Discovery of a Novel Oral Antithrombotic Drug Apixaban

Discovery of a Novel Oral Antithrombotic Drug ApixabanShuangyu MaBackgroundThrombosis, which is generally defined as the formation of parenthood choke off internal kin vessels, is a major health issue in western world. Thrombosis associated disease, such as ischemic stroke and high blood pressure, remain to be the leading causes of morbidity and mortality each course in essential countries 1. To make things worse, thrombosis is to a fault know as a chronic disease with episodic recurrence 2. According the American eye Association, thirty percent of the patients diagnosed with venous thromboembolism develop recurrence within the bordering 10 years 2.The balance between thrombosis formation and bleeding is providentially regulated by the blood coagulation shower, one of the most ethereal systems in human body ( issue 1, 3). The signal transmission and amplification on the cascade was conducted by a series of trypsin-like serine proteases with high specificity, often referre d as zymogens 4. The zymogens are cleaved by the previous coagulation figure to become mad proteases, which can thereby cleave the next zymogen along the cascade and achieve the signal amplification. Blood coagulation process can be triggered by intrinsic pathway or extrinsic pathway twain are followed by the activation of common pathway. The prothrombin is then cleaved by activated factor X to generate thrombin, eventually lead to the formation of stable fibrin clot. image 1. Blood coagulation cascade ( enrol extracted from Haematology (2nd edition) by C. J. Pallister and M. S. Watson 4)Until now, the most well-known oral anticoagulant is war uttermostin, which is the current standard therapy for clinic thrombosis treatment and prevention. Despite its widely application, warfarin has a huge draw stomach on its dosing issue. Many of the coagulation factors, including factors II, VII, IX and X are Vitamin K dependent the -carboxylation by Vitamin K is essential for their biolog ical activity. Targeting on vitamin-K renewing cycle, warfarin produces an anticoagulation effect by reducing activities of the four vitamin K-dependent coagulation factors. However, the responses to warfarin can be interfered dramatically by genetic and environment factors, such as the common mutation on cytochrome P450 encoding gene, disease states variation, do drugs-drug fundamental interaction or even diet change 5. Hence, warfarin treated patients expect blood test regularly (often once a week or even worse) to determine a safe dosage case by case. Despite handling with great cautious, the anticoagulant-associated intracerebral hemorrhage keeps increasing 2. Thus, numerous efforts ask been put on developing a novel antithrombotic drug with low try and high potency.From lead to drug the development of apixabanThe drug Apixaban, approved by FDA in 2012 for thromboembolism treatment and prevention, is developed by Pfizer and Bristol-Myers Squibb to shell out as an optimize d risk-benefit oral anticoagulant. The mechanism of apixaban is entirely different with warfarin it specifically posteriors on activated coagulation factor ten (FXa), rather than vitamin K. several(prenominal) clinical trial has proved its higher efficacy compared to asparin and lower risk compared to warfarin treatment 6.Given the central position in the blood coagulation cascade, FXa evolved as an attractive drug target for developing anticoagulants. The DuPont Merck pharmaceutic Company first launched a high throughput screening on FXa inhibitor in 1997 7. Because of the substrate similarity between FXa and the GPIIb/IIa receptor, they screened the mingled library which was originally designed to be GPIIb/IIa receptor antagonist. A hit was found with low micromolar FXa similarity (coumpound 1, Ki= 38.5 M). However, most of the reported FXa inhibitor in literature at that while posse the bisamindinoaryl coordinate, such as 2 and 3 (figure 2a). Inspired by these coalesces, t hey modified the hit to be bisamidines and obtained the first list of leads (including mix 4), as shown in figure 2b.Aiming at a higher potency, molecular casting studies were conducted to further optimize the lead compound, which resulted in the removal of methylene unit between the isoxazoline ring and the amidine carbonyl together with the substitution to the carbonyl. After the similitude of different substitution groups, they obtained compound 5 with Ki of 94 nM (Figure 3) 7.Fig 2 a. The structure of hit compound and several known FXa inhibitorb. The bisamidine lead compounds (figure extracted from 7).Fig 3. Effects of the amidine group position and substitution (figure extracted from 7)However, as an oral drug candidate, this dibasic lead compound might not have good oral absorption collect to its hydrophilicity. Therefore, the next goal is to slash its basicity by refilling one of the amidine groups with a neutral species. The molecular mannequin of compound 5 bound to FXa indicated that the p-amidine group fits in an hydrophobic aryl-binding carrier bag of FXa, which is consist of three evocative residues (Trp215, Tyr99 and Phe174, figure 4a) 8. Therefore, it is possible to replace the p-amidine with aromatic structural groups without lifesizely interrupt the affinity. They employed a biaryl group and obtained compound 6 with only 2-fold decrease in potency. Further investigation on different substitution at the terminal phenyl ring resulted in large enhancement on potency, such as compound 7 (Ki = 6.3 nM). (Figure 4b)Fig 4 a. Effects of substitutions on terminal phenyl ring (figures extracted from 8).b. Chemical structure of compound 6 and 7Nevertheless, another concern emerged to be the metabolic instability, as compound 7 bears an ester side chain, which is labile to esterase once it gets inside human body. Since the corresponding acid metabolite is three-fold less potent than the original ester 9, replacing the ester side chain appe ars becomes the imperative next step. Again, a list of compound 7 analogs bearing different side chains were synthesized and tested. Among all the functional groups, tetrazole substitution was found to be most potent, and compound 8 (Ki= 0.52 nM) was developed with additional modification on the biaryl ring motif (figure 5a). Variation on the position of amide moiety attachment and isoxazoline aromatization resulted in compound 9 (Ki= 0.15 nM, figure 5b) 10, while further investigation on a series of five-membered heterocyclic ring gives compound 10 with equal potency (Ki= 0.15 nM, figure 5c) and lower basicity 11 .Fig 5. Chemical structure of compound 8, 9 and 10 Despite the high potency and selectivity, compound 10 is unruffled far from perfect due to potential mutagenicity of the biarylanilines motif 12. Therefore, the cleavage of amide bond moldiness be prevented by either ligating or eliminating the nitrogen (figure 6). Compound 11 was developed to have the optimized pyrazole with an even higher FXa affinity (Ki = 0.03 nM). After the hold was settled, more compound 11 analogs with various substitution groups and terminal aromatic rings were synthesized and compared to afford the final drug candidate as compound 12 (apixaban, Ki= 0.08, figure 7a) 1. The optimized drug selectivity towards FXa was shown in figure 7b. The remedy index of the final drug compared to warfarin was demonstrated in run thrombosis models and shown figure 7c) 6.The drug candidate was sent for preclinical studies followed with several clinic trials, and eventually approved by FDA for venous thromboembolism prevention after hip/ stifle replacement and artrial fibrillation treatment. Another clinical trial for acute venous thromboembolism is still in progress.Fig 6. Compound 11 bearing the optimized pyrazole.Fig 7 a. Apixaban (compound 12)b. In vitro Ki values of apixaban for FXa and other human enzymes with structural or functional similarities (figure extracted from 6).c. Therapeu tic index of apixaban and warfarin in rabbit models (figure extracted from 6). certaintyComparing to the initial lead compound, the eventual drug changed dramatically in chemical structure, while its FXa inhibiting potency was enhanced by 107. Tracing punt to each modification step, the comprehensive consideration in drug potency, selectivity, absorption, transfiguration and toxicity is truly impressive and reveals the tremendous efforts embedded in a single drug. It is worth noting that molecular modeling with FXa structure played a critical role during the rational drug modification process.The baring of apixaban also demonstrated the importance of defining new drug target in modern drug development. The success of apixaban is largely attributed to the better drug target FXa. However, the clinical trial of apixaban on acute coronary syndrome was discontinued due to increased bleeding events 6, which suggests that other anticoagulants are still in pack for treating various type of thrombosis.Reference1 Pinto, D. J. Orwat, M. J. Koch, S. Rossi, K. A. Alexander, R. S. Smallwood, A. Wong, P. C. Rendina, A. R. Luettgen, J. M. Knabb, R. M. He, K. Xin, B. Wexler, R. R Lam, P. Y. 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