Compounds as mPGES-1 inhibitors

Abstract

The invention relates to amide-derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoic acid for use in a treatment for preventing or suppressing symptoms mediated by enhanced mPGES-1 expression or activity. In particular the invention relates the use of these compounds for treating diseases and conditions in which the inhibition of the enzyme mPGES-1 activity and/or expression would be beneficial such as inflammatory diseases, nociceptive pain, auto-immune diseases, breathing disorders, fever, cancer, inflammation related anorexia, Alzheimer's disease and cardiovascular disease.

Claims

1. A method for treating a disease or condition mediated by enhanced mPGES-1 expression or activity, wherein the method comprises the step of administering to a subject suffering from the disease or condition an effective amount of a compound represented by general structure (I): ##STR00030## wherein, T is represented by structure (IIIa) or (IIIb): ##STR00031## wherein each R.sup.7 is individually a C.sub.1-C.sub.6 alkyl moiety; L is a linker selected from: —(CH.sub.2).sub.2—, —(CH.sub.2).sub.2NHC(O)CH.sub.2—, —(CH.sub.2).sub.3—, —(CH.sub.2).sub.2NHC(NH.sub.2)═, —(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(NH.sub.2)═, —(CH.sub.2).sub.3NHC(NH.sub.2)═, —(CH.sub.2).sub.2NHC(Me)═, —(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(Me)═, —(CH.sub.2).sub.3NHC(Me)═, —(CH.sub.2).sub.2NR.sup.1′C(NH.sub.2)═, —C(CO.sub.2H)(CH.sub.2).sub.3—, —C(CO.sub.2H)(CH.sub.2).sub.3NHC(NH.sub.2)═, —C(CO.sub.2H)CH.sub.2—, —C(CO.sub.2H)(CH.sub.2).sub.2—, —C(CO.sub.2H)(CH.sub.2).sub.4—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —CHR.sup.2′C(O)—, —CHR.sup.2′CH.sub.2—, —CHR.sup.5CH.sub.2NR.sup.5′C(Me)═, —CHR.sup.2′(CH.sub.2).sub.2—, —(CH.sub.2).sub.2CHR.sup.1′—, —(CH.sub.2).sub.2CHR.sup.1′NHC(O)C(Me)—, —CH.sub.2CHR.sup.1′—, —CH.sub.2CHR.sup.1′NHC(Me)═, —CHR.sup.5(CH.sub.2).sub.2CHR.sup.5′—, —CHR.sup.2′CHR.sup.3′ (CH.sub.2).sub.2—, and —CR.sup.5═CH—CH═CR.sup.5′—CH.sub.2—, wherein R.sup.5 and R.sup.5′ represent the connection of a second linker between one backbone atom of the linker, bearing R.sup.5, and another backbone atom of the linker, bearing R.sup.5′, wherein R.sup.5′ is joined with R.sup.5 via the second linker, thus forming a 4-10-membered cyclic structure; N* is represented by structure (IIa) or (IIb) ##STR00032## R.sup.1 and R.sup.2 are each independently selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, or R.sup.1 and R.sup.2 are joined together and thus form a second linker between the amide nitrogen atom and the distal nitrogen atom, or R.sup.1 is joined with R.sup.1′ of the linker L in a cyclic structure and/or R.sup.2 is joined with R.sup.2′ of the linker L in a cyclic structure; R.sup.3 is selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties or (halo)alkoxy moieties, or R.sup.3 is absent when the distal nitrogen atom is part of an imine moiety; or optionally R.sup.3 is joined with R.sup.3′ of the linker L in a cyclic structure; and R.sup.4 is selected from hydrogen (H) or C.sub.1-C.sub.6 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties; X is an anion, wherein the disease or condition is selected from the group consisting of: acute and chronic inflammation, dermatitis, eczema, psoriasis, burns, acne vulgaris, hidradenitis suppurativa, tissue trauma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diverticulitis, irritable bowel disease (IBS), peptic ulcers, cystitis, prostatitis, pancreatitis, nephritis, influenza, rhinitis, pharyngitis, tonsillitis, conjunctivitis, iritis, scleritis, otitis, uveitis, inflammation related anorexia, an allergy, pelvic inflammatory disease, reperfusion injury, transplant rejection, tendinitis, vasculitis, phlebitis, acute pain, chronic pain, neuropathic pain, nociceptive pain, hyperalgesia, pain related to central sensitization, allodynia inflammatory pain, visceral pain, cancer pain, trauma pain, dental or surgery pain, postoperative pain, delivery pain, childbirth ache, persistent pain, peripheral mediated pain, central mediated pain, chronic headache, migraine, sinus headaches, tension headaches, phantom limb pain, peripheral nerve injury chemotherapy pain, cancer pain, arthritis, juvenile arthritis, ankylosing spondylitis, gout, rheumatic fever, bursitis, systemic lupus erythematosus (SLE), multiple sclerosis, sarcoidosis, pulmonary fibrosis, brain cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, melanoma, lymphoma, leukemia, skin T-cell lymphoma, skin B-cell lymphoma, diabetic vasculopathy, diabetic neuropathy, diabetic retinopathy, thrombosis, and coronary heart disease.

2. The method according to claim 1, wherein the compound is represented by structure (VI): ##STR00033## wherein N* is —NR.sup.3 or —N.sup.+R.sup.3R.sup.4X.sup.−, wherein T, X, R.sup.3, and R.sup.4 are as defined in claim 1.

3. The method according to claim 1 wherein the compound is represented by structure (VIIb): ##STR00034## wherein each R.sup.7 is methyl; N* is —NR.sup.3 or —N.sup.+R.sup.3R.sup.4X.sup.−; X is as defined in claim 1; R.sup.3 is as defined in claim 1; and R.sup.4 is as defined in claim 1.

4. The method according to claim 1, wherein symptoms mediated by enhanced mPGES-1 expression or activity at least include one or more of inflammation, pain, swelling, fever, angiogenesis and anorexia.

5. The method according to claim 1, wherein the total daily dose that is administered is in the range of about 5 to 2000 mg.

6. The method according to claim 1, wherein the compound is administered orally.

7. The method according to claim 1, wherein the compound is administered at least twice daily.

8. The method according to claim 7, wherein the interval between two administrations is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

9. The method according to claim 1, wherein the subject to be treated is a primate.

10. The method according to claim 1, wherein R.sup.5—R.sup.5′ is —CH═CH—.

11. The method according to claim 1, wherein T is represented by structure (IVa) or (IVb), N* is represented by structure (IIa), or by structure (IIb) wherein R.sup.4═H and X=Cl, and wherein: (A) L=—(CH.sub.2).sub.2—, R.sup.1—R.sup.2═—(CH.sub.2).sub.2—, R.sup.3═H; (B) L=—(CH.sub.2).sub.2—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (C) L=—(CH.sub.2).sub.2NHC(O)CH.sub.2—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (D) L=—(CH.sub.2).sub.3—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (E) L=—(CH.sub.2).sub.2NHC(NH.sub.2)═, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (F) L=—(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(NH.sub.2)═, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (G) L=—(CH.sub.2).sub.3NHC(NH.sub.2)═, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (H) L=—(CH.sub.2).sub.3—, R.sup.1 ═H, R.sup.2=Me, R.sup.3=Me; (I) L=—(CH.sub.2).sub.2—, R.sup.1 ═H, R.sup.2=Me, R.sup.3=Me; (J) L=—(CH.sub.2).sub.2NHC(Me)=, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (K) L=—(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(Me)=, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (L) L=—(CH.sub.2).sub.3NHC(Me)=, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (M) L=—(CH.sub.2).sub.2NR.sup.1′C(NH.sub.2)═, R.sup.1—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.2═H, R.sup.3=absent; (N) L=—C(CO.sub.2H)(CH.sub.2).sub.3—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (O) L=—C(CO.sub.2H)(CH.sub.2).sub.3NHC(NH.sub.2)═, R.sup.1 ═H, R.sup.2═H, R.sup.3=absent; (P) L=—C(CO.sub.2H)CH.sub.2—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (Q) L=—C(CO.sub.2H)(CH.sub.2).sub.2—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (R) L=—C(CO.sub.2H)(CH.sub.2).sub.4—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (S) L=—C(CO.sub.2H)(CH.sub.2).sub.3—, R.sup.1 ═H, R.sup.2=Me, R.sup.3=Me (T) L=—(CH.sub.2).sub.4—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (U) L=—(CH.sub.2).sub.5—, R.sup.1 ═H, R.sup.2═H, R.sup.3═H; (V) L=—(CH.sub.2).sub.4—, R.sup.1 ═H, R.sup.2=Me, R.sup.3=Me; (W) L=—CHR.sup.2′C(O)—, R.sup.1 ═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.3—, R.sup.3═H; (X) L=CHR.sup.1′CH.sub.2, R.sup.1 ═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.3—, R.sup.3═H; (Y) L=—CHR.sup.5CH.sub.2NR.sup.5′C(Me)=, R.sup.1 ═H, R.sup.2═H, R.sup.5—R.sup.5′═—(CH.sub.2).sub.3—, R.sup.3=absent; (Z) L=—CHR.sup.2′(CH.sub.2).sub.2—, R.sup.1═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.3═H; (AA) L=—(CH.sub.2).sub.2CHR.sup.1′—, R.sup.1—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.2═H, R.sup.3═H; (AB) L=—(CH.sub.2).sub.2CHR.sup.1′NHC(O)C(Me)-, R.sup.1—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.2═H, R.sup.3═H; (AC) L=—CH.sub.2CHR.sup.1′—, R.sup.1—R.sup.1′═—(CH.sub.2).sub.3—, R.sup.2═H, R.sup.3═H; (AD) L=—CH.sub.2CHR.sup.1′NHC(Me)=, R.sup.1—R.sup.1′═—(CH.sub.2).sub.3—, R.sup.2═H, R.sup.3=absent; (AE) L=—CHR.sup.5(CH.sub.2).sub.2CHR.sup.5′—, R.sup.1═H, R.sup.2═H, R.sup.5—R.sup.5′═—(CH.sub.2).sub.2—, R.sup.3═H; (AF) L=CHR.sup.2′CH.sub.2, R.sup.1 ═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.3—, R.sup.3=Me; (AG) L=CHR.sup.2′CH.sub.2, R.sup.1═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.3═H; (AH) L=—CHR.sup.2′(CH.sub.2).sub.2—, R.sup.1 ═H, R.sup.2—R.sup.1′═—(CH.sub.2).sub.2—, R.sup.3=Me; (AI) L=—CHR.sup.2′CHR.sup.3′(CH.sub.2).sub.2—, R.sup.1 ═H, R.sup.2—R.sup.2′═—CH.sub.2—, R.sup.3—R.sup.3′═—(CH.sub.2).sub.2—, R.sup.4═H, X═Cl; or (AJ) L=—CR.sup.5═CH—CH═CR.sup.5′—CH.sub.2—, R.sup.5—R.sup.5′ is —CH═CH—, R.sup.1═H, R.sup.2═H, R.sup.3═H, R.sup.4═H, X═Cl.

12. The method according to claim 1, wherein X is a pharmaceutically acceptable anion.

13. The method according to claim 1, wherein the compound is admixed with an aqueous solution or with an isotonic aqueous solution prior to administration.

14. The method according to claim 13, wherein the isotonic aqueous solution is saline.

15. The method according to claim 1, wherein the subject is human.

16. A method for inhibiting or suppressing functional activity of mPGES-1 in a subject, wherein the method comprises the step of administering to the subject an effective amount of a compound represented by general structure (I): ##STR00035## wherein, T is represented by structure (IIIa) or (IIIb): ##STR00036## wherein each R.sup.7 is individually a C.sub.1-C.sub.6 alkyl moiety; L is a linker selected from: —(CH.sub.2).sub.2—, —(CH.sub.2).sub.2NHC(O)CH.sub.2—, —(CH.sub.2).sub.3—, —(CH.sub.2).sub.2NHC(NH.sub.2)═, —(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(NH.sub.2)═, —(CH.sub.2).sub.3NHC(NH.sub.2)═, —(CH.sub.2).sub.2NHC(Me)═, —(CH.sub.2).sub.2NHC(O)CH.sub.2NHC(Me)═, —(CH.sub.2).sub.3NHC(Me)═, —(CH.sub.2).sub.2NR.sup.1′C(NH.sub.2)═, —C(CO.sub.2H)(CH.sub.2).sub.3—, —C(CO.sub.2H)(CH.sub.2).sub.3NHC(NH.sub.2)═, —C(CO.sub.2H)CH.sub.2—, —C(CO.sub.2H)(CH.sub.2).sub.2—, —C(CO.sub.2H)(CH.sub.2).sub.4—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —CHR.sup.2′C(O)—, —CHR.sup.2′CH.sub.2—, —CHR.sup.5CH.sub.2NR.sup.5′C(Me)═, —CHR.sup.2′(CH.sub.2).sub.2—, —(CH.sub.2).sub.2CHR.sup.1′—, —(CH.sub.2).sub.2CHR.sup.1′NHC(O)C(Me)—, —CH.sub.2CHR.sup.1′—, —CH.sub.2CHR.sup.1′NHC(Me)═, —CHR.sup.5(CH.sub.2).sub.2CHR.sup.5′—, —CHR.sup.2′CHR.sup.3′(CH.sub.2).sub.2—, and CR.sup.5═CH—CH═CR.sup.5′—CH.sub.2—, wherein R.sup.5 and R.sup.5′ represent the connection of a second linker between one backbone atom of the linker, bearing R.sup.5, and another backbone atom of the linker, bearing R.sup.5′, wherein R.sup.5′ is joined with R.sup.5 via the second linker, thus forming a 4-10-membered cyclic structure; N* is represented by structure (IIa) or (IIb) ##STR00037## R.sup.1 and R.sup.2 are each independently selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, or R.sup.1 and R.sup.2 are joined together and thus form a second linker between the amide nitrogen atom and the distal nitrogen atom, or R.sup.1 is joined with R″ of the linker L in a cyclic structure and/or R.sup.2 is joined with R.sup.2′ of the linker L in a cyclic structure; R.sup.3 is selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties or (halo)alkoxy moieties, or R.sup.3 is absent when the distal nitrogen atom is part of an imine moiety; or optionally R.sup.3 is joined with R.sup.3′ of the linker L in a cyclic structure; and R.sup.4 is selected from hydrogen (H) or C.sub.1-C.sub.6 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties; X is an anion, wherein the subject has enhanced or increased activity of mPGES-1 as a result of overexpression of mPGES-1, wherein enhanced activity of mPGES-1 or overexpression of mPGES-1 is higher than in corresponding normal subjects.

17. The method according to claim 1, wherein the compound is an mPGES-1 inhibitor.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Synthesis pathways of prostaglandins and the targeting strategies.

(2) FIG. 2. (A-C) Levels of prostaglandins (PG) PGE.sub.2 (A), PGD.sub.2 (B) and 6-keto PGF.sub.1a (C) in supernatant of RAW 264.7 murine macrophage cells exposed for 24 hours to vehicle or 1 μg/mL lipopolysaccharide (LPS), normalized on vehicle. (D-F) Levels of PGE.sub.2, PGD.sub.2 and 6-keto PGF.sub.1a in supernatant of RAW 264.7 murine macrophage cells exposed for 24 hours to vehicle or 1 μg/mL LPS, alone (set as 100%) or in combination with increasing concentrations of compound I-IVb-X (a compound of general structure (I) wherein T is of general structure (IVb), in the S,R-configuration, and wherein as per compound X the following apply: L=L.sup.19; R.sup.1═H; R.sup.2—R.sup.2′=L.sup.3; R.sup.3═H; indicated as KH) (D), non-selective COX inhibitor indomethacin (E) or COX-2 inhibitor celecoxib (F).

(3) FIG. 3. (A-B) Levels of PGE.sub.2 (A) and PGD.sub.2 (B) in supernatant of human primary skin fibroblasts exposed for 24 hours to vehicle, 1 μg/mL LPS or 1 μg/mL Interleukin-1β (IL-1β), normalized on vehicle. (C-D) Levels of PGE.sub.2 and PGD.sub.2 in supernatant of human primary skin fibroblasts exposed for 24 hours to 1 μg/mL LPS (C) or 1 μg/mL IL-1β (D) alone (set as 100%) or in combination with increasing concentrations of compound I-IVb-X (indicated as KH).

(4) FIG. 4. (A) Western blot analysis of mPGES-1, COX-2, cPGES, mPGES-2, and COX-1 enzymes in RAW 264.7 murine macrophage cells exposed for 24 hours to vehicle or 1 μg/mL LPS, alone or in combination with increasing concentrations of compound I-IVb-X (indicated as KH). (B-F) Quantification of the levels of mPGES-1 (B), COX-2 (C), mPGES-2 (D), cPGES (E) and COX-1 (F) enzymes as in (A) with Actin as a loading reference and normalized to the vehicle treated cells.

(5) FIG. 5. Quantification by qPCR of the RNA levels of mPGES-1 (A), COX-2 (B), cPGES (C), mPGES-2 (D), and COX-1 (E) enzymes in RAW 264.7 murine macrophage cells exposed for 24 hours to vehicle, 1 μg/mL LPS alone or in combination with increasing concentrations of compound I-IVb-X (indicated as KH).

(6) FIG. 6. mPGES-1 activity was assayed as conversion of PGH.sub.2 to PGE.sub.2 by microsomal fractions of RAW 264.7 cells treated for 24 hours with LPS 1 μg/mL to increase mPGES-1 expression. The microsomal fraction was exposed to increasing concentrations of compound to be tested, or 3 μM of know mPGES-1 inhibitors MK866 or PF9184 as positive controls. PGE.sub.2 level was normalized on the vehicle treated microsomal samples (100%). (A) compound I-IVb-X; (B) compound I-IVb-AE; (C) compound I-IVb-A-HCl; (D) compound I-IVb-I.

EXAMPLES

Example 1

(7) Methods and Materials

(8) Chemicals and Antibodies

(9) Antibodies directed against cPGES, mPGES-1 and mPGES-2 were purchased from Cayman chemicals. Antibodies directed against COX-1, COX-2 and Actin were purchased from R&D systems, Thermo Fisher Scientific and Sigma-Aldrich, respectively. COX inhibitors, Celecoxib and Indomethacin, and Lipopolysaccharide were purchased from Sigma-Aldrich. mPGES-1 inhibitors, MK866 and PF9184, and PGH.sub.2 were purchased from Cayman Chemicals. IL-1β was purchased at Cell Signaling Technologies.

(10) Compounds for use according to the invention were prepared as described in WO2014/011047 or in WO2017/060432.

(11) Raw 264.7 Cell Culture

(12) RAW264.7 cells (Sigma-Aldrich, St-Louis) were maintained in DMEM (Thermo Fisher Scientific) containing 10% FBS (Greiner Bio-one), and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) under a humidified atmosphere of 5% CO2 at 37° C. The cells were grown to 80% confluence, scraped and then cultured in either 96-well plates (2×10.sup.4 cells/well) or six-well plates (4×10.sup.5 cells/well) for 6-24 hours before an experiment.

(13) Human Primary Skin Fibroblast Culture

(14) The cells were maintained in M199, HEPES (Thermo Fischer Scientific) containing 10% FBS (#758093, Greiner Bio-one), 100 IU/ml penicillin and 100 μg/ml streptomycin (#30-002-CI, Corning) under a humidified atmosphere of 5% CO2 at 37° C. The cells were passaged by trypsinization every 4-5 days until they reached the passage number 20, and then discarded. The cells were grown to 80% confluence, trypsinized and then cultured in 96-well plates (4×10.sup.3 cells/well) 24 hours before an experiment.

(15) Prostaglandins Quantification

(16) The concentration of the PGE.sub.2, PGD.sub.2 and 6-keto-PGF.sub.1α in culture medium was determined by Enzyme-Linked Immuno Sorbent Assay (ELISA) using, respectively, the PGE.sub.2 high sensitivity EIA kit (Enzo Life Science), the Prostaglandin D.sub.2-MOX ELISA Kit (Cayman Chemicals) and the 6-keto Prostaglandin F.sub.1α ELISA Kit (Cayman Chemicals), according to the manufacturer's instructions. Samples (100 μl) of culture medium from each well were harvested and diluted with the assay buffer. The values of each prostanoid were calculated using a standard curve and normalized as indicated in the figure legends.

(17) Western Blot Analysis

(18) After treatment the cells were harvested by scrapping and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 0.2% Triton, 1× protease inhibitor, and 0.1 mg/ml DNAse). The protein concentration of the samples was determined by Bradford assay. Equal amount of proteins of each samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to PVDF membrane. The membranes were blocked for 1 h at room temperature in Odyssey blocking buffer (Li-Cor), and then incubated with primary antibody against mPGES-1 (1:200 dilution), mPGES-2 (1:200 dilution), cPGES (1:200 dilution), COX-1 (1:250 dilution), COX-2 (1:500 dilution), or β-actin (1:10000 dilution) in TBST at 4° C. overnight. After washing the membranes three times with TBST, secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 or Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647, Thermo Fisher scientific) was added at 1:10000 dilutions for 1 hour at room temperature. Fluorescence scanning of the blots was performed using an Odyssey Infrared Imaging System (Li-Cor).

(19) Microsomal PGES Activity

(20) PGES activity was measured by assessment of the conversion of PGH.sub.2 to PGE.sub.2. In brief, after 24 hours incubation with 1 μg/mL LPS the cells were harvested and lysed by sonication (three times for 10 seconds each, at 1 min intervals) in 300 μl of 1 M Tris-HCl (pH 8.0). After centrifugation of the lysate at 12,000 g for 10 min at 4° C., the supernatant was collected and further centrifuged at 100,000 g for 1 h at 4° C. The pellets (microsomal membranes) were resuspended in 100 μl of 0.1M Tris-HCl, pH 8.0, containing protease inhibitors) and subjected to measurement of microsomal PGES activity. For measurement of PGES activity, an aliquot of each sample equivalent to 50 μg of protein was incubated with 2 μg of PGH.sub.2 for 60 s at 24° C. in 0.1 ml of 1 M Tris-HCl containing 2 mM glutathione (Sigma-Aldrich) and 14 μM indomethacin, in the absence or presence of the tested compounds. The reaction was terminated by the addition of 100 mM FeCl2, and further incubated at 20-25° C. for 15 min. After centrifugation of the reaction mixture, the PGE.sub.2 concentration in the supernatant was measured with the PGE.sub.2 high sensitivity EIA kit (Enzo Life Science) according to manufacturer's instructions.

(21) RNA Quantification by qRT-PCR

(22) After 6 h of treatment with 1 μg/mL LPS, in the absence or presence of tested compounds, total RNA was isolated from the cells by Trizol reagent (Thermo Fisher Scientific). The RNA was transcribed into cDNA by first-strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA was denatured for 10 min at 95° C. Specific DNA fragments for COX-1, COX-2, mPGES-1, mPGES-2, cPGES, and PPIA were amplified by PCR with SYBR Green (Roche Life Science) Cycler for 40 cycles with 15 s at 95° C., 60 s at 60° C. The oligonucleotide primers used for COX1 were 5′-GATTGTACTCGCACGGGCTAC-3′ (forward) and 5′-GGATAAGGTTGGACCGCACT-3′ (reverse), for COX2 were 5′-AGGACTCTGCTCACGAAGGA-3′ (forward) and 5′-TGACATGGATTGGAACAGCA-3′ (reverse), for mPGES-1 were 5′-AGCA CACTGCTGGTCATCAA-3′ (forward) and 5′-CTCCACATCTGGGTCACTCC-3′ (reverse), for mPGES-2 were 5′-GCTGGGGCTGTACCACAC-3′ (forward) and 5′-GATTCACCTCCACCACCTGA-3′ (reverse), for cPGES were 5′-GGTAGAGACCGCCGGAGT-3′ (forward) and 5′-TCGTACCACTTTGCAGAAGCA-3′ (reverse), for PPIA were 5′-AGGGTGGTGACTTTACACGC-3′ (forward) and 5′-GATGCCAGGACCTGTATGCT-3′ (reverse). The PCR amplifications were also carried out in samples without cDNA as negative controls. The relative gene expression of COX-1, COX-2, mPGES-1, mPGES-2, and cPGES was determined by the ΔΔCT method comparing expression in vehicle and treated groups. PPIA was used as a reference gene.

(23) Results

(24) Compound I-IVb-X Selectively Decreases the Level of PGE.sub.2.

(25) RAW 264.7 macrophage cells were treated with the inflammatory stimulus LPS alone or in combination with increasing concentrations of compound I-IVb-X (a compound of general structure (I) wherein T is of general structure (IVb), in the S,R-configuration, and wherein as per compound X the following apply: L=L.sup.19; R.sup.1═H; R.sup.2—R.sup.2′=L.sup.3; R.sup.3═H), the NSAID indomethacin or the Coxib Celecoxib. After 24 hours incubation the levels of PGE.sub.2, PGD.sub.2 and 6-keto PGF.sub.1a were quantified in the cell supernatant by ELISA. As expected, LPS efficiently induced the production of the three prostaglandins (FIG. 2A-C) (Ikeda-Matsuo et al., 2005), which could all be dose-dependently reduced by the two COX inhibitors indomethacin and Celecoxib (FIG. 2E-F). Unexpectedly, while compound I-IVb-X could dose-dependently reduce the level of PGE.sub.2 it has no effect on the two other prostaglandins PGD.sub.2 and 6-keto PGF.sub.1a, a stable metabolite of PGI.sub.2 commonly measured as a surrogate of PGI.sub.2 (FIG. 2D). Similar results were obtained with compound I-IVb-X in human primary skin fibroblasts. After 24 hours incubation of fibroblast with either inflammatory stimulus LPS or IL-1β, PGE.sub.2 and PGD.sub.2 levels in the supernatant were strongly increased (FIGS. 3A&B). compound I-IVb-X could efficiently reduce the level of PGE.sub.2, but not PGD.sub.2, in supernatant of cells treated with either LPS (FIG. 3C) or IL-1β (FIG. 3D).

(26) Compound I-IVb-X Selectively Decrease the Expression of mPGES-1 Enzyme.

(27) RAW 264.7 macrophage cells were treated with the inflammatory stimulus LPS alone or in combination with increasing concentrations of compound I-IVb-X. After 24 hours incubation the levels of mPGES-1, mPGES-2, cPGES, COX-1 and COX-2 proteins and RNA were quantified in the cells by western-blot or qPCR, respectively. As expected, LPS efficiently induced the expression of the two inducible enzymes mPGES-1 and COX-2 at the protein (FIGS. 4 A-C)) and RNA (FIGS. 5A&B) levels, while had no effect on constitutively expressed enzymes mPGES-2, cPGES and COX-1 at the protein (FIGS. 4A,D-F) and RNA (FIGS. 5 C-E) levels. While compound I-IVb-X could dose-dependently reduce the LPS-induced expression of mPGES-1 protein (FIG. 4B) and RNA (FIG. 5A), it had no effect on COX-2 expression (FIGS. 4C&5B). Compound I-IVb-X had no effect on the other three constitutive enzymes mPGES-2, cPGES and COX-1 (FIGS. 4 D-F and FIG. 5 C-E). These results show that compound I-IVb-X can selectively inhibit the expression of mPGES-1 enzyme induced by inflammatory stimulus LPS, which explain its selectivity in reducing solely PGE.sub.2 and no other prostaglandins.

(28) Compound I-IVb-X Inhibits mPGES-1 Enzyme Activity.

(29) RAW 264.7 macrophage cells were treated with the inflammatory stimulus LPS to increase the expression of mPGES-1. After 24 hours incubation the microsomes were isolated and exposed to increasing concentration of compound I-IVb-X or a single concentration of previously described mPGES-1 inhibitors MK866 and PF9184 for 30 minutes. The activity of mPGES-1 was then assayed in the microsomes fraction as the conversion of PGH.sub.2 to PGE.sub.2. The results show that mPGES-1 activity in purified microsomes treated with compound I-IVb-X or the two positive controls MK866 and PF9184 was decreased (FIG. 6A).

Example 2—Inhibition of mPGES-1 Enzyme Activity

(30) Using the methods as described in Example 1, additional compounds were tested. Table 1 provides an overview of the compounds that were tested, along with corresponding IC.sub.50 values. FIGS. 6B, 6C, and 6D show corresponding inhibition curves for I-IVb-AE, I-IVb-A-HCl, and I-IVb-I, respectively.

(31) TABLE-US-00001 TABLE 1 exemplary compounds that inhibit mPGES-1 enzyme activity Compound IC.sub.50 reference Structure Formula I wherein: (x10.sup.−6) I-IVb-N T = IVb, N* = IIa L = L.sup.11, R.sup.1 = H, R.sup.2 = H, R.sup.3 = H 0.01041 I-IVb-AE T = IVb, N* = IIa L = L.sup.26, R.sup.1 = H, R.sup.2 = H, R.sup.5—R.sup.5′ = L.sup.1, R.sup.3 = H 0.01799 I-IVb-AB-HCl T = IVb, N* = IIb L = L.sup.23, R.sup.1—R.sup.1′ = L.sup.1, R.sup.2 = H, R.sup.3 = H R.sup.4 = H, X = Cl 0.2381 I-IVb-X T = IVb, N* = IIa L = L.sup.19, R.sup.1 = H, R.sup.2—R.sup.2′ = L.sup.3, R.sup.3 = H 0.4579 I-IVb-AI-HCl T = IVb, N* = IIb L = L.sup.27, R.sup.1 = H, R.sup.2—R.sup.2′ = —CH.sub.2—, R.sup.3—R.sup.3′ = L.sup.1, R.sup.4 = H, X = Cl 0.7262 I-IVb-A-HCl T = IVb, N* = IIb L = L.sup.1, R.sup.1—R.sup.2 = L.sup.1, R.sup.3 = H, R.sup.4 = H, X = Cl 1.024 I-IVb-AJ-HCl T = IVb, N* = IIb L = L.sup.28, R.sup.1 = H, R.sup.2 = H, R.sup.3 = H, R.sup.4 = H, X = Cl 1.05 I-IVb-I T = IVb, N* = IIa L = L.sup.1, R.sup.1 = H, R.sup.2 = Me, R.sup.3 = Me 1.118

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