2018 global small molecule marketing crown: the birth of apixaban

Johnson Apixaban is a direct factor Xa (FXa) inhibitor As an anticoagulant, it can be used to reduce the risk of stroke and systemic embolism in patients with non valvular atrial fibrillation, and also to prevent the formation of deep venous thrombosis (DVT) Apixaban is composed of Pfizer and Bristol Myers Squibb, BMS) joint R & D was approved by the European Drug Administration (EMA) on May 18, 2011, and then approved by the U.S Food and Drug Administration (FDA) on December 28, 2012, approved by the Japanese pharmaceutical medical device comprehensive agency (PMDA) on December 25, 2012, and approved by the China food and Drug Administration (CFDA) on January 22, 2013 In the past 2018, the global drug marketing champion is still sumerial, a macromolecular monoclonal antibody (although it's only a little less than 20 billion US dollars) Top 2 is a small molecule drug, but it's no longer lenalidomide, but the anticoagulant FXa inhibitor apixaban Apixaban has not only surpassed the first drug rivaroxaban at the same target, but also won No.1 in the global small molecule drug sales in 2018 Such achievements make the industry marvel! And behind it, how does this molecule get, and its process is more wonderful! Factor Xa is a proteolytic enzyme in the coagulation cascade Its function is to transform prothrombin into thrombin Its importance lies in the final link of the coagulation process caused by internal mechanism or external stimulation As an enzyme molecule, FXa has amplification effect One FXa molecule can hydrolyze tens of thousands of prothrombin to activate thrombin Therefore, FXa inhibitor is also considered as a more efficient antithrombotic drug FXa is a trypsin like protease, which directly inhibits the activity of FXa, thus inhibiting the production of thrombin, blocking thrombosis and coagulation FXa has no effect on platelet function, can reduce the risk of bleeding, and is superior to thrombin inhibitors and antiplatelet aggregation drugs in mechanism Figure 1 X-ray of interaction between FXa and apixaban In addition, let's take a look at rivaroxaban, the first drug on the market at the same target Rivaroxaban, an oral drug developed by Bayer company, was first listed in Canada and the European Union in 2008, and in China in 2009 Its trade name is bairitol ®, which is used for the prevention of VTE in patients undergoing selective hip or knee replacement surgery In terms of structure, Bayer started to set up a team in 1998 to develop a small molecule FXa inhibitor (FXa inhibitor originally originated from leech) Through high-throughput screening (hundreds of thousands of levels), we got the structure of the seedling compound, and then through a series of optimization, we finally got rivaroxaban Further comparing the structure of vaisaban and apixaban, we can find that apixaban is not the me too and me better of many traditional similar target drugs in structure, but has undergone a variety of structural transformation, skeleton transition and other operations, and finally obtained a new FXa inhibitor Figure 2 The structural differences between the livasha spot and apixaban From the beginning to the candidate and then to apixaban, this process involves a variety of principles and methods, such as skeleton transition, electronic platoon, molecular splicing and docking, which is difficult to operate and complex to work! The purpose of preliminary work is the same as that of most drug discovery, that is to improve the activity! At first, several companies reported the antithrombotic compounds of biaromatic amidines against FXa DuPont company screened the activity of IIb / IIIa receptor antagonists (the company has conducted in-depth research) on FXa It was found that the Ki value of the following compounds to FXa was 38.5 μ mol · L − 1, but the structure was monoaromatic amidines Fig 3 Structure of monoarylamidine obtained by universal sieve At that time, the diarylamine structure was the main structure The right tetrahydroisoquinoline butanone acid fragment was replaced by benzamidine, and the effect of the compound on thrombin (the stronger the better) and trypsin (the weaker the better) was further investigated to explore the selective activity Figure 4 Evolution of the structure from monoarylamidine to diarylamine In order to reduce the molecular flexibility, the methylene between isoxazole ring and amide was removed, and the side chain containing carbonyl group was introduced into isoxazole ring, but there was no breakthrough Figure 5 Introduction of carbonyl containing side chains It was found that the binding cavity S4 of biphenyl and FXa was composed of trp215, Tyr99 and phe174 amino acid residues The binding characteristics of molecular simulation and FXa show that the oxygen atom of sulfonyl group has strong hydrogen bond with Tyr99 and high selectivity Figure 6 Introduction of biphenyl structure At the same time, due to the presence of ester group in the structure, hydrolysis will occur in vivo, and there is a considerable proportion of free acid in the plasma of animals The activity of FXa in vivo is significantly lower than that of the unhydrogenated ester Figure 7 Near end benzene ring optimization + side chain ester transformation Isoxazoline is not an aromatic ring, but contains a chiral carbon atom It is also proved that the 5-position carbon chain has little effect on the activity of isoxazoline Then, pyrazol is used to replace isoxazole, the activity remains unchanged, and the synthesis is convenient After that, pyrazole structure was used as a new starting point (IC50 has reached 0.13 nm) Figure 8 Introduction of pyrazoles The inhibitory activity of 3-methyl-substituted pyrazoles to FXa was increased by one order of magnitude, and that of 3-trifluoromethyl-substituted pyrazoles was stronger Most of the compounds have low clearance distribution volume and long half-life, but oral bioavailability is less than 5%, which can not meet the requirements of oral administration There is a strong basic amidine group in the molecular structure (PKA is 10.7), which has a positive charge in the body and is not easy to be absorbed through the membrane, so the oral absorption is poor PK / PD data of some compounds are as follows Figure 9 Expansion from activity to more pharmaceutical properties The activity of benzylamine instead of benzamidine is slightly weaker than that of amidine compounds by 1-2 orders of magnitude, but because of the high activity of amidine at the level of PM, it can withstand the reduction of activity, so as to make up for the membrane absorption and optimize the overall effect of drug substitution + efficacy Because of reducing the basicity of the molecule, the absorption of the membrane is increased Among the benzamines, the compounds with trifluoromethyl substitution on pyrazol ring, 2-position near biphenyl substituted by fluorine and 4-methylsulfonyl substituted far benzene ring have the best oral bioavailability with half-life up to 7.5 H ID50 = 1.1 μ mol · kg − 1 · h − 1 and IC50 = 0.15 μ mol · L − 1 measured by the rabbit arteriovenous shunt thrombosis model Given these activity data and pharmacokinetic properties, DuPont has identified the candidate compound, No dpc-423, for development Figure 10 Chemical structure of dpc-423 The study of structure-activity relationship shows that the activity of dpc-423 is unchanged and its selectivity is improved, but its transmembrane property is significantly reduced because of the increase of molecular polarity; the activity of dpc-423 is unchanged and its transmembrane property is still low when the distal benzene ring is replaced by pyridine ring; when the distal benzene ring is replaced by midazole, tetrahydropyrrole or morpholine, the solubilizing group is increased and the transmembrane property is improved, but Therefore, the binding rate with plasma protein is more than 97%, which is also the reason for the decrease of activity In order to adjust the drug generation, maintain the activity intensity and selectivity, 1 - (2-methylimidazole) group should be used instead The substitution group on imidazole ring, hydroxymethyl and aminomethyl may reduce the activity due to the strong polarity Finally, it was found that razaxaban, in the form of hydrochloride, was used in clinical research for oral prevention and treatment Treatment of deep vein embolism Figure 11 Chemical structure of Reza Saban Considering that amide bond may be hydrolyzed into pyrazolic acid and biarylamine in vivo, the latter has the potential risk of mutagenicity, so it is necessary to avoid the toxic warning structure Based on the principle of structure transformation minimization, amide is cyclized onto pyrazol ring to form a combined heterocyclic structure, so as to improve the metabolic stability A series of new compounds were synthesized by linking azaxaban's aminobenzoisoxazole with heterocyclic pyrazoles The structure-activity relationships were summarized as follows: (1) the tertiary amino group, Dimethylaminomethyl group and 3-r-hydroxytetrahydropyrrole on the transformed distal benzene ring had the strongest anticoagulant activity and the strongest anticoagulant effect in vivo (2) The introduction of fluorine atom into the neighborhood of the benzene ring at the near end is weaker than the corresponding compound (3) The R-configuration activity of 3-hydroxytetrahydropyrrole is three times stronger than that of s-configuration, and its anticoagulant activity in vivo is also stronger than that of s-configuration (4) The activity of trifluoromethyl on pyrazol ring decreased when it was replaced by methyl group The pharmacokinetic study of beagle dogs showed that the following compounds had the best properties, namely bms740808 Figure 12 Chemical structure of bms740808 The candidate compounds are dpc423, Reza Saban and bms740808 The next step is to further optimize the combination of these fragments from the in vitro FXa activity, selectivity, in vivo anticoagulation and pharmacokinetic properties to determine the better structure A series of compounds were synthesized by splicing the preponderant fragments such as anisole, pyrazolpyridone, biphenyl tertiary amine and transforming the substituents on pyrazol ring Compared with trifluoromethyl compounds, compounds such as methanesulfonyl, carbamoyl, cyano and tetrazolyl have higher activity and selectivity The anticoagulant effect in vivo is significantly stronger than that in bms740808, suggesting that these groups can be transplanted on different mother nuclei In comparison, the following compounds have good pharmacokinetic properties, low clearance (CL = 0.32l · kg − 1 · h − 1), half-life T1 / 2 of 5.6h and oral bioavailability f = 100% Therefore, carbamoyl group is fixed and further optimized Figure 13 Fixation of carbamyl structure In the above structural optimization, another simultaneous study is to explore the influence of the transformation of the distal benzene ring substituents on the activity It is found that the para n-methylacetylamino substitution of the single benzene ring is unique, because the activity of the free amino group or other related groups is very poor, suggesting that the acylated secondary amine should be an important group participating in the binding The carbamyl substitution and the determination of the candidate corrected the over strong fat solubility of trifluoromethyl, combined with the n-methylacetylamino substitution of the distal benzene ring, the following compounds were obtained, with strong in vitro inhibition of FXa and in vivo anticoagulation and selectivity The activity of protease and CYP in human liver is very weak For example, the half-life of the far benzene ring containing lactam structure compound incubated with the temperature of liver microparticle is more than 100 min, the transmembrane of Caco-2 cell is also very high, the binding rate of plasma protein is 87%, the IC 50 of antithrombotic rabbit is 329 nmol · L − 1, the oral half-life is 5.8 h, and the oral bioavailability is f = 58% Finally, our small molecule medicine king, apixaban, was born! Figure 14 Birth of apixaban The molecular structure design process of apixaban can be described as improving! In the early stage of obtaining many compounds with very good activity, we did not rush to develop them, but continued to study the activity, in order to lay down enough "amount" of sacrificing activity for the subsequent structural transformation of the drug! The principle and method of structural modification seems to be broad-spectrum, but in fact, it shows a very good degree of organic binding, especially across the skeleton structure of rivaroxaban and the diamine structure of early seedling compounds This bold move to give up the "classical structure" is of great guiding significance for the development of domestic drug molecules Reference resources: 1 Design and synthesis of isoxazolinederivatives as factor Xa inhibitors J Med Chem.1999 DOI: 10.1021/jm980406a 2 Discovery of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]- 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(DPC423),