Patent Application
20080064705
The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for the purposes of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
The present invention discloses a serial of the theophylline-based nitrophenylpiperazine derivatives chemically with formula I for enhancing an aortic smooth muscle relaxation.
The endothelium plays a role in determining vascular tone by the release of vasoactive factors such as NO, prostacyclin, and EDHF. In general, the endothelium releases predominantly NO in large arteries such as rabbit carotid artery, although the contribution of EDHF assumes importance in smaller resistance arteries such as rat mesenteric artery. It is widely accepted that NO plays a central role in regulating the function of vascular smooth muscles. Its direct effect on vascular tone is brought about by cGMP formation in vascular smooth muscle cells. Several NO-related compounds, such as SNP and endogenous NO, activate sGC, elevate cGMP, and relax smooth muscles. The main finding of the present invention is that the vasorelaxant mechanisms of two KMUP-1 analogues, KMUP-3 and KMUP-4, are through NO/sGC/cGMP, AC/cAMP, and endothelium-dependent pathways, K+ channels, and PDE inhibitory activities.
KMUP-3 and KMUP-4 produce concentration-dependent aortic relaxations against the contraction induced by PE in endothelium-intact aortic rings. In endothelium-denuded aortic rings or during the inhibition of NOS, KMUP-3 and KMUP-4 are still able to evoke vasorelaxant effects. Therefore, it is suggested that KMUP-3 and KMUP-4 are more likely to have a direct action on a vascular smooth muscle component that does not involve the endothelium. However, at least part of the response to KMUP-3 or KMUP-4 is endothelium-dependent because a significant downward shift in the concentration-response curve is observed after endothelium removal. This concept is further supported by the evidence that KMUP-3 and KMUP-4 enhance the expression of eNOS in HUVECs. Increased eNOS protein is also found in a representative sGC activator YC-1, which appears to be less potent than KMUP-3 or KMUP-4. Considering the pEC50 and Emax values obtained from endothelium-denuded aortic rings, it is suggested that the vascular smooth muscle relaxant response, not interacting with endothelium, is more potent in KMUP-4 than in KMUP-3.
In endothelium-intact aortic rings, the vasorelaxant responses of KMUP-3 and KMUP-4 are attenuated by pretreatment with ODQ, the sGC inhibitor, or SQ 22536, the AC inhibitor, and they are also reduced by ODQ or SQ 22536 in endothelium-denuded aorta. Additionally, the relaxations of both agents are dramatically reduced by treating ODQ with SQ 22536 in endothelium-intact and endothelium-denuded aorta, but they are still not completely inhibited. The results of the present invention indicate that the relaxations of KMUP-3 and KMUP-4 not only activate the sGC/cGMP, AC/cAMP, and endothelium-dependent pathways, but they also have another direct action on vascular smooth muscles, which could involve K+-channel activation. Because the reduction of KMUP-3 relaxation by combined ODQ and SQ 22536 is more obvious than that of KMUP-4, it is suggested that KMUP-3, in contrast to KMUP-4, mainly acts on the cyclic nucleotide pathway, whereas KMUP-4 acts mainly on the vascular smooth muscle contractile mechanisms and/or the K+ channels.
Previous studies have shown that the vasodilators dependent on the K+-channel mechanism reduce their relaxant effects when exposed to high-K+ solutions because an increase in extracellular K+ attenuates the K+ gradient across the plasma membrane, thus rendering the K+ channel-activating mechanism ineffective; indeed, similar results were also found for KMUP-1 and KMUP-2. In the present invention, it is demonstrated that the vasorelaxations elicited by KMUP-3 and KMUP-4 are significantly reduced by increasing the extracellular concentration of K+ (80 mM). Accordingly, it is suggested that the relaxations of KMUP-3 and KMUP-4 involve smooth muscle hyperpolarization, and this action is more marked in KMUP-4 than in KMUP-3. The results further support the vasorelaxant action of KMUP-4 is predominantly on the K+-channel activation. Again, it is examined the contribution of K+-channels to KMUP-3- and KMUP-4-induced vasorelaxations in the following.
The vasorelaxant effects of KMUP-3 and KMUP-4 are significantly decreased by the above K+ channel blockers. The following results confirm that K+-channel activation also plays an important role on KMUP-3- and KMUP-4-induced vasorelaxations, especially for KMUP-4. KMUP-3 and KMUP-4 enhances the vasorelaxant responses not only to a cGMP-dependent vasodilator SNP but also to a cAMP-dependent vasodilator isoproterenol. On the other hand, it is observed that KMUP-3 and KMUP-4 also affect cyclic nucleotide breakdown at 10 mM because they inhibit the enzyme activities of PDE3, PDE4, and PDE5.
Furthermore, KMUP-3 and KMUP-4 significantly raise the intracellular cGMP and cAMP levels in RASMCs. Increased cGMP and cAMP levels are also markedly reduced by ODQ and SQ 22536, respectively. Therefore, it is suggested that KMUP-3 and KMUP-4 activate both NO/sGC/cGMP and AC/cAMP pathways and inhibit PDEs and thereby augment the intracellular cGMP and cAMP contents, leading to aortic smooth muscle relaxations.
In summary, the results of the present invention provide the evidence that the vascular smooth muscle relaxant activities of KMUP-3 and KMUP-4, KMUP-1 analogues, are most likely via cyclic nucleotide elevation, indomethacin-sensitive endothelium activation, K+-channel stimulation, and PDE inhibition. Although KMUP-3 and KMUP-4 show the effects similar to those of KMUP-1, neither AC/cAMP nor PG pathways were involved by KMUP-1 in rat vascular smooth muscles. Here, it is suggested that KMUP-3 and KMUP-4 raise cyclic nucleotides partly through PDE inhibition, produce vasorelaxing prostanoids, and therefore stimulate the K+ efflux, resulting in attenuation of Ca2+ influx-associated contractility in vascular smooth muscles. Because KMUP-3 and KMUP-4 have nonselective PDE-inhibitory activities, indeed, both of KMUP-3 and KMUP-4 act not only on vascular smooth muscle but also on non-vascular smooth muscle.
Pharmaceutical Activity
The pharmaceutical activities of the compounds, KMUP-3 and KMUP-4, of this invention have been proven by the following pharmaceutical experiments.
Wistar rats were provided from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). They were housed under conditions of constant temperature and controlled illumination (light on between 7:30 and 19:30). Food and water were available ad libitum. The study was approved by the Animal Care and Use Committee of the Kaohsiung Medical University.
Sildenafil citrate was kindly supplied by Cadila Healthcare Ltd. (Maninagar, India). Apamin, 4-aminopyridine (4-AP), charybdotoxin (ChTX), glibenclamide, indomethacin, Nv-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 9-(terahydro-2-furanyl)-9H-purin-6-amine (SQ 22536), tetraethylammonium (TEA), and YC-1 were all obtained from Sigma-Aldrich Chemical Co (St Louis, Mo.). All other reagents used were from E. Merck (Darmstadt, Germany). All drugs and reagents were dissolved in distilled water unless otherwise noted. Apamin was dissolved in 0.05 M acetic acid; indomethacin was dissolved in 100 mM sodium carbonate; ChTX, glibenclamide, ODQ, and YC-1 were dissolved in DMSO at 10 mM; KMUP-3 and KMUP-4 were dissolved in 10% absolute alcohol, 10% propylene glycol, and 2% 1 N HCl at 10 mM. Serial dilutions were made in distilled water.
Rat aortic smooth muscles were obtained as sterile surgical specimens. The tissue was washed and cut into 1- to 2-mm pieces and placed into culture dishes with Dulbecco modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 mg/mL streptomycin, and 2 mM glutamine. After these explants attached to the culture dish, usually in 1 to 2 days, DMEM supplemented with 10% FBS, penicillin, streptomycin, and glutamine was added. Rat aortic smooth muscle cells (RASMCs) migrated from the explants in 3-5 days. At this time, the explants were removed, and cells were allowed to achieve confluence. Cells were detached using 0.05% trypsin and 0.02% EDTA at 37° C. for 5 minutes to establish secondary cultures. Cultures were maintained for no more than 4 passages. To exclude contamination by endothelial cells and fibroblasts, the cell homogeneity was further confirmed by the presence of smooth muscle-specific α-actin and α-myosin. Indirect immunofluorescence staining for a variety of antigens was carried out by first plating the cells on chamber slides, fixing the cells in 3.7% formaldehyde in phosphate-buffered saline for 10 minutes, and then permeabilizing the cells with phosphate-buffered saline plus 0.1% Triton X-100. Cells were stained with a mouse monoclonal antibody directed against the amino-terminal 10 amino acids of a-smooth muscle actin and α-myosin (Boehringer Mannheim, Indianapolis, Ind.). All were stained with fluoresceinlabeled goat anti-mouse IgG antibody. Over 95% of the cell preparation was found to be composed of smooth muscle cells.
The aortic rings were prepared as follows. 24 Rats (200-300 g) were killed under mild anesthesia with ether, and their aortas were quickly excised. Thoracic aortas were cleaned of fat and connective tissue and cut into 3- to 4-mm-wide transverse rings, which were then mounted in a 10-mL organ bath and bathed at 37° C. in Krebs solution (NaCl: 118 mM, KCl: 4.8 mM, CaCl2: 2.5 mM, MgSO4: 1.2 mM, KH2PO4: 1.2 mM, NaHCO3: 24 mM, glucose: 11 mM), bubbled with a 95% O2+5% CO2 mixture.
Isometric tension was recorded with a force displacement transducer (Grass, Model FT03, Quincy, Mass.). The endothelium layer was removed mechanically by inserting the tip of a pair of forceps into the lumen and rolling the tissue back and forth several times on a paper towel moistened with physiological salt solution. At the beginning of each experiment, aortic rings were stretched to a resting tension of 1.5 g and then contracted with phenylephrine (PE, 10 mM), and once the contractions had reached a plateau, the endothelial integrity of the preparations (abbreviated as endothelium-intact in the following) or absence of endothelium (abbreviated as E− in the following) was verified by adding ACh (1 mM) to the superfusate. Only the aortic rings with a vasorelaxant response of >70% inhibition of preconstruction were considered endothelium-intact. The preparations were then washed and allowed to equilibrate with Krebs solution for 45 minutes before being contracted a second time with PE. When the stable vasoconstriction to PE (10 μM) was reached, concentration-response curves to KMUP-3 and KMUP-4 (1 nM-100 μM) were constructed. Additionally, aortic rings were preconstricted with 80 mM KCl. When the contraction reached a steady state, cumulative concentration-response curves to KMUP-3 and KMUP-4 (1 nM-100 μM) were determined. In this experiment, the high-K+ solution was prepared by replacing NaCl with KCl (80 mM) in an equimolar amount. In another experiment, the effects of KMUP-3 and KMUP-4 on the vasorelaxant responses to isoproterenol, a β-adrenoceptor-mediated cAMP-dependent vasodilator, or SNP, a NO-donor/cGMP-dependent vasodilator, were investigated by incubating the E− aortic rings with 1 mM of KMUP-3 and KMUP-4 respectively for 30 minutes before either isoproterenol (0.01 μM-10 mM) or SNP (1 nM-1 μM) was added.
To examine the possible mechanisms of vasorelaxant effects of KMUP-3 and KMUP-4, the aortic rings were pretreated with the sGC inhibitor ODQ (1 μM), the NOS inhibitor L-NAME (100 μM), the AC inhibitor SQ 22536 (100 μM), the PG inhibitor indomethacin (10 μM), the nonselective K+ channel blocker TEA (10 mM), the KATP channel blocker glibenclamide (1 μM), the voltage-dependent K+ (KV) channel blocker 4-AP (100 μM), the small-conductance Ca2+-dependent K+ (SKCa) channel blocker apamin (1 μM), or the BKCa channel blocker ChTX (0.1 μM) for 30 minutes before the addition of KMUP-3 or KMUP-4.
Please refer to
Furthermore, in order to compare the relaxant effects of KMUP-3 and KMUP-4 to the existing commercial agents, we choose theophylline which is the nonselective PDE inhibitor, milrinone which is the PDE3 selective inhibitor, rolipram which is the PDE4 selective inhibitor, and zaprinast which is the PDE5 selective inhibitor, as contrast agents. Please refer to
Please refer to
Please refer to
Please refer to
To investigate whether the inhibition of the NO/cGMP pathway results in cross-reduction of the AC/cAMP dependent pathway or vice versa, ODQ together with SQ 22536 are used to evaluate how the cross-linking between the two pathways may be influenced by KMUP-3 and KMUP-4. When ODQ was combined with SQ 22536 in endothelium-intact or endothelium-denuded aortic rings, there was an additive effect to diminish the vasorelaxations of KMUP-3 and KMUP-4 (accordingly illustrated in
Please refer to
PDE activities were determined by the method of Hidaka and Asano (Hidaka H, Asano T., Biochim Biophys Acta., 429, 485-497, 1976). Washed human platelets were used for both PDE3 and PDE5 analyses, and human U937 cells for PDE4. Purified protein containing PDE3, PDE4, or PDE5 enzyme was resuspended in 50 mM Tris-HCl containing 5 mM MgCl2 (pH 7.5). Subsequently, the enzyme (11.5 mg/mL, 10 mL) was incubated with Tris-HCl (80 mL), and 10 mM cGMP or cAMP substrate (final concentration 1 mM containing 0.1 μCi [3H]cGMP or [3H]cAMP) was added. After 20 minutes at 37° C., the samples were heated to 100° C. for 2 minutes. Ophiophagis Hannah snake venom (10 mg/mL, 10 mL) was then added and incubated at 37° C. for 10 minutes to convert the 5′-GMP and 5′-AMP to the uncharged nucleosides guanosine and adenosine, respectively. An ion-exchange resin (200 mL) was added to bind all unconverted cGMP or cAMP. After centrifuging, the supernatant was removed for determination of radiolabeled guanosine or adenosine by a liquid scintillation counter.
Human umbilical vein endothelial cells (HUVECs, American Type Culture Collection, Rockville, Md.) were cultured in F12 nutrient mixture medium supplemented with 10% FBS, 1.6 mM L-glutamine, 30 mg/mL endothelial cell growth supplement, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10 U/mL heparin. HUVECs of passages 3-5 were used for all experiments.
The enzyme inhibitory activities of KMUP-3 (10 mM) for PDE3, PDE4, and PDE5 were 55±3.1%, 48±2.4%, and 48±2.6% (n=3), respectively. Additionally, KMUP-4 (10 mM) for PDE3, PDE4, and PDE5 activities were 56±2.8%, 33±1.7%, and 15±3.1% (n=3), respectively. Under this condition, theophylline (10 mM) was used as a reference agent, and its inhibitory actions for PDE3, PDE4, and PDE5 were 8±1.0%, 8±1.2%, and 12±2.1% (n=3), respectively.
Intracellular cGMP and cAMP levels in RASMCs were described previously (Wu B N, Lin R J, Lin C Y, et al., Br J Pharmacol., 134, 265-274, 2001). In brief, cells were grown in 24-well plates (105 cells/well). At confluence, monolayer cells are washed with phosphate buffer solution (PBS) and then incubated with KMUP-3, KMUP-4, or other PDE inhibitors (10 or 100 μM) for 20 minutes. Incubation is terminated by the addition of 10% trichloroacetic acid (TCA). Cell suspensions are sonicated and then centrifuged at 2500 g for 15 minutes at 4° C. To remove TCA, the supernatants are extracted 3 times with 5 volumes of water-saturated diethyl ether. Then, the supernatants are lyophilized, and cGMP or cAMP of each sample is determined by using commercially available radioimmunoassay kits (Amersham Pharmacia Biotech, Buckinghamshire, England).
The results are expressed as mean±SEM. Statistical differences are determined by independent and paired Student t-test in unpaired and paired samples, respectively. Whenever a control group was compared with more than 1 treated group, the 1-way ANOVA or 2-way repeated-measures ANOVA was used. When the ANOVA manifested a statistical difference, the Dunnett or Tukey test is applied. A P-value of less than 0.05 is considered to be significant in all experiments. Analysis of the data and plotting of the figures are done with the aid of software (SigmaPlot Version 8.0 and SigmaStat Version 2.03, Chicago, Ill.) run on an IBM-compatible computer.
Please refer to Table 1. Table 1 is the effects of KMUP-3, KMUP-4, milrinone, rolipram and Zaprinast on cAMP and cGMP levels in RASMCs. The basal values of cGMP and cAMP are 32.14±2.61 fmol/105 cells and 6.31±0.53 pmol/105 cells (n=3) in RASMCs, respectively. KMUP-3, KMUP-4, and the PDE5 inhibitor zaprinast (100 μM) significantly increased the cGMP levels, but this is not observed for milrinone, the selective PDE3 inhibitor, and rolipram, the PDE4 inhibitor. Likewise, KMUP-3, KMUP-4, milrinone, and rolipram (10 μM) significantly elevated cAMP contents, but this is not found with zaprinast (10 μM). The rises of cGMP as a result of KMUP-3 and KMUP-4 were fully eliminated by pretreatment with ODQ (10 μM). Increased cAMP from KMUP-3 and KMUP-4 were partly attenuated by SQ 22536 (100 μM).
aMeasured from 10 μM of test agent each.
bMeasured from 100 μM of test agent each.
Please refer to Table 2. Table 2 is the effects of KMUP-3, KMUP-4, sildenafil (100 μM) on SNP (100 μM)-induced release of intracellular cGMP, respectively. As indicated in Table 3, KMUP-3, KMUP-4, and sildenafil (100 μM), the known PDE5 inhibitor, enhance the releases of intracellular cGMP by SNP (100 μM).
aMeasured from 10 μM of test agent each.
bMeasured from 100 μM of test agent each.
Enhanced eNOS Expression in HUVEC
Under normal condition, the intracellular NO activates sGC, which converts GTP into cGMP to relax smooth muscle. Nitric oxide synthase has three isomers including nNOS existing in nerve cells, eNOS existing in endothelia cells, and iNOS existing in macrophages. The nNOS and eNOS produce a small amount, and are Ca2+-dependent and consititutive-expressed. Therefore, the eNOS expression in HUVEC is enhanced which could be supported for the effect induced by KMUP-3 and KMUP-4.
Endothelial cells were processed, and eNOS protein abundance was determined by Western blot analysis using anti-eNOS antibody (BD Transduction Laboratories, San Diego, Calif.) in a manner that was similar to that described in our previous study.
Please refer to
The Synthesis of KMUP-3
Dissolve 1 mol theophylline into 2 mol 1,2-dibromoethane buffer to form a mixture. Stir the mixture until the temperature is raised to 100° C. on the mantle heater. After theophylline completely dissolves in the buffer, 125 ml, 1.6 N NaOH is added thereinto to react for 5-8 hours at approximately 100° C. Next, the mentioned reaction solution is filterated out the precipitated white NaBr under a reduced pressure and then concentrated to obtain an oil-like solution. Purify the oil-like solution with a solvent mixture of n-hexane and ethylacetate by a column having a packing gel of silica gel 60 to obtain an oil-like compound A. Dissolve the compound A in methanol and add piperazine for performing a reflux reaction to obtain a reaction solution. Further, the mentioned reaction solution is under a reduced pressure and then concentrated to obtain a first coarse crystal. Recrystallize the first coarse crystal with methanol and purify the first coarse crystal by a column having a packing gel of silica gel 60 to obtain a crystal compound B. Dissolve the crystal compound B in methanol to form a first solution. Then, dissolve 4-chloronitrobenzene in the first solution to form a second solution and perform a reflux reaction to obtain KMUP-3. KMUP-3 is further recrystalized in methanol.
The physical properties of KMUP-3:
1H NMR (CDCl3): δ 3.39 (s, 3H, NCH3), 3.57 (s, 3H, NCH3), 2.82 (t, 2H, NCH2), 4.43 (t, 2H, NCH2), 2.64 (t, 4H, 2×CH2), 3.10 (t, 4H, 2×CH2), 6.77-6.81 (m, 2H, 2×Ar—H), 7.15-7.19 (m, 2H, 2×Ar—H), 7.65 (s, 1H, imidazole-H); IR (KBr): 748.54 (ArC—Cl) & 1685.26 (C═O) cm−1; MS (m/s): 403 (Scan FAB+). Anal. (C19H23N4O7)C, H, N.
The Synthesis of KMUP-4
Dissolve 1 mol theophylline into 2 mol 1,2-dibromoethane buffer to form a mixture. Stir the mixture until the temperature is raised to 100° C. on the mantle heater. After the theophylline completely dissolves in the buffer, 125 ml, 1.6 N NaOH is added thereinto to react for 5-8 hours at approximately 100° C. Next, the mentioned reaction solution is filterated out the precipitated white NaBr under a reduced pressure and then concentrated to obtain an oil-like solution. Purify the oil-like solution by a column having a packing gel of silica gel 60 to obtain a compound A. Dissolve the compound A in methanol and add piperazine for performing a refluxation to obtain a reaction solution. Further, the mentioned reaction solution is under a reduced pressure and then concentrated to obtain a first coarse crystal. Recrystallize the first coarse crystal with methanol and purify the second coarse crystal by a column having a packing gel of silica gel 60 to obtain a crystal compound B. Dissolve the crystal compound B in methanol to form a first solution. Then, dissolve 2-chloronitrobenzene in the first solution to form a second solution and perform a reflux reaction to obtain KMUP-4. KMUP-4 is further recrystalized in methanol.
The physical properties of KMUP-4:
1H NMR (CDCl3): δ 3.60 (s, 3H, NCH3), 3.42 (s, 3H, NCH3), 4.45 (t, 2H, NCH2), 2.85 (t, 2H, NCH2), 2.70 (t, 4H, 2×CH2), 3.40 (t, 4H, 2×CH2), 6.82 (m, 2H, 2×Ar—H), 8.10 (m, 2H, 2×Ar—H), 7.69 (s, 1H, imidazole-H); IR (KBr): 1323.58 (NO2) & 1657.48 (C═O) cm−1; MS (m/s): 414 (Scan FAB+). Anal. (C19H23N4O7)C, H, N.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
