Study On The Design, Synthesis And Bioactivity Of Danshensu And Cysteine ​​Bodies And Phosphonate Analogues

Danshensu, (R)-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid as its chemical name, is one of the main hydrosoluble active ingredients of Danshen which has been applied in the therapy of coronary heart disease. Many research have been reported on its biological activities,5 such as dilating coronary arteries, inhibiting platelet aggregation, improving the activity of antioxidant enzyme, and reducing the production of active oxygen, inhibiting myocardial cell apoptosis and intracellular calcium overload. At present, Danshensu is obtained mainly from isolation and purification of Danshen, however, the procedure is complicated and time consuming due to the chemical instability of phenolic hydroxyl groups and low content in Danshen, which restrict the application of Danshensu in practice. So it is necessary to further investigate its structural modification to improve chemical stability and enhance pharmacological activities and then reveal the relationship between structure and activity. Danshensu (1)A series of (R)-Danshensu derivatives (32) were synthesized by asymmetric hydrogenation in good yields (>89%) and enantioselectivities (92-99%ee) in our previous work as well as their racemates (33) were also attained correspondingly.27 Preliminary pharmacological studies showed that these derivatives exhibited significant activity for cardiovascular protection by blocking oxidative stress and apoptosis pathway. The in vitro test showed that all these compounds could increase cell viability, and inhibit lipid hyperoxidation and apoptosis by regulating the expression of apoptosis-related molecule in gene and protein levels, up-regulating the expression of bcl-2 and down-regulating bax and caspase-3. The in vivo test indicated that 34 exhibited anti-myocardial ischemic effects featured by reducing infarction size and increasing the level of the intracellular enzymes detectable in serum. The esterified or etherified protection of Danshensu were not inclined to be oxidized, with high liposoluble and low toxic property (The median lethal dose of these Danshensu derivatives is less than 5g/kg, assayed by the National Shanghai Center for New Drug Safety Evaluation and Research). These results prompted us to further perform structural modifications of Danshensu and studies on their structure-activity relationships. R= H, CH3CO-,-CH2- R’= H, CH3CO-It has been confirmed that H2S possesses many important physiological functions in the regulation of vasodilation and vascular reconstruction in recent years.28 It is considered to be a new type of neural regulation factor and signaling transmitter besides NO and CO. Its cardiovascular bioactivities include relaxing vascular smooth muscle, inhibiting the proliferation of vascular smooth muscle cells and preventing the generation of transient hypertension. Endogenous H2S can also improve the ischemia-reperfusion injury by increasing the activity of antioxidant enzymes and direct removing part of the radical28 L-cysteine is human endogenous amino acid, non-toxic, stable and can release endogenous H2S through cystathionine a-decomposing enzyme and (3-cystathionine synthase by metabolism, which leads to inactivation of HMG-CoA reductase and accordingly inhibits the biosynthesis of cholesterol.34 As it Shows Danshensu derivatives and L-cysteine precursor which can produce endogenous H2S both have cardiovascular protection activity. By applying mutual prodrug strategy, a series of new type of conjugates of Danshensu and L-cysteine derivatives were designed and synthesized to improve their chemical stability and liposolubility as well as will be expected to enhance bioactivity through synergic effect.Moreover,α-substituted phosphonate compounds possess a wide range of biological activities, such as inhibition of the enzyme, antibacterial and anti-HIV activity.40 Based on the bioisosterism principle, the phosphonate analogs of Danshensu were designed and asymmetrically synthesized, then subjected to anti-myocardial ischemia bioassay to confirm whether the carboxylic group in Danshensu is pharmacophore.In this thesis, the designed amidated thioesterified and esterified conjugates of Danshensu and L-cysteine derivatives were obtained by using 3,4-hydroxybenzaldehyde and piperonal as the starting material respectively. In addition, the chiral phosphonate analog of Danshensu was synthesized from 3,4-dihydroxyphenylacetic acid. Totally 27 target compouds were attained and available for bioassay (see below) and their structures were confinned by’H NMR and mass spectra. The preliminary bioassay data indicated that some target compounds exhibited potent protective activity against hypoxia-induced cellular damage. At this stage, the following new concepts can be gained on the basis of the information attained from these preliminary bioassay results:(1) The amidated target compounds 62,107 and 117 significantly increased cell viability and reduced LDH leakage in a dose-dependent mannar, which signified that these compounds possessed potent protective effects against hypoxia-induced cellular damage. The activity of (R,R)-stereoisomer 62 is more potent than diastereoisomer 107, indicating that the configuration of chiral carbon exerted an obvious influence on their activities and (R,R)-was more favorable.(2) The thioesterified target compounds 127,128 and 132 demonstrated potent protective effects against hypoxia-induced cellular damage and 130 was inactive, indicating that a-acetoxy was the key pharmacophore responsible for the activity.(3) The thiazoline compounds 129 and 131 exhibited obvious protective effects against hypoxia-induced cellular damage and 129 showed a dose-dependent manner.(4) The phosphonate analogs of Danshensu exhibited no protective effects against hypoxia-induced cellular damage, suggesting the carboxylic group of Danshensu acting as an important pharmacophore.Using Hoechst staining and annexin-Ⅴ/PI, compounds 73、106、107 and 108 showed obvious anti-apoptotic activity.Further biological screening and the mechanism study on anti-myocardial ischemia activities are in progress. These promising pharmacological results mentioned above demonstrate that these conjugates of Danshensu and L-cysteine derivatives merit attention as potential anti-myocardial ischemia drugs and will undoubtedly be helpful in further optimization of anti-myocardial ischemia activities of this class of compounds.The deprotection of Boc group of S-2-(tert-Butoxycarbonylamino)ethyl 3-phenylpropanethioates (118-121) by trifluoroacetic acid (TFA) gave not only normal deprotecting products, but also 2-substituted thiazolines which were obtained by intramolecular dehydration of the corresponding compouds, whereas the deprotection of Boc group of S-2-(tert-butoxycarbonylamino)ethyl 3-phenylpropenethioates afforded only deprotecting products under the same condition due to their double bond rigid structures. The deprotection products and 2-substituted thiazolines could be obtained in high yields (85%-91%and 86%-89%) respectively by optimizing reaction conditions on the amount of TFA, the reaction temperature and time, which provided a facile and effective method to synthesize 2-substituted thiazolines.