The birth of Apixaban

Biaromatic amidine structure + isoxazole ring substitution

In order to reduce the flexibility of the molecule, the methylene group between the isoxazole ring and the amide was removed, and a carbonyl-containing side chain was introduced on the isoxazole ring, but there was no breakthrough
.

Biaromatic amidine structure → biphenyl structure

Going against the "big flow", the structure of biaryl amidine was changed to biphenyl, and it was found that the binding cavity S4 of biphenyl and FXa is a hydrophobic cavity composed of amino acid residues such as Trp215, Tyr99 and Phe174
.
The combination characteristics of molecular simulation and FXa show that the oxygen atom of the sulfonyl group has a strong hydrogen bond with Tyr99, and the selectivity is very high
.

Biphenyl fragment + near-end benzene ring optimization + side chain ester group transformation

Fix the methyl acetate group of the meta-amidine group and the isoxazole ring to investigate the influence of the aromatic ring in the middle on the activity; at the same time, due to the ester group in the structure, it will be hydrolyzed in the body, and there is a considerable proportion in the animal's plasma The free acid is present, and the in vivo activity of the free acid on FXa is significantly lower than that of the unhydrolyzed ester
.

Isoxazole ring → pyrazole ring

Isoxazoline is not an aromatic ring, but contains chiral carbon atoms.
It is also proved that the carbon chain at the 5-position has little effect on the activity.
Then, pyrazole is used to replace the isoxazole, the activity remains unchanged and the synthesis is convenient
.
The pyrazole structure was then used as a new starting point for optimization (IC50 has reached 0.
13 nM)
.

Pyrazole -CH3 & -CF3 → Activity & Selectivity

The 3-methyl-substituted pyrazole compound has an increased inhibitory activity on FXa by an order of magnitude, and the 3-trifluoromethyl-substituted pyrazole compound has stronger activity
.
Most compounds have a lower clearance rate of distribution and a longer half-life, but the oral bioavailability is less than 5%, which fails to meet the requirements of oral administration
.
The molecular structure contains a more basic amidine group (pKa of 10.
7).
The molecule in the body has a positive charge and is not easily absorbed through the membrane, so oral absorption is poor
.
The PK/PD data of some compounds are as follows
.

3

Identification of candidate compounds

DPC-423

Benzylamine replaces the benzamidine structure, and the activity is slightly weaker than the amidino compound by 1-2 orders of magnitude, but because the amidino has high activity at the pM level, it can withstand the reduction of the activity to compensate for the membrane absorption and optimize the pharmacokinetics + drug The total effect of the effect
.

As the alkalinity of the molecule is reduced, the absorption across the membrane is increased
.
Among the benzylamine compounds, compounds with trifluoromethyl substitution on the pyrazole ring, fluorine substitution at the 2-position of the proximal benzene ring of biphenyl, and 4-methylsulfonyl substitution on the distal benzene ring have the best oral bioavailability and half-life Up to 7.
5 hours, ID50 = 1.
1 μmol·kg−1·h−1 and IC50 = 0.
15 μmol·L−1 determined by the rabbit arteriovenous shunt thrombosis model
.
Given these activity data and pharmacokinetic properties, DuPont identified a candidate compound, numbered DPC-423, and entered development
.

Reza Shaban

The structure-activity relationship study shows that after the methylsulfonyl group of DPC-423 is transformed into a sulfamoyl group, its activity remains unchanged, and the selectivity is improved, but the membrane penetration property is significantly reduced due to the increased molecular polarity; remote benzene The ring is replaced with a pyridine ring, the activity remains unchanged, and the membrane penetration is still low; the replacement of the distal benzene ring with imidazole, tetrahydropyrrole or morpholine increases the solubilizing group and improves the membrane penetration, but it also affects the plasma The binding rate of protein>97% is also the reason for the decreased activity; in order to adjust the pharmacokinetics, maintain the activity strength and selectivity, it is advisable to use 1-(2-methylimidazole) group substitution; change the substituent on the imidazole ring, hydroxymethyl Radical and aminomethyl groups may reduce their activity due to their strong polarity; razaxaban was finally discovered, and clinical studies were carried out in the form of hydrochloride to prevent and treat deep vein thrombotic diseases orally
.

BMS740808

Considering that the amide bond may be hydrolyzed into pyrazolecarboxylic acid and bisarylamine in the body, the latter has the risk of potential mutagenicity.
Therefore, it is necessary to avoid the toxic warning structure.
Based on the principle of minimum structure change, the amide ring is combined with the pyridine ring.
On the azole ring, a combined heterocyclic structure is formed to improve metabolic stability
.

The aminobenzisoxazole of Razaxaban was connected to the heterocyclic pyrazole to synthesize a new class of compounds.
The structure-activity relationship is summarized as follows: (1) Transform the tertiary amino group on the remote benzene ring, two Methylaminomethyl and 3-R-hydroxytetrahydropyrrole have the strongest activity and the strongest anticoagulant effect in the body
.
(2) The introduction of fluorine atoms at the ortho position of the proximal benzene ring is less active than the corresponding compounds
.
(3) The R configuration activity of 3-hydroxytetrahydropyrrole is three times stronger than the S configuration, and the anticoagulant activity in vivo is also stronger than the S configuration
.
(4) The trifluoromethyl group on the pyrazole ring is replaced by a methyl group, which reduces the activity
.
The pharmacokinetic studies of Beagle dogs show that the following compound has the best properties, namely BMS740808
.

4

The birth of Apixaban

Candidates are the compounds DPC423, Razaxaban, and BMS740808.
The next step is to further optimize the combination of these fragments, and optimize them from multiple dimensions such as in vitro FXa activity, selectivity, in vivo anticoagulation and pharmacokinetic properties to determine more
。Excellent structure .

The dominant fragments such as anisole, pyrazolodihydropyridone, and tertiary benzidine are spliced ​​together, and the substituents on the pyrazole ring are changed to synthesize a series of compounds
.
Among them, compounds such as methylsulfonyl, carbamoyl, cyano and tetrazolyl have higher activity and selectivity than trifluoromethyl compounds, and their anticoagulant effect in vivo is significantly stronger than BMS740808, suggesting these groups Can be transplanted on different mother cores
.
In comparison, the compound in the figure below has good pharmacokinetic properties, low clearance rate (CL = 0.
32L·kg−1·h−1), half-life t1/2 of 5.
6 h, and oral bioavailability F=100 %, so the carbamoyl group is fixed for further optimization
.

In the above structural optimization, another study is to explore the effect of the substitution of the remote benzene ring on the activity.
It is found that the para-N-methylacetamido substitution of a single benzene ring is unique because of the free amino group or other related factors.
The activity of the groups is very poor, suggesting that the acylated secondary amine should be an important group involved in the binding
.

The carbamoyl substitution and the identification of candidates corrected the excessively strong fat solubility of trifluoromethyl, combined with the substitution of N-methylacetamido at the para-position of the distal benzene ring, the following compounds were obtained, which inhibited FXa in vitro and anticoagulant in vivo And the selectivity is very strong
.
The activity on human protease and liver CYP is very weak.
For example, the half-life of a compound containing a lactam structure in the distal benzene ring and liver microsomes is >100 min.
The transmembraneity of Caco-2 cells is also very high, and the plasma protein binding rate is 87%, rabbit antithrombotic IC50=329 nmol·L−1, oral half-life is 5.
8 h, oral bioavailability F=58%
.
Finally, our small molecule drug king Apixaban was born!

5

summary

The molecular structure design process of Apixaban can be described as excellence! When several compounds with very very good activity were obtained in the early stage, they did not advance rashly and were eager to develop.
Instead, they continued to study the activity, hoping to lay a sufficient "quantity" of sacrificing activity for the subsequent modification of the drug structure!

The principles and methods of its structural modification seem to be broad-spectrum, but in fact it shows a very good degree of organic integration, especially spanning the skeleton structure of rivaroxaban and the bisamidine structure of the early sprout compounds.
This bold abandonment of the "classical structure" The move is very instructive for the development of domestic drug molecules
.

Reference materials:

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), a highly potent, selective, and orally bioavailable inhibitor of blood coagulationfactor Xa.
2001.
DOI: 10.
1016/S0030-

4018(98)00117-5.

3.
Structure-based design of novel guanidine/benzamidinemimics:potent and orally bioavailable factor Xa inhibitors as novel anticoagulants.
2003.
DOI: 10.
1021/jm020578e.

4.
Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4 -c]pyridine-3-carboxamide(Apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.
2007.
DOI: org/10.
1021/jm070245n.

5.
Discovery of potent, efficacious, and orallybioavailable inhibitors of blood coagulation factor Xa with neutral P1 moieties.
DOI: 10.
1016/j.
bmcl.
2006.
08.
027.

6.
Multidimensional optimization of Apixaban creation.
Guo Zongru

7.
Research status and application of direct inhibitors of coagulation factor Xa.
2018.