Imidazoquinoline compound having Anti-inflammatory, antifungal, antiparasitic, and anticancer activity

Abstract

An imidazoquinoline compound having activity against inflammation, fungi, unicellular parasitic microorganisms, and cancer is described.

Description

The invention will be better understood by reference to the following examples, which illustrate but do not limit the invention described herein. CHEMICAL SYNTHESIS EXAMPLES EXAMPLE 1: Synthesis of 2-(3-Phenoxybenzyl)-l / / -imidazo[4,5-c]quinoline Step 1:        3-Nitroquinolin-4-ol OH NO2 70% Aqueous nitric acid (6.1 mL) was added dropwise to a mixture of 4-hydroxyquinoline (10 g, 69 mmol) and 100 mL of acetic acid heated at reflux. After 15 min, the mixture was allowed to cool to room temperature. Dilution with EtOH resulted in the formation of a precipitate, which was filtered and washed sequentially with EtOH, H2O, and EtOH. Drying of the filtrate in vacuo gave 4.62 g of a light yellow powder. 1H NMR (400 MHz, DMSO-d6) 8 9.2 (s, 1H), 8.3 (d, 1H), 7.9-7.7 (m, 2H), 7.5 (m, 1H). Step 2:       4-Chloro-3-nitroquinoline Cl NO2 Phosphorus oxychloride (2.5 mL, 27 mmol) was added dropwise to a mixture of 3-nitroquinolin-4-ol (4.6 g, 24 mmol) and 100 mL of DMF. The mixture was heated at 100 °C for 15 min, and then poured onto stirred ice. The slurry was neutralized with solid NaHCOs, and the precipitate was filtered and washed with saturated NaHCOa and H2O. The filtrate was taken up in DCM, dried over anhydrous Na2SO4, and concentrated to give 2.3 g of solid. Step 3:        A-( / er / -Butyl)-3-nitroquinolin-4-amine Step 4: A mixture of 4-chloro-3-nitroquinoline (6.30 g, 30.2 mmol), terZ-butylamine (6.40 mL, 60.5 mmol), TEA (8.50 mL, 60.6 mmol), and 40 mL of DCM was heated at reflux for 5 hr., when the starting material was consumed. The mixture was stirred overnight at room temperature. The mixture was partitioned between DCM and saturated NaHCOs, and the organic phase was dried over anhydrous Na2SO4 and concentrated to give the product. A4-( / er / -Butyl)quinoline-3,4-diamine NH2 A-( / er / -Butyl)-3-nitroquinolin-4-amine obtained above, 10% Pd-C (630 mg), 1.5 mL of TEA, and 50 mL of MeOH were stirred under an atmosphere of hydrogen until the starting material was consumed. The hydrogen was replaced by nitrogen, and the mixture was filtered through a pad of Celite and concentrated. The residue was dissolved in 1:1:1 MeOH, DCM, and toluene spiked with 0.5 mL of TEA and then concentrated to give 7.37 g of material. Rf 0.50 (7.5% MeOH / DCM + 1% TEA) Step 5:       A-(4-( / er / -Butylamino)quinolin-3-yl)-2-(3-phenoxyphenyl)acetamide EDC (6.5 g, 33.8 mmol) was added to a mixture of A4-( / er / -butyl)quinoline-3,4-diamine (4.91 g, 22.2 mmol), 3-phenoxyphenylacetic acid (5.2 g, 22.8 mmol), HOBt (3.49 g, 22.8 mmol), and DMAP (0.60 g, 4.9 mmol) in 60 mL of 1:1 DMF / DCM. After 17.5 hr., TLC of an aliquot of the mixture showed unconsumed starting material, so additional EDC (2.12 g, 11.0 mmol) and DMF (15 mL) was added, and the mixture was warmed to 45 °C and the DCM was allowed to boil off. After 68 hr., the mixture was cooled and partitioned between EtOAc (3x250 mL) and 5% Na2COa (2x150 mL) and brine (150 mL). The organic phases were dried over anhydrous Na2SO4 and concentrated to give the product. Step 6:       A-(4-Aminoquinolin-3-yl)-2-(3-phenoxyphenyl)acetamide A-(4-( / er / -Butylamino)quinolin-3-yl)-2-(3-phenoxyphenyl)acetamide was mixed with 1:1 TFA / DCM for 2 hr. at room temperature. The volatile components were evaporated, and the residue was dissolved in DCM and washed with 5% Na2COs, and the organic phase was dried over anhydrous Na2SO4 and then concentrated. Purification by flash chromatography (5% MeOH / DCM + 1% TEA) gave the product as a solid. The product was recrystallized from MeOH. LC-MS confirmed MW 369. R / 0.25 (10% MeOH / DCM) Step 7:       2-(3-Phenoxybenzyl)-lH-imidazo[4,5-c]quinoline A mixture of A-(4-aminoquinolin-3-yl)-2-(3-phenoxyphenyl)acetamide (1.76 g, 4.77 mmol) and NH4CI (21 mg, 0.39 mmol) in 25 mL of anisole was heated at reflux for 4.5 hr. The volatile material was evaporated, and the solid residue was partitioned between DCM and 5% Na2COs. The organic phase was dried over anhydrous Na2SO4 and concentrated. Purification by flash chromatography (5% MeOH / DCM ) gave 1.60 g of the product as a foamy solid. LC-MS confirmed MW 351. R / 0.46 (10% MeOH / DCM); ’H NMR (400 MHz, CDCI3) 8 9.04 (br s, 1H), 8.25 (br s, 1H), 8.14 (d, 1H, J=8.7 Hz), 7.62-7.58 (m, 1H), 7.52 (br s, 1H), 7.27-7.22 (m, 3H), 7.18-7.14 (m, 1H), 7.08-7.04 (m, 1H), 6.97-6.95 (m, 1H), 6.90-6.86 (m, 3H), 6.81-6.79 (m, 1H), 4.34 (s, 2H) BIOLOGICAL ACTIVITY EXAMPLES EXAMPLE A:      Anti-Cancer Activity In Vitro Compound AF has previously been identified as a potent lysosomotropic agent with anticancer activity against a number of cancer cell lines. The relative potency of Compunds GE and AF were compared in two cancer cell lines with very different genetic anomalies and oncogene drivers underlying for their tumorigenic properties. PC3 (prostate cancer) and A549 (lung cancer) cell lines were cultured in medium consisting of 90% F-12K Nutrient Mixture (Kaighn’s modification) with L-glutamine and 10% Hyclone Fetal Bovine Serum. No antibiotics were used in the medium. PC3 and A549 cells were plated in 96 well plates at a density of 20,000 and 10,000 cells per well, respectively, in a volume of 0.1 ml per well. Twenty four hours after plating the cells, the medium was replaced with 0.1 ml of medium containing either the AF or the GE compounds. The AF compound was tested at concentrations of 1, 0.5, 0.25, and 0.1 micromolar. The GE compound was tested at concentrations of 0.1, 0.05, 0.025, and 0.01 micromolar. The cells were then cultured for 72 hrs, and cell viability was evaluated with the WST-1 assay at 48 and 72 hours. For cell viability, the absorbance obtained in the WST-1 assay of cells exposed to the compounds was expressed as a percentage of the absorbance of wells of cells incubated in the absence of the compounds. All measurements are expressed as the average + SEM of 3 wells. Cancer cell viability as a function of dose (concentration) of GE and AF is shown in Figures 1-4. Compound GE reduced viability of cancer cells at doses approximately 1 / 10 of those required for similar reductions in cell viability by compound AF in both A549 lung cancer cells (Figures 1 and 2) and in PC3 prostate cancer cells (Figures 3 and 4). EXAMPLE B. Anti-inflammatory effects of Compound GE on psoriasiform dermatitis in mice Topical imiquimod (IMQ), a toll-like receptor agonist, has been established as a model of inflammatory skin diseases including psoriasis and atopic dermatitis that predicts clinical activity in human subjects. Dermal inflammatory changes and gene expression in mice treated with topical imiquimod mimic human psoriasiform dermatitis (van der Fits et al., 2009). The effect of Compound GE of the invention was tested in a mouse model of imiquimod-induced dermatitis. Compound GE was dissolved in ethanol at a concentration of 0.1% and then mixed with 9 volumes of petrolatum (melted on a heated water bath at 50 degrees C), yielding ointment containing 0.01% active drug. Petrolatum containing 10% ethanol was used as a control or vehicle treatment. Female Balb / C mice weighing approximately 20 grams were randomized and divided into three groups of 5 animals each. Polyethylene collars were affixed to the mice to prevent them from easily scratching their ears. 5% imiquimod was applied to both ears of each mouse (20 microliters per ear) daily for 5 days, and then every other day for the full duration of the study. Inflammatory changes, including an increase in ear thickness were apparent by day 5. On day 5 after initiation of imiquimod, treatment with Compound GE was started. Both ears of each mouse were treated with test ointments. Ear thickness and PASI assessments (Psoriasis Area and Severity Index, a standard psoriasis scoring system capturing swelling, erythema and scaling) were recorded twice per week throughout the study. Results Imiquimod treatment resulted in significant inflammatory changes, including an increase in ear thickness (Figure 5) and a change in PASI scores; control ears reached the maximum possible value in the PASI (12, reflecting severe swelling, erythema and scaling, each with a scale of 0-4) scoring system (Figures 6A-D). Compound GE, applied topically in an ointment base, reduced imiquimod-induced inflammatory damage to mouse ears, as assessed by caliper measurements of thickness and PASI scoring of appearance, reducing all three aspects of dermal inflammation captured by the PASI 5 scoring system. Compound GE was effective at a concentration of 0.01%, indicating high potency in this model of psoriasiform dermatitis. Reference van der Fits L, Mourits S, Voerman JS, Kant M, Boon L, Laman JD, Cornelissen 10 F, Mus AM, Florencia E, Prens EP, Lubberts E. (2009) Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23 / IL-17 axis. J Immunol. 182(9):5836-45.

Claims

1. A compound selected from the group consisting of:2-(3-Phenoxybenzyl)-1H-imidazo[4,5-c]quinoline or a pharmaceutically acceptable salt thereof.

2. A composition comprising the compound of claim 1.

3. The composition of claim 2, further comprising a pharmaceutically acceptable carrier.

4. A method for treating or preventing a condition in a mammalian subject; the conditionbeing selected from the group consisting of an inflammatory disease, a fungal infection, an infection with a unicellular parasitic microorganism, and a neoplastic disease; comprising administering to the subject an effective amount of the compound of claim 1 or the composition of claim 2 or claim 3.

5. Use of the compound of claim 1 or the composition of claim 2 or claim 3 for treating orpreventing a condition in a mammalian subject; the condition being selected from the group consisting of an inflammatory disease, a fungal infection, an infection with a unicellular parasitic microorganism, and a neoplastic disease.

6. Use of the compound of claim 1 in the manufacture of a medicament for treating orpreventing a condition in a mammalian subject; the condition being selected from the group consisting of an inflammatory disease, a fungal infection, an infection with a unicellular parasitic microorganism, and a neoplastic disease.

7. The method or use of any one of claims 4-6, wherein the mammalian subject is a humansubject.

8. The method or use of any one of claims 4-6, wherein the condition is an inflammatorydisease.2021396262   26 Jun 20269.     The method or use of claim 8, wherein the inflammatory disease is an inflammatory skincondition, optionally wherein the inflammatory skin condition is selected from the group consisting of psoriasis, psoriatic dermatitis, eczema, atopic dermatitis, and impetigo.

10. The method or use of claim 8, wherein the inflammatory disease is a systemic autoimmune disorder, optionally wherein the systemic autoimmune disorder is selected from the group consisting of rheumatoid arthritis, systemic and discoid lupus erythematosis, psoriatic arthritis, vasculitis, Sjogrens syndrome, scleroderma, autoimmune hepatitis, and multiple sclerosis.

11. The method or use of any one of claims 4-6, wherein the condition is a fungal infection, optionally wherein the fungal infection is caused by a fungus selected from the group consisting of Candida, Saccharomyces, Trichophyton, Cryptococcus, Aspergillus, and Rhizopus, further optionally wherein:the Candida is Candida albicans or Candida glabrata;the Saccharomyces is Saccharomyces cerevisiae;the Trichophyton is Trichophyton rubrum;the Cryptococcus is Cryptococcus neoformans, optionally Cryptococcus neoformans serotype D or Cryptococcus neoformans serotype A;the Aspergillus is Aspergillus fumigatus.

12. The method or use of any one of claims 4-6, wherein the condition is infection with a unicellular parasitic microorganism, optionally wherein the parasitic microorganism is a parasitic microorganism that resides within acidic vacuoles in cells of the subject.

13. The method or use of claim 12, wherein the parasitic microorganism is selected from the group consisting of mycobacteria, gram positive bacteria, amoebae, gram negative bacteria, tuberculosis, listeria, leishmania, a trypanosome, Coxiella burnetii, and a Plasmodium.

14. The method or use of any one of claims 4-6, wherein the condition is a neoplastic disease.2021396262   26 Jun 202615. The method or use of claim 14, wherein the neoplastic disease is a hematologic cancer or a solid tumor.

16. The method or use of any one of claims 4-6, wherein the compound or composition is administered topically to the subject.

17. The method or use of any one of claims 4-6, wherein the compound or composition is administered systemically to the subject, optionally wherein the systemic administration is administered orally, rectally, parenterally, or nasally.

18. A method of inhibiting a fungus ex vivo, comprising contacting a surface or the fungus with the compound of claim 1 or the composition of claim 2 or claim 3.

19. The method of claim 18, wherein the fungus is selected from the group consisting of Candida, Saccharomyces, Trichophyton, Cryptococcus, Aspergillus, and Rhizopus, optionally wherein:the Candida is Candida albicans or Candida glabrata;the Saccharomyces is Saccharomyces cerevisiae;the Trichophyton is Trichophyton rubrum;the Cryptococcus is Cryptococcus neoformans, optionally Cryptococcus neoformans serotype D or Cryptococcus neoformans serotype A;the Aspergillus is Aspergillus fumigatus.

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