Synthesis and Biological Evaluation of N-aryl-4-aryl-1,3-Thiazole-2-Amine Derivatives as Direct 5-Lipoxygenase Inhibitors

Authors

  • Jeehee Suh,

    1. Center for Metabolic Syndrome Therapeutics, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Korea
    2. Department of Chemistry, Chungnam National University, Yuseong-gu, Daejeon 305-764, Korea
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  • Eul Kgun Yum,

    1. Department of Chemistry, Chungnam National University, Yuseong-gu, Daejeon 305-764, Korea
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  • Hyae Gyeong Cheon,

    Corresponding author
    1. Department of Pharmacology and Pharmaceutical Sciences, Gachon University of Medicine and Science, Incheon 406-799, Korea
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  • Young Sik Cho

    Corresponding author
    1. Department of Pharmacy, Keimyung University, 1000 Sindang-dong, Dalseo-gu, Daegu 704-701, Korea
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Corresponding authors: Young Sik Cho, yscho123@kmu.ac.kr;
Hyae Gyeong Cheon, hgcheon@gachon.ac.kr

Abstract

Biological evaluation of N-aryl-4-aryl-1,3-thiazole-2-amine derivatives was examined for anti-inflammatory activity in in vitro and in vivo assays. The thiazole compounds showed direct inhibition of 5-lipoxygenase (LOX) that is a key enzyme of leukotrienes synthesis and involved in the inflammation-related diseases, including asthma and rheumatoid arthritis. To optimize biological activity, we synthesized 1,3-thiazole-2-amine derivatives and investigated for structure and activity relationship. Especially, N-(3,5-dimethylphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine was shown to have a potent anti-inflammatory activity as a 5-LOX inhibitor.

Abbreviations
5-LO,

5-lipoxygenase

LTB4,

leukotriene B4

SAR,

structure and activity relationship

AA,

arachidonic acid

COX-2,

cyclooxygenase-2

RBL-1,

rat basophilic leukemia-1

Starting from arachidonic acid (AA) released from cytoplasmic membrane upon external stimulation, inflammatory chemical mediators are generated by a cascade of enzymes (1). Among them, prostaglandins (PGs) and leukotrienes (LTs) are, respectively, generated by cyclooxygenase (COX) and lipoxygenase (LOX) at the bifurcation of metabolic pathway using AA (2). Usage of classical NSAIDs was limited because of their non-selective inhibition of COX enzymes, which leads to side-effects like ulceration that resulted in the search for selective COX-2 inhibitors (COXibs). In the beginning, a variety of approaches targeting cyclooxygenase-2 (COX-2) have been successful for interfering with inflammatory progression, but found soon to be inappropriate because of the adverse effect of heart attacks (3,4). Afterward, special attentions on alternative pathway to produce inflammatory messengers have been given to promote the drug development for the treatment of inflammation-related diseases. In fact, LTs produced by LOX have potent pro-inflammatory properties including bronchoconstriction and chemotaxis of polymorphonuclear leukocytes (5,6). LTB4 is a well-known chemoattractant for inflammatory cells (7), and other three LTC4, D4, and E4, which contain glutathione conjugation, are collectively referred as the slow-reacting substances of anaphylaxis (8). Besides most notable asthma, rheumatoid arthritis and inflammatory bowel disease (9–11), these LTs can also play a role in the generation of psoriasis and atopic dermatitis, in which topical application of LOX products causes psoriasis-like lesions, suggesting their inflammatory roles in the pathogenesis of skin disorders (12,13).

5-Lipoxygenase (5-LOX) is a key enzyme involved in the first step of the LTs synthesis, and its dysregulation causes various inflammatory diseases mentioned earlier (14,15). Therefore, 5-LOX inhibitors to intervene LTs production can be developed as the effective therapeutic agents to treat inflammatory diseases. Presently, the only drug approved by FDA as a direct 5-LOX inhibitor is zileuton (16), while there are mostly drugs based on the abrogation of the ligand–receptor interaction or indirect interference in the process of 5-LOX activation (17,18).

In an effort to discover new chemical entities with selective inhibitory activities of 5-LOX, in-house chemical library was screened by high-throughput screening (HTS) method as described previously (19). As a result, we identified a new series of aminothiazole derivatives, and further characterized biochemical and pharmacological profiles of a representative compound, N-(3,5-dimethylphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine, 14. The potent compound 14 used at 1 micromolar inhibited 5-LOX enzyme activity in vitro and LTB4 production from intact RBL cells by 96% and 98%, respectively, (Table 1). Structure and activity relationship (SAR) study revealed that the presence of either OH or NH2 group at 2- or 4-position of N-aryl moiety of aminothiazole scaffold was mandatory for its inhibitory activity, while 4-aryl moiety of it was relatively tolerable to a variety of substituents. Intriguingly, the presence of 3,5-diCH3 group adjacent to OH group attached to N-aryl of aryl aminothiazole was more potent than was that of 3,5-diCl or 3-Cl-5-CH3, although compound 13 and 16 bearing Cl and F group at 4-aryl moiety of N-aryl-4-aryl-1,3-thiazole-2-amine analogs remained still effective. Apart from thiazole derivatives, a class of thiazolinone scaffold has been studied as selective 5-LOX inhibitors from other researchers (20).

Table 1.   Inhibitory effects of N-aryl-4-aryl-1,3-thiazole-2-amines on enzymatic 5-lipoxygenase (LOX) activity Thumbnail image of

Consistent with those in vitro and cell-based assays, topical application of the compound 14 onto skin in AA-induced ear edema model improved inflammatory skin lesions as determined by phenotypic and biochemical parameters. Furthermore, it was demonstrated that the compound 14 had a longer-lasting effect upon topical application than zileuton. Its successful topical application rather than oral route proposed a potential use as a therapeutic agent for inflammation-related skin disorders.

Methods and Materials

Cell culture and animal care

RBL-1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, glutamine, penicillin, and streptomycin. For in vivo efficacy, Balb/c mice (male, 8 weeks of age) were bred, cared in the barrier system under temperature- and humidity-controlled room with a 12:12 h light cycle, and acclimated for 1 week before experiments. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH) and approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Chemical Technology (Daejeon, Korea).

Reagents and enzymes

Chemicals and enzymes used in this experiment were available commercially or were prepared by in-house synthesis or partial isolation. Xylenol, iron sulfate, ATP, hexadecyltrimethylammonium bromide (HTAB), α-phophatidyl choline (PC) were obtained from Sigma (St. Louis, MO, USA). AA and A23187 were obtained from Calbiochem (La Jolla, CA, USA). The LT B4 (LTB4) enzyme immunoassay (EIA) kit was obtained from Amersham (Piscataway, NJ, USA). Zileuton and a series of chemical derivatives were synthesized in Korea Research Institute of Chemical Technology (KRICT; Daejeon). Soybean 15-LOX enzyme was obtained from Cayman Chemical (Ann Arbor, MI, USA), and 12-LOX was prepared from platelets-rich plasma as described before (21,22). All other reagents were of analytical grade from Sigma.

Synthesis of N-(4-hydroxyphenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (3)

About 0.60 g (2.76 mmol) of 2-bromo-4′-fluoroacetophenone was dissolved in 10 mL of ethanol, 0.29 g (3.59 mmol) of sodium thiocyanate and 0.33 g (3.04 mmol) of p-aminophenol were added at room temperature, and the mixture was refluxed at 80 °C for 12 h. After the reaction was completed, the reaction mixture was diluted with 10 mL of ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, and concentrated under a reduced pressure. The residue was purified using silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain the title compound (0.490 g, 62%).

1 H NMR (300 MHz, DMSO-d6) δ 6.76 (d, = 8.73 Hz, 2H), 7.19–7.27 (m, 3H), 7.48 (d, = 8.73 Hz, 2H), 7.90–7.95 (m, 2H), 9.13 (s, 1H), 9.90 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 101.55, 115.26, 115.48, 115.55, 119.25, 127.54, 127.65, 131.29, 131.33, 133.25, 149.00, 152.35, 159.93, 163.17, 164.35.

HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0580.

MS (EI) m/e 286 (100%) [M]+, 152 (25%), 134 (10%).

The following compounds were prepared according to the general procedures described previously employing the appropriate 2-bromo-acetophenone.

Synthesis of N-(4-hydroxyphenyl)-4-(4-bromophenyl)-1,3-thiazole-2-amine (4)

1 H NMR (300 MHz, DMSO-d6) δ 6.54 (d, = 8.8 Hz, 2H), 7.08 (s, 1H), 7.24 (d, = 8.8 Hz, 2H), 7.38 (d, = 8.5 Hz, 2H), 7.62 (d, = 8.5 Hz, 2H), 8.90 (s, 1H), 9.71 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 102.76, 115.48, 119.31, 120.40, 127.64, 131.50, 133.18, 133.86, 148.85, 152.40, 164.40.

HR MS (M+) calcd. for C15H11 BrN2OS: 345.9775; found: 345.9758.

MS (EI) m/e 346 (100%) [M]+, 348 (98%), 134 (15%), 89 (20%).

Synthesis of N-(4-hydroxyphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (5)

1 H NMR (300 MHz, DMSO-d6) δ 6.75 (d, = 8.8 Hz, 2H), 7.28 (s, 1H), 7.45 (d, = 8.8 Hz, 2H), 7.48 (d, = 8.6 Hz, 2H), 7.89 (d, = 8.6 Hz, 2H), 9.12 (s, 1H), 9.92 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 102.66, 115.48, 119.30, 127.32, 128.59, 131.81, 133.19, 133.52, 148.80, 152.40, 164.39.

HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0274.

MS (EI) m/e 302 (100%) [M]+, 304 (33%), 168 (17%), 134 (23%).

Synthesis of N-(4-hydroxyphenyl)-4-(4-nitrophenyl)-1,3-thiazole-2-amine (6)

1 H NMR (300 MHz, DMSO-d6) δ 6.68 (d, = 8.8 Hz, 2H), 7.40 (d, = 8.8 Hz, 2H), 7.55 (s, 1H), 8.07 (d, = 8.9 Hz, 2H), 8.21 (d, = 8.9 Hz, 2H), 9.09 (s, 1H), 9.97 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 106.89, 115.53, 119.50, 124.10, 126.43, 132.98, 140.62, 146.15, 148.04, 152.59, 164.66.

HR MS (M+) calcd. for C15H11 N3O3S: 313.0521; found: 313.0526.

MS (EI) m/e 313 (100%) [M]+, 267 (25%), 89 (18%).

Synthesis of N-(4-hydroxyphenyl)-4-(2-fluorophenyl)-1,3-thiazole-2-amine (7)

1 H NMR (300 MHz, DMSO-d6) δ 6.74 (d, = 8.8 Hz, 2H), 7.13 (s, 1H), 7.23–7.36 (m, 3H), 7.45 (d, = 8.8 Hz, 2H), 8.09 (m, 1H), 9.16 (s, 1H), 9.93 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 106.42, 106.62, 115.49, 115.81, 116.11, 119.32, 122.12, 122.27, 124.69, 124.74, 128.91, 129.03, 129.54, 129.58, 133.19, 143.70, 143.73, 152.41, 157.89, 161.19, 163.55.

HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0570.

MS (EI) m/e 286 (100%) [M]+, 152 (18%).

Synthesis of N-(4-hydroxyphenyl)-4-(3-fluorophenyl)-1,3-thiazole-2-amine (8)

1 H NMR (500 MHz, DMSO-d6) δ 6.74 (d, = 8.9 Hz, 2H), 7.11 (m, 1H), 7.34 (s, 1H), 7.45 (m, 3H), 7.64 (d, = 10.8 Hz, 1H), 7.72 (d, = 8.7 Hz, 1H), 9.14 (s, 1H), 9.93 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 103.36, 111.94, 112.24, 113.94, 114.23, 115.51, 119.35, 120.05, 121.66, 130.53, 130.64, 133.16, 137.01, 137.12, 148.74, 148.78, 152.44, 160.91, 164.13, 164.36.

HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0576.

MS (EI) m/e 286 (100%) [M]+, 152 (18%).

Synthesis of N-(2-hydroxyphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (9)

1H NMR (300 MHz, DMSO-d6) δ 6.83 (m, 3H), 7.33 (s, 1H), 7.46 (d, = 8.52 Hz, 1H), 7.89 (d, = 8.52 Hz, 1H), 8.26 (m, 1H), 9.50 (s, 1H), 9.89 (d, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 104.06, 115.00, 119.03, 119.26, 122.41, 127.24, 128.64, 129.19, 131.81, 133.49, 146.39, 148.289, 164.31.

HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0281.

MS (EI) m/e 302 (100%) [M]+, 304 (33%), 269 (40%), 165 (403%).

Synthesis of N-(3-hydroxyphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (10)

1 H NMR (300 MHz, DMSO-d6) δ 6.37 (d, = 8.1 Hz, 1H), 7.01 (d, = 8.1 Hz, 1H), 7.11 (t, = 8.1 Hz, 1H), 7.32 (s, 1H), 7.39 (s, 1H), 7.48 (d, = 8.7 Hz, 2H), 7.95 (d, = 8.7 Hz, 2H), 9.43 (s, 1H), 10.17 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 103.62, 104.08, 107.87, 108.58, 127.42, 128.62, 129.64, 131.94, 133.40, 142.12, 148.84, 157.95, 163.20.

HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0282.

MS (EI) m/e 302 (100%) [M]+, 304 (33%), 168 (17%), 134 (23%).

Synthesis of N-(4-hydroxyphenyl)-4-(2,5-dichlorophenyl)-1,3-thiazole-2-amine (11)

1 H NMR (500 MHz, DMSO-d6) δ 6.73 (d, = 8.8 Hz, 2H), 7.36–7.42 (m, 4H), 7.56 (d, = 8.6 Hz, 1H), 7.93 (s, 1H), 9.19 (s, 1H), 9.95 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 108.01, 115.50, 119.61, 128.54, 129.31, 130.32, 131.79, 132.17, 133.00, 134.70, 145.16, 152.61, 163.75.

HR MS (M+) calcd. for C15H10Cl2N2OS: 335.9891; found: 335.9897.

MS (EI) m/e 336 (100%) [M]+, 338 (79%), 340 (19%), 301 (17%), 166 (17%).

Synthesis of N-(4-hydroxyphenyl)-4-(2,4-difluorophenyl)-1,3-thiazole-2-amine (12)

1 H NMR (300 MHz, DMSO-d6) δ 6.72 (d, = 8.67 Hz, 2H), 7.11 (s, 1H), 7.20 (t, 1H), 7.34 (t, 1H), 7.47 (d, = 8.67 Hz, 2H), 8.11 (q, = 8.1 Hz, 1H), 9.15 (s, 1H), 9.95 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 104.12, 104.47, 104.82, 105.85, 106.04, 111.69, 112.01, 115.50, 118.99, 119.04, 119.19, 119.36, 130.63, 130.69, 130.76, 130.82, 133.14, 142.90, 152.45, 157.77, 157.93, 159.40, 159.56, 161.11, 161.27, 162.67, 162.83, 163.66.

HR MS (M+) calcd. for C15H10 F2N2OS: 304.0482; found: 304.0475.

MS (EI) m/e 304 (100%) [M]+, 170 (20%).

Synthesis of N-(3,5-dichloro-4-hydroxyphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (13)

1 H NMR (300 MHz, DMSO-d6) δ 7.44 (s, 1H), 7.51 (d, = 8.4 Hz, 2H), 7.68 (s, 2H), 7.90 (d, = 8.4 Hz, 2H), 9.71 (br, 1H),

10.33 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 104.15, 116.89, 122.74, 127.20, 128.75, 132.10, 133.24, 134.46, 143.30, 148.77, 162.97.

HR MS (M+) calcd. for C15H9 Cl3N2OS: 369.9501; found: 369.9503.

MS (EI) m/e 370 (83%) [M]+, 168 (33%), 133 (53%), 89 (100%).

Synthesis of N-(3,5-dimethyl-4-hydroxyphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (14)

1 H NMR (300 MHz, CDCl3) δ 2.27 (s, 6H), 7.29 (s, 1H), 7.36 (s, 1H), 7.56 (d, = 8.3 Hz, 2H), 7.99 (d, = 8.3 Hz, 2H), 8.04 (s, 1H), 9.91 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 17.00, 102.51, 118.21, 124.94, 127.27, 128.62, 131.80, 133.18, 133.57, 148.34, 148.79, 164.62.

HR MS (M+) calcd. for C17H15 ClN2OS: 330.0594; found: 330.0609.

MS (EI) m/e 330 (100%) [M]+, 332 (33%), 162 (9%).

Synthesis of N-(3-chloro-4-hydroxy-5-dimethyl phenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (15)

1 H NMR (300 MHz, CDCl3) δ 2.25 (s, 3H), 4.68 (s, 1H), 6.67 (s, 1H), 6.95–6.96 (m, 1H), 7.02–7.29 (m, 4H), 7.74–7.79 (m, 2H), 7.97 (m, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 17.21, 41.64, 102.29, 112.79, 115.38, 115.67, 115.73, 115.91, 116.21, 118.55, 120.73, 127.47, 127.57, 127.67, 131.17, 131.21, 131.67, 131.80, 134.03, 145.42, 148.98, 160.01, 163.25, 163.53, 167.29, 190.98.

HR MS (M+) calcd. for C16H12Cl FN2OS: 334.0343; found: 334.0340.

MS (EI) m/e 334 (100%) [M]+, 336 (33%), 152 (25%), 123 (22%).

Synthesis of N-(3,5-dichloro-4-hydroxyphenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (16)

1 H NMR (300 MHz, DMSO-d6) δ 7.15 (s, 1H), 7.20 (m, 2H), 7.69 (s, 2H), 7.83 (m, 2H), 10.2 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 103.05, 115.45, 115.73, 116.84, 122.74, 127.44, 127.55, 134.52, 143.25, 148.95, 162.90.

HR MS (M+) calcd. for C15H9Cl2 FN2OS: 353.9797; found: 353.9789.

MS (EI) m/e 354 (81%) [M]+, 356 (52%), 358 (11%), 152 (100%).

Synthesis of N-(2,5-dimethyl-4-hydroxyphenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (17)

1 H NMR (300 MHz, CDCl3) δ 2.18 (s, 3H), 2.21 (s, 3H), 6.62 (s, 1H), 6.72 (s, 1H), 7.04 (m, 2H), 7.15 (s, 1H), 7.67 (s, 1H), 7.75 (m, 2H), 8.55 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 15.67, 17.48, 25.13, 30.42, 67.02, 101.25, 115.18, 115.47, 116.59, 121.96, 127.43, 127.54, 127.66, 130.61, 131.29, 131.48, 131.52, 149.24, 153.24, 159.86, 163.09, 168.94.

HR MS (M+) calcd. for C17H15 FN2OS: 314.0889; found: 314.0895.

MS (EI) m/e 314 (100%) [M]+, 281 (37%), 160 (26%).

Synthesis of N-(2,3,5-trimethyl-4-hydroxyphenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (18)

1 H NMR (300 MHz, CDCl3) δ 2.22 (s, 6H), 2.24 (s, 3H), 6.59 (s, 1H), 7.02 (m, 3H), 7.73 (m, 2H).

13 C NMR (75 MHz, DMSO-d6) δ 12.94, 14.41, 16.63, 101.15, 115.16, 115.44, 122.41, 124.22, 125.36, 127.43, 127.54, 130.80, 131.13, 132.21, 149.31, 151.21, 169.63.

HR MS (M+) calcd. for C18H17FN2OS: 328.1046; found: 328.1043.

MS (EI) m/e 328 (100%) [M]+, 295 (20%).

Synthesis of N-(4-aminophenyl)-4-(phenyl)-1,3-thiazole-2-amine (23)

1 H NMR (300 MHz, DMSO-d6) δ 4.94 (br, 2H), 6.60 (d, = 8.4 Hz, 2H), 7.17 (s, 1H), 7.30 (m, 3H), 7.43 (t, = 7.53 Hz, 2H), 7.89 (d, = 8.4 Hz, 2H), 9.69 (s,1H).

13 C NMR (75 MHz, DMSO-d6) δ 101.33, 114.41, 120.09, 125.61, 127.39, 128.56, 130.88, 134.79, 144.16, 150.11, 165.20.

HR MS (M+) calcd. for C15H13 N3S: 267.0830; found: 267.0846.

MS (EI) m/e 267 (100%) [M]+, 133 (18%).

Synthesis of N-(4-aminophenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (24)

Synthesis of N-(4-acetaminophenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine

About 0.45 g (2.00 mmol) of 2-bromo-4′-fluoroacetophenone was dissolved in 10 mL of ethanol, 0.21 g (2.59 mmol) of sodium thiocyanate and 0.33 g (2.20 mmol) of p-aminophenol were added at room temperature, and the mixture was refluxed at 80 °C for 12 h. After the reaction was completed, the reaction mixture was diluted with 10 mL of ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, and concentrated under a reduced pressure. The residue was purified using silica gel column chromatography (hexane:ethyl acetate = 1:1) to obtain the title compound (0.38 g, 58%).

1H NMR (300 MHz, DMSO-d6) δ 2.08 (s, 3H), 7.28 (m, 3H), 7.58 (d, 2H), 7.66 (d, 2H), 7.99 (dd, 1H), 9.89 (s, 1H), 10.22 (s,1H).

MS (EI) m/e 327 (100%) [M]+, 285 (60%), 152 (13%).

Synthesis of N-(4-aminophenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine (24)

About 0.23 g (0.70 mmol) of N-(4-acetaminophenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine was dissolved in 2 mL of 6 N HCl solution. The mixture was refluxed at 100 °C under a nitrogen atmosphere for 2 h. After cooling to ambient temperature, the mixture was basified with 2 N NaOH, extracted into ethyl acetate, dried over MgSO4, and filtered and distilled under a reduced pressure. The resulting residue was purified using silica gel column chromatography (n-hexane:ethyl acetate = 1:1) to obtain the title compound (0.19 g, 96%).

1 H NMR (300 MHz, DMSO-d6) δ 4.90 (br, 2H), 6.57 (d, = 8.37 Hz, 2H), 7.13 (s, 1H), 7.20 (m, 4H), 7.90 (m, 2H), 9.68 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 101.05, 114.36, 115.22, 115.51, 120.11, 127.49, 127.59, 130.78, 131.38, 131.42, 144.20, 149.03, 159.94, 163.20, 165.29.

HR MS (M+) calcd. for C15H12 FN3S: 285.0736; found: 285.0738.

MS (EI) m/e 285 (100%) [M]+, 152 (10%), 133 (22%).

Synthesis of N-(4-aminophenyl)-4-(4-bromophenyl)-1,3-thiazole-2-amine (21)

1 H NMR (300 MHz, DMSO-d6) δ 4.86 (br, 2H), 6.57 (d, = 8.6 Hz, 2H), 7.23 (s, 1H), 7.27 (d, = 8.6 Hz, 2H), 7.59 (d, = 8.4 H, 2H), 7.82 (d, = 8.4Hz, 2H), 9.70 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 102.24, 114.36, 120.18, 120.31, 127.60, 130.70, 131.47, 133.96, 144.26, 148.88, 165.35.

HRMS (M+) calcd. for C15H12BrN3S: 344.9935; found: 344.9939.

MS (EI) m/e 345 (100%) [M]+, 347 (98%), 164 (12%), 133 (30%).

Synthesis of N-(4-aminophenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (22)

1 H NMR (300 MHz, DMSO-d6) δ 4.89 (br, 2H), 6.58 (d, = 8.6 Hz, 2H), 7.22 (s, 1H), 7.29 (d, = 8.6 Hz, 2H), 7.45 (d, = 8.5 Hz, 2H), 7.87 (d, = 8.5 Hz, 2H), 9.70 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 102.13, 114.36, 120.15, 127.27, 128.54, 130.71, 131.71, 133.61, 144.23, 148.82, 165.33.

HR MS (M+) calcd. for C15H12 ClN3S: 301.0440; found: 301.0439.

MS (EI) m/e 301 (100%) [M]+, 303 (33%), 168 (13%).

Synthesis of N-(4-aminophenyl)-4-(4-nitrophenyl)-1,3-thiazole-2-amine (25)

1 H NMR (300 MHz, DMSO-d6) δ 4.67 (brs, 2H), 6.36 (d, = 8.6 Hz, 2H), 7.05 (d, = 8.6 Hz, 2H), 7.31 (s, 1H), 7.89 (d, = 8.7 Hz, 2H), 8.04 (d, = 8.7 Hz, 2H), 9.59 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 106.407, 114.38, 120.37, 124.08, 126.38, 130.47, 140.72, 144.47, 146.09, 148.10, 165.63.

HR MS (M+) calcd. for C15H12 N4O2S: 312.0681; found: 312.0682.

MS (EI) m/e 312 (100%) [M]+, 266 (33%), 133 (8%).

Synthesis of N-(2-aminophenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (26)

1 H NMR (300 MHz, DMSO-d6) δ 5.00 (s, 2H), 6.61 (t, = 7.1 Hz, 1H), 6.75 (d, = 7.1 Hz, 1H), 6.91 (t, = 7.1 Hz, 1H), 7.25 (s, 1H), 7.44 (d, = 8.5 Hz, 2H), 7.50 (d, = 7.1 Hz, 1H), 7.86 (d, = 8.5 Hz, 1H), 9.11 (s, 1H).

HR MS (M+) calcd. for C15H11 FN2OS: 301.0440; found: 301.0428.

MS (EI) m/e 301 (88%) [M]+, 303 (33%), 268 (100%), 164 (84%).

Synthesis of N-(3-aminophenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine (27)

1 H NMR (300 MHz, DMSO-d6) δ 5.13 (s, 2H), 6.22 (d, = 8.7 Hz, 1H), 6.79 (d, = 8.7 Hz, 1H), 6.93 (m, 2H), 7.35 (s, 1H), 7.45 (d, = 8.5 Hz, 2H), 7.94 (d, = 8.5 Hz, 2H), 9.98 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 103.72, 107.65, 111.69, 112.10, 127.52, 128.58, 129.09, 131.92, 133.39, 141.39, 148.96, 163.15, 168.29.

HR MS (M+) calcd. for C15H12ClN3S: 301.0440; found: 301.0454.

MS (EI) m/e 301 (88%) [M]+, 303 (33%), 268 (100%), 164 (84%).

Synthesis of N-(4-aminophenyl)-4-(2,5-dichlorophenyl)-1,3-thiazole-2-amine (31)

1 H NMR (300 MHz, DMSO-d6) δ 4.87 (s, 2H), 6.56 (d, = 8.6 Hz, 2H), 7.22 (d, = 8.6 Hz, 2H), 7.33 (s, 1H), 7.43 (d, = 8.5 Hz, 1H), 7.56 (d, = 8.5 Hz, 1H), 7.95 (s, 1H), 9.73 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 107.53, 114.41, 120.47, 128.40, 129.22, 130.32, 130.54, 131.76, 132.15, 134.68, 144.40, 145.14, 164.68,

HRMS (M+) calcd. for C15H11 Cl2N3S: 335.0051; found: 335.0052.

MS (EI) m/e 335 (100%) [M]+, 337 (79%), 379 (19%).

Synthesis of N-(4-aminophenyl)-4-(2,4-difluorophenyl)-1,3-thiazole-2-amine (32)

1 H NMR (300 MHz, DMSO-d6) δ 4.86 (s, 2H), 6.57 (d, = 8.6 Hz, 2H), 7.04 (s, 1H), 7.16–7.36 (m, 4H), 8.04 (m, 1H), 9.72 (s, 1H).

13 C NMR (75 MHz, DMSO-d6) δ 104.08, 104.43, 104.78 105.35, 105.54, 111.69, 111.96, 114.36, 119.05, 119.21, 119.25, 120.21, 130.66, 131.69, 131.76, 131.82, 133.14, 142.90, 144.30, 158.73, 158.93, 160.34, 160.56, 162.11, 162.27, 163.67, 163.83, 164.59.

HR MS (M+) calcd. for C15H11 F2N3S: 303.0642; found: 303.0655.

MS (EI) m/e 303 (100%) [M]+, 133 (25%).

In vitro 5-LOX assay

5-LOX enzyme assay was carried out with some modifications of ferric oxidation of xylenol orange (FOX) assay, which is based on the complex formation of Fe3+/xylenol orange with absorption at visible light (19,25). The enzyme sources were prepared from insect cell (Sf21) lysates highly expressing rat 5-LOX as described previously (19). Cell lysates (15 μg protein) were preincubated with various concentrations of either some selected chemicals or zileuton (in DMSO) in 50 μL of 50 mm Tris-HCl buffer (pH 7.4) containing 0.4 mm CaCl2, 24 μg/mL phosphatidylcholine (PC) and 20 μm ATP at room temperature for 4 min. The reactions were started by the addition of 40 μm AA, kept for another 4 min, and terminated by the addition of 100 μL FOX reagent: sulfuric acid (25 mm), xylenol orange (100 μm), iron (II) sulfate (100 μm), methanol:water (9:1, v/v). Blank assay was carried out without substrate during incubation, after that substrate was added before mixing with FOX reagent.

12- and 15-lipoxygenases assays for the determination of specificity

Catalytic activities of 12-LOX and 15-LOX were tested as the same way as 5-LOX assay except that ATP was absent in the assay system. As reference compounds of 12- and 15-LOXs, hinokitiol, and ebselen were used, respectively.

LTB4 secretion in RBL-1 cells activated by A23187

Cell-based assay was carried out according to the methods as previously reported (26). In brief, RBL-1 cells were plated at a density of 4 × 104 cells into 96 well plates. Various concentrations of either compounds or zileuton dissolved in DMSO (final 0.5%) was added to plates and kept incubation for 10 min at 37 °C prior to the stimulation with Ca2+ ionophore A23187 (final 10 μm in DMSO). After further incubation for 15 min, culture media were collected and LTB4 content in the supernatant was measured by using an EIA kit (Amersham).

Arachidonic acid-induced ear inflammation in mice

To induce inflammatory response, 2 mg of AA in 20 μL of acetone was painted to the inner surface of the right ear of Balb/c mouse (8 weeks of age, male, n = 5–6/group). A potent derivative 14 and zileuton were painted directly on the ears or orally given to mice 1 h before AA treatment. For vehicle control of p.o administration, equal volume of 0.5% CMC to treated groups was given. One hour after AA stimulation, ear thickness of the treated- and untreated-ears was measured with a dial thickness gauge (Mitutoyo, Tokyo, Japan). For long-lasting effect of topical application against ear edema animal model, mouse ears were preloaded topically with either zileuton or compound 14 over the indicated times, followed by AA administration. The effects of chemical derivatives were calculated as the percent difference in ear thickness from the mean values of the contralateral untreated ear. Myeloperoxidase (MPO) activity in the inflamed ear tissue was determined according to the method described by Bradley et al. (27). Briefly, punched ears (2 mm in diameter) were chopped and homogenized in 300 μL of 50 mm potassium phosphate buffer, pH 6.0 containing 0.5% HTAB. The homogenate was then centrifuged and the resulting supernatant (100 μL) was allowed to react with 0.167 mg/mL o-dianisidine and 0.0005% H2O2 in order to determine the absorbance at 450 nm.

Statistical analysis

Results were expressed as means ± SEM of two experiments each carried out as triplicates. Statistical significance was evaluated using Student’s t-test, and p < 0.05 was considered as statistically significant.

Results and Discussion

Chemistry

To delineate the pharmacological moiety for 5-LOX inhibition, a series of 1,3-thiazole derivatives were synthesized and evaluated by in vitro enzymatic assay and further cell-based format. The N-aryl-4-aryl-1,3-thiazole-2-amine analogs 332 were prepared according to Hantzsch thiazole synthesis (28) and a literature procedure (29) as shown in Scheme 1. Briefly, A series of compounds were prepared from the substitution of 2-bromoacetophenone derivatives 1 with excess sodium thiocynate and sequential cyclization with various aniline derivatives 2 in refluxing ethyl alcohol. The N-aryl-4-aryl-1,3-thiazole-2-amine analogs 332 were synthesized with substituent variation at phenyl groups as described in Experimental Section.

Figure Scheme 1:.

 Sodium thiocyanate, EtOH, reflux, 40–83%.

Biological activity

In this work, we pursued to identify a novel scaffold that is able to inhibit directly the enzyme activity of 5-LOX from in-house chemical library of Korea Research Institute of Chemical Technology (KRICT; Daejeon). Primarily, a highly diverse chemical library was tested using enzymatic assay as previously described (19). Eventually, we identified N-aryl-4-aryl-1,3-thiazole-2-amine derivatives as the potent inhibitor of 5-LOX catalytic activity. For further SAR analyses, chemical derivatives bearing a variety of diverse substituents were synthesized and then their inhibitory potencies against enzymatic 5-LOX were listed in Table 1. From the in vitro enzymatic results above, we found that either OH or NH2 group at N-aryl moiety of N-aryl-4-aryl-1,3-thiazole-2-amine analogs was critical for conferring inhibitory activity against 5-LOX. By comparing IC50, the compounds 318 with OH group at N-aryl moiety present in N-aryl-4-aryl-1,3-thiazole-2-amine analogs exhibited a little more potent in vitro inhibitory activity for 5-LOX than did the compounds 24–32 bearing NH2 group (Table 1). On the other hand, a variety of substituents (F, Cl, Br, OCH3, NO2, CH3) at 4-aryl moiety of N-aryl-4-aryl-1,3-thiazole-2-amine analogs did not show any drastic changes with regard to 5-LOX inhibition. As OH group was differentially introduced at N-aryl moiety in thiazole compounds (5, 9 and 10), the inhibitory activity on 5-LOX was significantly influenced by OH position at N-aryl. Specifically, 4-substituted OH at N-aryl-1,3-thiazole compound 5 (98% inhibition at 1 μm) showed good inhibitory activity. However, 3-substituted OH at N-aryl compound 10 (19% inhibition at 1 μm) abolished completely the 5-LOX inhibitory potency. On the other hand, 2-substituted OH at N-aryl compound 9 (90% inhibition at 1 μm) was comparable with compound 5 regarding 5-LOX inhibitory power. Variations in position of NH2 group into N-aryl exhibited inhibitory profiles similar to those observed in OH group attached to N-aryl. Placement of NH2 at 2- or 4-position of N-aryl moiety remained active, whereas attachment of it at 3-position severely diminished its inhibitory activity. Based on these results, we found that OH or NH2 group at the 2- or 4-position of N-aryl moiety was pharmacologically necessary for 5-LOX inhibition. Aside from NH2 or OH, the introduction of –OCH3, –CO2CH3, –CF3, –CH2OH or –NHCOCH3 group at 4-position of N-aryl compounds 1923 deteriorated severely their inhibitory activities against 5-LOX. The synthesized N-(aminophenyl)-4-aryl-1,3-thiazole-2-amines analogs 2432 with various substituents at 4-aryl moiety were also examined. When NH2 group was located at a 4-position of N-aryl moiety, the SAR in these analogs revealed a very similar trend to the N-hydroxyphenyl-4-aryl-1,3-thiazole-2-amine analogs. Interestingly, the positioning of an electron donor NH2 at N-aryl of compound 27 (IC50 = 160 nm, 77% inhibition at 1 μm) was remarkable in improving IC50 value, unlike compound 23 containing NHCOCH3 at N-aryl (39% inhibition at 1 μm). Additionally, an in vitro enzymatic assay demonstrated that the 2,5-, or 3,5-disubstituted compounds 1317 with electron donors or electron acceptors around OH group at N-aryl did not affect significantly 5-LOX inhibitory activity, comparable with parent compound (4131JH0380) and 2,3,5-trisubstituted compound 18.

Subsequently, the potent N-hydroxyphenyl-4-aryl-1,3-thiazole-2-amine analogs selected through in vitro enzymatic assay were further validated in a cell-based assay (Tables 1 and 2), in which LTs B4 (LTB4) is released into culture media from Ca2+ionophore (A23187)-stimulated rat basophilic leukemia-1 (RBL-1) cells. We simply reasoned that additional methyl group around OH of R2 can make readily penetrable to cell membrane. Importantly, pharmacophore that we found is the structure bearing thiazole ring with OH or NH2 at 4 position of R2 and the presence of this functional group is especially crucial for 5-LO inhibitory activity. It may be expected that substituents can improve uptake into cells or protect from intracellular metabolism of OH or NH2 of benzene ring because OH or NH2 group of R2 is likely to be metabolized in vivo (our unpublished pharmacokinetics data). Based on this rationale, selected compounds 1318 were tested as a function of dose in a cell-based format to achieve the IC50 values of those compounds (Table 2). Some of the derivatives were comparable or superior to zileuton with respect to interference with LTB4 generation. Intriguingly, we found that modification of substituent around OH group at the 4- position of N-aryl moiety influenced very strongly the ability of compounds to inhibit LTB4 production. As shown in Table 2, compound 14 (IC50 = 25 nm, 98% inhibition at 1 μm) with 3,5-diCH3 group around OH group was shown to be the most potent in this series of 1,3-thiazole analogs, while compound 17 with 2,5-diCH3 was relatively weak. However, compound 13 (IC50 = 77 nm, 99% inhibition at 1 μm), 15 (IC50 = 348 nm, 93% inhibition at 1 μm) and 16 (IC50 = 324 nm, 81% inhibition at 1 μm) with Cl group at N-aryl moiety were less potent than the compound 14, suggesting that introduction of halogen group may interfere with being permeable to cell membrane or may affect its cellular metabolism. As shown in in vivo efficacies of the compound 14 via different routes (Table 3), the compound 14 had a protective effect against AA-induced edema when topically, but not orally administered, as predicted strongly from pharmacokinetic data demonstrating that derivatives with pharmacological moiety of thiazole did not exhibit good bioavailability in rat (30). It is demonstrated that a specific compound KR-33749, chosen through in vitro and cell-based assays, improves inflammatory skin lesions such as atopic dermatitis. This study highlights a perspective series of processes from screening, SAR through in vivo efficacy over previous report performed in our group. Through SAR study, specifically, we could propose pharmacophore responsible for inhibitory potency. Typically, administration of selected compound 14 via p.o route was less effective than that via topical application or intraperitoneal route (data not shown), implying that the compound 14 is readily susceptible to hepatic metabolism. Topical application of the compound 14 to mice reduced the inflammatory responses such as ear swelling and MPO activity following AA spreading onto ear. More intriguingly, it not only had more potent anti-inflammatory activity than had zileuton, but also exhibited sustainable protective effect in AA-inflamed ear model. The in vivo efficacy of compound 14 in mice given at a low dose considerably improved the two indications of acute inflammation such as MPO and ear edema. Of note, preapplied zileuton did not retain its anti-inflammatory effect, while compound 14 remained relatively potent even if applied topically 2 days before AA painting. It suggests that compound 14 resides longer around skin tissue or is metabolically stable compared with zileuton (Figure 1). Lastly, we checked the selectivity of compound 14 for 5-LOX over the other isozymes (Table 4) because other LOXs shares partly with 5-LOX with regard to the structural or non-heme iron constraint for their activities. More favorably, it inhibited preferentially 5-LOX to 12-LOX or 15-LOX so that IC50 values were 10–100 times higher for 12-(15-)LOX than that for 5-LOX, thereby minimizing side-effects. On the other hand, 12-LOX and 15-LOX have, respectively, been known to be implicated in tumor angiogenesis and colorectal tumorigenesis (23,24). Therefore, dual inhibition of 5-LOX/12-LOX or 5-LOX/15-LOX might be beneficial than single inhibition of 5-LOX under specific circumstances.

Table 2.   The inhibitory effects of N-aryl-4-aryl-1,3-thiazole-2-amines on LTB4 secretion from A23187-stimulated RBL-1 cells
CompoundsIC50 (nm)a
  1. aCellular 5-lipoxygenase activities were assayed for potent chemical derivatives in a wide range of concentrations. IC50 was calculated by fitting the dose–response curves using GraFit software, achieving relative potencies of compounds under investigation.

Zileuton430
1377
1425
15348
16324
17576
18121
Table 3.   Comparative in vivo potency of compounds via p.o and topical applications.
CompoundsaEar edema (% inhibition)b
ThicknessMPO
  1. aMice (5–6 mice/group) were orally given with each compound at a dose of 100 mg/kg (mpk) 30 min before arachidonic acid (AA) painting onto ear.

  2. bAA-treated ear tissues were assessed by two inflammation indications reflecting tissue swelling and neutrophil infiltration, so that ears applied with test compounds were relatively compared with those treated with vehicle.

  3. cTo test topical effects of chemical derivatives, 100 μg of each compound were directly applied to ears of mice, and 30 min later, AA was painted to induce inflammation. Ear edema and neutrophil infiltration were measured by ear thickness and myeloperoxidase (MPO) activity in the extracted tissues and then evaluated by the inhibitory power in chemical-treated groups relative to vehicle-treated group.

Zileuton7582
144562
Compounds (100 μg)Ear edema (% inhibition)c
ThicknessMPO
Zileuton2638
146060
Figure 1.

 The sustainable anti-inflammatory activity of compound 14 and zileuton when applied topically to ear. Chemical dissolved in acetone was applied on the right ear 30 min before arachidonic acid (AA) painting (2 mg in 20 μL acetone) on the same ear. One hour after AA application, ear thickness of inflamed right ear was measured and then inflamed ears were taken for myeloperoxidase (MPO) activity. Ear thickness and MPO activity in ear homogenates. Data are mean ± SEM from three experiments (n = 5 in each experiment). To determine the duration of action of compound 14, the compound was applied and kept for 48, 24 or 0.5 h. After the indicated times, AA was applied, and ear thickness and MPO activity were measured 1 h later.

Table 4.   Selective inhibition of compound 14 against 12-lipoxygenase (LOX) and 15-LOX.
CompoundIC50m)a
12-LOXb15-LOXb
  1. aIC50 of compound 14 for LOXs was calculated from % inhibitory activity as a function of concentration.

  2. bBoth 12-LOX and 15-LOX assays were carried out according to protocol as previously reported (19).

1412.5>100

Conclusions and Future Directions

Taken together, we identified a novel series of skeleton with anti-inflammatory activities targeting 5-LOX through high-throughput screening. As a result, we synthesized and evaluated N-aryl-4-aryl-1,3-thiazole-2-amine derivatives as direct 5- LOX inhibitors. Through SAR and chemical optimization, a compound 14 was proven to be more effective against AA-induced ear edema than zileuton when topically applied, but not orally. In fact, the in vivo efficacy of 14 on 5-LOX in mice given at low doses considerably improved two indications of acute inflammation such as MPO and ear edema, superior to zileuton which is the only drug approved by FDA as a direct 5-LOX inhibitor. Although its precise mechanism remains unclear, it might be estimated that skin penetration and metabolic transformation of 14 would be primary for its therapeutic success. In fact, we have not checked the mechanism yet. We will further elucidate the action mechanism of compound 14 by which it would inhibit in either reversible or irreversible. Regarding redox properties, we examined indirectly the possibilities of redox inhibitor by carrying out methemoglobin induction (data not shown). Compound 14 did not induce methemoglobin formation, indicating that it does not inhibit 5-LO in a redox manner. Besides in vivo efficacy, it exerted more selective inhibition against 5-LOX over other isozymes (Table 4). Therefore, it is encouraged from its selectivity and in vivo efficacy data that this compound will be applicable to a wide range of skin models associated with 5-LOX in the future.

Acknowledgments

The present research has been conducted by the Settlement Research Grant of Keimyung University in 2011.

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