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Current Medicinal Chemistry, 2012, 19, 4348-4358
Design, Synthesis and Biological Activity of New Polyenolic Inhibitors of Matrix Metalloproteinases: A Focus on Chemically-Modified Curcumins Yu Zhang1, Ying Gu2, Hsi-Ming Lee3, Elena Hambardjieva4, Kveta Vranková4, Lorne M. Golub3,*, and Francis Johnson1,4,* 1
Department of Chemistry; 2Department of General Dentistry; 3Department of Oral Biology and Pathology; 4Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11790, United States Abstract: Matrix metalloproteinases (MMPs) are essential for the degradation and turnover of components of the extracellular matrix (ECM) and, when pathologically elevated, mediate connective tissue loss (including bone destruction) in various inflammatory and other diseases. Tetracyclines (TCs) are known inhibitors of mammalian-derived MMPs, and non-antibiotic formulations of Doxycycline are FDA-approved to treat periodontitis and the chronic inflammatory skin disease, rosacea. Because the C-11/ C-12 diketonic moiety of the tetracyclines is primarily responsible, through zinc-binding, for MMP inhibition, we have uniquely modified curcumin as a “core” molecule, since it contains a similar enolic system and is known to have beneficial effects in diseases where connective-tissue loss occurs. Specifically we have developed new congeners which exhibit improved zinc-binding and solubility, and potent reduction of excessive MMP levels and activity. We now describe a series of curcuminoid bi- and tri-carbonylmethanes in which all of these properties are substantially improved. An N-phenylaminocarbonyl derivative of bis-demethoxycurcumin (CMC2.24) was selected as the “lead” substance because it showed superior potency in vitro (i.e., the lowest IC 50) against a series of neutral proteases (MMPs) associated with tissue erosion. Moreover, CMC2.24 administered to diabetic rats orally (30mg/kg), reduced the secretion of pathologically-excessive levels of MMP-9 to normal in cultured peritoneal macrophages with no evidence of toxicity. Thus, this (and other similar novel) compound(s) may be useful in various diseases of connective-tissue loss.
Keywords: Matrix metalloproteinases, chemically-modified-curcumin, connective tissue degradation, zinc-binding, enolic -diketone moiety. A. INTRODUCTION Matrix metalloproteinases (MMPs) are a group of more than twenty-five structurally-related zinc-containing neutral proteolytic enzymes. The first of these, namely MMP-1 or collagenase-1, was discovered by Gross et al. in 1962 [1]. While examining resorbing tail tissue from a tadpole undergoing metamorphosis, they found that it secreted a neutral proteinase uniquely able to degrade the triple-helical collagen macromolecule and native collagen fibrils under physiologic conditions of pH and temperature. It is now known that the various MMPs (Table. 1) can collaboratively degrade essentially all of the constituents of the extracellular matrix (ECM) and, when expressed and activated in pathologicallyexcessive levels, can exacerbate a variety of diseases, causing the destruction of connective tissue including cartilage and bone [2-8]. Because collagen and the other constituents of the ECM, such as the proteoglycans, elastin and fibronectin, exist virtually everywhere in the body, it is not surprising that the development of MMP-inhibitors, to treat human diseases ranging from arthritis and periodontal disease to postmenopausal osteoporosis, cardiovascular disease and cancer, has been a target of both academic groups and the pharmaceutical industry for the past several decades [9-11]. However, it has been recognized recently that excessive inhibition of MMPs, especially those that are constitutive, is undesirable and can cause significant complications such as musculoskeletal sideeffects. Basal levels of these enzymes are required for physiologic functions including (but not limited to) normal connective tissue turnover and bone-remodeling, as well as activation of growth factors and anti-inflammatory cytokines [3, 11]. In this regard, in 1983 Golub et al. [5, 8, 12] described their discovery of a previously unrecognized new therapeutic use for an old family of drugs, namely the ability of tetracyclines (TCs) to inhibit MMPs, and by a mechanism unrelated to their antibiotic activity. This discovery was translated into the development of two FDA-approved, orallyadministered drugs, namely Periostat® for the treatment of chronic inflammatory oral-bone-destructive periodontitis, and Oracea® for *Address correspondence to these authors at the Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA; Tel: (631) 632-8866; Fax: (631)632-9721; E-mail:
[email protected] and Department of Oral Biology and Pathology, School of Dental Medicine, Stony Brook University, Stony Brook, NY 11794-8702, USA; Tel: (631) 632-8912; Fax: (631)-632-9705; E-mail:
[email protected] -;/12 $58.00+.00
chronic inflammatory skin disease, both formulated at an FDA mandated sub-antimicrobial dose of Doxycycline (1a). Following this lead, a series of chemically-modified tetracyclines (CMTs) were developed, no longer possessing anti-bacterial activity, but which still behaved as MMP inhibitors [5]. One of these (CMT-3, 1b) was found to be effective as an anti-angiogenic agent in phase II clinical trials on patients with Kaposi’s sarcoma [13, 14]. However, a significant side-effect of the tetracyclines and their nonantimicrobial congeners such as CMT-3, is increased photosensitivity, particularly at higher doses, and this limits their long-term use [14-16]. Given that the enolic -diketone assembly on the tetracycline molecule at carbons 11, 11a and 12 appears to be responsible for the MMP inhibition via zinc-binding [5, 12], we selected curcumin as a possible lead framework because, like the TCs, it also contains a similar metal ion-binding moiety (Scheme. 1), and has shown evidence of safety and efficacy as an inhibitor of both proinflammatory mediators and MMPs, in a variety of disease conditions [17-22]. Our current investigations have focused on a series of enolic bi-and tri-carbonyl compounds derived from curcumin (2), seeking to improve both metal binding and the solubility characteristics, in addition to potentially increasing the anti-inflammatory and MMP-inhibitory properties. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-hepta diene-3,5-dione; 2], is a natural dietary ingredient found in turmeric, a coloring compound derived from the perennial herb Curcuma longa L. and was isolated initially in 1815, first crystallized in 1870, and its chemical structure was elucidated in 1910 by Lampe et al. [23]. It has been synthesized by several methods, including those of Pabon [24] and of Pedersen et al. [25]. The natural material has been used throughout history, principally as an herbal treatment for a variety of pulmonary, gastrointestinal and liver diseases, as well as a remedy for non-healing wounds, and has been shown to exhibit a number of other therapeutic effects in both in vitro and in vivo model systems [26-28]. Despite these many beneficial effects, poor oral absorption and the need to use extremely high oral doses of the compound in either animals or humans, has limited its clinical application [29]. With this background in mind, our group has synthesized a series of novel chemically-modified curcumins (CMCs) with improved zincbinding and other characteristics compared to curcumin. In the current report, we describe twenty-three CMCs in addition to curcumin, including their MMP-inhibitory properties, and identify a “lead” © 2012 Bentham Science Publishers
New Polyenolic Inhibitors of Matrix Metalloproteinases
Table 1.
Current Medicinal Chemistry, 2012 Vol. 19, No. 25
Matrix Metalloproteinases (MMPs) and their Principle Substrates Modified from References [2-4, 11]
Group
Enzyme
Collagenases
Fibroblast Collagenase
MMP-1
Fibrillar Collagens, PG Core Protein
Neutrophil Collagenase
MMP-8
Fibrillar Collagens, 1 Proteinase Inhibitor (Serpin)
MMP
Principle Substrates
Collagenase-3
MMP-13
Fibrillar Collagens, PG Core Protein
Collagenase-4
MMP-18
Collagen I
Gelatinases
Gelatinase A
MMP-2
Gelatin, Elastin, Fibronectin, Collagens IV, V and VI, Cardiac Myocyte Intracellular Contractile Proteins
Gelatinase B
MMP-9
Gelatin, Elastin, Fibronectin, Collagen I, IV and V
Stromelysins
Stromelysin-1
MMP-3
Fibronectin, Non-Fibrillar Collagens, Laminin
Stromelysin-2
MMP-10
Fibronectin, Non-Fibrillar Collagens, Laminin
Membrane-type
Unclassified
4349
Stromelysin-3
MMP-11
Gelatin, Fibrillar Collagens, 1 Proteinase Inhibitor (Serpin)
MT1-MMP
MMP-14
Pro-MMP-2, Fibrillar Collagens, Gelatin, Elastin, Casein, Fibronectin, Vitronectin, Aggrecan
MT2-MMP
MMP-15
Pro-MMP-2, Gelatin, Fibronectin, Laminin, Nidogen, Tenascin
MT3-MMP
MMP-16
Pro-MMP-2, Collagen III, Gelatin
MT4-MMP
MMP-17
Pro-MMP-2, Gelatin
MT5-MMP
MMP-24
Pro-MMP-2
MT6-MMP
MMP-25
Pro-MMP-2
Matrilysin
MMP-7
Fibronectin, Collagen IV and X, Laminin, Aggrecan, Casein, Decorin, Insulin
Macrophage Metalloelastase
MMP-12
Elastin, Fibronectin, Proteoglycan, 1-Antitrypsin
RASI-1
MMP-19
Gelatin
Enamelysin
MMP-20
Amelogenin, Aggrecan
CA-MMP
MMP-23
Mca-peptide
Matrilysin-2
MMP-26
Fibrinogen, Fibronectin, Vitronectin
Epilysin
MMP-28
Casein
CH3 5
7 8
OH 4
6 10 OH
7
11 O
6
12 OH
5
2
1 O
11a 10 OH
11 O
O 1 2
Doxycycline (1a)
O
OH
3
9
NH2
4
8
H3CO
OH
3 OH
11a
9
N(CH3)2
12 OH
3
OH 5
4
6
OH
CMT-3 (1b) 2
1 O
NH2 O
Curcumin (2) 7
HO
OCH3 OH
Scheme 1. Structural comparison of the zinc binding sites of Tetracycline and Curcumin.
compound, i.e., an N-phenylaminocarbonyl analogue of bisdemethoxycurcumin, which exhibits superior therapeutic potential. B. MATERIALS AND METHODS 1. Synthesis of Curcumin Analogues All reagents and solvents employed in this experimental work were reagent grade and were used as such unless otherwise specified. Melting points were taken on a Thomas-Hoover open capillary melting point apparatus and are uncorrected. 1H NMR spectra were recorded on a Varian Gemini 300 spectrometer either in CDCl3 or
DMSO-d6. Chemical shifts are reported in parts per million (ppm) relative to TMS. Mass spectra were recorded on either a Thermo Electron DSQ GC/MS equipped with a solid probe inlet and EI ionization or an Agilent 1100LC (API-ES)/MSD-VL(m/z=50-1500) using electrospray ionization. Thin-layer chromatography (TLC) was performed on silica gel sheets (Tiedel-deHaën, Sleeze, Germany). After appropriate purification all new products showed a single spot on TLC analysis in the following solvent systems: 25% ethyl acetate in hexanes and 10% methanol in dichloromethane. Components were visualized by UV light (l=254 nm). Flash column chromatographic separations were carried out on 60A (230-400 mesh) silica gel (TSI Chemical Co., Cambridge, MA). All reactants
4350 Current Medicinal Chemistry, 2012 Vol. 19, No. 25
that were moisture or air-sensitive were conducted under dry nitrogen. The starting materials and reagents, unless otherwise specified, were the best grade commercially available (Sigma-Aldrich, Milwaukee, WI or Fluka Chemie GmbH, Sigma-Aldrich, Germany) and were used without further purification. General Procedure for the Synthesis of 3-Substituted-2,4Pentandiones 2,4-Pentandione (1.00g, 10mmol) was added to a suspension of magnesium chloride (1.35g, 1.2eq) in 20mL methylene chloride, followed by pyridine (2.13mL, 2.5eq), and the mixture was stirred at 0oC for 1h, then either an alkyl chloroformate or a phenyl isocyanate (1.0eq) was added dropwise at 0oC. The mixture was allowed to warm up to room temperature during 5hours, and then was poured into 3N aqueous HCl solution (10mL) and extracted with methylene chloride (20mL). The organic layer was washed with 20mL brine solution, dried over sodium sulfate and the solvent was removed under vacuo. The product was then either distilled or recrystallized from an appropriate solvent as required [30, 31]. The essential data for each individual compound are given below. 3-Methoxycarbonyl-2,4-pentandione (4a): Colorless liquid, 68.1% yield. Distilled at 45oC, 0.5mmHg.( lit.[30], bp 75-76oC, 0.3mmHg) 1H NMR (DMSO-d6, 300MHz): 3.00(s, 6H, -CH3), 3.73(s, 3H, -OCH3), 17.76(s, 1H, -OH enol). ESI (-ve) MS m/z 157.1[M-H]-. 3-Ethoxycarbonyl-2,4-pentandione (4b): Colorless liquid, 60.5% yield. Distilled at 60oC, 1.4mmHg.(lit.[31], bp 88-91oC, 8mmHg) 1H NMR (DMSO-d6, 300MHz): 1.25(t, 3H, -CH3) , 2.30(s, 6H, -CH3), 4.20(q, 2H, -CH2), 17.75(s, 1H, -OH enol). ESI (-ve) MS m/z 171.2[M-H]-. 3-Phenylaminocarbonyl-2,4-pentandione (4c): White solid, 72.0% yield, mp 118-119oC.(lit.[32], mp 117-119oC) 1H NMR (DMSO-d6, 300MHz): 2.15(s, 6H, -CH3), 7.08(t, 1H, Ar-H), 7.32(t, 2H, Ar-H), 7.64(d, J=8.1Hz, 2H, Ar-H), 10.36(s, 1H, -NH), 16.46(s, 1H, -OH, enol). ESI (-ve) MS m/z 218.1[M-H]-. General Method for the Synthesis of the Curcumin Analogues 2,4-Pentandione or a 3-substituted 2,4-pentandione (10mmol) and very finely powdered boron oxide (0.49g, 7mmol, 0.7eq) were placed in a 50mL flask and heated to 120oC for 5min to form a pale-yellow viscous liquid which was cooled to room temperature. The appropriate aldehyde (20mmol, 2.0eq) and trimethyl borate (4.16g, 40mmol, 4.0eq) were dissolved in ethyl acetate (10mL) and gradually added to the reaction mixture over 10min. Thereafter, with stirring, 0.05mL butylamine and 0.2mL butylammonium acetate (0.136g/mL) in dimethylformamide solution was then added. After 1 hour, a red-colored precipitate began to form and stirring was continued at room temperature for 48 hours. The precipitate was removed by filtration, washed with dry ether and air-dried, then dissolved in methanol (50mL) and boiled for 30 min at 60oC. The methanol was removed by rotary evaporation and the solid crude product was purified by recrystallization from dichloromethane (20mL) and methanol (20mL). 4-(4-methylpiperazinyl-1-yl)benzaldehyde (5p): Potassium carbonate (3.50g, 25.3mmol) was added to a solution of 4fluorobenzaldehyde (2.71mL, 3.14g, 25.3mmol) and Nmethylpiperazine (2.55mL, 2.30g, 23mmol) in DMSO (23mL) and the mixture was heated under reflux (120oC) for 5h. The reaction mixture was then poured into water (450mL) and extracted with ether (420mL). The combined organic layers were washed with water, dried over Na2SO4 and evaporated. The crude material was crystallized from a hexane:ethyl acetate (3:1) to afford 5p as a paleyellow crystalline solid, yield 46.0 %, mp 56-57oC.(lit.[33]) 1 H NMR (CDCl3, 300MHz) 2.35(s, 3H, -CH3), 2.55(t, J = 5.1Hz, 4H, -CH2), 3.42(t, J = 5.1Hz, 4H, -CH2), 6.90-6.94(m, 2H, Ar-H), 7.74-
Zhang et al.
7.77(m, 2H, Ar-H), 9.78(s, 1H, -CHO). MS (EI) m/z (%) 204 (M˙+, 100), 133(30), 132(35). 1,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1E,6E-dien-3,5dione (2): From 2,4-pentandione (3) and 4-hydroxy-3methoxybenzaldehyde (5a); orange crystals, yield 77.0%, mp 175176oC.(lit. [24], mp 177-178oC) 1H NMR (DMSO-d6, 300MHz): 3.83(s, 6H, -OCH3), 6.05(s, 1H, =CH), 6.75(d, J=15.6Hz, 2H, =CH), 6.81(d, J=7.8Hz, 2H, Ar-H), 7.14(d, J=8.4Hz, 1H, Ar-H), 7.15(d, J=8.1Hz, 1H, Ar-H), 7.31(d, J=2.1Hz, 2H, Ar-H), 7.54(d, J=15.9 Hz, 2H, =CH), 9.66(s. 2H, -OH phenol), 16.40 (s, 1H, -OH enol); ESI (-ve) MS m/z 367.2[M-H]-. 1,7-Diphenyl-hepta-1E,6E-dien-3,5-dione (6a): From 2,4pentanedione (3) and benzaldehyde (5b); orange crystals, yield 65.1%, mp 141-142oC. (lit. [24] mp 140.5oC) 1H NMR (DMSO-d6, 300MHz): 6.21(s, 1H, =CH), 6.96(d, J=15.9Hz, 2H, =CH), 7.417.48(m, 6H, Ar-H), 6.65(d, J=15.9Hz, 2H, =CH), 7.71-7.75(m, 4H, Ar-H), 16.08(s, 1H, -OH enol). ESI (-ve) MS m/z 275.2 [M-H]-. 1,7-Bis(3-pyridyl)-hepta-1E,6E-dien-3,5-dione (6b): From 2,4-pentandione (3) and 3-pyridinecarboxaldehyde (5c); yellow solid, yield 62.3%, mp 171-172oC.1H NMR (DMSO-d6, 300MHz): 6.20(s, 1H, =CH), 7.12(d, J=16.2Hz, 2H, =CH), 7.45-7.49(m, 2H, Ar-H), 7.68(d, J=15.9Hz, 2H, =CH), 8.18(d, J=8.4Hz, 2H, Ar-H), 8.57-8.59(m, 2H, Ar-H), 8.90(d, J=1.8Hz, 2H, Ar-H), 15.97(s, 1H, -OH enol). ESI (-ve) MS m/z 277.2[M-H]-. 1,7-Bis-phenyl-4-methoxycarbonylhepta-1E,6E-dien-3,5dione (6c) : From 3-methoxycarbonyl-2,4-pentandione (4a) and benzaldehyde (5d); white solid, yield 52.1%, mp 126-127oC. 1H NMR (DMSO-d6, 300MHz): 3.92(s. 3H, -OCH3), 7.26(d, J=15.6Hz, 2H, =CH), 7.45-7.47(m, 6H, Ar-H), 7.72-7.75(m, 4H, Ar-H), 7.82(d, J=15.6Hz, 2H, =CH), 18.07(s, 1H, -OH enol). ESI (ve) MS m/z 333.2[M-H]-. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6d): From 3-methoxycarbonyl-2,4pentandione (4a) and 4-hydroxy3-methoxy-benzaldehyde (5a); yellow crystals, yield 72.0%, mp 175-176o C. 1H NMR (DMSOd6, 300MHz): 3.82(s, 3H, -OCH3 ), 3.91(s, 6H, Ar-OCH3 ), 6.84(d, J=7.2Hz, 2H, Ar-H), 7.03(d, J= 15.6Hz, 2H, =CH), 7.19(d, J=8.1Hz, 1H, Ar-H), 7.20(d, J=8.4Hz, 1H, Ar-H), 7.28(d, J=1.5Hz, 2H, Ar-H), 7.73(d, J=15.6Hz, 2H, =CH), 9.81(s. 2H, OH phenol), 18.31(s, 1H, -OH enol); ESI (-ve) MS m/z 425.2[MH]-. 1,7-Bis(3-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6Edien-3,5-dione (6e): From 3-methoxycarbonyl-2,4-pentandione (4a) and 3-Hydroxybenzaldehyde (5f); yellow crystals, yield 40.2%, mp 188-189oC. 1H NMR (DMSO-d6 , 300MHz): 3.91(s, 3H, -OCH3 ), 6.86(d, J=7.8Hz, 2H, =CH), 7.08-7.28(m, 8H, ArH), 7.73(d, J=15.3Hz, 2H, =CH), 9.70(s, 2H, -OH phenol), 18.11(s, 1H, -OH enol). ESI (-ve) MS m/z 365.2[M-H]-. 1,7-Bis(4-N,N-dimethylaminophenyl)-hepta-1E,6E-dien3,5-dione (6f): From 2,4-pentandione (3) and 4-(Dimethylamino) benzaldehyde (5g); purple crystals, yield 61.1%, mp 212214oC.(lit. [24] mp 214-215oC) 1 H NMR (DMSO-d6 , 300MHz): 2.98(s, 12H, -N(CH3)2 ), 5.95(s, 1H, =CH), 6.59(d, J=15.9Hz, 2H, =CH), 6.72(d, J=9.0Hz, 4H, Ar-H), 7.50(d, J=15.6Hz, 2H, =CH), 7.53(d, J=8.7Hz, 4H, Ar-H), 16.62(s, 1H, -OH enol). ESI (-ve) MS m/z 365.1[M-H]-. 1,7-Bis(3-pyridyl)-4-methoxycarbonylhepta-1E,6E-dien3,5-dione (6g): From 3-methoxycarbonyl-2,4-pentandione (4a) and 3-Pyridinecarboxaldehyde (5c); yellow solid, yield 38.7%, mp 195-196o C. 1H NMR (DMSO-d6 , 300MHz): 3.92(s, 3H, OCH3 ), 7.38(d, J= 15.9Hz, 2H, =CH), 7.49(t, 2H, Ar-H), 7.86(d, J=15.3Hz, 2H, =CH), 8.19(d, J=7.5Hz, 2H, Ar-H), 8.61(d, J=4.2Hz, 2H, Ar-H), 8.90(s, 2H, Ar-H), 17.89(s, 1H, -OH enol). ESI (-ve) MS m/z 335.2[M-H]-.
New Polyenolic Inhibitors of Matrix Metalloproteinases
1,7-Bis(2-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6Edien-3,5-dione (6h): From 3-methoxycarbonyl-2,4-pentandione (4a) and 2-Hydroxybenzaldehyde (5i); yellow crystals, yield 46.3%, mp 165-166oC. 1H NMR (DMSO-d6, 300MHz): 3.88(s, 3H, -OCH3), 6.84-6.94(m, 4H, Ar-H), 7.26(d, J=15.3Hz, 1H, ArH), 7.27(d, J=15.3Hz, 1H, Ar-H), 7.29(d, J=15.6Hz, 2H, =CH), 7.58(d, J=7.5Hz,1H, Ar-H), 7.58(d, J=8.1Hz, 1H, Ar-H), 8.01(d, J=15.6Hz, 2H, =CH), 10.42 (s, 2H, -OH phenol), 18.11(s, 1H, -OH enol); ESI (-ve) MS m/z 365.2[M-H]-. 1,7-Bis(4-N,N-dimethylaminophenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6i) From 3-methoxycarbonyl-2,4pentandione (4a) and 4-(Dimethylamino)benzaldehyde (5g); purple crystals, yield 45.1%, mp 224-225oC. 1H NMR (DMSO-d6, 300MHz): 3.01(s, 12H, -N(CH3)2), 3.89(s, 3H, -OCH3), 6.74(d, J=8.7Hz, 4H, Ar-H), 6.88(d, J=15.3 Hz, 2H, =CH), 7.53(d, J=9.0 Hz, 4H, Ar-H), 7.70(d, J=15.3 Hz, 2H, =CH), 18.47(s, 1H, -OH enol); ESI (-ve) MS m/z 419.2[M-H]-. 1,7-Bis(3-nitro-4-hydroxy-5-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6j): From 3-methoxy carbonyl-2,4-pentandione (4a) and 4-hydroxy-3-methoxy-5nitrobenzaldehyde (5k); orange solid, yield 26.0%, mp 207-208oC. 1 H NMR (DMSO-d6, 300MHz): 3.92(s, 3H, -OCH3), 3.94(d, J=1.5Hz, 6H, Ar-H), 6.98(d, J=15.9Hz, 1H, =CH), 7.21(d, J=13.5Hz, 1H, Ar-H), 7.61(d, J=15.9Hz, 1H, =CH), 7.63(d, J=13.5Hz, 2H, Ar-H), 7.77(d, J=15.6Hz, 1H, Ar-H), 7.84(d, J=9.3Hz, 2H, Ar-H), 11.03(s, 2H, -OH phenol), 18.06(s, 1H, -OH enol). ESI (-ve) MS m/z 515.0 [M-H]-. 1,7-Bis(4-hydroxy-3,5-dimethoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6k): From 3-methoxycarbonyl-2,4pentandione (4a) and 4-hydroxy-3,5- dimethoxybenzaldehyde (5l); yellow crystals, yield 77.0%, mp 179-180oC. 1H NMR (DMSO-d6, 300MHz): 3.82(s, 12H, Ar-OCH3), 3.92(s, 3H, -OCH3), 7.03(s, 5H, Ar-H), 7.09(s, 1H, Ar-H), 7.73(d, J=15.6Hz, 2H, =CH), 9.18(s, 2H, -OH phenol), 18.30(s, 1H, -OH enol); ESI (-ve) MS m/z 485.2[M-H]-. 1,7-Bis(4-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6Edien-3,5-dione (6l): From 3-methoxycarbonyl-2,4-pentandione (4a) and 4-hydroxybenzaldehyde (5m); yellow crystals, yield 49.2%, mp 214-216oC. 1H NMR (DMSO-d6, 300MHz): 3.90(s, 3H, -OCH3), 6.83(d, J=8.4Hz, 4H, Ar-H), 6.99(d, J=15.6Hz, 2H, =CH), 7.58(d, J=8.7Hz, 4H, Ar-H), 7.73(d, J=15.6Hz, 2H, =CH), 10.17(s, 2H, OH phenol), 18.27(s, 1H, -OH enol); ESI (-ve) MS m/z 365.2[MH]-. 1,7-Bis(4-acetoxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6m) : From 3-methoxycarbonyl-2,4pentandione (4a) and 4-acetoxy-3-methoxybenzaldehyde (5n); pale-yellow crystals, yield 46.0%, mp 169-170oC. 1H NMR (DMSO-d6, 300MHz): 2.27(s, 6H, -COCH3), 3.84(s, 6H, ArOCH3), 3.92(s, 3H, -OCH3), 7.17(d, J=8.4Hz, 2H, Ar-H), 7.26(d, J=15.9Hz, 2H, =CH), 7.36(d, J=8.1Hz, 2H, Ar-H), 7. 49(s, 2H, ArH), 7.81(d, J=15.3 Hz, 2H, =CH), 18.04(s, 1H, -OH enol); ESI (ve) MS m/z 509.2[M-H]-. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-ethoxycarbonylhepta-1E,6E-dien-3,5-dione (6n): From 3-ethoxycarbonyl-2,4pentandione (4b) and 4-hydroxy-3- methoxybenzaldehyde (5a); yellow crystals, yield 44.6%. mp 158-159oC. 1H NMR (DMSO-d6, 300MHz): 1.32-1.37(t, 3H, -CH3), 3.82(s, 6H, Ar-OCH3), 4.39(q, 2H, -CH2), 6.84(d, J=8.1Hz, 2H, Ar-H), 7.08(d, J=15.3Hz, 2H, =CH), 7.17(d, J=8.1Hz, 1H, Ar-H), 7.17(d, J=8.1Hz, 1H, Ar-H), 7.26(d, J=1.5Hz, 2H, Ar-H), 7.72(d, J=15.9Hz, 2H, =CH), 9.80(s, 2H, -OH phenol), 18.30(s, 1H, -OH enol); ESI (-ve) MS m/z 439.2[M-H]-. 1,7-Bis(4-(4-methylpiperazinyl-1-yl)phenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (6o): From 3methoxycarbonyl-2,4-pentandione (4a) and 4-(4-methylpiperazinyl-
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1-yl) benzaldehyde (5p); dark-red crystals, 49.2% yield, mp 191192 oC. 1H NMR (CDCl3, 300MHz) 3.04(s, 6H, -CH3), 3.94(s, 3H, -OCH3), 6.65-6.69(m, 4H, Ar-H), 7.00(d, J = 14.4Hz, 2H, =CH), 7.46-7.49 (m, 4H, Ar-H), 7.78 (d, J = 15.3Hz, 2H, =CH), 15.69 (br s, 1H, -OH enol). ESI (-ve) MS m/z 529.2[M-H]-. 1,7-Bis(2-hydroxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5- dione (6p): From 3-methoxycarbonyl-2,4pentandione (4a) and 2-hydroxy-3-methoxybenzaldehyde (5q); yellow crystals, yield 25.8%, mp 201-202oC. 1H NMR (DMSO-d6, 300MHz): 3.82(s, 6H, Ar-OCH3), 3.88(s, 3H, -OCH3), 6.83(t, 2H, Ar-H), 7.04(d, J=7.5Hz, 2H, Ar-H), 7.18(d, J=7.5Hz, 2H, Ar-H), 7.26(d, J=15.9Hz, 2H, =CH), 8.04(d, J=15.6Hz, 2H, =CH), 9.62(s, 2H, -OH phenol), 18.13(s, 1H, -OH enol); ESI (-ve) MS m/z 425.2[M-H]-. 1,7-Bis(4-(4-methylpiperazinyl-1-yl))hepta-1E,6E-dien-3,5dione (6q): From 3-methoxycarbonyl-2,4-pentandione (4a) and 4(4-methylpiperazinyl-1-yl)benzaldehyde (5p); orange-red crystals, yield 46.4%, mp 239-240oC. 1H NMR (CDCl3, 300MHz) 2.36(s, 6H, -NCH3), 2.56(t, J = 5.1Hz, 8H, -CH2), 3.32(t, J = 5.0Hz, 8H, CH2), 5.74(s, 1H, =CH), 6.46(d, J = 15.6Hz, 2H, =CH), 6.876.91(m, 4H, Ar-H), 7.45-7.48(m, 4H, Ar-H), 7.58(d, J = 15.6Hz, 2H, =CH), 16.20(br s, 1H, -OH enol); ESI (-ve) MS m/z 471.2[MH]-. 1,7-Bis(4-dimethylaminophenyl)-4-N-phenylaminocarbonylhepta-1E,6E-dien-3,5-dione (6r): From 3-phenylaminocarbonyl2,4-pentandione (4c) and 4-(Dimethylamino)benzaldehyde (5g); purple crystals, yield 51.7%, mp 208-209oC. 1H NMR (DMSO-d6, 300MHz): 2.96(s, 12H, -N(CH3)2), 6.59(d, J=15.6Hz, 2H, =CH), 6.69(d, J=8.1Hz, 4H, Ar-H), 7.13(t, 1H, Ar-H), 7.39-7.42(m, 6H, Ar-H), 7.66(d, J=15.3Hz, 2H, =CH), 7.73(d, J=8.1Hz, 2H, Ar-H), 10.56(s, 1H, -NH), 17.78(s, 1H, -OH enol). ESI (-ve) MS m/z 480.2[M-H]-. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-N-phenylaminocarbonylhepta-1E,6E-dien-3,5-dione (6s): From 3-phenylamino carbonyl-2,4-pentandione (4c) and 4-hydroxy-3-methoxybenzaldehyde (5a); orange crystals, yield 46.6%, mp 193-194oC. 1H NMR (DMSO-d6, 300 MHz): 3.70(s, 6H, Ar-OCH3), 6.73(d, J=15.6 Hz, 2H, =CH), 6.79(d, J=8.1Hz, 2H, Ar-H), 7.08-7.15(m, 5H, Ar-H), 7.37(t, 2H, Ar-H), 7.69(d, J=3.9Hz, 2H, Ar-H), 7.72(d, J=3.9Hz, 2H, Ar-H), 9.79(s, 2H, -OH phenol), 10.59(s, 1H, -NH), 17.56(s, 1H, -OH enol); ESI (-ve) MS m/z 486.2[M-H]-. 1,7-Bis(4-hydroxyphenyl)-4-N-phenylaminocarbonylhepta1E,6E-dien-3,5-dione (6t): From 3-phenylaminocarbonyl-2,4pentandione (4c) and 4-hydroxybenzaldehyde (5m); yellow crystals, yield 46.2%, mp 220-221oC. 1H NMR (DMSO-d6, 300MHz): 6.68(d, J=15.6Hz, 4H, =CH), 6.79(d, J=8.7Hz, 4H, Ar-H), 7.13(t, 1H, Ar-H), 7.38(t, 2H, Ar-H), 7.45(d, J=9.0 Hz, 2H, Ar-H), 7.70(d, J=6.3Hz, 2H, Ar-H), 7.72(d, J=9.0 Hz, 2H, Ar-H), 10.14(s, 2H, OH phenol), 10.61(s, 1H, -NH), 17.56(s, 1H, -OH enol); ESI (-ve) MS m/z 426.2[M-H]-. 1,7-bis(4-hydroxy-3-methoxyphenyl)-4-methoxycarbonylheptane-3,5-dione (7): 6d (0.426g, 1mmol) was added to 10mL ethyl acetate, followed by catalytic amount of 10% Pd/C. The reaction mixture was stirred under the atmosphere of H2 for 2 hours, and then filtered through Celite. The solvent was removed by rotary evaporation, and the solid crude product was purified by recrystallization from dichloromethane (4 mL) and methanol (6 mL). Paleyellow solid, yield 52.1%, mp 92-94oC. 1H NMR (DMSO-d6, 300MHz): 2.62-2.84(m, 8H, -CH2), 3.71(s, 3H, -OCH3), 3.72(s, 6H, Ar-OCH3), 6.54(d, J=1.8Hz, 1H, Ar-H), 6.57(d, J=2.1Hz, 1H, Ar-H), 6.63(d, J=2.7Hz, 1H, Ar-H), 6.65(d, J=2.7Hz, 1H, Ar-H), 6.74(d, J=1.5Hz, 2H, Ar-H), 8.73(s, 2H, -OH phenol), 17.57(s, 1H, -OH enol); ESI (-ve) MS m/z 429.2[M-H]-. (3Z,5E)-6-(4-dimethylaminophenyl)-4-hydroxyhexa-3,5dien-2-one (8): 2,4-Pentandione (10mmol) and boron oxide (0.49 g,
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7mmol, 0.7eq) in a 50 mL flask were heated to 120oC for 5min to form pale-yellow suspension. Aldehyde (10mmol, 1.0eq) and trimethyl borate (4.16g, 40mmol, 4.0eq) were dissolved in ethyl acetate (10mL) and gradually added to reaction mixture. While stirring, 0.05mL of butylamine and 0.2mL of butylammonium acetate in dimethylformamide solution (0.136g/mL) were added. After 1hour, a red precipitate started to form. The whole reaction was stirred at room temperature for 48hours. The precipitate was filtered and dried, then dissolved in methanol (50mL) and boiled for 30 min at 60oC. Methanol was removed by rotary evaporation and the crude product (8) was purified by crystallization from dichloromethane (20mL) and methanol (20mL); orange-red crystals; mp 136-137oC; 1H NMR (CDCl3, 300 MHz) 2.13(s, 3H, -CH3), 3.03(s, 6H, -N(CH3)2), 5.60(s, 1H, =CH), 6.27(d, J = 15.6 Hz, 1H, =CH), 6.65-6.69(m, 2H, Ar-H), 7.41-7.44(m, 2H, Ar-H), 7.56(d, J=15.9 Hz, 1H, =CH), 18.46(br s, 1H, -OH enol).
of 1mM CaCl2, 0.2M NaCl and 50mM Tris/HCl (pH=7.6) buffer and 10L of enzyme, were added first to the 96-well plate, followed by the addition of different concentrations of Inhibitor (1L) and substrate (10L) to a final reaction volume now containing 1% DMSO. The whole mixture was incubated at 37oC for 4h, and Fluoro Count (Packard Instrument Co., CT) was used to measure the increase in fluorescence when the substrate was cleaved. By comparison with the uninhibited degradation from enzyme and substrate, the percentage of inhibition was determined by subtracting the degradation of the enzyme, substrate and inhibitor mixture. The fluorescence of blank plates containing the reaction mixture minus enzyme was subtracted from each assay. The IC50 for each compound was obtained from a plot of the percentage of inhibition versus the inhibitor concentration.
1-(4-Dimethylaminophenyl)-7-(4-hydroxy-3-methoxyphe nyl)-hepta-1E,6E-dien-3,5-dione (9a): From 4-hydroxy-3-me thoxybenzaldehyde(3a) and (3Z,5E)-6-(4-dimethylamino phenyl)4-hydroxyhexa-3,5-dien-2-one (6); orange-red crystals; mp 171172oC; 1H NMR (CDCl3, 300 MHz) 3.07(s, 6H, -N(CH3)2), 3.95(s, 3H, Ar-OCH3), 5.77(s, 1H, =CH), 5.83(br s, 1H, Ar-H), 6.43(d, J=15.9Hz, 1H, =CH), 6.47(d, J=15.9Hz, 1H, =CH), 6.666.70(m, 2H, Ar-H), 6.93(d, J=9.0Hz, 1H, Ar-H), 7.05(d, J=2.1Hz, 1H, Ar-H), 7.11 (dd, J=8.2, 1.7Hz, 1H, Ar-H), 7.44-7.48(m, 4H, Ar-H), 7.55(d, J=15.9Hz, 1H, =CH), 7.63(d, J = 15.9Hz, 1H, =CH), 16.20(br s, 1H, -OH enol); MS (EI) m/z (%) 364 (M˙+, 60), 267 (100), 188 (35), 174 (100), 147 (50), 146 (50), 134 (100).
As discussed previously [36], human peripheral blood mononuclear cells (PBMC) were isolated and purified from blood (Red Cross Blood Bank) by density gradient centrifugation over Lymphoprep and adherence. PBMC cells at 5x105cells/mL were then cultured in serum-free macrophage media (37oC, 5% CO2) overnight with LPS (P. gingivalis, 50ng/mL) or vehicle alone. Curcumin or CMC2.24 was added at final concentrations of 2 or 5μM, and conditioned media were analyzed for MMP-9 by gelatin zymography.
1-(4-Dimethylaminophenyl)-7-((4-N-methylpiperazin-1-yl) phenyl)-hepta-1E,6E-dien-3,5-dione (9b): From 4-(4-N-methyl piperazin-1-yl)benzaldehyde (3p) and (3Z,5E)-6-(4-dime thylaminophenyl)-4-hydroxyhexa-3,5-dien-2-one (6); orange-red crystals; mp 242-243oC; 1H NMR (CDCl3, 300 MHz) 2.36(s, 3H, NCH3), 2.56(t, J = 5.2Hz, 4H, -CH2), 3.03(s, 6H, -N(CH3)2), 3.31(t, J = 5.0Hz, 4H, -CH2), 5.74(s, 1H, =CH), 6.43(d, J = 15.6Hz, 1H, =CH), 6.46(d, J = 15.6Hz, 1H, =CH), 6.66-6.70(m, 2H, Ar-H), 6.87-6.91(m, 2H, Ar-H), 7.44-7.47(m, 4H, Ar-H), 7.57 (d, J = 15.9Hz, 1H, =CH), 7.61 (d, J = 15.9Hz, 1H, =CH), 16.27 (br s, 1H, -OH enol); MS (EI) m/z (%) 417 (M˙+, 40), 321 (75), 266 (30), 189 (40), 188 (40), 174 (95), 147 (45), 146 (50), 134 (100).
The gelatin zymography system was purchased from Invitrogen Corp. (Carlsbad, CA), and SDS-PAGE gels containing polyacrylamide copolymerized gelatin at a final concentration of 1mg/ml were prepared. After electrophoresis, the gels were washed in 2.5% Triton X-100 and incubated at 370C overnight in calcium assay buffer (40mM Tris, 200mM NaCl, 10mM CaCl2, pH 7.5). After incubation, the gels were stained with Coomassie Brilliant Blue R-250. Clear zones of lysis against a blue background indicate gelatinolytic activity, as described by Brown et al. [37].
2. MMP Inhibition Assay MMP-1, -2, -3, -7, -8, -12, -13 and -14 were all recombinant human enzymes and were purchased from R&D Systems, Inc. (Minneapolis, MN), whereas MMP-9, human neutrophil monomer was purchased from Calbiochem, EMD Biosciences, Inc. (La Jolla, CA). MMP-1, -2, -3, -7, -8, -9, -12 and -13 were activated by adding p-aminophenylmercuric acetate (APMA) purchased from Sigma-Aldrich (St. Louis, MO) to achieve a final concentration of 1mM in DMSO purchased from Sigma-Aldrich (St. Louis, MO). MMP-14 was activated by recombinant human furin, purchased from R&D Systems, Inc. (Minneapolis, MN). The substrate for MMP-1, -2, -7, -8, -9, -12, -13 and -14 was Mca-Pro-Leu-Gly-LeuDpa-Ala-Arg-NH2 fluorogenic peptide substrate IX, purchased from R&D Systems, Inc. (Minneapolis, MN) [40]. [Mca: (7Methoxycoumarin-4-yl)acetyl, Dpa: N-3-(2, 4-Dinitrophenyl)-L2,3-diaminopropionyl]. The substrate for MMP-3 was Mca-ArgPro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2 fluorogenic peptide substrate II, purchased from R&D Systems, Inc. (Minneapolis, MN) [34, 35]. [Mca: (7-Methoxycoumarin-4-yl)acetyl, Nval: Norvaline, Dnp: 2, 4-Dinitrophenyl]. Cleavage of the fluorogenic substrate was measured by the differences in intensities of the excitation and emission wavelengths at 320nm and 405nm, respectively. Stock solutions of 1,10-phenanthroline, curcumin and curcumin analogues were prepared in DMSO at concentrations of 1, 5, 10, 25, 50, 100, 250 and 500M. 100L solution containing 80L
3. Cell Assay Method and Materials
4. Gelatin Zymography
5. Preliminary in vivo Study on MMP-9 Adult male Sprague-Dawley rats (275g average body weight, viral antibody free, Charles River Labs, Wilmington, MA) were distributed into three groups (three rats per group): non-diabetic controls, rats that were made type I diabetic by STZ injection (70mg/kg) into the tail vein after an overnight fast, and diabetic rats that were orallyadministered the most potent of the CMC derivatives, 6t (30mg/kg) once/day by oral intubation. After three weeks, blood samples were obtained from each rat (non-fasting) from each of the three groups and were analyzed for glucose levels by standard technique (OneTouch Ultra Glucometer, Johnson and Johnson, New Brunswick, NJ). The rats were then sacrificed by CO2 asphyxiation. Five days prior to sacrifice, the rats were injected intraperitoneally with 3% thioglycollate medium, and the peritoneal exudates were collected on the day of sacrifice. The macrophages were isolated by density gradient centrifugation over Lymphoprep [36]. Cells at 1x106cells/mL were incubated in macrophage serum-free media (37oC, 5%CO2/95%O2) for overnight and conditioned media were collected, and aliquots examined for MMP-2 and MMP-9 by gelatin zymography as described above. The band densities from the gelatin zymogram were quantitated by scanning on a laser densitometer, and Image analysis was performed using Image J. C. RESULTS In general, 2 (curcumin) and its analogues (6a-6t) required for the structure-activity relationship (SAR) studies were synthesized by a modified Pabon reaction [24]. The synthetic route is shown in (Scheme 2), and a list of the resulting compounds is documented in (Table 2).
New Polyenolic Inhibitors of Matrix Metalloproteinases
Current Medicinal Chemistry, 2012 Vol. 19, No. 25
O
OH
3
R'COCl or R''NCO MgCl2 / Pyridine 0oC, overnight R.T., 5h 4a-4c R2
MeOH
Aldehyde 5a-5t B2O3 / (CH3O)3B n-Butylamine Cat. nBuNH3+OAcEthyl Acetate 48h, R.T.
O
R3
OH
R1
O
OH
R2
X
X R1
R4
4353
R3
R4
R5
R5 6a-6t X=C or N
Scheme 2. Synthesis of Curcumin Analogues with a C-4 substituent. Table 2.
Curcumin Analogues Synthesized by the Modified Pabon Reaction
Compound
Name
2
Curcumin
6a
CMC2.2
R1
R2
R3
R4
R5
-H
-H
-OCH3
-OH
-H
-H
-H
-H
-H
-H
6b
CMC2.3
-H
-H
Pyridin-3-yl
-H
-H
6c
CMC2.4
-CO2CH3
-H
-H
-H
-H
6d
CMC2.5
-CO2CH3
-H
-OCH3
-OH
-H
6e
CMC2.6
-CO2CH3
-H
-OH
-H
-H
6f
CMC2.7
-H
-H
-H
-N(CH3)2
-H
6g
CMC2.8
-CO2CH3
-H
Pyridin-3-yl
-H
-H
6h
CMC2.10
-CO2CH3
-OH
-H
-H
-H
6i
CMC2.11
-CO2CH3
-H
-H
-N(CH3)2
-H
6j
CMC2.12
-CO2CH3
-H
-OCH3
-OH
-NO2
6k
CMC2.13
-CO2CH3
-H
-OCH3
-OH
-OCH3
6l
CMC2.14
-CO2CH3
-H
-H
-OH
-H
6m
CMC2.15
-CO2CH3
-H
-OCH3
-O2CCH3
-H
6n
CMC2.16
-CO2C2H 5
-H
-OCH3
-OH
-H
6o
CMC2.17
-CO2CH3
-H
-H
4-Methyl Piperazinyl
-H
6p
CMC2.18
-CO2CH3
-OH
-OCH3
-H
-H
6q
CMC2.19
-H
-H
-H
4-Methyl Piperazinyl
-H
6r
CMC2.22
-CONHPh
-H
-H
-N(CH3)
-H
6s
CMC2.23
-CONHPh
-H
-OCH3
-OH
-H
6t
CMC2.24
-CONHPh
-H
-H
-OH
-H
For those analogues in which there is a 4-substituent, the syntheses were accomplished using the appropriate 3-alkoxycarbonyl (or arylaminocarbonyl)-substituted-2,4-pentandione, prepared according to a procedure described by Bingham and Tyman [38]. In brief, a 2,4-pentandione was heated with boron trioxide at 100oC until the mixture became a clear viscous oil. This complex was then treated with a solution of trimethyl borate in ethyl acetate with stirring until complete dissolution had occurred and the mixture was homogeneous. Two equivalents of an aromatic aldehyde were then added followed by a catalyst consisting of n-butylamine and nbutylamine acetate. The presence of the acetic acid salt improves yields by facilitating the dehydration step of the aldol intermediates in an acid/base-catalysed process. One tetrahydro-curcumin analogue (7) also was synthesized by hydrogenation of 6d (Scheme 3). Two unsymmetrical curcumin analogues were also synthesized by a modified Pabon procedure (Scheme 4), namely 9a (CMC2.21) and 9b (CMC2.22). In these cases 2,4-pentandione was allowed to react with one equivalent of 4-dimethylaminobenzaldehyde to give 8,
which after isolation, was sequentially treated with one equivalent of either aldehyde 5a or 5p to give 9a or 9p respectively. MMP inhibition assays were then carried out with MMPs 1, -2, 3, -7, -8, -9, -12, -13 and -14. Each enzyme individually was incubated in vitro with the MMP-susceptible fluorogenic peptide substrate, Mca-K-P-L-G-Dpa-A-R-NH2 as previously described [39]. For each compound the micromolar (μM) concentration that was required to inhibit 50% of the proteolytic activity of the MMP (IC50; see Table 3) was determined from a plot of the concentration of the inhibition versus the concentration of the inhibitor. Our current lead compound, 6t (a novel bis-(demethoxy) phenylaminocarbonyl derivative of curcumin; CMC2.24), exhibited inhibitory IC50 values in vitro, ranging from 2-8 μM against two collagenases (MMP-8, -13), two gelatinases (MMP-2, -9), and MMP- 3, -7 and -12. In addition, the membranetype MMP, namely MMP-14, was also effectively inhibited more by 6t (CMC2.24) than by the other known MMP inhibitors, 1,10phenanthroline (IC50=43.8M) and curcumin (IC50=29.5M), since its IC50 was determined to be 15.3M.
4354 Current Medicinal Chemistry, 2012 Vol. 19, No. 25
O
Zhang et al.
O
OH
H3CO
OCH3 CO2CH3
HO
H3CO
H2/Pd-C Ethyl Acetate, R.T., 2h
OH
OH OCH3 CO2CH3
HO
6d
OH
7
Scheme 3. Preparation of the tetrahydro-curcumin analogue. OHC O
O
N(CH3)2
OH
B2O3 / (CH3O)3B n-Butylamine Cat. nBuNH3+OAcEthyl Acetate 48h, R.T.
3
CHO
OH
N(CH3)2
8
O
OH
R'' 5a, 5p
R'' N(CH3)2
B2O3 / (CH3O)3B n-Butylamine Cat. nBuNH3+OAcEthyl Acetate 48h, R.T.
9a,9b
Scheme 4. Preparation of unsymmetric curcumin analogues. Table 3.
In vitro potency (IC50 in M concentration) of curcumin and selected chemically-modified curcumins (CMCs), as inhibitors of nine different mammalian (human-derived) MMPs. In this experiment, a fluorimetric assay of MMP activity was used, and 1, 10-phenanthroline, a zinc cation binding-agent, traditionally used to quench in vitro MMP activity reactions, was used as the positive control for the experiment. IC50s were measured using a synthetic fluorescent peptide substrate (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2), for MMPs and which has a cleavage site between Gly and Leu. Each value represents the mean of 3 or 4 analyses (± S.E.M.) Compounds
1,10Phenanthroline
2 (Curcumin)
6d (CMC2.5)
6l (CMC2.14)
6s (CMC2.23)
6t (CMC2.24)
MMP-1
42.0±1.1
MMP-8
31.3±0.5
85.8±1.8
74.0±3.5
76.3±6.5
68.0±3.2
69.8±2.0
6.8±1.0
30.8±1.5
20.0±2.0
2.5±0.3
4.5±0.5
MMP-13 MMP-2
50.0±10.4
3.7±0.3
28.3±4.4
26.7±1.7
3.3±0.3
2.7±0.7
73.8±1.0
5.0±0.7
25.3±1.3
23.8±0.9
6.3±0.9
4.8±0.5
MMP-9
45.0±12.6
30.0±2.9
55.0±17.3
43.3±4.4
8.7±0.7
8.0±0.6
MMP-3
77.0±3.2
4.7±0.8
32.5±2.8
28.3±1.0
5.3±0.7
2.9±0.4
MMPs
Collagenases
Gelatinases
Others
MMP-7
196.8±8.8
51.8±2.5
48.8±0.5
57.5±4.6
21.5±1.0
5.0±0.7
MMP-12
29.5±1.3
2.6±0.2
27.8±1.7
5.3±0.3
4.5±0.5
2.0±0.4
MMP-14
43.8±4.2
29.5±3.2
48.5±4.3
40.0±8.4
41.3±4.9
15.3±3.1
These MMPs are generally significantly up-regulated during a variety of tissue-destructive diseases such as (but certainly not limited to) arthritis, cardiovascular disease, periodontal disease, and cancer. In contrast, MMP-1 is often considered constitutive due, in part, to its involvement in the degradation of the triple-helical collagen molecule during physiologic connective-tissue turnover, and this collagenase requires much higher concentrations of 6t to inhibit its activity (IC50=69.8M). Regarding the effect on viable cells, as illustrated in Fig. (1a and 1b), human peripheral blood monocytes were cultured in serum-free media with and without lipopolysaccharide (LPS). Compound 2 (curcumin) at 2 or 5M did not appear to decrease the MMP-9 levels in the conditioned media Fig. (1a). In contrast, when 6t (CMC2.24) was
added to the culture at the same concentration either 2 or 5M, the extracellular MMP-9 levels were reduced in a dose-dependent manner Fig. (1b) with a lesser effect at 2M, but dramatically reduced at the 5M concentration. Regarding in vivo efficacy of 6t (CMC2.24), when rats were rendered severely diabetic, we found that these animals exhibited a 4-fold increase in MMP-9 levels Fig. (2b) produced by peritoneal macrophages in cell culture, compared to the non-diabetic control rats; and when the diabetic rats were orally administered 6t (30mg/kg), the pathologically-elevated levels of MMP-9 were reduced to essentially normal levels Fig. (2b) in spite of the severely hyperglycemic state of these animals, which remained unchanged
New Polyenolic Inhibitors of Matrix Metalloproteinases
Current Medicinal Chemistry, 2012 Vol. 19, No. 25
4355
a Std
Cells
Cells
Cells + LPS + 2 (curcumin)
+LPS
2μM
2μM
5μM
5μM
92 kD
b Std
Cells
Cells
Cells + LPS + 6t (CMC2.24)
+LPS
2μM
2μM
5μM
5μM
92 kD Fig. (1). Effect of curcumin Fig. (1a) and CMC2.24 Fig. (1b) on MMP-9 levels in conditioned media from human monocytes stimulated by LPS in cell culture. Human peripheral mononuclear cells (PBMC) (5x 10 5 cells/well) were cultured in serum-free media (37oC, 5% CO2) overnight with LPS (P. gingivalis, 50 ng/mL) containing vehicle alone or 2 (curcumin) or 6t (CMC2.24) at different concentrations Fig. (1a and 1b). Conditioned medium from both experiments were analyzed for MMP-9 levels by gelatin zymography.
2D Std
N N
D D
D+2.24
92 kD 72 kD
2b
Densitometric Scanning (Units)
500 450 400 350 300 250 200 150 100 50 0 Normal
Diabetic
Diabetic +2.24
4356 Current Medicinal Chemistry, 2012 Vol. 19, No. 25
Zhang et al.
2c Blood Glucose 700
Glucose (mg/dL)
600 500 400 300 200 100 0 Normal
Diabetic
Diabetic + 2.24
Fig. (2). Effect of in vivo 6t (CMC2.24) on levels of MMP-9 secreted by peritoneal macrophages from diabetic rats. Thioglycollate-induced peritoneal macrophages derived from N, D and D rats treated with 6t daily for three weeks were isolated as described in Methods section. Cells at 1 x 106 cells/mL were cultured in serum-free media (37oC, 5%CO2/95%O2 ) overnight, and conditioned media were analyzed for MMP-9 levels by gelatin zymography. Std: MMP standards; N: Non-diabetic control; D: Diabetic control; D+2.24: diabetics orally administered 6t daily for three weeks. Pro-MMP-9 has a MW of 92kD, while Pro-MMP2 is 72kD (Representative zymogram is shown in Fig. 2a). The band densities from the gelatin zymogram were measured by scanning on a laser densitometer, and Image analysis was performed using Image J Fig. (2b). Each value represents the mean of 3 analyses ± S.E.M. Blood glucose levels (mg/100mL) for the three groups of rats are shown in Fig. (2c). Each value represents the mean ± S.E.M. for 3 analyses per group.
Fig. (2c). It has to be recognized that these biological data, in cell culture and in vivo, are preliminary, and are currently being expanded. It should also be recognized that the in vivo experiments on the cohorts of three rats (normal, diabetic and diabetic+2.24) have been repeated three times with slight variations each time, and the pattern of change was similar; therefore, the data in Fig. (2a, 2b and 2c) are representative. D. DISCUSSION The first curcumin analogues that were prepared contained unsubstituted phenyl or pyridin-3-yl rings (6a and 6b respectively), but these were found to lack any significant MMP inhibitory activity as judged from an assay that had been described in previous work on the tetracyclines [40]. In an attempt to increase the solubility and potentially to improve the zinc-binding ability, a series of analogues having a carbonyl substituent at the C-4 position was synthesized. Such analogues, having an additional electronwithdrawing group at this location, were chosen to try to improve these properties and possibly to augment the biological activity. By way of example, the MMP inhibition assay of 4-methoxy carbonylcurcumin (6d), one of the first analogues that was synthesized, showed that it was more active than 6c, was as potent as curcumin itself, but had greater solubility than either. Thereafter, we also synthesized a series of analogues in which different substituents were introduced onto the terminal 1,7-aromatic rings. These included groups such as methoxy-, dimethylamino-, nitro, 4methylpiperazinyl etc. Also, since curcumin undergoes metabolic reduction to tetrahydrocurcumin and other more highly reduced substances, in rats and mice in vivo [41], a tetrahydro-curcumin analogue (7) also was synthesized by hydrogenation of 6d. However, the latter compound lacked inhibitory activity against several selected MMPs, namely MMP-2, -8 and -9. Thus we suggest that intrinsically, the double bonds are a requirement for the MMP inhibition and that the 1,3-diketone assembly is not sufficient by itself. The corresponding ethoxycarbonyl compound (6n) is no more soluble than 6d and has similar biological activity. When a 4-Nmethylpiperazinyl substituent was introduced as in 6o or 6q, in an attempt to decrease the liposolubility, the inhibitory activity fell
dramatically. Again, neither of the unsymmetrical curcumin analogues, namely 9a and 9b, showed improved inhibitory activity. Of the curcumin analogues having a 4-methoxycarbonyl substituent, only 6d and 4-methoxycarbonyl bis(demethoxy)curcumin (6l) showed any significantly better activity. Nevertheless, when a phenylaminocarbonyl group was introduced at the 4-position, the MMP inhibitory activity was increased substantially as is evident from the data for 6s and 6t. The latter proved to be significantly more potent than curcumin, specifically against the MMPs -7, -9, -12, and -14, all of which are known to be inducible and involved in various inflammatory diseases and cancer, whereas the constitutive collagenase enzyme MMP-1, was much less affected by at least an order of magnitude. These results parallel those observed in the case of the tetracyclines [11]. The introduction of the phenylaminocarbonyl group at C4 resulted also in improved solubility, probably associated both with the ~10-fold greater acidity (pKa data will be reported elsewhere), (versus 2, curcumin) and its amidic character. The data shown in (Table 3), indicate that all of the curcumin analogues are generally better as MMP-inhibitors than the standard zinc chelating agent, 1,10-phenanthroline. However, in general, the two most potent chemically-modified curcumin analogues are 6s and 6t. Of these, 6t proved to be the more potent and the less toxic in cell and tissue culture and in in vivo studies. It is significant that MMP-12 is produced by osteoclasts [42] and its inhibition (as described in the current study) by 6t at least partially explains the ability of this compound (together with inhibition of other MMPs such as MMP-13) to prevent bone-destruction in the diabetic rats. Compound 6s was not pursued further because, (a) unlike 6t, it did not reduce diabetic complications in the rat in vivo (data not shown), and (b) it was less effective in reducing cartilage destruction in tissue culture [45]. The effects of compounds 6d and 6t in cell culture (human peripheral blood monocytes), tissue culture (bovine cartilage) and in vivo on diabetic rats, have already been described (in abstract form) [43-45] and indicate several potential therapeutic areas of application as described below: (i) Cell Culture Studies It should be recognized that both 6d and 6t have been found by our group (abstract only) to inhibit the production of various inflam-
New Polyenolic Inhibitors of Matrix Metalloproteinases
matory mediators, induced in human monocytes either by endotoxin from E. coli, or by a complex of CRP oxidized LDL cholesterol [43]. These mediators include not only pro-inflammatory cytokines and mediators such as IL-1 and TNF- but also PGE2, MCP-1 and others. The expression by monocytes in culture, of pathologically- excessive levels of MMP-2,-8 and -13, was also reduced to normal levels by treatment with either 6d or 6t [43, 44]. (ii) Tissue Culture Studies We reported recently (abstract only) that IL-1-induced excessive proteoglycan (S35-labeled) degradation of bovine cartilage in culture (a model for osteo-arthritis) was reduced to basal/control levels by 6s and 6t but not by 6d [45].
Current Medicinal Chemistry, 2012 Vol. 19, No. 25
ACKNOWLEDGEMENTS This research was supported by grants from the Center for Advanced Biotechnology (CAT) at Stony Brook (Award # 42065-11092236); the Stony Brook Bioscience Technology Commercialization Fund (BTCF) (Award # A37298-30-1074970); and Stony Brook Research Foundation (Award # 37298-1-1050308). We would also like to thank Chem-Master International Inc. for the generous gift of chemical supplies. We are grateful to Dr. James Marecek for assistance with NMR determinations, and Dr. Béla Ruzsicska, Dr. Charles Iden and Mrs. Irina Zaitseva for determining the mass spectra. ABBREVIATIONS MMP
=
Matrix Metalloproteinases
(iii) In Vivo Studies
ECM
=
Extracellular Matrix
We have previously demonstrated that either inducing diabetes with streptozotocin (STZ), or causing periodontitis locally by endotoxin injection into the gingiva, increases the production of cytokines and MMPs. In these studies, both 6d and 6t were found to inhibit the production of pathologically-excessive levels of these inflammatory mediators and MMPs in tissue from rats that had been administered these curcumin analogues by the oral route. Of particular significance, administration of 6d or 6t by oral gavage, in doses as high as 500mg/kg body weight, showed no evidence of toxicity in these already seriously-ill animals. This suggests that the continuing tissue degradation associated with diabetes, might be arrested by the use of 6t (CMC2.24). Additional studies describing the actions of these inhibitors on specific disease conditions will be presented in future publications. In addition, curcumin and its analogues have also been tested for their therapeutic potential as inhibitors of inflammatory mediators (cytokines and prostaglandins) in in vivo models of various diseases including osteoarthritis, periodontitis, acute respiratory disease syndrome (ARDS) and diabetes. Of extreme interest, 6t seems efficacious and safe regardless whether it is administered systemically, by either the oral or intraperitoneal routes, or topically, e.g., in successful wound-healing studies [44].
FDA
=
U.S. Food and Drug Adminstration
TCs
=
Tetracyclines
CMT
=
Chemically-Modified Tetracycline
CMC
=
Chemically-Modified Curcumin
E. CONCLUSIONS A series of curcumin analogues were synthesized by a variation of the Pabon reaction and examined for their ability to inhibit a battery of matrix metalloproteinases. This limited structure-activity relationship study revealed that those compounds containing an additional electron-withdrawing carbonyl function at C-4 can, at low M levels, inhibit matrix metalloproteinases. These inhibitors are, in general, much more active than the parent curcumin (2) and do not share the latter’s insolubility characteristics. Significantly, they appear to have the ability to reduce the pathologic levels of only the inducible MMPs to essentially normal homeostatic levels, which may be one of the reasons that these substances show little or no demonstrable toxicity at oral doses as high as 500mg/kg bodyweight [43]. We expect these compounds to have no antibiotic (preliminary data not shown) or photosensitizing properties (given that curcumin itself does not cause photosensitivity), and we are currently studying the potential of these compounds to control diseases of tissue loss/breakdown, especially those associated with aging, inflammation and cancer.
TLC
=
Thin-Layer Chromatography
DMSO
=
Dimethyl Sulfoxide
NMR
=
Nuclear Magnetic Resonance
ESI-MS
=
Electrospray Ionization Mass Spectrometry
bp
=
Boiling Point
mp
=
Melting Point
APMA
=
P-Aminophenylmercuric Acetate
Mca
=
(7-Methoxycoumarin-4-yl)acetyl,
Dpa
=
N-3-(2, 4-Dinitrophenyl)-L-2,3-diamino pro pio nyl
Nval
=
Norvaline
Dnp
=
2, 4-Dinitrophenyl
PBMC
=
Peripheral Blood Mononuclear Cells
SAR
=
Structure-Activity Relationship
LPS
=
Lipopolysaccharide
CRP
=
C-Reactive Protein
LDL
=
Low-Density Lipoprotein
STZ
=
Streptozotocin
REFERENCES [1] [2] [3] [4] [5]
CONFLICT OF INTEREST Dr. Francis Johnson and Dr. Lorne M. Golub are listed as inventors on patent applications describing some of the compounds in their study. These patent applications have been fully assigned to their institutions, the Research Foundation of Stony Brook University and to Chem-Master International Inc.
4357
[6] [7] [8]
Gross, J.; Lapiere, C.M. Collagenolytic activity in amphibian tissues – a tissue culture assay. Proc. Natl. Acad. Sci. U.S.A., 1962, 48, 1014-1022 Visse R.; Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res., 2003, 92, 827-839 Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Design and Therapeutic Application of Matrix Metalloproteinase Inhibitors. Chem. Rev., 1999, 99, 2735-2776 Peterson, J.T. The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors. Cardiovasc. Res., 2006, 69, 677-687. Golub, L.M.; Greenwald, R.A.; Ramamurthy, N.S.; McNamara, T.F.; Rifkin, B.R. Tetracyclines inhibit connective tissue breakdown: New therapeutic implications for an old family of drugs. Crit. Revs. Oral Biol. Med., 1991, 2, 297-322 Peterson, J.T. The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors. Cardio. Res., 2006, 69, 677-687 Golub, L.M.; Wolff, M.; Roberts, S.;Lee, H.M.; Leung, M.; Payonk, G.S. Treating periodontal diseases by blocking tissue-destructive enzymes. J Am Dent Assoc., 1994,125,163-169 Golub, L.M.; Lee, H.M.; Lehrer, G.; Nemiroff, A.; McNamara, T.F.; Kaplan, R.; Ramamurthy, N.S. Minocycline reduces gingival collagenolytic activity
4358 Current Medicinal Chemistry, 2012 Vol. 19, No. 25
[9] [10] [11]
[12] [13]
[14] [15] [16] [17]
[18]
[19]
[20] [21]
[22] [23] [24] [25] [26] [27] [28] [29] [30]
Zhang et al.
during diabetes: Preliminary observations and a proposed new mechanism of action. J. Periodont. Res., 1983, 18, 516-526 Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science, 2002,295, 2387-2392 Overall, C. M.; Lopez-Otin, C. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nature Rev. Cancer, 2002, 2, 657-672 Sorsa T.; Tjäderhane L.; Konttinen Y.T.; Lauhrio A.; Salo, T.; Lee, H.M.; Golub, L.M.; Brown, D.L.; Mäntylä, P. Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment for periodontal inflammation. Ann. Med., 2006, 38, 306-321 Golub, L.M.; Lee, H.M.; Ryan, M.E.; Giannobile, W.V.; Payne, J.; Sorsa, T. Tetracyclines Inhibit Connective Tissue Breakdown by Multiple NonAntimicrobial Mechanisms. Adv. Dent. Res., 1998, 12, 12-26 Dezube, B.J.; Krown, S.E.; Lee, J.Y.; Bauer, K.S.; Aboulafia, D.M. Randomized Phase II Trial of Matrix Metalloproteinase Inhibitor COL-3 in AIDSRelated Kaposi's Sarcoma: An AIDS Malignancy Consortium Study. Journal of Clinical Oncology, 2006, 24, 1389-1394 Richards, C.; Pantanowitz, L.; Dezube, B.J. Antimicrobial and nonantimicrobial tetracyclines in human cancer trials. Pharmacological Research, 2011, 63, 151-156 Sandberg, S.; Glette, J.; Hopen, G.; Solberg, C.O. Doxycycline induced photodamage to human neutrophils and tryptophan. Photochem Photobiol., 1984, 39, 43-48 Riaz, M.; Pilpel, N. Effects of ultraviolet light on lecithin monolayers in the presence of fluorescein dyes and tetracycline drugs. J Pharm Pharmacol., 1983, 35, 215-218 Bachmeier, B.E.; Mohrenz, I.V.; Mirisola, V.; Schleicher, E.; Romeo, F.; Höhneke, C.; Jochum, M.; Nerlich, A.G.; Pfeffer, U. Curcumin downregulates the inflammatory cytokines CXCL1 and -2 in breast cancer cells via NFkB. Carcinogenesis, 2008, 29, 779-789 Kaur, G.; Tirkey, N.; Bharrhan, S.; Chanana, V.; Rishi, P. ; Chopra, K. Inhibition of oxidative stress and cytokine activity by curcumin in amelioration of endotoxin-induced experiemental hepatotoxicity. Clin. Exp. Immunol., 2006, 145, 313-321 Begum, A.N. Jones, M.R.; Lim, G.P.; Morihara, T.; Kim, P.;Heath, D.D.; Rock, C.L.; Pruitt, M.A.; Yang F.; Hudspeth, B.; Hu, S.; Faull, K.F.; Teter, B.; Cole, G.M.; Frautschy, S.A. Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease. J Pharmacol Exp Ther., 2008, 326, 196-208 Banerji, A.; Chakrabarti, J.; Mitra, A.; Chatterjee, A. Effect of curcumin on gelatinase A (MMP-2) activity in B16F10 melanoma cells. Cancer Lett., 2004, 211, 235-242 Woo, M.S.; Jung, S.H.; Kim, S.Y.; Hyun, J.W.; Ko, K.H.; Kim, W.K.; Kim, H.S. Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells. Biochem Biophys Res Commun., 2005, 335, 1017-1025 Baum L.; Ng, A. Curcumin interaction with copper and iron suggests one possible mechanism of action in Alzheimer's disease animal models. J. Alzheimers Dis., 2004 , 6, 367-377 Lampe, V.; Milobedzka, J.; Kostanecki, St. V. Zur Kenntnis des Curcumins. Berichte., 1910, 43, 2163 Pabon, H.J.J. A synthesis of curcumin and related compounds. RECUEIL, 1964, 83, 379-386 Pedersen, U.; Rasmussen, P.B.; Lawesson, S.O. Synthesis of naturally occurring curcuminoids and related compounds. Liebigs Ann. Chem., 1985, 15571569 Aggarwal, B.B.; Kumar, A; Bharti, A.C. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res., 2003, 23, 363-398 Sharma, O.P. Antioxidant activity of curcumin and related compounds. Biochem. Pharmacol., 1976, 25, 1811-1812; Ruby, A.J.; Kuttan, G; Babu, K.D.; Rajasekharan, K.N.; Kuttan, R. Antitumour and antioxidant activity of natural curcuminoids. Cancer Lett., 1995, 94, 79-83 Anand, P.; Kunnumakkara, A.B.; Newman, R.A.; Aggarwal, B.B. Bioavailability of Curcumin: Problems and Promises. Molecular Pharmaceutics, 2007,4, 807-818 Cameron, D.W.; Deutscher, D. J.; Feutrill, G.I.; Griffiths, P.G. Chemistry of the Coccoidea. VIII. Synthesis of the ancient dyestuff kermesic acid and of
Received: June 07, 2012
Revised: July 17, 2012
Accepted: July 18, 2012
[31] [32]
[33]
[34]
[35] [36] [37]
[38] [39] [40]
[41] [42] [43]
[44]
[45]
related anthraquinones Australian Journal of Chemistry, 1981 , 34, 24012421 Buechi,G.; Wuest,H. Synthetic Studies on Damascenones. Helvetica Chimica Acta, 1971, 54, 1767-1775 Eckberg, R.P.;Nelson, J.H.; Kenney, J.W.; Howells, P.N.; Henry, R.A.Reactions of bis(2,4-pentanedionato)nickel(II) with isocyanates and other electrophiles. Electrophilic addition to 2,4-pentanedione catalyzed by bis(2,4-pentanedionato)nickel(II). Inorganic Chemistry, 1977, 16, 3128-3129 O'Brien, P.M.; Sliskovic, D.R.; Blankley, C. J.; Roth, B.D.; Wilson, M.W.; Hamelehle, K.L.; Krause, B.R.; Stanfield, R.L. Inhibitors of AcylCoA:Cholesterol O-Acyl Transferase (ACAT) as Hypocholesterolemic Agents. 8. Incorporation of Amide or Amine Functionalities into a Series of Disubstituted Ureas and Carbamates. Effects on ACAT Inhibition in vitro and Efficacy in vivo. J. Med. Chem., 1994, 37, 1810-1822 Nagase, H.; Enghild, J.J.; Suzuki, K.; Salvesen, G. Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry, 1990, 29, 5783-5789 Nagase, H.; Fields, C.G.; Fields, G.B. Design and Characterization of a Fluorogenic Substrate Selectively Hydrolyzed by Stromelysin 1 (Matrix Metalloproteinase-3). J. Biol. Chem., 1994, 269, 20952-20957 Liao, G.J.; Simone, J.; Simon, S.R. Paracrine downregulation of FcrRIII in human monocyte-derived macrophages induced by phagocytosis of nonopsonized particles. Blood, 1994, 83, 2294-2304 Brown, D.L.; Desai, K.K.; Vakili, B.A.; Nouneh, C.; Lee, H.M.; Golub, L.M. Clinical and biochemical results of the metalloproteinase inhibition with subantimicrobial doses of doxycycline to prevent acute coronary syndromes (MIDAS) pilot trial. Arterioscler. Thromb. Vasc. Biol., 2004, 24, 733-738 Bingham, S.J.; Tyman, J.H.P. Improved synthesis of alkyl diacetylacetates. Organic Preparations and Procedures INT., 2001, 33, 357-409 Knight, C.G.; Willenbrock, F; Murphy, G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett., 1992, 296, 263-266 Golub, L.M.; Lee, H.M.; Stoner, J.A.; Sorsa, T.; Reinhardt, R.A.; Wolff, M.S.; Ryan, M.E.; Nummikoski, P.V.; Payne, J.B. Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women. J Periodontol., 2008, 79, 1409-1418 Sugiyama, Y; Kawakishi, S; Osawa, T. Involvement of the -diketone moiety in the antioxidative mechanism of tetrahydrocurcumin. Biochem. Pharmacol. 1996, 52, 519-525 Morimoto, T.; Yamasaki, M.; Nakata, K.; Tsuji, M.; Nakamura, H. The expression of macrophage and neutrophil elastases in rat periradicular lesions. J. Endod., 2008, 34, 1072-1076 (a). Clemens M.; Kang, D.; Napolitano, N.; Lee, H.M.; Golub, L.M.; Gu, Y. LPS or CRP/oxLDL Cholesterol-Complex: Temporal Study of Cytokine/MMP Production. J. Dent. Res., 2011, 90, Abstract # 2284; b. Napolitano, N.;Kang, D.; Clemems, M.; Lee, H.M.; Johnson, F.; Golub, L.M.; Gu, Y. Chemically Modified Curcumins Suppress Human Mononuclear Cell Cytokines. J. Dent. Res., 2011, 90, Abstract # 2292 (a). Clemens, M.; Napolitano, N.; Lee, H.M.; Zhang, Y.; Johnson,F.; Golub, L.M.; Gu, Y. Chemically modified Curcumin Normalizes Chronic Inflammation in Diabetic Rats. J. Dent. Res., 2012, 91, (spec. issue), Abstract # 1526; b. Napolitano, N.; Clemems, M.; Lee, H.M.; Zhang, Y.; Johnson, F.; Golub, L.M.; Gu, Y. Novel Curcumin Derivatives Suppress Inflammatory Mediators in Periodontally-relevant Cells. J. Dent. Res., 2012, 91, (spec. issue), Abstract # 1527; c. Elburki, M.; Lee, H.M.; Gupta, N.; Balacky, P.; Zhang, Y.; Johnson, F.; Zhang, Y.; Golub, L.M. Chemically-Modified Curcumins Inhibit Alveolar Bone Loss in Diabetic Rats with Periodontitis. J. Dent. Res., 2012, 91, (spec. issue), Abstract # 1528; d. Yu, H.W.; Zhang, Y.; Zhang, Y.; Lee, H.M.; McClain, S.A.; Johnson, F.; Golub, L.M. A Novel Chemicallymodified Curcumin (CMC2.24) Improves Diabetic Wound-Healing. J. Dent. Res., 2012, 91, (spec. issue), Abstract # 1529 Katzap, E.; Goldstein, M.J.; Shah, N.V.; Schwartz, J.; Razzano, P.; Golub, L.M.; Johnson, F.; Greenwald, R.; Grande, D. The chondroprotective capability of curcumin (curcuma longa) and its derivatives against IL-1 and OsMmediated chrondrolysis. Trans. Orthoped. Res. Soc., 2011, Abstract # 36.
