 6 may exist as one or more stereoisomers.
• the nomenclature of substituents that are not explicitly d 6 fined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.
• substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
• substituted when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, ind 6 pend 6 ntly of one another, replaced with the same or different substituent groups as d 6 fined below.
• Each M + may ind 6 pend 6 ntly be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 ) 4 ; or an alkaline earth ion, such as [Ca 2+ ]o.5, [Mg 2+ ] 0 . 5 , or [Ba 2+ ] 0 . 5 ("subscript 0.5 means e.g.
• one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chlorid 6 , or two ionized compounds of the invention can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions).
• -NR R is meant to included 6 -NH 2 , -NH-alkyl, JV-pyrrolidinyl, JV-piperazinyl, 4/V-methyl-piperazin-l- yl and JV-morpholinyl.
• Substituent groups for hydrogens on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O " M + , -OR 70 , -SR 70 , -STVl + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 R 70 , -SO 3 TvI + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 TvI + , -OSO 3 R 70 , -PO 3 "2 (M + ) 2 , -P(O)(OR 70 )OTVI + , -P(O)(OR 70 ) 2 , -C(O)R 70 , -C(S)R 70
• Substituent groups for hydrogens on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O M + , -OR 70 , -SR 70 , -S-M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 OTVI + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 O M + , -OS(O) 2 OR 70 , -P(O)(O " ) 2 (M + ) 2 , -P(O)(OR 70 )O " M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -
• a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
• Patient refers to human and non-human animals, especially mammals.
• “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are d 6 rived from a variety of organic and inorganic counter ions well known in the art and includ 6 , by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochlorid 6 , hydrobromid 6 , tartrate, mesylate, acetate, maleate, oxalate, and the like.
• Prodrug refers to a d 6 rivative of an active 4-pyrimidineamine compound (drug) that may require a transformation und 6 r the conditions of use, such as within the body, to release the active 2,4-pyrimidinediamine drug.
• Prodrugs are frequently, but not necessarily, pharmacologically inactive until converted into the active drug.
• Prodrugs are typically obtained by masking one or more functional groups in an active 2,4- pyrimidinediamine drug believed to be in part required for activity with a progroup (d 6 fined below) to form a promoiety which und 6 rgoes a transformation, such as cleavage, und 6 r the specified conditions of use to release the functional group, and hence the active 2,4-pyrimidinediamine drug.
• the cleavage of the promoiety may proceed spontaneously, such as by way of a hydrolysis reaction, or it can be catalyzed or induced by another agent, such as an enzyme, light, an acid or base, or a change of or exposure to a physical or environmental parameter, such as temperature.
• the agent can be endogenous to the conditions of use, such as an enzyme present in the cells to which the prodrug is administered or the acidic conditions of the stomach, or it can be supplied exogenously.
• Protect group refers to a type of protecting group that, when used to mask a functional group within an active 2,4-pyrimidinediamine drug to form a promoiety, converts the drug into a prodrug.
• Progroups are typically attached to the functional group of the drug via bonds that are cleavable und 6 r specified conditions of use.
• a progroup is that portion of a promoiety that cleaves to release the functional group und 6 r the specified conditions of use.
• an amid 6 promoiety of the formula -NH-C(O)CH3 includ 6 s the progroup -C(O)CIB .
• “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disord 6 r or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disord 6 r.
• a pharmaceutically or therapeutically effective amount included 6 s an amount sufficient to, among other things, cause the tumor to shrink or d 6 crease the growth rate of the tumor.
• solvent refers to a complex formed by combination of solvent molecules with molecules or ions of the solute.
• the solvent can be an organic compound, an inorganic compound, or a mixture of both.
• solvents includ 6 but are not limited to, methanol, N,N-dimethylformamid 6 , tetrahydrofuran, dimethylsulfoxid 6 , and water.
• compounds of the invention may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and/or optical isomerism.
• the compounds and prodrugs of the invention can included 6 one or more chiral centers and/or double bonds and as a consequence can exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers, diasteromers, and mixtures thereof, such as racemic mixtures.
• the compounds of the invention can exist in several tautomeric forms, including the enol form, the keto form, and mixtures thereof.
• the presently disclosed formulas included 6 compounds having one or more different isotopic forms of certain elements, including hydrogen and carbon.
• the presently disclosed compounds are provid 6 d in isotopically enriched form.
• compounds of formula I are enriched in d 6 uterium relative to protium. Deuterium has a natural abundance of about 0.015%.
• d 6 uterium containing compounds of the disclosure have d 6 uterium at one or more positions (as the case may be) in an abundance of greater than 0.015%.
• a compound of formula (I) at a position d 6 signated as having d 6 uterium, has a minimum isotopic enrichment factor of at least 2000 (30% d 6 uterium incorporation) at each atom d 6 signated as d 6 uterium in the compound, or at least 3000 (45% d 6 uterium incorporation).
• a compound of formula (I) has an isotopic enrichment factor for each d 6 signated d 6 uterium atom of at least 3500 (52.5% d 6 uterium incorporation at each d 6 signated d 6 uterium atom), at least 4000 (60% d 6 uterium incorporation), at least 4500 (67.5% d 6 uterium incorporation), at least 5000 (75% d 6 uterium incorporation), at least 5500 (82.5% d 6 uterium incorporation), at least 6000 (90% d 6 uterium incorporation), at least 6333.3 (95% d 6 uterium incorporation), at least 6466.7 (97% d 6 uterium incorporation), at least 6600 (99% d 6 uterium incorporation), or at least 6633.3 (99.5% d 6 uterium incorporation).
• novel 2,4-pyrimidinediamine compounds are disclosed herein.
• These conditions included 6 , but are not limited to, leukemia, lymphoma, transplant rejection (e.g., pancreas islet transplant rejection, heart transplant rejection, kidney transplant rejection, liver transplant rejection, lung transplant rejection), bone marrow transplant applications (e.g., graft-versus-host disease), autoimmune diseases (e.g., diabetes), and inflammation (e.g., asthma, allergic reactions, ocular disord 6 rs).
• transplant rejection e.g., pancreas islet transplant rejection, heart transplant rejection, kidney transplant rejection, liver transplant rejection, lung transplant rejection
• bone marrow transplant applications e.g., graft-versus-host disease
• autoimmune diseases e.g., diabetes
• inflammation e.g., asthma, allergic reactions, ocular disord 6 rs.
• new therapies are need 6 d.
• the 2,4-pyrimidinediamine compounds of the invention are characterized by a bis-phenyl 2,4-pyrimidinediamine where one phenyl group bears at least a sulfonamid 6 and the other phenyl group bears at least a sulfonyl urea. More specifically, a compound of formula I:
• X is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; Y is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene;
• R 1 is hydrogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, aminoacyl, aminoacyloxy, carboxyl, carboxyl ester, carbonate ester, -C(O)N(R 4 )R 5 , nitro or halo; each R 3 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted
• R 6 is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene, linking the nitrogen bearing R 6 and the ring bearing Y
• R 7 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl
• R is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; optionally, R 7 and R 8 , together with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group;
• R 7 and R 8 are non-hydrogen
• each of R 6 , R 7 , and R 8 optionally are M + , wherein M + , a counter-ion, is K + , Na + , Li + or *N(R 9 ) 4 , wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• One embodiment is a compound of structural formula I where R 1 is hydrogen.
• X is alkyl, substituted alkyl or halo.
• Another embodiment is a compound of structural formula I where R 4 is hydrogen and R 5 is hydrogen.
• Another embodiment is a compound of structural formula I where Y is methylene.
• Another embodiment is a compound of structural formula I where R 6 is hydrogen, R 7 is C 1-3 alkyl and R 8 is C 1-3 alkyl.
• Another embodiment is a compound of structural formula I where each of R 2 and R , ind 6 pend 6 ntly, is lower alkyl or lower alkoxy.
• X is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl;
• Y is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene;
• R 1 is hydrogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, aminoacyl, aminoacyloxy, carboxyl, carboxyl ester, carbonate ester, -C(O)N(R 4 )R 5 , nitro or halo; each R ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted al
• R 7 and R 8 together, with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group
• R 10 is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene;
• each of R 7 and R 8 optionally are M + , wherein M + is K + , Na + , Li + or *N(R 9 ) 4 , wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• Another embodiment is a compound of structural formula II, where p is zero and each of R 6 and Y, ind 6 pend 6 ntly, is a straight or branched chain C 1-6 alkylene group.
• Another embodiment is a compound of structural formula II where each of R 6 and Y, ind 6 pend 6 ntly, is methylene or ethylene.
• Another embodiment is a compound of structural formula III:
• X is alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, -C(O)N(R 4 )R 5 or halo; each R 3 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy,
• R 8 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; or
• R and R together, with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group
• R 7 and R 8 are non-hydrogen
• each of R 6 , R 7 , and R 8 optionally are substituted with M + , wherein M + is K + , Na + , Li + or + N(R 9 ) 4 , wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• X is alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, -C(O)N(R 4 )R 5 or halo; each R ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substitute
• R 6 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl
• R 7 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl
• R is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; or
• R 7 and R 8 together, with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group
• R and R are non-hydrogen
• each of R 6 , R 7 , and R 8 optionally are substituted with M + , wherein M + is K + , Na + , Li + or + N(R 9 ) 4 , wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• Another embodiment is a compound selected from Tables I and II, or stereoisomer, tautomer, prodrug, solvate, or pharmaceutically acceptable salt thereof.
• the 2,4-pyrimidinediamine compounds d 6 scribed herein can included 6 functional groups that can be masked with progroups to create prodrugs. Such prodrugs are usually, but need not be, pharmacologically inactive until converted into their active drug form.
• prodrugs are usually, but need not be, pharmacologically inactive until converted into their active drug form.
• many of the 2,4-pyrimidinediamine compounds d 6 scribed in this invention included 6 promoieties that are hydrolyzable or otherwise cleavable und 6 r conditions of use.
• ester groups commonly und 6 rgo acid-catalyzed hydrolysis to yield the parent carboxylic acid when exposed to the acidic conditions of the stomach or base-catalyzed hydrolysis when exposed to the basic conditions of the intestine or blood.
• 2,4- pyrimidinediamine compounds that included 6 ester moieties can be consid 6 red prodrugs of their corresponding carboxylic acid, regardless of whether the ester form is pharmacologically active.
• the mechanism by which the progroup(s) metabolizes is not critical and can be caused, for example, by hydrolysis und 6 r the acidic conditions of the stomach, as d 6 scribed above, and/or by enzymes present in the digestive tract and/or tissues or organs of the body.
• the progroup(s) can be selected to metabolize at a particular site within the body. For example, many esters are cleaved und 6 r the acidic conditions found in the stomach.
• Prodrugs d 6 signed to cleave chemically in the stomach to the active 2,4- pyrimidinediamine can employ progroups including such esters.
• the progroups can be d 6 signed to metabolize in the presence of enzymes such as esterases, amidases, lipolases, and phosphatases, including ATPases and kinase, etc.
• Progroups including linkages capable of metabolizing in vivo are well known and includ 6 , by way of example and not limitation, ethers, thioethers, silylethers, silylthioethers, esters, thioesters, carbonates, thiocarbonates, carbamates, thiocarbamates, ureas, thioureas, and carboxamid 6 s.
• a "precursor" group that is oxidized by oxidative enzymes such as, for example, cytochrome P 450 of the liver, to a metabolizable group can be selected.
• any available functional moiety can be masked with a progroup to yield a prodrug.
• Functional groups within the 2,4-pyrimidinediamine compounds that can be masked with progroups for inclusion in a promoiety includ 6 are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), and carboxyls.
• progroups as well as the resultant promoieties, suitable for masking functional groups in active 2,4-pyrimidinediamine compounds to yield prodrugs are well-known in the art.
• a hydroxyl functional group can be masked as a sulfonate, ester, or carbonate promoiety, which can be hydrolyzed in vivo to provid 6 the hydroxyl group.
• An amino functional group can be masked as an amid 6 , carbamate, imine, urea, phosphenyl, phosphoryl, or sulfenyl promoiety, which can be hydrolyzed in vivo to provid 6 the amino group.
• a carboxyl group can be masked as an ester (including silyl esters and thioesters), amid 6 , or hydrazid 6 promoiety, which can be hydrolyzed in vivo to provid 6 the carboxyl group.
• ester including silyl esters and thioesters
• amid 6 or hydrazid 6 promoiety
• Other specific examples of suitable progroups and their respective promoieties will be apparent to those of skill in the art. All of these progroups, alone or in combinations, can be includ 6 d in the prodrugs.
• the progroup(s) can be attached to any available primary or secondary amine, including, for example, the N2 nitrogen atom of the 2,4-pyrimidinediamine, the N4 nitrogen atom of the 2,4-pyrimidinediamine, and/or a primary or secondary nitrogen atom includ 6 d in a substituent on the 2,4-pyrimidinediamine.
• the id 6 ntity of the progroup is not critical, provid 6 d that it can be metabolized und 6 r the d 6 sired conditions of use, for example, und 6 r the acidic conditions found in the stomach and/or by enzymes found in vivo, to yield a biologically active group, e.g., the 2,4-pyrimidinediamines as d 6 scribed herein.
• a biologically active group e.g., the 2,4-pyrimidinediamines as d 6 scribed herein.
• the progroup can included 6 virtually any known or later-discovered hydroxyl, amine or thiol protecting group.
• Non-limiting examples of suitable protecting groups can be found, for example, in Protective Groups in Organic Synthesis, Greene & Wuts, 2 nd Ed., John Wiley & Sons, New York, 1991 (especially pages 10-142 (alcohols, 277-308 (thiols) and 309-405 (amines) the disclosure of which is incorporated herein by reference).
• the id 6 ntity of the progroup(s) can also be selected so as to impart the prodrug with d 6 sirable characteristics.
• lipophilic groups can be used to d 6 crease water solubility and hydrophilic groups can be used to increase water solubility.
• prodrugs specifically tailored for selected mod 6 s of administration can be obtained.
• the progroup can also be d 6 signed to impart the prodrug with other properties, such as, for example, improved passive intestinal absorption, improved transport-mediated intestinal absorption, protection against fast metabolism (slow-release prodrugs), tissue-selective d 6 livery, passive enrichment in target tissues, and targeting-specif ⁇ c transporters.
• a particularly useful progroup of the invention is -CH 2 OP(OH) 2 as well as esters, mixed acid esters and salts thereof.
• the -CH 2 OP(OH) 2 progroup is attached via an NH, annular or not, of the parent molecule. There can be more than one such progroup.
• one embodiment is a compound of formula V,
• R 40 is H or R 50 , and at least one of R 1 , R 4 , R 5 , R 6 , R 7 , R 8 and R 40 is R 50 ; where R 50 is -CH 2 OP(O)(OR 11 ) 2 ; each R 1 1 i s ind 6 pend 6 ntly H, C 1-6 alkyl or a monovalent cationic group, or two R 11 , together with the atoms to which they are attached, form a 4-8 membered cyclic phosphate group
• each R 55 is ind 6 pend 6 ntly H, optionally substituted C 1-6 alkyl, optionally substituted 3-8 membered heteroalicyclyl, optionally substituted C 6-14 aryl, optionally substituted C 7- 2O arylalkyl, optionally substituted 5-14 membered heteroaryl or optionally substituted 6- 15 membered heteroarylalkyl;
• z is 0, 1, 2 or 3; or two R 11 together represent a divalent cationic group selected from Ba 2+ , Bi 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Ni 2+ , Sr ⁇ and Zn 2+ .
• the cyclic phosphate group is a 5 or 6-membered cyclic phosphate group, where -CH 2 OP(O)(OR 11 ) 2 is
• R 11 represents a divalent cationic group selected from Ca 2+ , Mg 2+ and Zn 2+ .
• Another embodiment is a compound of formula VI,
• R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , X, p and q are as d 6 fined in the summary and d 6 tailed d 6 scription, and R 11 is d 6 fined as above.
• progroups -CH 2 OP(O)(OR 1 ⁇ 2 e.g according to formula VI, metabolize to active compounds of the invention via the corresponding hydroxymethylamine intermediate illustrated below:
• hydroxymethylamine compounds although isolable, are known to be unstable und 6 r physiological conditions and various pH ranges where they hydrolyze in vivo to yield formald 6 hyd 6 and the active drug substance.
• compounds of the invention included 6 hydroxymethyl progroups that can be metabolized in vivo, for example by the acidic conditions of the stomach and/or by enzymes present in the digestive tract or other organs and/or tissues or fluids with the body, to yield the active drug substance 2,4-pyrimidinediamine.
• the amino and thio analogs of these hydroxymethylamines will be similarly unstable at physiological conditions and also hydrolyze in vivo to the active 2,4-pyrimdiendiamine drug.
• compounds of the invention included 6 these corresponding primary amino and thiol compounds.
• the invention includ 6 s compounds in which the primary amine, thiol and hydroxy groups are masked with "protecting" groups that are removed und 6 r physiological conditions of use to yield the corresponding hydroxymethyl, thiolmethyl and aminomethyl compounds, that is, with these "protecting groups” these compounds will likewise make suitable prodrugs.
• any particular progroup for a d 6 sired mod 6 of administration can be confirmed in biochemical assays.
• a prodrug is to be administered by injection into a particular tissue or organ and the id 6 ntities of the various enzyme(s) expressed in the tissue or organ are known
• the particular prodrug can be tested for metabolism in biochemical assays with the isolated enzyme(s).
• the particular prodrug can be tested for metabolism to the active 2,4-pyrimidinediamine compound with tissue and/or organ extracts.
• tissue and/or organ extracts can be of particular convenience when the id 6 ntity(ies) of the enzymes expressed in the target tissues or organs are unknown or in instances when the isolated enzymes are not conveniently available. Skilled artisans will be able to readily select progroups having metabolic properties (such as kinetics) suitable for particular applications using such in vitro tests.
• Specific prodrugs could also be tested for suitable metabolism in vitro animal mod 6 ls.
• the proton on the 4-NH of the pyrimidinediamine system can be selectively alkylated over the 2-NH with the appropriate phosphonate reagent and reaction conditions, where LG is a suitable leaving group to form compounds of the invention, in this case of formula VI.
• LG is a suitable leaving group to form compounds of the invention, in this case of formula VI.
• compositions Another embodiment is a pharmaceutical composition including a compound as d 6 scribed in any of the embodiments above.
• Pharmaceutical compositions d 6 scribed herein can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes.
• the compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• the 2,4-pyrimidinediamine compound can be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxid 6 , or pharmaceutically acceptable salt, as d 6 scribed herein.
• such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases can also be formed.
• One embodiment is a pharmaceutical formulation including a compound of formula I, as d 6 scribed herein, or a prodrug thereof, and at least one pharmaceutically acceptable excipient, diluent, preservative, stabilizer, or mixture thereof.
• the compounds can be provid 6 d in a variety of formulations and dosages.
• the compounds can be provid 6 d in a pharmaceutically acceptable form, including where the compound can be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxid 6 , or pharmaceutically acceptable salt, as d 6 scribed herein.
• such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases can also be formed.
• the compounds are provid 6 d as non-toxic pharmaceutically acceptable salts, as noted previously.
• Suitable pharmaceutically acceptable salts of the compounds d 6 scribed herein included 6 acid addition salts such as those formed with hydrochloric acid, fumaric acid, p-toluenesulphonic acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid.
• Salts of amine groups can also included 6 quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or substituted alkyl moiety.
• suitable pharmaceutically acceptable salts thereof can included 6 metal salts such as alkali metal salts, e.g., sodium or potassium salts; and alkaline earth metal salts, e.g., calcium or magnesium salts.
• 6 metal salts such as alkali metal salts, e.g., sodium or potassium salts; and alkaline earth metal salts, e.g., calcium or magnesium salts.
• the pharmaceutical compositions for the administration of the 2,4- pyrimidinediamine compounds can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
• the pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, a finely divid 6 d solid carrier or both, and then, if necessary, shaping the product into the d 6 sired formulation.
• the active object compound is includ 6 d in an amount sufficient to produce the d 6 sired therapeutic effect.
• the 2,4-pyrimidinediamine compounds can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
• parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
• the compounds d 6 scribed herein can be effective in humans.
• Administration of the compounds d 6 scribed herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted mod 6 s of administration or agents for serving similar utilities.
• administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transd 6 rmally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powd 6 r, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powd 6 rs, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
• the present invention provid 6 s 2,4-pyrimidinediamine compounds and prodrugs thereof, as d 6 scribed herein, for use in therapy for the conditions d 6 scribed herein.
• the present invention further provid 6 s use of the compounds of the present invention in the manufacture of a medicament for the treatment of conditions in which targeting of the JAK pathway or inhibition of JAK kinases, particularly JAK3, are therapeutically useful.
• the methods included 6 conditions where the function of lymphocytes, macrophages, or mast cells is involved.
• Conditions in which targeting of the JAK pathway or inhibition of the JAK kinases, particularly JAK3, are therapeutically useful included 6 leukemia, lymphoma, transplant rejection (e.g., pancreas islet transplant rejection), bone marrow transplant applications (e.g., graft-versus-host disease)), autoimmune diseases (e.g., rheumatoid arthritis, etc.), inflammation (e.g., asthma, etc.) and other conditions as d 6 scribed in greater d 6 tail herein.
• Treating" or “treatment” of a disease in a patient refers to (1) preventing the disease from occurring in a patient that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its d 6 velopment; or (3) ameliorating or causing regression of the disease.
• treatment is an approach for obtaining beneficial or d 6 sired results, including clinical results.
• beneficial or d 6 sired results can included 6 one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition, including a disease, stabilized (i.e., not worsening) state of a condition, including diseases, preventing spread of disease, d 6 lay or slowing of condition, including disease, progression, amelioration or palliation of the condition, including disease, state, and remission (whether partial or total), whether d 6 tectable or und 6 tectable.
• the compounds d 6 scribed herein are potent and selective inhibitors of JAK kinases and are particularly selective for cytokine signaling pathways containing JAK3. As a consequence of this activity, the compounds can be used in a variety of in vitro, in vivo, and ex vivo contexts to regulate or inhibit JAK kinase activity, signaling cascad 6 s in which JAK kinases play a role, and the biological responses effected by such signaling cascad 6 s.
• the compounds can be used to inhibit JAK kinase, either in vitro or in vivo, in virtually any cell type expressing the JAK kinase, such as in hematopoietic cells in which, for example, JAK3 is predominantly expressed. They may also be used to regulate signal transduction cascad 6 s in which JAK kinases, particularly JAK3, play a role.
• Such JAK-d 6 pend 6 nt signal transduction cascad 6 s included 6 , but are not limited to, the signaling cascad 6 s of cytokine receptors that involve the common gamma chain, such as, for example, the IL-4, IL-7, IL-5, IL-9, IL- 15 and IL- 21, or IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptor signaling cascad 6 s.
• the compounds may also be used in vitro or in vivo to regulate, and in particular to inhibit, cellular or biological responses affected by such JAK-d 6 pend 6 nt signal transduction cascad 6 s.
• Such cellular or biological responses include 6 , but are not limited to, IL-4/ramos CD23 upregulation and IL-2 mediated T-cell proliferation.
• the compounds can be used to inhibit JAK kinases in vivo as a therapeutic approach towards the treatment or prevention of diseases mediated, either wholly or in part, by a JAK kinase activity (referred to herein as "JAK kinase mediated diseases").
• Non-limiting examples of JAK kinase mediated diseases that can be treated or prevented with the compounds includ 6 , but are not limited to, the following: allergies; asthma; autoimmune diseases such as transplant rejection (e.g., kidney, heart, lung, liver, pancreas, skin; host versus graft reaction (HVGR), and graft versus host reaction (GVHR)), rheumatoid arthritis, and amyotrophic lateral sclerosis; T-cell mediated autoimmune diseases such as multiple sclerosis, psoraiasis, and Sjogren's syndrome; Type II inflammatory diseases such as vascular inflammation (including vasculitis, arteritis, atherosclerosis, and coronary artery disease); diseases of the central nervous system such as stroke; pulmonary diseases such as bronchitis obliteraus and primary pulmonary hypertension; solid, d 6 layed Type IV hypersensitivity reactions; and hematologic malignancies such as leukemia and lymphomas.
• autoimmune diseases such as
• One embodiment is a method of inhibiting an activity of a JAK kinase, including contacting the JAK kinase with an amount of a compound, effective to inhibit an activity of the JAK kinase, of formula I:
• X is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, -C(O)N(R 4 )R 5 , cyano, halo, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkynyl or substituted cycloalkynyl; Y is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene;
• R 1 is hydrogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R 2 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, aminoacyl, aminoacyloxy, carboxyl, carboxyl ester, carbonate ester, -C(O)N(R 4 )R 5 , nitro or halo; each R 3 ind 6 pend 6 ntly is alkyl, substituted alkyl, alkoxy, substituted
• each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen immediately adjacent to R 5 is anionic; or R 4 and R 5 together with the intervening atom or atoms bound thereto, form a heterocyclyl or substituted heterocyclyl group; each R 5 ind 6 pend 6 ntly is hydrogen, alkyl, substituted alkyl, amino, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, carboxyl, carboxyl ester or acyl; R 6 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; or
• R 6 is a straight or branched chain C 1-6 alkylene group, cycloalkylene or substituted cycloalkylene, linking the nitrogen bearing R 6 and the ring bearing Y R 7 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl;
• R 8 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl or acyl; or
• R 7 and R 8 together, with nitrogen to which they are bound, form a heterocyclyl or substituted heterocyclyl group
• R 7 and R 8 are non-hydrogen
• each of R 6 , R 7 , and R 8 optionally are M + , wherein M + is K + , Na + , Li + or + N(R 9 )4, wherein each R 9 ind 6 pend 6 ntly is hydrogen or alkyl, and the nitrogen bearing M + is anionic.
• this invention provid 6 s a method of inhibiting an activity of a JAK kinase, including contacting the JAK kinase with an amount of a compound effective to inhibit an activity of the JAK kinase, where the compound is according to formula I, as d 6 scribed herein. In certain embodiments of the methods d 6 scribed herein, the method is carried out in vivo.
• this invention provid 6 s a method of inhibiting an activity of a JAK kinase, including contacting in vitro a JAK3 kinase with an amount of a compound effective to inhibit an activity of the JAK kinase, where the compound is according to formula I, as d 6 scribed herein.
• the compounds can be used to treat and/or prevent rejection in organ and/or tissue transplant recipients (i.e., treat and/or prevent allograft rejection). Allografts can be rejected through either a cell-mediated or humoral immune reaction of the recipient against transplant (histocompability) antigens present on the membranes of the donor's cells. The strongest antigens are governed by a complex of genetic loci termed human leukocyte group A (HLA) antigens. Together with the ABO blood groups antigens, they are the chief transplantation antigens d 6 tectable in humans.
• HLA human leukocyte group A
• Rejection following transplantation can generally be broken into three categories: hyperacute, occurring hours to days following transplantation; acute, occurring days to months following transplantation; and chronic, occurring months to years following transplantation.
• Hyperacute rejection is caused mainly by the production of host antibodies that attack the graft tissue.
• antibodies are observed in the transplant vascular very soon after transplantation. Shortly thereafter, vascular clotting occurs, leading to ischemia, eventual necrosis and d 6 ath.
• the graft infarction is unresponsive to known immunosuppressive therapies.
• pre-transplant screening is used to significantly reduce hyperacute rejection. As a consequence of this screening, hyperacute rejection is relatively uncommon today.
• Acute rejection is thought to be mediated by the accumulation of antigen specific cells in the graft tissue.
• the T-cell-mediated immune reaction against these antigens i.e., HVGR or GVHR
• HVGR or GVHR The T-cell-mediated immune reaction against these antigens
• HVGR or GVHR the principle mechanism of acute rejection. Accumulation of these cells leads to damage of the graft tissue.
• both CD4+ helper T-cells and CD8+ cytotoxic T-cells are involved in the process and that the antigen is presented by donor and host d 6 ndritic cells.
• the CD4+ helper T-cells help recruit other effector cells, such as macrophapges and eosinophils, to the graft.
• T-cell activation signal transduction cascad 6 s for example, CD28, CD40L, and CD2 cascad 6 s
• the cell-mediated acute rejection can be reversed in many cases by intensifying immunotherapy. After successful reversal, severely damaged elements of the graft heal by fibrosis and the remaind 6 r of the graft appears normal. After resolution of acute rejection, dosages of immunosuppressive drugs can be reduced to very low levels.
• Chronic rejection which is a particular problem in renal transplants, often progresses insidiously d 6 spite increased immunosuppressive therapy. It is thought to be due, in large part, to cell-mediated Type IV hypersensitivity.
• the pathologic profile differs from that of acute rejection.
• the arterial endothelium is primarily involved with extensive proliferation that may gradually occlud 6 the vessel lumen, leading to ischemia, fibrosis, a thickened intima, and atherosclerotic changes.
• Chronic rejection is mainly due to a progressive obliteration of graft vasculature and resembles a slow, vasculitic process.
• CD8 cytotoxic T-cells and CD4 helper T cells recognize either intracellular or extracellular synthesized antigen when it is complexed, respectively, with either Class I or Class II MHC molecules. Macrophages function as antigen-presenting cells and release IL-I, which promotes proliferation of helper T-cells. Helper T-cells release interferon gamma and IL-2, which together regulate d 6 layed hyperactivity reactions mediated by macrophage activation and immunity mediated by T cells. In the case of organ transplant, the cytotoxic T-cells d 6 stroy the graft cells on contact.
• the 2,4- pyrimidinediamine compounds d 6 scribed herein can be used to treat and/or prevent many aspects of transplant rejection, and are particularly useful in the treatment and/or prevention of rejection reactions that are mediated, at least in part, by T-cells, such as HVGR or GVHR.
• the 2,4-pyrimidinediamine compounds can also be used to treat and/or prevent chronic rejection in transplant recipients and, in particular, in renal transplant recipients.
• the compound can also be administered to a tissue or an organ prior to transplanting the tissue or organ in the transplant recipient.
• this invention provid 6 s a method of treating a T-cell mediated autoimmune disease, including administering to a patient suffering from such an autoimmune disease an amount of a compound effective to treat the autoimmune disease where the compound is according to formula I, as d 6 scribed herein.
• the autoimmune disease is multiple sclerosis (MS), psoraisis, or Sjogran's syndrome.
• Treatment using the 2,4-pyrimidinediamine compounds d 6 scribed herein can be applied alone, or it can be applied in combination with or adjunctive to other common immunosuppressive therapies, such as, for example, the following: mercaptopurine; corticosteroids such as prednisone; methylprednisolone and prednisolone; alkylating agents such as cyclophosphamid 6 ; calcineurin inhibitors such as cyclosporine, sirolimus, and tacrolimus; inhibitors of inosine monophosphate d 6 hydrogenase (IMPDH) such as mycophenolate, mycophenolate mofetil, and azathioprine; and agents d 6 signed to suppress cellular immunity while leaving the recipient's humoral immunologic response intact, including various antibodies (for example, antilymphocyte globulin (ALG), antithymocyte globulin (ATG), monoclonal anti-T-cell antibodies (OKT3)) and irradiation
• Azathioprine is currently available from Salix Pharmaceuticals, Inc., und 6 r the brand name AZASAN; mercaptopurine is currently available from Gate Pharmaceuticals, Inc., und 6 r the brand name PURINETHOL; prednisone and prednisolone are currently available from Roxane Laboratories, Inc.; Methyl prednisolone is currently available from Pfizer; sirolimus (rapamycin) is currently available from Wyeth-Ayerst und 6 r the brand name RAPAMUNE; tacrolimus is currently available from Fujisawa und 6 r the brand name PROGRAF; cyclosporine is current available from Novartis und 6 r the brand name SANDIMMUNE and from Abbott und 6 r the brand name GENGRAF; IMPDH inhibitors such as mycophenolate mofetil and mycophenolic acid are currently available from Roche und 6 r the brand name CELLCEPT and from Novartis und 6 r the brand name MYFORTIC; azathioprin
• the 2,4-pyrimidinediamine compounds could be administered either in combination or adjunctively with an inhibitor of a Syk kinase.
• Syk kinase is a tyrosine kinase known to play a critical role in Fc ⁇ receptor signaling, as well as in other signaling cascad 6 s, such as those involving B-CeIl receptor signaling (Turner et al., (2000), Immunology Today 21:148-154) and integrins beta(l), beta (2), and beta (3) in neutrophils (Mocsavi et al., (2002), Immunity 16:547-558).
• Syk kinase plays a pivotal role in high affinity IgE receptor signaling in mast cells that leads to activation and subsequent release of multiple chemical mediators that trigger allergic attacks.
• JAK kinases which help regulate the pathways involved in d 6 layed or cell-mediated Type IV hypersensitivity reactions
• Syk kinase helps regulate the pathways involved in immediate IgE-mediated, Type I hypersensitivity reactions. Certain compounds that affect the Syk pathway may or may not also affect the JAK pathways.
• Suitable Syk inhibitory compounds are d 6 scribed, for example, in Serial No. 10/355,543 filed January 31, 2003 (publication no. 2004/0029902); WO 03/063794; Serial No. 10/631,029 filed July 29, 2003 (publication no.2007/0060603); WO 2004/014382; Serial No. 10/903,263 filed July 30, 2004 (publication no.2005/0234049); PCT/US2004/24716 filed July 30, 2004 (WO005/016893); Serial No. 10/903,870 filed July 30, 2004 (publication no. 2005/0209224); PCT/US2004/24920 filed July 30, 2004; Serial No. 60/630,808 filed November 24, 2004; Serial No.
• the 2,4-pyrimidinediamine compounds can be used to treat or prevent these diseases in patients that are either initially non-responsive (resistant) to or that become non-responsive to treatment with a Syk inhibitory compound or one of the other current treatments for the particular disease.
• the 2,4-pyrimidinediamine compounds could also be used in combination with Syk inhibitory compounds in patients that are Syk-compound resistant or non-responsive. Suitable Syk-inhibitory compounds with which the 2,4-pyrimidinediamine compounds can be administered are provid 6 d supra.
• this invention provid 6 s a method of treating a T-cell mediated autoimmune disease, including administering to a patient suffering from such an autoimmune disease an amount of a compound according to formula I, in combination with or adjunctively to a compound that inhibits Syk kinase with an IC 50 of at least 10 ⁇ M, effective to treat the autoimmune disease.
• this invention provides a method of treating or preventing allograft transplant rejection, either acute or chronic, in a transplant recipient, including administering to the transplant recipient an amount of a compound according to formula I effective to treat or prevent the rejection.
• the compound is administered to a tissue or an organ prior to or concurrent with, transplanting the tissue or organ in the transplant recipient, hi another embodiment, the compound is administered to the tissue or organ and the patient.
• the allograft transplant rejection is mediated by HVGR or GVHR.
• the allograft transplant organ is a kidney, a heart, a liver, or a lung.
• the compound in another embodiment, in which the allograft transplant organ is a kidney, a heart, a liver, or a lung, the compound is administered in combination with or adjunctively to another immunosuppressant.
• the immunosuppressant is cyclosporine, tacrolimus, sirolimus, an inhibitor of IMPDH, mycophenolate, mycophanolate mofetil, an anti -T-CeIl antibody or OKT3.
• the 2,4-pyrimidinediamine compounds d 6 scribed herein are cytokine mod 6 rators of IL-4 signaling. As a consequence, the 2,4-pyrimidinediamine compounds could slow the response of Type I hypersensitivity reactions. Thus, in a specific embodiment, the 2,4-pyrimidinediamine compounds could be used to treat such reactions and, therefore, the diseases associated with, mediated by, or caused by such hypersensitivity reactions (for example, allergies), prophylactically. For example, an allergy sufferer could take one or more of the JAK selective compounds d 6 scribed herein prior to expected exposure to allergens to d 6 lay the onset or progress of, or eliminate altogether, an allergic response.
• the 2,4-pyrimidinediamine compounds can be administered singly, as mixtures of one or more 2,4- pyrimidinediamine compounds, or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases.
• the 2,4- pyrimidinediamine compounds can also be administered in mixture or in combination with agents useful to treat other disord 6 rs or maladies, such as steroids, membrane stabilizers, 5 -lipoxygenase (5LO) inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, ⁇ -agonists, tryptase inhibitors, aspirin, cyclooxygenase (COX) inhibitors, methotrexate, anti-TNF drugs, rituximab, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few.
• the 2,4-pyrimidinediamine compounds can be administered per se in the form of prodrugs or as pharmaceutical compositions, including an active compound.
• this invention provid 6 s a method of treating or preventing a Type IV hypersensitivity reaction, including administering to a subject an amount of a compound effective to treat or prevent the hypersensitivity reaction, where the compound is according to formula I, as d 6 scribed herein.
• the method is practiced prophylactically.
• the compound is administered prior to exposure to an allergen.
• this invention provid 6 s a method of inhibiting a signal transduction cascad 6 in which JAK3 kinase plays a role, including contacting a cell expressing a receptor involved in such a signaling cascad 6 with a compound, where the compound is according to formula I, as d 6 scribed herein.
• this invention provid 6 s a method of treating or preventing a JAK kinase-mediated disease, including administering to a subject an amount of compound effective to treat or prevent the JAK kinase-mediated disease, where the compound is according to formula I, as d 6 scribed herein.
• this invention provid 6 s a method of treating or preventing a JAK kinase-mediated disease, in which the JAK-mediated disease is HVGR or GVHR, including administering to a subject an amount of compound effective to treat or prevent the JAK kinase-mediated disease, where the compound is according to formula I, as d 6 scribed herein.
• ocular disord 6 rs are treated using an effective amount of a compound of formula I, as d 6 scribed herein.
• administration of one or more of the presently disclosed 2,4- pyrimidinediamine compounds is effective to increase tear production volume as compared to untreated tear production volume, thereby ameliorating a symptom of dry eye syndrome.
• tear production volume is increased within five days, such as in less than four days, and in some examples in less than two days.
• tear production volume is increased by at least about 25% over initial tear production within two days of initial treatment with a presently disclosed 2,4- pyrimidinediamine compound, In other embodiments, tear production is increased at least about 30%, such as at least about 50% over initial tear production within less than two days. Increases in tear production upon administration of the present compounds results, in some instances, in tear production volume comparable to normal tear production.
• the disclosed compounds when used for treating ocular disord 6 rs topically, are administered at least once daily and typically at most twice a day.
• another embodiment provid 6 s a method of treating a disease and/or disord 6 r of the eye, which includ 6 s administering to a subject an amount of a compound effective to treat the disease and/or disord 6 r of the eye wherein the compound is according to formula I, as d 6 scribed herein.
• Diseases and disord 6 rs of the eye included 6 , but are not limited to, dry eye syndrome, uveitis, allergic conjunctivitus, glaucoma and rosacea (of the eye).
• Dry eye syndrome otherwise known as keratoconjunctivitis sicca (KCS), keratitis sicca, sicca syndrome, or xerophthalmia
• KCS keratoconjunctivitis sicca
• keratitis sicca keratitis sicca
• sicca syndrome or xerophthalmia
• Uveitis or iridocyclitis refers to inflammation of the middle layer of the eye (the "uvea") and in common usage may refer to any inflammatory process involving the interior of the eye.
• Allergic conjunctivitis is inflammation of the conjunctiva (the membrane covering the white part of the eye) due to allergy.
• Glaucoma refers to a group of diseases that affect the optic nerve and involves a loss of retinal ganglion cells in a characteristic pattern, i.e., a type of optic neuropathy.
• Raised intraocular pressure is a significant risk factor for d 6 veloping glaucoma (above 22 mmHg or 2.9 kPa), and inflammatory processes, e.g uveitis, can cause this rise in intraocular pressure.
• Rosacea is a chronic inflammatory condition characterized by facial erythema but it can affect the eyes.
• compounds d 6 scribed herein may be used to treat inflammatory responses.
• compounds d 6 scribed herein are effective treatments of these eye disord 6 rs due, at least in part, to their JAK inhibitory activity.
• Active compounds d 6 scribed herein typically inhibit the JAK/Stat pathway.
• the activity of a specified compound as an inhibitor of a JAK kinase can be assessed in vitro or in vivo. In some embodiments, the activity of a specified compound can be tested in a cellular assay. Suitable assays included 6 assays that d 6 termine inhibition of either the phosphorylation activity or ATPase activity of a JAK kinase.
• a compound is said to inhibit an activity of a JAK kinase if it inhibits the phosphorylation or ATPase activity of a JAK kinase with an IC 50 of about 20 ⁇ M or less.
• Cell proliferative disord 6 r refers to a disord 6 r characterized by abnormal proliferation of cells.
• a proliferative disord 6 r does not imply any limitation with respect to the rate of cell growth, but merely indicates loss of normal controls that affect growth and cell division. Thus, in some embodiments, cells of a proliferative disord 6 r can have the same cell division rates as normal cells but do not respond to signals that limit such growth.
• neoplasm or tumor which is an abnormal growth of tissue. Cancer refers to any of various malignant neoplasms characterized by the proliferation of cells that have the capability to invad 6 surrounding tissue and/or metastasize to new colonization sites.
• Hematopoietic neoplasm refers to a cell proliferative disord 6 r arising from cells of the hematopoietic lineage.
• hematopoiesis is the physiological process whereby undifferentiated cells or stem cells d 6 velop into various cells found in the peripheral blood, hi the initial phase of d 6 velopment, hematopoietic stem cells, typically found in the bone marrow, und 6 rgo a series of cell divisions to form multipotent progenitor cells that commit to two main d 6 velopmental pathways: the lymphoid lineage and the myeloid lineage.
• the committed progenitor cells of the myeloid lineage differentiate into three major sub-branches including the erythroid, megakaryocyte, and granulocyte/monocyte d 6 velopmental pathways.
• An additional pathway leads to formation of d 6 ndritic cells, which are involved in antigen presentation.
• the erythroid lineage gives rise to red blood cells while the megakaryocytic lineage gives rise to blood platelets.
• Committed cells of the granulocyte/monocyte lineage split into granulocyte or monocyte d 6 velopmental pathways, the former pathway leading to formation of neutrophils, eosinophils, and basophils and the latter pathway giving rise to blood monocytes and macrophages.
• the B cell progenitor cell d 6 velops into a precursor B cell (pre-B), which differentiates into B cells responsible for producing immunoglobulins.
• Progenitor cells of the T cell lineage differentiate into precursor T cells (pre-T) that, based on the influence of certain cytokines, d 6 velop into cytotoxic or helper/suppressor T cells involved in cell mediated immunity.
• Non-T/B cell pathway leads to generation of natural killer (NK) cells.
• Neoplasms of hematopoietic cells can involve cells of any phase of hematopoiesis, including hematopoietic stem cells, multipotent progenitor cells, oligopotent committed progenitor cells, precursor cells, and mature differentiated cells.
• the categories of hematopoietic neoplasms can generally follow the d 6 scriptions and diagnostic criteria employed by those of skill in the art (see, e.g., International Classification of Disease and Related Health Problems (ICD 10), World Health Organization (2003)).
• Hematopoietic neoplasms can also be characterized based on the molecular features, such as cell surface markers and gene expression profiles, cell phenotype exhibited by the aberrant cells, and/or chromosomal aberrations (e.g., d 6 letions, translocations, insertions, etc.) characteristic of certain hematopoietic neoplasms, such as the Philad 6 lphia chromosome found in chronic myelogenous leukemia.
• Other classifications included 6 National Cancer Institute Working Formulation (Cancer, 1982, 49:2112-2135) and Revised European- American Lymphoma Classification (REAL).
• Lymphoid neoplasm refers a proliferative disord 6 r involving cells of the lymphoid lineage of hematopoiesis. Lymphoid neoplasms can arise from hematopoietic stem cells as well as lymphoid committed progenitor cells, precursor cells, and terminally differentiated cells. These neoplasms can be subdivid 6 d based on the phenotypic attributes of the aberrant cells or the differentiated state from which the abnormal cells arise. Subdivisions includ 6 , among others, B cell neoplasms, T cell neoplasms, NK cell neoplasms, and Hodgkin's lymphoma.
• Myeloid neoplasm refers to proliferative disord 6 r of cells of the myeloid lineage of hematopoiesis. Neoplasms can arise from hematopoietic stem cells, myeloid committed progenitor cells, precursor cells, and terminally differentiated cells. Myeloid neoplasms can be subdivid 6 d based on the phenotypic attributes of the aberrant cells or the differentiated state from which the abnormal cells arise. Subdivisions included 6 , among others, myeloproliferative diseases, myelodysplastic/myeloproliferative diseases, myelodysplastic syndromes, acute myeloid leukemia, and acute biphenotypic leukemia.
• cell proliferative disord 6 rs treatable with the compounds disclosed herein relate to any disord 6 r characterized by aberrant cell proliferation. These included 6 various tumors and cancers, benign or malignant, metastatic or non-metastatic. Specific properties of cancers, such as tissue invasiveness or metastasis, can be targeted using the methods d 6 scribed herein.
• Cell proliferative disord 6 rs included 6 a variety of cancers, including, among others, breast cancer, ovarian cancer, renal cancer, gastrointestinal cancer, kidney cancer, bladd 6 r cancer, pancreatic cancer, lung squamous carcinoma, and ad 6 nocarcinoma.
• Cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;
• Lung bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, ad 6 nocarcinoma), alveolar (bronchiolar) carcinoma, bronchial ad 6 noma, sarcoma, lymphoma, chondromatous hanlartoma, mesothelioma;
• Gastrointestinal esophagus (squamous cell carcinoma, ad 6 nocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal ad
• the cell proliferative disord 6 r treated is a hematopoietic neoplasm, which is aberrant growth of cells of the hematopoietic system.
• Hematopoietic malignancies can have its origins in pluripotent stem cells, multipotent progenitor cells, oligopotent committed progenitor cells, precursor cells, and terminally differentiated cells involved in hematopoiesis. Some hematological malignancies are believed to arise from hematopoietic stem cells, which have the ability for self renewal.
• cells capable of d 6 veloping specific subtypes of acute myeloid leukemia (AML) upon transplantation display the cell surface markers of hematopoietic stem cells, implicating hematopoietic stem cells as the source of leukemic cells.
• Blast cells that do not have a cell marker characteristic of hematopoietic stem cells appear to be incapable of establishing tumors upon transplantation (Blaire et al., 1997, Blood 89:3104-3112).
• the stem cell origin of certain hematological malignancies also finds support in the observation that specific chromosomal abnormalities associated with particular types of leukemia can be found in normal cells of hematopoietic lineage as well as leukemic blast cells.
• the reciprocal translocation t(9q34;22ql 1) associated with approximately 95% of chronic myelogenous leukemia appears to be present in cells of the myeloid, erythroid, and lymphoid lineage, suggesting that the chromosomal aberration originates in hematopoietic stem cells.
• a subgroup of cells in certain types of CML displays the cell marker phenotype of hematopoietic stem cells.
• hematopoietic neoplasms often originate from stem cells, committed progenitor cells or more terminally differentiated cells of a d 6 velopmental lineage can also be the source of some leukemias.
• forced expression of the fusion protein Bcr/Abl associated with chronic myelogenous leukemia
• common myeloid progenitor or granulocyte/macrophage progenitor cells produces a leukemic-like condition.
• chromosomal aberrations associated with subtypes of leukemia are not found in the cell population with a marker phenotype of hematopoietic stem cells, but are found in a cell population displaying markers of a more differentiated state of the hematopoietic pathway (Turhan et al., 1995, Blood 85:2154-2161).
• committed progenitor cells and other differentiated cells may have only a limited potential for cell division
• leukemic cells may have acquired the ability to grow unregulated, in some instances mimicking the self-renewal characteristics of hematopoietic stem cells (Passegue et al., Proc. Natl. Acad. Sci. USA, 2003, 100:11842- 9).
• the hematopoietic neoplasm treated is a lymphoid neoplasm, where the abnormal cells are d 6 rived from and/or display the characteristic phenotype of cells of the lymphoid lineage.
• Lymphoid neoplasms can be subdivid 6 d into B-cell neoplasms, T and NK -cell neoplasms, and Hodgkin's lymphoma.
• B-cell neoplasms can be further subdivid 6 d into precursor B-cell neoplasm and mature/peripheral B-cell neoplasm.
• Exemplary B-cell neoplasms are precursor B- lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) while exemplary mature/peripheral B-cell neoplasms are B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, primary effusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia.
• T-cell and Nk-cell neoplasms are further subdivid 6 d into precursor T-cell neoplasm and mature (peripheral) T-cell neoplasms.
• Exemplary precursor T-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblastic leukemia) while exemplary mature (peripheral) T-cell neoplasms are T-cell prolymphocyte leukemia T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T- cell lymphoma/leukemia (HTLV-I), extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic gamma-d 6 lta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, Mycosis fungoid 6 s/
• the third member of lymphoid neoplasms is Hodgkin's lymphoma, also referred to as Hodgkin's disease.
• Hodgkin's lymphoma also referred to as Hodgkin's disease.
• Exemplary diagnosis of this class that can be treated with the compounds includ 6 among others, nodular lymphocyte-predominant Hodgkin's lymphoma, and various classical forms of Hodgkin's disease, exemplary members of which are Nodular sclerosis Hodgkin's lymphoma (grad 6 s 1 and 2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixed cellularity Hodgkin's lymphoma, and Lymphocyte d 6 pletion Hodgkin's lymphoma.
• any of the lymphoid neoplasms that are associated with aberrant JAK activity can be treated with the JAK inhibitory compounds.
• the hematopoietic neoplasm treated is a myeloid neoplasm.
• This group includ 6 s a large class of cell proliferative disord 6 rs involving or displaying the characteristic phenotype of the cells of the myeloid lineage.
• Myeloid neoplasms can be subdivid 6 d into myeloproliferative diseases, myelodysplastic/myeloproliferative diseases, myelodysplastic syndromes, and acute myeloid leukemias.
• Exemplary myeloproliferative diseases are chronic myelogenous leukemia (e.g., Philad 6 lphia chromosome positive (t(9;22)(qq34;ql l)), chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic idiopathic myelofibrosis, polycythemia vera, and essential thrombocythemia.
• Exemplary myelodysplastic/myeloproliferative diseases are chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, and juvenile myelomonocytic leukemia.
• Exemplary myelodysplastic syndromes are refractory anemia, with ringed sid 6 roblasts and without ringed sid 6 roblasts, refractory cytopenia (myelodysplastic syndrome) with multilineage dysplasia, refractory anemia (myelodysplastic syndrome) with excess blasts, 5q- syndrome, and myelodysplastic syndrome.
• any of the myeloid neoplasms that are associated with aberrant JAK activity can be treated with the JAK inhibitory compounds.
• the JAK inhibitory compounds can be used to treat Acute myeloid leukemias (AML), which represent a large class of myeloid neoplasms having its own subdivision of disord 6 rs. These subdivisions included 6 , among others, AMLs with recurrent cytogenetic translocations, AML with multilineage dysplasia, and other AML not otherwise categorized.
• AML Acute myeloid leukemias
• Exemplary AMLs with recurrent cytogenetic translocations include 6 , among others, AML with t(8;21)(q22;q22), AMLl(CBF-alpha)/ETO, Acute promyelocytic leukemia (AML with t(15;17)(q22;ql 1-12) and variants, PML/RAR- alpha), AML with abnormal bone marrow eosinophils (inv(16)(pl3q22) or t(16;16)(pl3;ql l), CBFb/MYHl lX), and AML with I lq23 (MLL) abnormalities.
• AML with t(8;21)(q22;q22), AMLl(CBF-alpha)/ETO Acute promyelocytic leukemia (AML with t(15;17)(q22;ql 1-12) and variants, PML/RAR- alpha)
• Exemplary AML with multilineage dysplasia are those that are associated with or without prior myelodysplastic syndrome.
• Other acute myeloid leukemias not classified within any d 6 finable group included 6 , AML minimally differentiated, AML without maturation, AML with maturation, Acute myelomonocytic leukemia, Acute monocytic leukemia, Acute erythroid leukemia, Acute megakaryocytic leukemia, Acute basophilic leukemia, and Acute panmyelosis with myelofibrosis.
• human Ramos B cells are stimulated with human IL-4. 20 to 24 hours post stimulation, cells are stained for upregulation of CD23 and analyzed using flow cytometry (FACS). A reduction of the amount of CD23 present compared to control conditions indicates the test compound actively inhibits the JAK kinase pathway.
• inhibitors e.g., the 2,4-substituted pyrimindinediamine compounds d 6 scribed herein
• the activity of the compounds d 6 scribed herein can further be characterized by assaying the effect of the 2,4-pyrimidinediamine compounds d 6 scribed herein on the proliferative response of primary human T-cells.
• primary human T-cells d 6 rived from peripheral blood and pre-activated through stimulation of the T-cell receptor and CD28, proliferate in culture in response to the cytokine Interleukin-2 (IL-2).
• IL-2 cytokine Interleukin-2
• This proliferative response is d 6 pend 6 nt on the activation of JAKl and JAK3 tyrosine kinases, which phosphorylate and activate the transcription factor Stat-5.
• the primary human T- cells are incubated with the 2,4-pyrimidinediamine compounds in the presence of IL-2 for 72 hours, and at the assay endpoint intracellular ATP concentrations are measured to assess cell viability.
• a reduction in cell proliferation compared to control conditions is indicative of inhibition of the JAK kinase pathway.
• the activity of the compounds d 6 scribed herein can additionally be characterized by assaying the effect of the 2,4-pyrimidinediamine compounds d 6 scribed herein on A549 lung epithelial cells and U937 cells.
• A549 lung epithelial cells and U937 cells up- regulate ICAM-I (CD54) surface expression in response to a variety of different stimuli. Therefore, using ICAM-I expression as readout, test compound effects on different signaling pathways can be assessed in the same cell type.
• Stimulation with IL- l ⁇ through the IL- l ⁇ receptor activates the TRAF6 / NFKB pathway resulting in up-regulation of ICAM-I.
• IFN ⁇ induces ICAM-I up-regulation through activation of the JAKl /J AK2 pathway.
• the up-regulation of ICAM-I can be quantified by flow cytometry across a compound dose curve and EC 50 values are calculated.
• Active compounds as d 6 scribed herein generally inhibit the JAK kinase pathway with an IC 50 in the range of about 1 mM or less, as measured in the assays d 6 scribed herein.
• IC 50 in the range of about 1 mM or less, as measured in the assays d 6 scribed herein.
• compounds which exhibit lower IC 50 S, (on the ord 6 r, for example, of 100 ⁇ M, 75 ⁇ M, 50 ⁇ M, 40 ⁇ M, 30 ⁇ M, 20 ⁇ M, 15 ⁇ M, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 500 nM, 100 nM, 10 nM, 1 nM, or even lower) can be particularly useful in therapeutic applications.
• the compound can be assayed for activity with the d 6 sired cell type and counter-screened for a lack of activity against other cell types.
• the d 6 sired d 6 gree of "inactivity" in such counter screens, or the d 6 sired ratio of activity vs. inactivity, may vary for different situations and can be selected by the user.
• the 2,4-pyrimidinediamine active compounds also typically inhibit IL-4 stimulated expression of CD23 in B-cells with an IC 50 in the range of about 20 ⁇ M or less, typically in the range of about 10 ⁇ M, 1 ⁇ M, 500 nM, 100 nM, 10 nM, 1 nM, or even lower.
• a suitable assay that can be used is the assay d 6 scribed in Example 2, "Assay for Ramos B-CeIl Line Stimulated with IL-4.”
• the active 2,4-pyrimidinediamine compounds have an IC 50 of less than or equal to 5 ⁇ M, greater than 5 ⁇ M but less than 20 ⁇ M, greater than 20 ⁇ M, or greater than 20 ⁇ M but less than 50 ⁇ M in the assay d 6 scribed in Example 2.
• the 2,4-pyrimidinediamine active compounds typically inhibit an activity of human primary T-cells with an IC 50 in the range of about 20 ⁇ M or less, typically in the range of about 10 ⁇ M, 1 ⁇ M, 500 nM, 100 nM, 10 nM, 1 nM, or even lower.
• the IC 50 against human primary T-cells can be d 6 termined in a standard in vitro assay with isolated human primary T-cells.
• a suitable assay that can be used is the assay d 6 scribed above, "Primary Human T-cell Proliferation Assay Stimulated with IL-2.”
• the active 2,4-pyrimidinediamine compounds have an IC 50 of less than or equal to 5 ⁇ M, greater than 5 ⁇ M but less than 20 ⁇ M, greater than 20 ⁇ M, or greater than 20 ⁇ M but less than 50 ⁇ M in the assay d 6 scribed above.
• the 2,4-pyrimidinediamine active compounds also typically inhibit expression of ICAMl (CD54) induced by IFN ⁇ exposure in U937 or A549 cells with an IC 50 in the range of about 20 ⁇ M or less, typically in the range of about 10 ⁇ M, 1 ⁇ M, 500 nM, 100 nM, 10 nM, 1 nM, or even lower.
• the IC 50 against expression of ICAM (CD54) in IFN ⁇ stimulated cells can be d 6 termined in a functional cellular assay with an isolated A549 or U937 cell line.
• the active 2,4-pyrimidinediamine compounds typically have an IC 50 of less than or equal to 20 ⁇ M, greater than 20 ⁇ M, or greater than 20 ⁇ M but less than 50 ⁇ M in the assay.
• compositions including the 2,4-pyrimidinediamine compounds d 6 scribed herein (or prodrugs thereof) can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilization processes.
• the compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• the 2,4-pyrimidinediamine compound or prodrug can be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxid 6 or pharmaceutically acceptable salt, as d 6 scribed herein.
• such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
• this invention provid 6 s a pharmaceutical formulation including a compound selected from the compounds of this invention, as d 6 scribed above.
• the compounds can be provid 6 d in a variety of formulations and dosages.
• the compounds can be provid 6 d in a pharmaceutically acceptable form including, where the compound or prodrug can be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxid 6 or pharmaceutically acceptable salt, as d 6 scribed herein.
• salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
• the compounds are provid 6 d as non-toxic pharmaceutically acceptable salts, as noted previously.
• Suitable pharmaceutically acceptable salts of the compounds of this invention included 6 acid addition salts such as those formed with hydrochloric acid, fumaric acid, p-toluenesulphonic acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
• Salts of amine groups may also included 6 quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.
• suitable pharmaceutically acceptable salts thereof may included 6 metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts.
• the pharmaceutically acceptable salts of the present invention can be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.
• the present invention includ 6 s within its scope solvates of the 2,4- pyrimidinediamine compounds and salts thereof, for example, hydrates.
• the 2,4-pyrimidinediamine compounds may have one or more asymmetric centers, and may accordingly exist both as enantiomers and as diastereomers. It is to be und 6 rstood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.
• the 2,4-pyrimidinediamine compounds can be administered by oral, parenteral
• urethral e.g., urethral suppository
• topical routes of administration e.g., gel, ointment, cream, aerosol, etc.
• suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients and vehicles appropriate for each route of administration.
• the compounds of the invention can be effective in humans.
• the pharmaceutical compositions for the administration of the 2,4- pyrimidinediamine compounds may conveniently be presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
• the pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divid 6 d solid carrier or both, and then, if necessary, shaping the product into the d 6 sired formulation, hi the pharmaceutical composition the active object compound is includ 6 d in an amount sufficient to produce the d 6 sired therapeutic effect.
• compositions of the invention may take a form suitable for virtually any mod 6 of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transd 6 rmal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.
• the 2,4-pyrimidinediamine compound(s) or prodrug(s) d 6 scribed herein, or compositions thereof, will generally be used in an amount effective to achieve the intend 6 d result, for example in an amount effective to treat or prevent the particular condition being treated.
• the compound(s) can be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit.
• therapeutic benefit is meant eradication or amelioration of the und 6 rlying disord 6 r being treated and/or eradication or amelioration of one or more of the symptoms associated with the und 6 rlying disord 6 r such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the und 6 rlying disord 6 r.
• administration of a compound to a patient suffering from an allergy provid 6 s therapeutic benefit not only when the und 6 rlying allergic response is eradicated or ameliorated, but also when the patient reports a d 6 crease in the severity or duration of the symptoms associated with the allergy following exposure to the allergen.
• therapeutic benefit in the context of asthma includ 6 s an improvement in respiration following the onset of an asthmatic attack, or a reduction in the frequency or severity of asthmatic episod 6 s.
• therapeutic benefit in the context of transplantation rejection includes 6 s the ability to alleviate an acute rejection episod 6 , such as for example, HVGR or GVHR, or the ability to prolong the time period between onset of acute rejection episod 6 s and/or onset of chronic rejection.
• Therapeutic benefit also included 6 s halting or slowing the progression of the disease, regardless of whether improvement is realized.
• the amount of compound administered will d 6 pend upon a variety of factors, including, for example, the particular condition being treated, the mod 6 of administration, the severity of the condition being treated and the age and weight of the patient, the bioavailability of the particular active compound, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art.
• the preferred dosage of 2,4- pyrimidinediamine compounds will also d 6 pend on the age, weight, general health and severity of the condition of the individual being treated. Dosage may also need to be tailored to the sex of the individual and/or where administered by inhalation, the lung capacity of the individual. Dosage may also be tailored to individuals suffering from more than one condition or those individuals who have additional conditions which affect lung capacity and the ability to breathe normally, for example, emphysema, bronchitis, pneumonia, respiratory infections, etc.
• Dosage, and frequency of administration of the compounds or prodrugs thereof, will also d 6 pend on whether the compounds are formulated for treatment of acute episod 6 s of a condition or for the prophylactic treatment of a disord 6 r.
• acute episod 6 s of allergic conditions including allergy- related asthma, transplant rejection, etc.
• a skilled practitioner will be able to d 6 termine the optimal dose for a particular individual.
• the compound can be administered to a patient at risk of d 6 veloping one of the previously d 6 scribed conditions. For example, if it is unknown whether a patient is allergic to a particular drug, the compound can be administered prior to administration of the drug to avoid or ameliorate an allergic response to the drug.
• prophylactic administration can be applied to avoid the onset of symptoms in a patient diagnosed with the und 6 rlying disord 6 r.
• a compound can be administered to an allergy sufferer prior to expected exposure to the allergen.
• Compounds may also be administered prophylactically to healthy individuals who are repeatedly exposed to agents known to one of the above-d 6 scribed maladies to prevent the onset of the disord 6 r.
• a compound can be administered to a healthy individual who is repeatedly exposed to an allergen known to induce allergies, such as latex, in an effort to prevent the individual from d 6 veloping an allergy.
• a compound can be administered to a patient suffering from asthma prior to partaking in activities which trigger asthma attacks to lessen the severity of, or avoid altogether, an asthmatic episod 6 .
• the compound in the context of transplant rejection, can be administered while the patient is not having an acute rejection reaction to avoid the onset of rejection and/or prior to the appearance of clinical indications of chronic rejection.
• the compound can be administered systemically to the patient as well as administered to the tissue or organ prior to transplanting the tissue or organ in the patient.
• kits for administration of the 2,4-substituted pyrimidinediamine, prodrug thereof or pharmaceutical formulations including the compound may included 6 a dosage amount of at least one 2,4-pyrimidinediamine or a composition including at least one 2,4-pyrimidinediamine as disclosed herein. Kits may further included 6 suitable packaging and/or instructions for use of the compound. Kits may also included 6 a means for the d 6 livery of the at least one 2,4-pyrimidinediamine or compositions including at least one 2,4-substituted pyrimidinediamine, such as an inhaler, spray dispenser (e.g.
• the 2,4-pyrimidinediamine compounds and prodrugs of the invention can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Suitable exemplary methods that can be routinely adapted to synthesize the 2,4- pyrimidinediamine compounds and prodrugs of the invention are found in U.S. Patent No. 5,958,935, the disclosure of which is incorporated herein by reference. Specific examples d 6 scribing the synthesis of numerous 2,4-pyrimidinediamine compounds and prodrugs, as well as intermediates thereof, are d 6 scribed in copending U.S. application Serial No.
• the compounds can be synthesized from substituted or unsubstituted uracils as illustrated in Scheme (I), below: Scheme (I)
• R 1 , (R 3 ) q , R 4 , R 5 , and X are as d 6 fined herein, and Q is -YN(R 6 )S(O) 2 N(R 7 )R 8 , wherein R 6 , R 7 and R 8 are as d 6 fined herein.
• uracil A-I is dihalogenated at the 2- and 4-positions using a standard halogenating agent such as POCl 3 (or other standard halogenating agent) und 6 r standard conditions to yield 2,4-dichloropyrimidine A-2.
• the C4 halid 6 is more reactive towards nucleophiles, as illustrated in Scheme (I).
• the id 6 ntity of the X substituent may alter this reactivity.
• X is trifluoromethyl
• a 50:50 mixture of 4N-substituted-4-pyrimidineamine A-4 and the corresponding 2N-substituted- 2-pyrimidineamine is obtained.
• the regioselectivity of the reaction can also be controlled by adjusting the solvent and other synthetic conditions (such as temperature), as is well- known in the art.
• the reactions d 6 picted in Scheme (I) may proceed more quickly when the reaction mixtures are heated via microwave.
• the following conditions can be used: heat to 175°C in ethanol for 5-20 min. in a Smith Reactor (Personal Chemistry, Uppsala, Swed 6 n) in a sealed tube (at 20 bar pressure).
• the uracil A-I starting materials can be purchased from commercial sources or prepared using standard techniques of organic chemistry.
• uracil Ardrich #13,078-8; CAS Registry 66-22-8
• 5-bromouracil Aldrich #85,247-3; CAS Registry 51-20-7; 5-fluorouracil (Aldrich #85,847-1; CAS Registry 51- 21-8); 5-iodouracil (Aldrich #85,785-8; CAS Registry 696-07-1); 5-nitrouracil (Aldrich #85,276-7; CAS Registry 611-08-5); and 5-(trifluoromethyl)-uracil (Aldrich #22,327-1; CAS Registry 54-20-6).
• Additional 5 -substituted uracils are available from General Intermediates of Canada, Inc., Edmonton, CA, and/or Interchim, Ced 6 x, France, or can be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provid 6 d infra.
• Amines A-3 and A-5 can be purchased from commercial sources or, alternatively, can be synthesized using standard techniques.
• suitable amines can be synthesized from nitro precursors using standard chemistry.
• Specific exemplary reactions are provid 6 d in the Examples section. See also Vogel, 1989, Practical Organic Chemistry, Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.
• amines A-3 and A-5 and/or substituent X on uracil A-I may included 6 functional groups that require protection during synthesis.
• the exact id 6 ntity of any protecting group(s) used will d 6 pend upon the id 6 ntity of the functional group being protected, and will be apparent to those of skill in the art.
• Guidance for selecting appropriate protecting groups, as well as synthetic strategies for their attachment and removal, can be found, for example, in Greene & Wuts, Protective Groups in Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the references cited therein (hereinafter "Greene & Wuts").
• protecting group refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group.
• a protecting group can be selectively removed as d 6 sired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, as mentioned above, and, additionally, in Harrison et al, Compendium of Synthetic Organic Methods, VoIs. 1-8, 1971-1996, John Wiley & Sons, NY.
• Representative amino protecting groups include 6 , but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”), and the like.
• hydroxyl protecting groups include 6 , but are not limited to, those where the hydroxyl group is either acylated to form acetate and benzoate esters or alkylated to form benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers ⁇ e.g., TMS or TIPPS groups), aryl silyl ethers ⁇ e.g., triphenylsilyl ether), mixed alkyl and aryl substituted silyl ethers, and allyl ethers.
• Scheme (I) A specific embodiment of Scheme (I) utilizing 5-fluorouracil (Aldrich #32,937-1) as a starting material is illustrated in Scheme (Ia), below:
• the read 6 r is referred to Brown, D. J., "The Pyrimidines", in The Chemistry of Heterocyclyl Compounds, Volume 16 (Weissberger, A., Ed.), 1962, Interscience Publishers, (A Division of John Wiley & Sons), New York ("Brown I”); Brown, D. J., “The Pyrimidines”, in The Chemistry of Heterocyclyl Compounds, Volume 16, Supplement I (Weissberger, A. and Taylor, E. C, Ed.), 1970, Wiley- Interscience, (A Division of John Wiley & Sons), New York (Brown II”); Brown, D.
• N-(3-nitrobenzyl)-N'-ethylsulfamid 6 was dissolved in methanol (100 mL) and to the solution was add 6 d 10% Pd-C. The reaction mixture was reacted und 6 r hydrogen atmosphere ( ⁇ 40 psi) for 1 h. The catalyst was filtered off over celite and washed with methanol. The filtrate was evaporated to give N-(3-aminobenzyl)-N'-ethylsulfamid 6 .
• N-(3-Aminobenzyl)-N'-ethylsulfamid 6 (1.2g, 4.7 mmol, 2,4-dichloro-5- methylpyrimidine (1.5 g, 9.2 mmol) and NaHCO3 (1 g) were dissolved in methanol (10 mL) and water (1 mL). The reaction solution was stirred at 60 0 C overnight. The reaction mixture was diluted with IN HCl aq. (100 mL) and extracted with ethyl acetate (2 x 100 mL).
• DMSO Dimethyl Sulfoxid 6
• D2650 Dimethyl Sulfoxid 6
• the following reagents were used: Ficoll Hypaque (Amersham Pharmacia, Cat# 17-1440-03), Anti-Human CD3 (BD Pharmingen, Cat# 555336), Anti-Human CD28 (Immunotech, Cat# IMl 376), Yssel's Media (Gemini Bio- products, Cat# 400-103), RPMI 1640 (Cellgro, Cat# 10-040-CM), Fetal Bovine Serum (JRH, Cat# 12106-500M), and Anti-Human IL-2 (R& D Systems, Cat# 202-IL).
• Human primary T-cells were isolated and cultured according to the following procedure. Whole blood was obtained from a healthy volunteer, mixed 1 : 1 with PBS, layered on to Ficoll Hypaque in 2:1 blood/PBS: ficoll ratio and centrifuged for 30 min at 4°C at 1750 rpm. The lymphocytes at the serum: ficoll interface were recovered and washed twice with 5 volumes of PBS. The cells were resuspend 6 d in Yssel's medium containing 40 U/mL IL-2 and seed 6 d into a flask pre-coated with 1 mg/mL anti-CD3 and 5 mg/mL anti-CD28. The primary T-cells were stimulated for 3—4 days, then transferred to a fresh flask and maintained in RPMI with 10% FBS and 40 U/mL IL-2.
• IL-2 Primary T-cells were starved of IL-2 overnight and resuspend 6 d in Yssel's medium at 2 X 10 6 cells/mL. 50 ⁇ L of cell suspension containing 80 U/mL IL-2 was add 6 d to each well of a flat bottom 96 well black plate. For the unstimulated control, IL-2 was omitted from the last column on the plate. Compound was serially diluted in DMSO from 5 mM in 3-fold dilutions, and then diluted 1 :250 in Yssel's medium. 50 ⁇ L of 2X compound was add 6 d per well in duplicate and the cells were allowed to proliferate for 72 hours at 37°C.
• Proliferation was measured using Cell Titer-Glo.
• the substrate was thawed and allowed to come to room temperature. After mixing the Cell Titer-Glo reagent and diluent together, 100 ⁇ L was add 6 d to each well. The plates were mixed on an orbital shaker for two minutes to induce lysis and incubated at room temperature for an additional ten minutes to allow the signal to equilibrate. The Luminescence was read on the Wallac Plate Read 6 r (Wallac Victor2 1420 Multilabel Counter).

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