# Patent

## Title
Prostaglandin transporter inhibitors and uses thereof

## Abstract

Disclosed are compounds for inhibiting prostaglandin transporter (PGT) activity, pharmaceuticals compositions including the compounds, and methods of treating subjects using the compounds.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national stage entry under 35 U.S.C. §371 of PCT International Patent Application No. PCT/US2010/002555, filed Sep. 20, 2010, and claims priority to U.S. Provisional Patent Application No. 61/277,291, filed Sep. 23, 2009, the contents of which are incorporated herein by reference in their entirety.


BACKGROUND OF THE INVENTION
Various publications are referred to in parentheses throughout this application. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Prostaglandins (PGs) are synthesized from arachidonic acid by cyclooxygenases (COX1 and COX2) and corresponding synthases (Helliwell et al. 2004). PGs play an important role in physiology and clinical settings. Their biological effects include triggering inflammation, fever and pain (Blatteis and Sehic, 1997; Bley et al., 1998; Vanegas and Schaible, 2001; Samad et al., 2002); induction of labor (Ulmann et al., 1992); modulation of renal hemodynamics and of water and solute reabsorption (Epstein, 1986; Wang et al., 1998; Yokoyama et al., 2002); arterial vasodilatation (Clyman et al., 1978; Coceani and Olley, 1988; Smith et al., 1994); stimulation of cell proliferation and angiogenesis (Ferrara et al, 1997; Tsujii et al, 1998; Young, 2004; Mann et al, 2006; Sheng et al, 2001; Shao et al, 2006); and mediating sensitization of sensory neurons (Southall and Vasko, 2000; Southall and Vasko, 2001; Seybold et al., 2003). PG analogues, such as latanoprost and unoprostone, have been used to treat glaucoma (Stjemschantz, 1995; Alm, 1998; Susanna et al., 2002; Stjemschantz, 2004). At the cellular level, PGs are involved in several major signaling pathways, including the mitogen-activated protein (MAP) kinase and protein kinase A pathways by upregulation of cAMP (Narumiya et al., 1999; Bos et al., 2004).
The magnitude of PG effects depends not only on their production but also their metabolism. The prostaglandin transporter (PGT) (Kanai et al., 1995; U.S. Pat. No. 5,792,851) removes PGs from the extracellular compartment and thereby terminates their interactions with receptors on cell membranes. PGT delivers PGs to cytoplasmic 15-OH PG dehydrogenase (Schuster, 2002; Nomura et al., 2004), resulting in oxidation and inactivation. Because PGT is highly expressed in the tissues and organs where PGs are synthesized (Bao et al., 2002), and because PGT regulates a broad and complex PG signaling system, inhibitors of PGT are important for manipulating signaling. Inhibition of PGT lowers blood pressure by vasodilation and natriuresis and inhibits platelet aggregation (Chi et al., 2009).
Known PGT blockers include inhibitors of the organic anion transporters (OATS), such as bromcresol green and bromosulfophthalein, and some COX2 inhibitors, such as indomethacin and ibuprofen (Bito and Salvador, 1976; Kanai et al., 1995). One of the main problems with these inhibitors is that they are not specific for PGT (Jacquemin et al., 1994; Sweet et al., 1997). Recently, specific PGT inhibitors have been developed (Chi et al., 2005; WO 2007/136638). The present invention addresses the need for even more potent specific inhibitors of PGT.
SUMMARY OF THE INVENTION
The invention provides compounds that inhibit prostaglandin transporter (PGT) activity, where the compounds are represented by the structure




where the variables R1, R2, R3, R4 and W are defined herein below.

The invention provides pharmaceutical compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable carrier.
The invention is further directed to methods of inhibiting prostaglandin transporter (PGT) activity in a subject comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit PGT activity.
The invention also provides methods of inhibiting cyclooxygenase 2 (COX2) activity in a subject comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit COX2 activity.
The invention further provides a method of treating a subject with a disease or disorder associated with prostaglandin activity and/or COX2 activity comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit prostaglandin transporter (PGT) activity and/or COX2 activity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1B. Prostaglandin transporter (PGT) inhibitors accelerate wound healing. A. Inhibitor T26A applied directly to the wound accelerates wound healing in mice (bottom row) compared to treatment with vehicle (Veh) (top row). B. Averaged wound areas of 4 mice, each of them had 2 wounds and received topically applied vehicle Veh1 (2% DMSO+2% cremophor in water) on one wound and T26A on the other. On the wounds of a separate group of 4 mice, vehicle Veh2 (2% EtOH) was applied to one wound and 200 μM PGE2 was applied to the other.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides compounds that inhibit prostaglandin transporter (PGT) activity, where the compounds are represented by the structure:




wherein

    W is O or NR5;
    R1 is H, —CH3, —(CH2)2OH,






    R2 is






    R3 is —(CH2)5CH3, —(CH2)6CO2H, —(CH2)6CO2CH3, —(CH2)dNHCO-Ph, —(CH2)6CONH-Ph, —(CH2)6CONHCH2-Ph,






    R4 and R5 are independently H or —CH3;
    R6 is O or NR9;
    R7 is H, —CH3, —C(CH3)3, —CH2OH, —(CH2)2OH, —(CH2)2O(CH2)2OH, —(CH2CH2O)3CH3, —(CH2CH2O)2CH2CO2CH3, —(CH2)5CH3,






    R8 is H, —OH, —CH2OH, —CO2H, —CO2CH2CH3, —CO(CH2)6CH3, —OCH3, —NH2, —SO2NH2, —CONH-Bn or






    R9 is H or —CH3;
    R10 is —CH2NH2, —CO2H or —CO2CH3;
    R11 is —SO2-Ph, —CH2-Ph, —CONN-Ph, —COCH3,






and

    R12 is —CH3,






where X1, X2, X3, X4, X5, X6 and X7 are independently H, halogen, —OH, —CH3, —CF3, —OCH3, —CO2H, —CO2CH3, —CH2CO2H, —CH2CO2CH3, phenyl or —O-Bn; and where a=0-2; b=1-6; c=0-1; d=4-7; and e=0-1; or a pharmaceutically acceptable salt thereof.

As used herein in chemical structures, “Ph” stands for phenyl, “Bn” stands for benzyl (—CH2Ph), “Bz” stands for benzoyl (—(C═O)Ph), and “Me” stands for methyl (—CH3). The point of attachment of the side group substitution to the main part of the compound is indicated by “( )” The terms ortho, meta and para refer to the positions of substitutions in relation to the main part of the compound.
In preferred compounds, W is NR5. In preferred compounds, R6 is NR9.
In preferred compounds, at least one of R4, R5 and R9 is H, or all of R4, R5 and R9 are H. Preferably, at least R5 (out of R4, R5 and R9) is H.
In preferred compounds, one of X1 and X2 is H, and the other is halogen, —CF3, —CH3, —CO2H, —CO2CH3, —OCH3 or phenyl; or both X1 and X2 are halogen. In preferred compounds, one of X3 and X4 is H, and the other is halogen, —CO2H, —CO2CH3, —CH2CO2H, —CH2CO2CH3, —OH, —OCH3 or —O-Bn; or one of X3 and X4 is —OH, and the other is halogen, —CO2H or —CO2CH3. In preferred compounds, one of X5 and X6 is H, and the other is halogen, —CF3, —OCH3 or phenyl; or both X5 and X6 are halogen. In preferred compounds, X7 is H, —CF3 or —OCH3.
In preferred compounds, R8 is located in para position. In preferred compounds, one or both of X1 and X2 are located in ortho position, or one or both of X1 and X2 are located in meta position, or X1 is located in meta position and X2 is located in para position, or X1 is located in ortho position and X2 is located in para position. In preferred compounds, X3 is in meta position and X4 is in para position. In preferred compounds, X5 or X6 is in meta position, or X5 or X6 is in para position.
Preferred compounds have the structure:




or a pharmaceutically acceptable salt thereof.

In preferred compounds,

    R1 is






    R2 is






and

    R3 is






where X1, X2, X3, X4, X5 and X6 are independently H, halogen, —OH, —CH3, —CF3, —OCH3, —CO2H, —CO2CH3, —CH2CO2H or —CH2CO2CH3; and where a=1-2; and b=1-5; or a pharmaceutically acceptable salt thereof.

In preferred compounds,

    R1 is






    R2 is






and

    R3 is






where X1 is H or halogen; where X4 is H, halogen or —CO2H; and where X5 is H, halogen or —OCH3; or a pharmaceutically acceptable salt thereof.

Preferred compounds have the structure:




or a pharmaceutically acceptable salt thereof.
In the compounds described herein, W—R1 can be replaced with halogen.
Halogens are F, Cl, Br, I and At. Preferred halogens are Br, Cl and F.
The invention provides a compound having the structure:












































































or a pharmaceutically acceptable salt thereof.

Preferred compounds have the structure:







or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising any of the compounds disclosed herein and a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” it is meant a material that (i) is compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions, and the like.
The invention provides a method of inhibiting prostaglandin transporter (PGT) activity in a subject comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit PGT activity.
The invention also provides a method of inhibiting cyclooxygenase 2 (COX2) activity in a subject comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit COX2 activity.
The invention further provides a method of treating a disease or disorder in a subject associated with prostaglandin activity and/or COX2 activity comprising administering to the subject any of the compounds disclosed herein in an amount effective to inhibit prostaglandin transporter (PGT) activity and/or COX2 activity. The disease or disorder can be, for example, arthritis, fever, common cold, hypertension, glaucoma, a wound, initiation of labor, dysmenorrhea, menstrual cramps, inflammatory bowel disease, Crohn's disease, emphysema, acute respiratory distress syndrome, asthma, bronchitis, chronic obstructive pulmonary disease, Alzheimer's disease, organ transplant toxicity, cachexia, allergic reactions, allergic contact hypersensitivity, cancer, tissue ulceration, peptic ulcers, gastritis, regional enteritis, ulcerative colitis, diverticulitis, recurrent gastrointestinal lesion, gastrointestinal bleeding, coagulation, anemia, synovitis, gout, ankylosing spondylitis, inflammation, restenosis, periodontal disease, epidermolysis bullosa, osteoporosis, loosening of artificial joint implants; atherosclerosis, aortic aneurysm, periarteritis nodosa, congestive heart failure, myocardial infarction, stroke, cerebral ischemia, head trauma, spinal cord injury, neuralgia, neuro-degenerative disorders, autoimmune disorders, Huntington's disease, Parkinson's disease, migraine, depression, peripheral neuropathy, pain, gingivitis, cerebral amyloid angiopathy, nootropic or cognition enhancement, amyotrophic lateral sclerosis, multiple sclerosis, ocular angiogenesis, corneal injury, macular degeneration, conjunctivitis, abnormal wound healing, muscle or joint sprains or strains, tendonitis, skin disorders, myasthenia gravis, polymyositis, myositis, bursitis, burns, diabetes, tumor invasion, tumor growth, tumor metastasis, corneal scarring, scleritis, immunodeficiency diseases, sepsis, premature labor, hyporothrombinemia, hemophilia, thyroiditis, sarcoidosis, Behcet's syndrome, hypersensitivity, kidney disease, rickettsial infections, protozoan diseases, reproductive disorders or septic shock. Preferably, the disease or disorder is inflammation, pain, a wound, or a cardiovascular disease, such as hypertension or atherosclerosis.
The above-described compounds can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.
Accordingly, compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, cornstarch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like.
The compounds can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compounds into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Rectal administration includes administering the compound, in a pharmaceutical composition, into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C., dissolving the composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches (such as the well-known nicotine patch), ointments, creams, gels, salves and the like.
Topical administration may be preferred for localized application of the compound, for example, for promoting wound healing or for ocular administration (e.g., eye drops).
The present invention includes nasally administering to the mammal a therapeutically effective amount of the compound, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream powder, or using a nasal tampon or nasal sponge.
Where the compound is administered peripherally such that it must cross the blood-brain barrier, the compound is preferably formulated in a pharmaceutical composition that enhances the ability of the compound to cross the blood-brain barrier of the mammal. Such formulations are known in the art and include lipophilic compounds to promote absorption. Uptake of non-lipophilic compounds can be enhanced by combination with a lipophilic substance. Lipophilic substances that can enhance delivery of the compound across the nasal mucus include but are not limited to fatty acids (e.g., palmitic acid), gangliosides (e.g., GM-1), phospholipids (e.g., phosphatidylserine), and emulsifiers (e.g., polysorbate 80), bile salts such as sodium deoxycholate, and detergent-like substances including, for example, polysorbate 80 such as TweenTM, octoxynol such as TritonTM X-100, and sodium tauro-24,25-dihydrofusidate (STDHF).
In particular embodiments of the invention, the compound is combined with micelles comprised of lipophilic substanceS. Alternatively, the compound can be combined with liposomes (lipid vesicles) to enhance absorption. The compound can be contained or dissolved within the liposome and/or associated with its surface. Suitable liposomes include phospholipids (e.g., phosphatidylserine) and/or gangliosides (e.g., GM-1). Bile salts and their derivatives and detergent-like substances can also be included in the liposome formulation.
The invention also provides for the use of any of the compounds disclosed herein for treating a subject and for the use of any of the compounds disclosed herein for the preparation of a pharmaceutical composition for treating a subject, where the subject is being treated to inhibit prostaglandin transporter (PGT) activity or inhibit cyclooxygenase 2 (COX2) activity or the subject has a disease or disorder associated with prostaglandin activity and/or COX2 activity.
The present invention is illustrated in the following Experimental Details section, which is set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims that follow thereafter.
EXPERIMENTAL DETAILS
A. Preparation of Chemical Compounds
Compounds were made according to one of 11 generic schemes described below. For each scheme, a specific example with a corresponding experimental description is given. Other compounds made via that same scheme are listed in tabular form beneath the experimental description. The compounds were synthesized at Provid Pharmaceuticals, Inc., North Brunswick N.J.
General procedures. HPLC was performed on Rainin SD-300 or Varian ProStar equipped with a single wavelength UV detector at 214 nm and linear gradients. Analytical HPLC was performed on a Varian C18 column (microsorb 60-8, 4.6×250 mm) at a flow rate of 1 mL/min. Semi-preparative HPLC was performed on a Varian C18 column (microsorb 60-8, 10.0×250 mm) at a flow rate of 5 mL/min. Preparative HPLC was routinely performed on a Varian C18 column (microsorb 60-8, 21.4×250 mm) at a flow rate of 20 mL/min. The solvent system used on linear gradients was water with 0.075% TFA (solvent A) vs Acetonitrile with 0.075% TFA (solvent B). Silica gel used in flash column chromatography was obtained from Sorbent Technologies (Atlanta, Ga.). LC-MS spectra were taken on Waters ZQ LC/MS-ESI or APCI.




Experimental:





Preparation of amine 1: To a solution of Boc-1-amino-3,6-dioxa-8-octane diamine (1 eq.) and benzoyl chloride (1.2 eqs.) in CH2Cl2 (40 mL) was added TEA (2.5 eqs.). The reaction mixture was stirred overnight. Subsequently, the reaction mixture was partitioned between saturated aqueous NaHCO3 and CH2Cl2, the layers separated, and the aqueous layer extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification. The obtained residue was dissolved in TFA (20 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was concentrated in vacuo and amine 1 was used without further purification.




Preparation of triazine 2a: To a solution of cyanuric chloride (1.14 eqs) and aniline (1.0 eq.) in THF (27 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was then partitioned between water and EtOAc, the layers separated, and the aqueous layer was extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and 4-(1H-tetrazol-5-yl)aniline (1.1 eqs.) in THF (3.0 mL) was added iPr2NEt (5.0 eqs.). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 1 (1.1 eqs.) in THF (3.3 mL) was added iPr2NEt (2.5 eqs.). Some methanol was added to help solubilize the starting material. The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The obtained residue was purified via reverse phase HPLC to yield triazine 2a (4.3 mg, 2.2% overall yield). MS (LCMS, ESI): Rt=7.58 mins (>90% pure) m/z=582 (M+H)+.
The following compounds were or could be made by the procedure described for triazine 2a:
















 












Compound
W
R1
R2
R4
(M + H)+





2b
NH
Ph—




H
544


 


2c
NH
Ph—




H
620


 


2d
NMe
Me—




H
572


 


2e
NH








H
588


 


2f
NH








H
690


 


2g
NH
Ph—




H
554


 


2h
NH
Ph—




H
586


 


2i
NH
Ph—




H
572


 


2j
NH
Ph—




H
554


 


2k
NH
Ph—




H
555


 


2l
O
H—




H
455













Experimental:





To a solution of cyanuric chloride (1.14 eqs.) and tbutyl 4-aminobenzoate (1 eq.) in THF (81 mL) was added iPr2NEt (1.1 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1 eq.) and 5-aminoindazole (1.1 eqs.) in THF (22 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1 eq.) and amine 1 (1.1 eqs.) in THF (7.2 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified via column chromatography over silica gel (2:1/hexanes:EtOAc→95:5/CH2Cl2:MeOH) to yield a residue (0.115 g, 24% overall yield) that was used without further purification.
The residue (1 eq.) was dissolved in TFA (1.8 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was then concentrated in vacuo and the obtained residue used without any further purification.
To a mixture of the residue (1 eq.), benzyl amine (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1.0 mL). Subsequently, iPr2NEt (10 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo and the residue purified via reverse-phase HPLC to yield triazine 3a (9.4 mg, 20% yield over 2 steps). MS (LCMS, ESI): Rt=7.56 mins (>90% pure) m/z=687 (M+H)+.
The following compounds were or could be made by the procedure described for triazine 3a:
















 














Com-









pound
W
R6
R7
R4
R2
R3
(M + H)+





3b
NH
NH
Me(CH2)5—
H








681


 


3c
NH
NH




H








705


 


3d
NH
NH




H








647


 


3e
NH
NH




H








719


 


3f
NH
NH




H








719













Experimental:





Preparation of triazine 4a: To a solution of cyanuric chloride (1.14 eqs.) and 4-benzyloxy aniline hydrochloride (1 eq.) in THF (2.7 mL) was added iPr2NEt (5.0 eqs.). The reaction mixture was allowed to stir at ambient temperature for 1.0 hour. Subsequently, p-anisidine (1.05 eqs.) was added to the reaction mixture and the reaction stirred overnight at 50° C. The reaction mixture was cooled to ambient temperature then partitioned between water and EtOAc, the layers separated, and the aqueous layer extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1 eq.) and amine 1 (1.1 eqs.) in THF (2.4 mL) was added iPr2NEt (1.5 eqs.). The reaction mixture was heated to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue filtered over a plug of silica gel (CH2Cl2→49:1/CH2Cl2:MeOH). The organics from the 49:1/CH2Cl2:MeOH elution were collected and concentrated in vacuo. The obtained residue was used without further purification.
A solution of benzyl ether (1 eq.) in TFA (1.2 mL) and DMS (1.2 mL) was heated to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 4a (18.0 mg, 13.6% overall yield). MS (LCMS, ESI): Rt=6.21 mins (>90% pure) m/z=560 (M+H)+.
The following compounds were or could be made by the procedure described for triazine 4a:
















 














Com-






(M + 


pound
W
R1
R3
R4
X3
X4
H)+





4b
NH
Ph—
P
H
OH
H
530


4c
NMe
Me—
P
H
OH
H
482


 


4d
NH




P
H
OH
H
498


 


4e
NH




P
H
OH
H
600


 


4f
NH




P
H
OH
H
574


 


4g
NH




P
H
OH
H
588


 


4h
NH




P
H
OH 
H
615


 


4i
NH




P
H
OH
H
545


 


4j
NH




P
H
OH
H
560


 


4k
NH




P
H
OH
H
536


 


4l
NH




P
H
OH
H
546


 


4m
NH
Ph—




H
OH
H
426


 


4n
NH




P
H
OH
H
609


 


4o
NH
Ph—




H
OH
H
455


 


4p
NH
Ph—




H
OH
H
437


 


4q
NH
Ph—




H
OH
H
379


 


4r
NH
Ph—
P
H
OH
Cl
564


 


4s
NH
Ph—
P
H




H
572


 


4t
NH
Ph—
P
H
—CO2H
H
558


 


4u
NH
Ph—




H
OH
H
486


 


4v
NH




P
H
OH
H
656


 


4w
NH




P
H
OH
H
599


 


4x
NH
Ph—




H
OH
H
498


 


4y
NH
Ph—




H
OH
H
512


 


4z
NH
P
P
H
OH
H
689













Experimental:





Preparation of amine 5: To a solution of 2-[2-(Boc-amino)ethoxy]ethoxy acetic acid dicyclohexylamine salt (1 eq.) in benzene (8 mL) and MeOH (2 mL) was added TMSCHN2 (2.0 eqs.). The reaction mixture was stirred for 1.0 h, after which the reaction mixture was concentrated in vacuo. The obtained residue was then treated with saturated methanolic HCl. The resulting reaction mixture was stirred for 1 hour before being concentrated in vacuo. The residue was used without further purification.




Preparation of triazine product 6a: A solution of cyanuric chloride (1 eq.) in THF (5 mL) was treated with 4-benzyloxyaniline hydrochloride (0.85 eq.) and iPr2NEt (2.0 eqs.) with stirring at ambient temperature for 0.5 h. The reaction mixture was poured into 50 mL EtOAc and the organic solution was washed with H2O (2×) and with brine (1×). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The obtained residue was used without further purification.
To a solution of the residue (1 eq.) and aniline (1.1 eqs.) in THF (7.2 mL) was added iPr2NEt (2.0 eqs.). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of chlorotriazine (1 eq.) and amine 5 (1.1 eqs.) in THF (3.5 mL) was added iPr2NEt (3.0 eqs.). Subsequently, CH2Cl2 (1.0 mL) was added in order to make the reaction homogenous. The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of benzyl ether (1.0 eq.) in TFA (1.1 mL) was added DMS (1.1 mL). The reaction mixture was warmed to 35° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue purified via reverse-phase HPLC to yield a residue that was used without further purification.
A heterogeneous mixture of methyl ester (1.0 eq.) and LiOH (30 eqs.) in THF (0.3 mL), MeOH (0.3 mL) and H2O (0.3 mL) was stirred at ambient temperature overnight. The reaction mixture was then acidified via addition of TFA and the reaction mixture concentrated in vacuo. The residue was purified via reverse phase HPLC to yield triazine 6a (14.0 mg, 9.6% overall yield). MS (LCMS, ESI): Rt=5.55 mins (>90% pure) m/z=441 (M+H)+.
The following compounds were or could be made by the procedure described for triazine 6a:
















 














Compound
W
R1
R3
R4
X3
X4
(M + H)+





6b
NH
Ph—




H
OH
H
423













Experimental:





Preparation of amine 7: To a solution of bis(2-hydroxyethyl)ether (1.0 eq) and iPr2NEt (4.0 eqs.) in CH2Cl2 (24 mL) was added MsCl (2.2 eqs.). The reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was partitioned between water and CH2Cl2, the layers separated, and the aqueous layer extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and NaI (0.2 eqs.) in DMF (24 mL) was added NaN3 (2.3 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was cooled to ambient temperature then partitioned between water and ether. The layers were separated and the aqueous extracted with ether (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) in THF (0.6 mL), ether (3 mL) and 1.0 M HCl (3 mL, aqueous) was added PPh3 (1.05 eqs.). The reaction mixture was stirred at ambient temperature for 60 hours. Subsequently, the organics were removed from the reaction mixture in vacuo. Diethyl ether was added and the layers separated and the ethereal layer washed with 4.0 M HCl in water (50 mL). The combined aqueous layers were extracted with ether (2×) and the aqueous layer basicified to a pH ̃14 by addition of solid NaOH. The aqueous layer was then extracted with CH2Cl2 (3×) and the combined dichloromethane layers dried over Na2SO4, filtered and concentrated in vacuo. The obtained residue was used without further purification.
To a solution of the residue (1.0 eq.) and benzoyl chloride (1.1 eqs.) in CH2Cl2 (20 mL) was added TEA (2.5 eqs.). The reaction mixture was stirred overnight. Subsequently, the reaction mixture was partitioned between saturated aqueous NaHCO3 and CH2Cl2, the layers separated, and the aqueous layer extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue (0.182 g, 33% crude yield over 4 steps) was used without further purification.
To a solution of the residue (1.0 eq.) in THF (3.2 mL) and H2O (1.0 mL) was added PMe3 (3.0 eqs.). The reaction mixture was stirred for 2 hours. The reaction mixture was concentrated in vacuo and the desired product used without further purification.




Preparation of triazine 8a: To a solution of cyanuric chloride (1.14 eqs.) and 4-aminobenzoate (1.0 eq.) in THF (81 mL) was added iPr2NEt (1.1 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and 4-benzyloxyaniline (1.1 eqs.) in THF (52 mL) was added iPr2NEt (3.0 eqs). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 7 (1.2 eqs.) in THF (1.5 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified via column chromatography over silica gel (2:1/hexanes:EtOAc→98:2/CH2Cl2:MeOH). The obtained residue (0.024 g, 26% yield over 3 steps) was used without further purification.
The residue (1.0 eq.) was dissolved in TFA (1.0 mL) and DMS (1.0 mL). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a mixture of the residue (1.0 eq.), 4-fluorobenzylamine (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1 mL). Subsequently, iPr2NEt (3.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 8a (2.0 mg, 8.7% over 2 steps). MS (ESI): m/z=637 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.61 mins (>92% pure).
The following compounds were or could be made by the procedure described for triazine 8a:
















 








 















Com-










pound
W
X4
X7
R3
R4
R6
R7
(M + H)+





8b
NH
H
H
P
H
NH
Me—
587


8c
NH
H
H
P
H
NMe
Me—
601


8d
NH
H
H
P
H
NH
tBu—
629


 


8e
NH
H
H
P
H
NH




661


 


8f
NH
H
H
P
H
NH




719


 


8g
NH
H
H
P
H
NH
H—
573


8h
NHpara
H
H
P
H
NH
Bn-
663


8i
NH
H
H
P
H
NH
MeO2C(CH2)6—
715


8j
NH
H
H
P
H
NH
Me(CH2)5—
657


 


8k
NH
H
H
P
H
NH




733


 


8l
NH
H
H
P
H
NH




691


 


8m
NH
H
H
P
H
NH




664


 


8n
NH
H
H
P
H
NMe
Bn-
677


8o
NH
H
H
P
H
O
Bn-
664


 


8p
NH
H
H
P
H
NH




664


 


8q
NH
H
H
P
H
NH




664


 


8r
NH
H
H
P
H
NH




721


 


8s
NH
H
H
P
H
NH




721


 


8t
NHmeta
H
H
P
H
NH
Bn-
663


8u
NH
H
—OMe
P
H
NH
Bn-
693


8v
NH
H
—CF3
P
H
NH
Bn-
731


8w
O
H
H
P
H
NH
Bn-
664


 


8x
NH
H
H
P
H
NH




739


 


8y
NH
H
H
P
H
NH




697


 


8z
NH
H
H
P
H
NH




693


 


8aa
NH
H
H
P
H
NH




713


 


8bb
NH
H
H
P
H
NH




739


 


8cc
NH
H
H
P
H
NH




697


 


8dd
NH
H
H
P
H
NH




693


 


8ee
NH
H
H
P
H
NH




739


 


8ff
NH
H
H
P
H
NH




702


 


8gg
NH
H
H




H
NH




653


 


8hh
NH
H
H
P
H
NH




681


 


8ii
NH
H
H
P
H
NH




731


 


8jj
NH
H
H
P
H
NH




731


 


8kk
NH
H
H
P
H
NH




677


 


8ll
NH
H
H
P
H
NH
Ph—
649


 


8mm
NH
H
H
P
H
NH




731


 


8nn
NH
H
H
P
H
NH




681


 


8oo
NH
H
H
P
H
NH




697


 


8pp
NH
H
H
P
H
NH




677


 


8qq
NH
H
H
P
H
NH




731


 


8rr
NH
H
H
P
H
NH




741


 


8ss
NH
H
H
P
H
NH




681


 


8tt
NH
H
H
P
H
NH




677


 


8uu
NH
H
H
P
H
NH




731


 


8vv
NH
H
H
P
H
NH




669


 


8ww
NH
H
H




H
NH
Bn-
619


 


8xx
NH
H
H




H
NH
Me(CH2)5—
613


 


8yy
NH
H
H
—(CH2)5Me
H
NH
Bn-
512


8zz
NH
H
H
P
Me
NH
Bn-
677


8aaa
NMe
H
H
P
H
NH
Bn-
677


 


8bbb
NH
H
H
P
H
NH




699


 


8ccc
NH
H
H
P
H
NH




699


 


8ddd
NH
H
H
P
H
NH




699


 


8eee
NH
H
H
P
H
NH




699


 


8fff
NH
H
H
P
H
NH




699


 


8ggg
NH
H
H
P
H
NH




699


 


8hhh
NH
F
H
P
H
NH




699


 


8iii
NH
CO2Me
H
P
H
NH




739


 


8jjj
NH
H
H




H
NH




538


 


8kkk
NH
H
H




H
NH




637


 


8lll
NH
H
H




H
NH




673


 


8mmm
NH
H
H
P
H
NH




730













Experimental:





Preparation of aniline 9: To a solution of methyl 5-amino salicylate (1.0 eq.), in MeOH (10 mL) was added Boc2O (1.1 eqs.) followed by TEA (1.1 eqs.). The reaction mixture was allowed to stir for 1 hour. Subsequently, imidazole (0.5 eqs.) was added and the reaction mixture stirred for 10 mins at ambient temperature. The reaction mixture was concentrated in vacuo and the residue partitioned between CH2Cl2 and water. The layers were separated and the aqueous extracted with CH2Cl2 (3×). The combined organics were then washed with 0.1 M HCl (lx, aqueous), dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) in DMF (10 mL) was added K2CO3 (1.2 eqs.) followed by BnBr (1.1 eqs.). The reaction mixture was heated to 60° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue partitioned between CH2Cl2 and water. The layers were separated and the aqueous extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
The residue was dissolved EtOAc (3 mL) and concentrated HCl (3 mL) was added. The reaction mixture was stirred for 1 hour at ambient temperature before the reaction mixture was concentrated in vacuo. The residue was used without further purification.




To a solution of cyanuric chloride (1.14 eqs.) and tbutyl 4-aminobenzoate (1.0 eq.) in THF (81 mL) was added iPr2NEt (1.1 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and aniline 9 (1.1 eqs.) in THF (52 mL) was added iPr2NEt (3.0 eqs). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 1 (1.2 eqs.) in THF (1.5 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified via column chromatography over silica gel (2:1/hexanes:EtOAc→98:2/CH2Cl2:MeOH). The obtained residue (0.116 g, 78% yield over 3 steps) was used without further purification.
The residue (1.0 eq.) was dissolved in TFA (1.0 mL) and DMS (1.0 mL). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a mixture of the residue (1.0 eq.), 4-fluorobenzylamine (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1 mL). Subsequently, iPr2NEt (3.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 8iii (25.0 mg, 18% yield over 2 steps).
A heterogeneous mixture of triazine 8iii (1.0 eq.) and LiOH (30 eq.) in THF (0.3 mL), MeOH (0.3 mL) and H2O (0.3 mL) was stirred at ambient temperature overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 10a (13.0 mg, 59% yield):MS (ESI): m/z=725 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.91 mins (>96% pure).
The following compounds were or could be made by the procedure described for triazine 10a:
















 








 
















Compound
W
X3
X4
C
R3
R4
R6
R7
(M + H)+





10b
NH
OH
H
0
P
H
NH
CO2H(CH2)6—
701


 


10c
NH
OH
H
0
P
H
NH




707


 


10d
NH
OH
H
0
P
H
NH




707


 


10e
NH
OH
H
0
P
H
NH




707


 


10f
NH
CO2H
H
1
P
H
NH
Bn-
705


10g
NH
CH2CO2H
H
0
P
H
NH
Bn-
705


 


10h
NH
CO2H
H
0
P
H
NH




709













Experimential:





Preparation of amine 11: To a solution of ethanolamine (1.0 eq.) and imidazole (2.0 eqs.) in CH2Cl2 (8.0 mL) was added TBSCl (1.5 eqs.). The reaction mixture was quenched by addition of 1.0 M NaHSO4 in water. The layers were separated and the aqueous extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.




Preparation of triazine 12: To a solution of cyanuric chloride (1.14 eqs.) and tbutyl 4-aminobenzoate (1.0 eq.) in THF (81 mL) was added iPr2NEt (1.1 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was partitioned between water and EtOAc, the layers separated and the aqueous layer was extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and 4-benzyloxyaniline (1.1 eqs.) in THF (52 mL) was added iPr2NEt (3.0 eqs). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 1 (1.2 eqs.) in THF (1.5 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified via column chromatography over silica gel (2:1/hexanes:EtOAc→98:2/CH2Cl2:MeOH). The obtained residue (0.116 g, 78% yield over 3 steps) was used without further purification.
The residue (1.0 eq.) was dissolved in TFA (1.0 mL) and DMS (1.0 mL). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a mixture of the residue (1.0 eq), amine 11 (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1.0 mL). Subsequently, iPr2NEt (3.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo. The residue was dissolved in THF (1.0 mL) and to the solution was added TBAF (1.1 eqs.). The reaction mixture was stirred for 1 hour. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 12 (13.0 mg, 41% yield): MS (LCMS, ESI): Rt=5.24 mins (>90% pure) m/z=617(M+H)+.




Experimental:





A solution of cyanuric chloride (1 eq.) in THF (5 mL) was treated with 4-benzyloxyaniline hydrochloride (0.85 eq.) and iPr2NEt (2.0 eqs.) with stirring at ambient temperature for 0.5 h. The reaction mixture was poured into 50 mL EtOAc and the organic solution was washed with H2O (2×) and with brine (1×). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The obtained residue was used without further purification.
To a solution of the residue (1.0 eq.) in THF (29 mL) was added iPr2NEt (2.0 eqs.) followed by aniline (1.1 eqs.). The reaction mixture was heated to 50° C. and stirred for 4 hours. The reaction mixture was cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1 eq.) and Boc-1-amino-3,6-dioxa-8-octanediamine (1.1 eqs.) in THF (29 mL) was added iPr2NEt (2.5 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1 eq.) in CH2Cl2 (29 mL) was added TFA (9.5 mL). The reaction mixture was stirred for 1 h after which the reaction mixture was concentrated in vacuo. The residue was used without further purification.
Preparation of triazine 13a: To a solution of the residue (1 eq.) and 4-methoxybenzoyl chloride (1.1 eqs.) in CH2Cl2 (1.0 mL) was added iPr2NEt (4 eqs.). The reaction mixture was stirred overnight. The reaction mixture was then concentrated in vacuo and the residue used without further purification.
A solution of the residue (1 eq.) in DMS (0.5 mL) and TFA (0.5 mL) was heated to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 13a (6.6 mg, 25% overall yield). MS (LCMS, ESI): Rt=6.43 mins (>90% pure) m/z=560 (M+H)+.
Preparation of triazine 13b: To a solution of the residue (1 eq.) and benzenesulfonyl chloride (1.1 eqs.) in CH2Cl2 (1.0 mL) was added iPr2NEt (4 eqs.). The reaction mixture was stirred overnight. The reaction mixture was then concentrated in vacuo and the residue used without further purification.
A solution of the residue (1 eq.) in DMS (0.5 mL) and TFA (0.5 mL) was heated to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 13b (5.8 mg, 22% overall yield). MS (LCMS, ESI): Rt=6.98 mins (>90% pure) m/z=566 (M+H)+
Preparation of triazine 13c: To a mixture of the residue (1.0 eq.), α, α, α-trifluoromethyl toluic acid (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1.0 mL). Subsequently, iPr2NEt (10 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
A solution of the residue (1 eq.) in DMS (0.5 mL) and TFA (0.5 mL) was heated to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 13c (11.0 mg, 32% overall yield). MS (LCMS, ESI): Rt=7.05 mins (>90% pure) m/z=598 (M+H)+
The following compounds were or could be made by the procedures described for triazines 13a, 13b or 13c:
















 












Compound
W
X4
R3
R4
(M + H)+





13d
NH
H




H
531


 


13e
NH
H




H
548


 


13f
NH
H




H
608


 


13g
NH
H




H
531


 


13h
NH
H




H
548


 


13i
NH
H




H
566


 


13j
NH
H




H
608


 


13k
NH
H




H
560


 


13l
NH
H




H
536


 


13m
NH
H




H
606


 


13n
NH
H




H
468


 


13o
NH
H




H
545


 


13p
NH
H




H
470


 


13q
NH
H




H
484


 


13r
NH
H




H
498


 


13s
NH
H




H
512













Experimental:





Preparation of ester 14: To a solution of 4-(aminomethyl)benzoic acid (1 eq.) in benzene (0.3 mL) and MeOH (0.3 mL) was added TMSCHN2 (1.3 eqs.). The reaction mixture was stirred for 1.0 h, after which the reaction mixture was concentrated in vacuo. Ester 14 was used without further purification.




Preparation of triazine 15: To a solution of cyanuric chloride (1.14 eqs.) and i butyl 4-aminobenzoate (1.0 eq.) in THF (81 mL) was added iPr2NEt (1.1 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and 4-benzyloxyaniline.HCl (1.1 eqs.) in THF (52 mL) was added iPr2NEt (3.0 eqs). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc (3×), and the combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 1 (1.2 eqs.) in THF (1.5 mL) was added iPr2NEt (3.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified via column chromatography over silica gel (2:1/hexanes:EtOAc →98:2/CH2Cl2:MeOH). The obtained residue (0.116 g, 78% yield over 3 steps) was used without further purification.
The residue (1.0 eq.) was dissolved in TFA (1.0 mL) and DMS (1.0 mL). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a mixture of the residue (1.0 eq.), amine 14 (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1 mL). Subsequently, iPr2NEt (3.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was then concentrated in vacuo and the used without further purification.
A heterogeneous mixture of the residue (1 eq.) and LiOH (30 eqs.) in THF (0.3 mL), MeOH (0.3 mL) and H2O (0.3 mL) was stirred at ambient temperature overnight. The reaction mixture was then acidified via addition of TFA and the reaction mixture concentrated in vacuo. The reaction mixture was concentrated in vacuo and the residue purified via reverse-phase HPLC. The obtained residue was then stirred in methanolic HCl (1.0 mL) overnight. Upon completion, the reaction mixture was concentrated in vacuo to yield triazine 15 (2.0 mg, 6.5% overall yield):MS (ESI): m/z=721 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.42 mins (>90% pure).




Experimental:





Preparation of amine 16: To a solution of bis(2-hydroxyethyl)ether (1.0 eqs) and iPr2NEt (4.0 eqs.) in CH2Cl2 (24 mL) was added MsCl (2.2 eqs.). The reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was partitioned between water and CH2Cl2, the layers separated, and the aqueous layer extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and NaI (0.2 eqs.) in DMF (24 mL) was added NaN3 (2.3 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was cooled to ambient temperature and partitioned between water and ether. The layers were separated and the aqueous extracted with ether (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) in THF (0.6 mL), ether (3 mL) and 1.0 M HCl (3 mL, aqueous) was added PPh3 (1.05 eqs.). The reaction mixture was stirred at ambient temperature for 60 hours. Subsequently, the organics were removed from the reaction mixture in vacuo. Diethyl ether was added and the layers separated and the ethereal layer washed with 4.0 M HCl in water (50 mL). The combined aqueous layers were extracted with ether (2×) and the aqueous layer basicified to a pH ̃14 by addition of solid NaOH. The aqueous layer was then extracted with CH2Cl2 (3×) and the combined dichloromethane layers dried over Na2SO4, filtered and concentrated in vacuo. The obtained residue was used without further purification.
A solution of the residue (1.0 eq.) and Boc2O (1.1 eqs.) in CH2Cl2 (70 mL) was added TEA (2.0 eqs.). The reaction mixture was stirred at ambient temperature for 2.0 hours. The reaction mixture was concentrated in vacuo. The residue was filtered over a plug of silica gel (2:1/hex:EtOAc) and the obtained residue (1.27 g, 38%) used with any further purification.
To a biphasic solution of the residue (1.0 eq.) in THF (23 mL) and water (6.6 mL) was added PMe3 (5.0 eqs.). The reaction mixture was stirred for 2 hours. Subsequently, the reaction mixture was concentrated in vacuo and the residue dissolved in CH2Cl2 (53 mL). To the reaction mixture was added trifluoroacetic anhydride (1.2 eqs.) followed by TEA (2.0 eqs.). The reaction mixture was stirred at ambient temperature overnight. The mixture was then partitioned between water and CH2Cl2, the layers separated and the aqueous extracted with CH2Cl2 (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without any further purification.
To a solution of the residue (1.0 eq.) in EtOAc (40 mL) was added concentrated HCl (13.6 mL). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was then concentrated in vacuo and the obtained amine 16 was used without any further purification.




Preparation of triazines 17a, 17b and 17c: To a solution of cyanuric chloride (1.14 eqs.) and tbutyl 4-amino benzoate (1.0 eq.) in THF (51 mL) was added iPr2NEt (2.0 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. To the solution was then added 4-benzyloxy aniline hydrochloride (1.3 eqs.) and iPr2NEt (2.0 eqs.). The reaction mixture was warmed to 50° C. and stirred overnight. The reaction mixture was then cooled to ambient temperature and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and amine 16 (1.07 eqs.) in THF (51 mL) was added iPr2NEt (6.0 eqs.). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue filtered over a plug of silica gel (2:1/hexanes:EtOAc→98:2/CH2Cl2:MeOH). The organics from the 98:2/CH2Cl2:MeOH were collected and concentrated in vacuo. The obtained residue (1.69 g, 49% crude yield) was used without further purification.
The obtained residue (1.0 eq.) was dissolved in TFA (10 mL) and the solution was stirred for 1 hour at ambient temperature. The reaction mixture was concentrated in vacuo and the residue dissolved in DMF (25 mL). To the reaction mixture was added benzylamine (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.). Subsequently, iPr2NEt (5.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a solution of the residue (1.0 eq.) in MeOH (22 mL) and water (4.0 mL) was added K2CO3 (5.0 eqs.). The heterogeneous mixture was heated to 45° C. and stirred vigorously overnight. Subsequently, the reaction mixture was concentrated in vacuo and the residue used without further purification.
For 17a: To a mixture of the residue (1.0 eq.) and 4-fluorobenzoyl chloride (1.1 eqs) was added DMF (1.0 mL) followed by iPr2NEt (2.0 eqs.). The reaction mixture was stirred overnight. Subsequently, the reaction mixture was concentrated in vacuo and the residue used without further purification. The residue (1.0 eq.) was dissolved in TFA (1 mL) and DMS (1 mL) and the obtained reaction mixture was warmed to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 17a (3.0 mg, 9.0% overall yield). MS (ESI): ink=637 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.57 mins (>90% pure).
For 17b: To a mixture of the residue (1.0 eq.), α, α, α-trifluoromethyl toluic acid (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.) was added DMF (1.0 mL). Subsequently, iPr2NEt (2.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification. The residue (1.0 eq.) was dissolved in TFA (1 mL) and DMS (1 mL) and the obtained reaction mixture was warmed to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 17b (3.4 mg, 10.7% overall yield). MS (ESI): m/z=687 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.83 mins (>90% pure).
For 17c: To a solution of the residue (1.0 eq.) in DCE/EtOH (2 mL, 1:1/v:v) was added benzaldehyde (1.2 eqs.) and AcOH (3 drops). The reaction mixture was stirred for 1 hour before sodium trisacetoxy borohydride (5.0 eqs.) was added. The reaction mixture was concentrated in vacuo and the residue used without further purification. The residue (1.0 eq.) was dissolved in TFA (1 mL) and DMS (1 mL) and the obtained reaction mixture was warmed to 40° C. and stirred overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 17c (2.4 mg, 8.0% overall yield). MS (ESI): m/z=605 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=12.85 mins (90% pure).
The following compounds were or could be made by the procedure described for triazines 17a, 17b or 17c:
















 













Compound
W
R4
R6
R7
R11
(M + H)+





17d
NH
H
NH
Bn-




648


 


17e
NH
H
NH
Bn-




662


 


17f
NH
H
NH
Bn-




620


 


17g
NH
H
NH
Bn-




649


 


17h
NH
H
NH
Bn-




655


 


17i
NH
H
NH
Bn-




634


 


17j
NH
H
NH
Bn-




557













Experimental:





To a solution of cyanuric chloride (1.14 eqs.) and methyl 4-amino benzoate (1.0 eq.) in THF (3.0 mL) was added iPr2NEt (4.0 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour. To the solution was then added 4-[(N-Boc)aminomethyl]aniline (1.3 eqs.) and the reaction mixture warmed to 50° C. and stirred overnight. The reaction mixture was then cooled and partitioned between EtOAc and water. The layers were separated and the aqueous extracted with EtOAc (3×). The combined organics were dried over Na2SO4, filtered and concentrated in vacuo. The residue was used without further purification.
To a solution of the residue (1.0 eq.) and 4-benzyloxy aniline hydrochloride (2.0 eqs.) in THF (3.5 mL) was added iPr2NEt (4.0 eqs). The reaction mixture was warmed to 65° C. and stirred overnight. The reaction mixture was concentrated in vacuo and the residue filtered over a plug of silica gel (2:1/hexanes:EtOAc→98:2/CH2Cl2:MeOH). The organics collected from the 98:2/CH2Cl2:MeOH elution were concentrated in vacuo and the obtained residue was used without further purification.
A heterogeneous mixture of the residue (1.0 eq.) and LiOH (30 eqs.) in THF (1.0 mL), MeOH (1.0 mL) and H2O (1.0 mL) was stirred at ambient temperature overnight. The reaction mixture was concentrated in vacuo and the residue dissolved in DMF (3.0 mL). To the solution was added 4-fluorobenzylamine (1.1 eqs.), EDAC.HCl (1.2 eqs.) and anhydrous HOBT (1.2 eqs.). Subsequently, iPr2NEt (4.0 eqs.) was added to the reaction mixture and the resulting solution stirred overnight. The reaction mixture was concentrated in vacuo and the residue used without further purification.
The residue (1.0 eq.) was dissolved in TFA (3.5 mL) and the reaction mixture stirred at ambient temperature for 1 hour. The reaction mixture was concentrated in vacuo and the residue used without further purification.
To a solution of the residue (1.0 eq.) in DCE:EtOH (1.5 mL:1.5 mL) was added benzylaldehyde (1.2 eqs.). The reaction mixture was stirred at ambient temperature for 1 hour before sodium trisacetoxy borohydride (5.0 eqs.) was added. The reaction mixture was allowed to stir an additional 3 hours at ambient temperature before being concentrated in vacuo. The residue was then dissolved in TFA (1.5 mL) and DMS (1.5 mL) and stirred at 45° C. overnight. The reaction mixture was then concentrated in vacuo and the residue purified via reverse phase HPLC to yield triazine 18 (5.2 mg, 2.4% overall yield). MS (ESI): m/z=641 (M+H)+; analytical HPLC (10-90% MeCN in H2O, 20 mins, flow rate=1.0 mL/min.) Rt=14.33 mins (95% pure).
B. Inhibitory Properties of Compounds
MDCK cells stably transfected with rat PGT (Endo et al., 2002) were seeded at 15-20% confluence on 24-well plates. The day on which the cells were seeded was considered day 1. PGE2 uptake experiments were conducted on day 4. All of the PGE2 uptake experiments were conducted at room temperature. On day 4, cells were washed twice with Waymouth buffer (135 mM NaCl, 13 mM H-Hepes, 13 mM Na-Hepes, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.8 mM MgSO4, 5 mM KCl, and 28 mM D-glucose). Then 200 μL of Waymouth buffer containing [3H]PGE2 (purchased from Perkin Elmer) was added to each well. At the designed time, the uptake of [3H]PGE2 was stopped by aspiration of uptake buffer; this was followed by immediate washing twice with 500 μL of chilled Waymouth buffer. Cells were then lysed with 100 μL lysis buffer containing 0.25% SDS and 0.05 N NaOH. 1.5 mL of scintillation solution was added to each well, and intracellular [3H]PGE2 was counted by MicroBeta Counter.
For preliminary testing of the compounds, 20 μL of Waymouth buffer containing the compound was added to each well; this was immediately followed by the addition of 180 μL of Waymouth buffer containing [3H]PGE2. In each well, the total volume of uptake medium was 200 μL. Organic compounds were first dissolved in EtOH and then diluted in Waymouth buffer. The percent inhibition of [3H]PGE2 uptake by compounds was calculated as [(uptakevehicle−uptakeinhibitor)÷(uptakevehicle)]×100.
To determine IC50 of each compound, 20 μof Waymouth buffer containing various concentrations of the compound was added to each well; this was immediately followed by the addition of 180 μL of Waymouth buffer containing [3H]PGE2. IC50 was calculated by fitting an equation of y=m1−m1*(m0/(m2+m0)).
Results for the compounds are presented in Table 1, Inhibitory Activities of PGT Inhibitors. Abbreviations: Bn=benzyl (—CH2Ph), Bz=benzoyl (—(C═O)Ph), Me=methyl (—CH3), Ph=phenyl.







TABLE 1







Inhibitory Activities of PGT Inhibitors




















Inh
Inh
Inh
Inh
Inh
Inh






(% of
(% of
(% of
(% of
(% of
(% of


Cpd

Mol.
IC50
Ctl)
Ctl)
Ctl)
Ctl)
Ctl)
Ctl)


ID
Structure
Weight
(nM)
0.1 μM
0.5 μM
1 μM
2.5 μM
5 μM
10 μM



















T26A





378

72.65






 


1




543.62


9.6


8.5



 


2




619.71


1.6


6.5



 


3




563.05


0.4


1.7



 


4




529.59
4850

7.0
32.4

67.6
81.5


 


5




571.67


6.6


9.6



 


6




587.67


3.6


11.7



 


7




689.80


4.8


22.5



 


8




843.81
223.8

52.3






 


9




481.55


1.3


18.8



 


10




497.55


1.4


15.0



 


11




599.68


11.4


29.9



 


12




568.05


2.8


 4.7



 


13




687.62


19.2


19.3



 


14




700.66


10.6


35.9



 


15




714.69


15.0


16.6



 


16




742.74
9770

11.8


44.8



 


17




701.65
6570

22.0


55.6



 


18




774.74


21.1


48.9



 


19




832.82


41.3


75.0



 


20




686.63


9.6


14.4



 


21




730.69


15.7


68.2



 


22




776.76
42.7

62.4


88.1
95.5


 


23




673.64


14.4


38.4



 


24




728.71


1.7


 9.2



 


25




658.62


12.9


27.7



 


26




673.64


2.6


35.2



 


27




649.66


28.2


53.8



 


28




659.61


2.4


56.5



 


29




758.62



15.9


49.6


 


30




661.6



26.1


78.25


 


31




722.51



12.2


69.2


 


32




673.64



29.0


71.0


 


33




758.62



13.2


54.6


 


34




661.6



25.8


81


 


35




679.59



20.4


75.6


 


36




722.51



30.3


81.1


 


37




673.64



32.4


79.0


 


38




679.66



22.8


63.0


 


39




649.64



42.9


86.2


 


40




719.71



16.3


46.9


 


41




581.54



0.3


 6.6


 


42




658.62



22.8


57.6


 


43




828.83
1300


34.8


84.1


 


44




770.79
760


65.0


77.5


 


45




653.52



5.0


12.9


 


46




722.69



4.1


32.4


 


47




846.8



22.6


76.5


 


48




700.78



14.2


62.8


 


49




568.5



1.7


12.5


 


50




550.53



9.3


15.1


 


51




440.45



17.4


29.8


 


52




422.48



0.1


 1.8


 


53




667.63
4040


65.2

83.4
86.4


 


54




695.65



33.7


52.2


 


55




492.49



12.7


26.1


 


56




678.06



38.0


80.5


 


57




571.63
5570


27.4

29.5
56.6


 


58




557.6



16.25

41.0
52.5


 


59




599.56
2460


36.8


75.8


 


60




699.67



 7.2


34.5


 


61




685.65



 7.5


46.1


 


62




611.61



20.4


50.1


 


63




625.75



11.0


44.1


 


64




800.78
115

15.5
71.6

77.2
90.2


 


65




794.82
1380

18.4
66.4

84.0
90.0


 


66




804.81


10.9


64.4



 


67




891.77


13.4


76.1



 


68




790.78


15.0


70.8



 


69




777.74
890

24.9


83.2



 


70




891.77
1050

21.2


84.0



 


71




891.77


16.7


78.4



 


72




711.61



19.7

66.1
75.3


 


73




834.79




38.9
75.3



 


74




834.79

45.0
51.6

56.9
82.0



 


75




667.63



8.4

10.1
12.5


 


76




769.81


13.5

35.9
70.5



 


77




712.63


1.8

 4.3
23.2



 


78




776.76


15.5

24.5
83.2



 


79




706.75


5.4

29.2
54.9



 


80




706.75


0.7

21.8
57.2



 


81




806.78


0.4

16.2
80.1



 


82




844.76


2.1

5.0
47.6



 


83




777.74
609

4.1

10.4
42.0



 


84




583.56



5.7

13.7
37.1


 


85




597.58



7.6

12.2
49.6


 


86




806.78
870
50.0
70.7

81.4
86.4



 


87




826.82
710
55.1
71.8

83.5
90.6



 


88




842.85
820
61.8
67.6

82.6
88.2



 


89




811.2


33.8

76.5
86.0



 


90




806.78


23.4

60.6
72.7



 


91




852.85
620
60.8
72.1

84.0
88.0



 


92




815.79


33.7

71.3
80.6



 


93




806.78


24.6

43.3
51.1



 


94




826.82


36.0

66.7
76.1



 


95




668.62



1.9

23.4
50.4


 


96




611.61



8.6

23.9
51.9


 


97




625.64



13.1

19.4
30.8


 


98




767.15


6.7

24.9
75.1



 


99




794.75
23.7
81.3
61.8

77.1
89.4



 


100




845.65

41.1
56.4

57.6
86.0



 


101




844.76

47.4
54.0

71.0
91.4



 


102




790.78


41.9

65.7
88.0



 


103




762.73


38.3

62.2
83.7



 


104




845.65

46.6
61.6

87.8
91.6



 


105




794.75
47.5
67.9
64.6

81
92.45



 


106




811.2

68.4
64.8

68.8
93.1



 


107




790.78

65.2
56.3

80.7
91.3



 


108




845.65

70.3
60.8

78.3
76.6



 


109




855.65

49.5
55.6

59.8
85.1



 


110




794.75
31.3
69.6
60.6

78.3
93.6



 


111




790.78

52.2
59.1

81.5
93.2



 


112




706.75


17.5

32.2
49.4



 


113




844.76

36.8
49.2

84.1
92.4



 


114




834.79

41.0
61.4






 


115




782.81

54.4
76.8






 


116




760.76

36.4
59.4






 


117




832.82

 8.1
36.7






 


118




732.7
20.9
74.2
90.7






 


119




726.74

57.2
81.4






 


120




625.64

11.2
29.1






 


121




704.77

 1.0
28.8






 


122




832.82

 5.8
38.1






 


123




790.78

12.0
36.6






 


124




790.78
34
68.8
85.4






 


125




704.77

3.2
24.3






 


126




812.74

24.0
56.3
70.0





 


127




812.74

17.0
52.3
70.2





 


128




812.74

36.6
71.8
82.2





 


129




812.74

 8.9
45.1
65.6





 


130




812.74

41.6
73.2
81.7





 


131




812.74

22.3
47.0
74.3





 


132




875.77

15.0
22.1
32.4





 


133




889.79

 4.3
12.6
19.4





 


134




800.7

14.0
27.7
46.7





 


135




847.71

13.2
28.4
49.6





 


136




750.7
23.9
33.3
64.1
77.9





 


137




762.73
36.7
42.0
64.9
75.6





 


138




768.76

17.8
22.5
34.5





 


139




747.72

16.3
30.7
39.0





 


140




670.64

11.5
4.5
10.9





 


141




718.72

12.7
22.0
43.5





 


142




932.79

8.4
20.1
39.2





 


143 (85% pure)




812.74
16.5
33.7
57.8
69.5





 


144




852.78

 3.4
5.8
10.8





 


145




651.56

7.1
3.1
6.9





 


146




724.74
44.8
28.5
59.8
70.4





 


147




750.7
31.7
44.5
70.2
79.5





 


148




786.7

 8.4
12.9
20.7





 


149




868.75

 3.8
9.1
17.6





 


150




708

4.2
14.3
23.9





 


151







0.7
8.4









C. Acceleration of Wound Healing by Inhibitors of Prostaglandin Transporter (PGT)

Studies were conducted using compound T26A (Table 1). Two full-thickness wounds with diameter of 5 mm were created on the back of mice symmetrically under the shoulder blades. 50 μL vehicle (2% DMSO+2% cremophor in water) was applied to one wound (FIG. 1A, top panel), and that of 2 mM T26A was applied to the other (FIG. 1A, bottom panel), immediately after surgery and every other day afterwards. T26A accelerates wound healing as shown over a 10 day period in FIG. 1A. T26A increased vascularization and blood flow in the wound. FIG. 1B shows averaged wound areas of 4 mice, each of them had 2 wounds and received topically applied vehicle Veh1 (2% DMSO+2% cremophor in water) on one wound and T26A on the other. On the wounds of a separate group of 4 mice, vehicle Veh2 (2% EtOH, yellow) was applied to one wound and 200 μM PGE2 (green) was applied to the other. Results were statistically significant (p<0.05, T26A vs. vehicle).
REFERENCES
Alm A (1998) Prostaglandin derivates as ocular hypotensive agents. Progress in Retinal and Eye Research 17:291-312.
Bao Y, Pucci M L, Chan B O, Lu R, Ito S and Schuster V L (2002) Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids. American Journal of Physiology 282:F1103-1110.
Bito L Z and Salvador E V (1976) Effects of anti-inflammatory agents and some other drugs on prostaglandin biotransport. J. Pharmacol. Exp. Ther. 198:481-488.
Blatteis C M and Sehic E (1997) Fever: How may circulating pyrogens signal the brain? News in Physiological Sciences. 12:1-9.
Bley K R, Hunter J C, Eglen R M and Smith J A (1998) The role of IP prostanoid receptors in inflammatory pain. Trends Pharmacol Sci 19:141-147.
Bos C L, Richel D J, Ritsema T, Peppelenbosch M P and Versteeg H H (2004) Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 36:1187-1205.
Chi Y, Khersonsky S M, Chang Y T, Schuster V L. Identification of a new class of prostaglandin transporter inhibitors and characterization of their biological effects on prostaglandin E2 transport. J Pharmacol Exp Ther. 2006 March; 316(3):1346-50. Epub 2005 November 3.
Chi Y, Min J, Jasmin J-F, Duranski M, Seki Y, Nomura T, Folkert I W, Charron M J, Lisanti M, Lefer D J, Chang Y-T, Schuster V L. A small molecule inhibitor of the prostaglandin transporter PGT lowers blood pressure by vasodilation and natriuresis and inhibits platelet aggregation. Manuscript, 2009.
Clyman R1, Mauray F, Roman C and Rudolph A M (1978) PGI2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGF1alpha. Prostaglandins 16:259-264.
Coceani F and Olley P M (1988) The control of cardiovascular shunts in the fetal and perinatal period. Can J Physiol Pharmacol 66:1129-1134.
Endo S, Nomura T, Chan B S, Lu R, Pucci M L, Bao Y and Schuster V L (2002) Expression of PGT in MDCK cell monolayers: polarized apical localization and induction of active PG transport. American Journal of Physiology 282:F618-F622.
Epstein M (1986) Prostaglandins and the kidney. American journal of medicine; v. 80, no. 1A, 1986. Technical Publishing, New York, N.Y.
Ferrara N and Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocr Rev 18: 4-25.
Helliwell R J, Adams L F and Mitchell M D (2004) Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins Leukotrienes and Essential Fatty Acids 70:101-113.
Jacquemin E, Hagenbuch B, Stieger B, Wolkoff A W and Meier P J (1994) Expression cloning of a rat liver Na+-independent organic anion transporter. Proc Natl Acad Sci USA 91:133-137.
Kanai N, Lu R, Satriano J A, Bao Y, Wolkoff A W and Schuster V L (1995) Identification and characterization of a prostaglandin transporter. Science 268:866-869.
Mann J R, Backlund M G, Buchanan F G, Daikoku T, Holla V R, Rosenberg D W, Dey S K, and DuBois R N (2006) Repression of prostaglandin dehydrogenase by epidermal growth factor and snail increases prostaglandin E2 and promotes cancer progression. Cancer Res 66: 6649-56.
Narumiya S, Sugimoto Y and Ushikubi F (1999) Prostanoid receptors: structures, properties, and functions. Physiological Reviews 79:1193-1226.
Nomura T, Lu R, Pucci M L and Schuster V L (2004) The two-step model of prostaglandin signal termination: in vitro reconstitution with the prostaglandin transporter and prostaglandin 15 dehydrogenase. Mol Pharmacol 65:973-978.
Samad T A, Sapirstein A and Woolf C J (2002) Prostanoids and pain: unraveling mechanisms and revealing therapeutic targets. Trends Mol Med 8:390-396.
Seybold V S, Jia Y P, and Abrahams L G. Cyclo-oxygenase-2 contributes to central sensitization in rats with peripheral inflammation. Pain. 2003 September; 105(1-2):47-55.
Schuster VL (2002) Prostaglandin Transport. Prostaglandins and Other Lipid Mediators 68-69:633=647.
Shao J, Sheng G G, Mifflin R C, Powell D W, and Sheng H (2006) Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis. Cancer Res 66: 846-55.
Sheng H, Shao J, Washington M K, and DuBois RN (2001) Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 276: 18075-81.
Smith G C S, Coleman R A and McGrath J C (1994) Characterization of dilator prostanoid receptors in the fetal rabbit ductus arteriosus. Journal of Pharmacology & Experimental Therapeutics 271:390-396.
Southall M D and Vasko M R (2000) Prostaglandin E(2)-mediated sensitization of rat sensory neurons is not altered by nerve growth factor. Neurosci Lett 287: 33-36.
Southall M D and Vasko M R (2001) Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2-induced cAMP production and sensitization of sensory neurons. J Biol Chem 276: 16083-91.
Stjernschantz J (1995) Prostaglandins as ocular hypotensive agents; development of an analogue for glaucoma treatment. Advances in Prostaglandin Thromboxane and Leukotriene Research 23:63-68.
Stjernschantz J (2004) Studies on ocular inflammation and development of a prostaglandin analogue for glaucoma treatment. Experimental Eye Research 78:759-766.
Susanna R, Jr., Chew P and Kitazawa Y (2002) Current status of prostaglandin therapy: latanoprost and unoprostone. Survey in Ophthalmology 47 Suppl 1:S97-104.
Sweet D H, Wolff N A and Pritchard J B (1997) Expression cloning and characterization of ROAT1. The basolateral organic anion transporter in rat kidney. J. Biol. Chem. 272:30088-30095.
Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois R N (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93: 705-716.
Ulmann A, Silvestre L, Chemama L, Rezvani Y, Renault M, Aguillaume C J. and Baulieu E E (1992) Medical termination of early pregnancy with mifepristone (RU 486) followed by a prostaglandin analogue. Study in 16,369 women. Acta Obstet. Gynec. Scand. 71:278-283.
Vanegas H and Schaible H G (2001) Prostaglandins and cyclooxygenases [correction of cycloxygenases] in the spinal cord. Prog Neurobiol 64:327-363.
Wang J L, Cheng H F, Zhang M Z, McKanna J A and Harris R C (1998) Selective increase of cyclooxygenase-2 expression in a model of renal ablation. Am. J. Physiol. 275:F613-F622.
Yokoyama C, Yabuki T, Shimonishi M, Wada M, Hatae T, Ohkawara S, Takeda J, Kinoshita T, Okabe M and Tanabe T (2002) Prostacyclin-deficient mice develop ischemic renal disorders, including nephrosclerosis and renal infarction. Circulation 106:2397-2403.
Young M R (2004) Tumor-derived prostaglandin E2 and transforming growth factor-beta stimulate endothelial cell motility through inhibition of protein phosphatase-2A and involvement of PTEN and phosphatidylinositide 3-kinase. Angiogenesis 7: 123-131.
U.S. Pat. No. 5,792,851, issued Aug. 11, 1998, Human Prostaglandin Transporter, Schuster et al.
PCT International Patent Application Publication No. WO 2007/136638 A2, published Nov. 29, 2007, Prostaglandin Transporter Inhibitors.

## Claims

1. A compound having the structure:



wherein W is O or NR5;




R4 and R5 are independently H or —CH3;
R6 is O or NR9;
R7 is H, —CH3, —C(CH3)3, —CH2OH, —(CH2)2OH, —(CH2)2O(CH2)2OH, —(CH2CH2O)3CH3, —(CH2CH2O)2CH2CO2CH3, —(CH2)5CH3,




R8 is H, —OH, —CH2OH, —CO2H, —CO2CH2CH3, —CO(CH2)6CH3, —OCH3, —NH2, —SO2NH2, —CONH-Bn or




R9 is H or —CH3;
R10 is —CH2NH2, —CO2H or —CO2CH3;
R11 is —SO2-Ph, —CH2-Ph, —CONH-Ph, —COCH3,





and
R12 is —CH3,




where X1, X2, X3, X4, X5 and X6 are independently H, halogen, —OH, —CH3, —CF3, —OCH3, —CO2H, —CO2CH3, —CH2CO2H or —CH2CO2CH3 ;
where X7 is independently H, halogen, —OH, —CH3, —CF3, —OCH3, —CO2H, —CO2CH3, —CH2CO2H, —CH2CO2CH3, phenyl or —O-Bn; and
where a=1-2; b=1-5; c=0-1; d=4-7; and e=0-1;
or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein W is NR5.

3. The compound of claim 1, wherein R6 is NR9.

4. The compound of claim 1, wherein at least one of R4, R5 and R9 is H.

5. The compound of claim 1, wherein all of R4, R5 and R9 are H.

6. The compound of claim 1, wherein R8 is located in para position.

7. The compound of claim 1, wherein one of X1 and X2 is H, and the other is halogen, —CF3, —CH3, —CO2H, —CO2CH3, —OCH3 or phenyl.

8. The compound of claim 1, wherein both X1 and X2 are halogen.

9. The compound of claim 1, wherein one of X3 and X4 is H, and the other is halogen, —CO2H, —CO2CH3, —CH2CO2H, —CH2CO2CH3, —OH, —OCH3 or —O-Bn.

10. The compound of claim 1, wherein one of X3 and X4 is —OH, and the other is halogen, —CO2H or —CO2CH3.

11. The compound of claim 1, wherein one of X5 and X6 is H, and the other is halogen, —CF3, —OCH3 or phenyl.

12. The compound of claim 1, wherein both X5 and X6 are halogen.

13. The compound of claim 1, wherein X7 is H, —CF3 or —OCH3.

14. The compound of claim 1, wherein one or both of X1 and X2 are located in ortho position.

15. The compound of claim 1, wherein one or both of X1 and X2 are located in meta position.

16. The compound of claim 1, wherein X1 is located in meta position and X2 is located in para position.

17. The compound of claim 1, wherein X1 is located in ortho position and X2 is located in para position.

18. The compound of claim 1, wherein X3 is in meta position and X4 is in para position.

19. The compound of claim 1, wherein X5 or X6 is in meta position.

20. The compound of claim 1, wherein X5 or X6 is in para position.

21. The compound of claim 1 having the structure:



or a pharmaceutically acceptable salt thereof.

22. The compound of claim 1 having the structure:



or a pharmaceutically acceptable salt thereof.

23. The compound of claim 1 having the structure:



or a pharmaceutically acceptable salt thereof.

24. The compound of claim 1, wherein
R1 is




R2 is





and
R3 is





and
where X1 is H or halogen;
where X4 is H, halogen or —CO2H; and
where X5 is H, halogen or —OCH3;
or a pharmaceutically acceptable salt thereof.

25. The compound of claim 1, where R5 is H.

26. The compound of claim 1 having the structure:



or a pharmaceutically acceptable salt thereof.

27. The compound of claim 1, wherein the halogen is Br, Cl or F.

28. The compound of claim 1 having the structure:






or a pharmaceutically acceptable salt thereof.

29. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

30. A method of treating rheumatoid arthritis in a subject comprising administering a compound of claim 1 to the subject in an amount effective to treat rheumatoid arthritis.

31. A compound having the structure:


















or a pharmaceutically acceptable salt thereof.