Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`Contents lists available at ScienceDirect
`
`Journal of Molecular Structure: THEOCHEM
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t h e o c h e m
`
`Molecular structure, lipophilicity, solubility, absorption, and polar surface area
`of novel anticoagulant agents
`
`Milan Remko *
`
`Comenius University Bratislava, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Odbojarov 10, SK-832 32 Bratislava, Slovakia
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 28 July 2009
`Received in revised form 4 September 2009
`Accepted 6 September 2009
`Available online 12 September 2009
`
`Keywords:
`Factor Xa inhibitors
`Direct inhibitors of thrombin
`Molecular structure
`Solvent effect
`Lipophilicity
`Solubility
`
`The methods of theoretical chemistry have been used to elucidate molecular properties of factor Xa
`inhibitors (rivaroxaban, apixaban, otamixaban, betrixaban, razaxaban, and DX-9065a) and direct inhibi-
`tor of thrombin (dabigatran). The geometries and energies of these drugs have been computed using HF/
`6-31G(d), Becke3LYP/6-31G(d) and Becke3LYP/6-31++G(d,p) model chemistries. In the case of the Xa
`inhibitors (rivaroxaban, apixaban, otamixaban, betrixaban, razaxaban, and DX-9065a) the fully optimized
`most stable conformers possess characteristic L-shape structure. Water has a remarkable effect on the
`geometry of the anticoagulants studied. The anticoagulant drugs exhibit the largest stability in solvent
`as expected. Computed partition coefficients (ALOGPS method) for drugs studied varied between 1.7
`and 3.9. Neutral compounds are described as lipophilic drugs. Rivaroxaban is drug with lowest lipophil-
`icity. The anticoagulants studied are only slightly soluble in water, their computed solubilities from inter-
`val between 5 and 70 mg/L are sufficient for fast absorption. Experimentally determined solubility of
`rivaroxaban (8 mg/L) is very well interpreted by calculation. Rivaroxaban with PSA value 88 belongs to
`the anticoagulants with increased absorption. Direct thrombin inhibitor dabigatran is molecule with high
`total number of proton donor and proton acceptor groups (15), high PSA (150) and lowest absorption of
`the compounds studied.
`
`Ó 2009 Elsevier B.V. All rights reserved.
`
`1. Introduction
`
`Anticoagulants are key drugs for the prophylaxis and treatment
`of thromboembolic disorders [1–5]. Commonly used anticoagu-
`lants include parenterally administered unfractionated heparin
`and low molecular weight heparins, and the orally administered
`vitamin K antagonists (warfarin) [1,6]. These drugs are not tar-
`geted, i.e. they inhibit more than one enzyme in the coagulation
`cascade [1–6]. Heparin-based anticoagulants are indirect inhibitors
`that enhance the proteinase inhibitory activity of a natural antico-
`agulant, antithrombin [6]. Although effective, their use has been
`hampered by numerous limitations [4]. There is a growing interest
`in new, orally active anticoagulants with significant advantages to
`current agents such as heparin and warfarin for the treatment and
`prevention of thrombotic diseases. The new anticoagulants under
`investigation for venous thromboembolism treatment target factor
`Xa (fXa) or thrombin [1–5]. Inhibitors of factor Xa block thrombin
`generation, whereas thrombin inhibitors block the activity of
`thrombin, the enzyme that catalyses the conversion of fibrinogen
`to fibrin [1,3]. Besides synthetic indirect factor Xa inhibitors from
`the family of glycosaminoglycans (fondaparinux,
`idraparinux)
`
`* Tel.: +421 2 50117225; fax: +421 2 50117100.
`E-mail address: remko@fpharm.uniba.sk
`
`0166-1280/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
`doi:10.1016/j.theochem.2009.09.011
`
`numerous direct, selective factor Xa inhibitors are currently at var-
`ious stages of development in different therapeutical indications
`[1–4]. Small-molecule synthetic compounds such as rivaroxaban,
`razaxaban, apixaban, betrixaban are members of a new class of or-
`ally available active-site-directed factor Xa inhibitors. Small-mole-
`cule direct thrombin inhibitors (dabigatran etexilate)
`inhibit
`thrombin directly by directly binding to the active catalytic site
`[1–3].
`Despite a great deal of experimental evidence for the relationship
`betweenthechemicalandpharmaceuticalpropertiesofnewanticoag-
`ulants targeting factor Xa or thrombin and their biological activity,
`there is no single experimental study concerned with the systematic
`comparativeexperimentalinvestigationofthephysico-chemicaland
`pharmacokinetic parameters of these medicinally useful new antico-
`agulants. Quantitative structure activity relationships of factor Xa
`inhibitorswerediscussedquiterecently[7],andtheX-raycrystalstruc-
`turesofrivaroxaban[8],DX-9065a[9],apixaban[10],otamixaban[11]
`andrazaxaban[12]incomplexeswithfactorXawereusedtoclarifythe
`binding mode of these ligands. The molecular structure of six mono-
`meric structural units (1-OMe DIdoA-2SNa2 (unit A), 1-OMe GlcN-
`S6SNa2(unitD),1,4-DiOMeGlcNa(unitE),1,4-DiOMeGlcN-S3S6SNa3
`(unitF),1,4-DiOMeIdoA-2SNa2(unitG),and1,4-DiOMeGlcN-S6SNa2
`(unitH)),fourdimericstructuralunits(D–E,E–F,F–G,andG–H),twotri-
`mericstructuralunits(D–E–F,andF–G–H)andpentamerD–E–F–G–H
`
`MYLAN EXHIBIT 1021
`
`

`

`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`77
`
`(fondaparinux) of heparin has been previously investigated [13–17]
`using thedensityfunctionaltheory.
`The absence of experimental data of novel synthetic anticoagu-
`lants targeting factor Xa or thrombin presents a challenge to the
`application of computational modeling techniques in order to en-
`hance our understanding of the subtle biological effects of these
`anticoagulants. In this paper we have used the results of large-
`scale theoretical calculations for the study of the molecular struc-
`ture, pKa, lipophilicity, solubility, absorption, and polar surface area
`of factor Xa inhibitors (rivaroxaban, apixaban, otamixaban, betrix-
`aban, razaxaban, and DX-9065a) and direct inhibitors of thrombin
`(dabigatran). The results of theoretical studies of these drugs were
`compared with the available experimental data and discussed in
`relation to the present theories of action of these agents.
`
`2. Computational details
`
`Theoretical calculations of the rivaroxaban, apixaban, otamix-
`aban, betrixaban, razaxaban, DX-9065a, dabigatran, and dabigatran
`etexilate (Fig. 1) were carried out with the Gaussian 03 computer
`code [18] at the ab initio SCF (HF [19]) and density functional the-
`ory (DFT, Becke3LYP [20–24]) levels of theory using the 6–31G(d)
`and 6–31++G(d,p) basis sets. In order to evaluate the conforma-
`tional behavior of these systems in solvent, we carried out optimi-
`zation calculations in the presence of water. The methodology used
`in this work is centered on two solvation methods, PCM [25,26]
`and Onsager [27] models. The structures of all gas-phase species
`were fully optimized at the HF/6–31G(d) and Becke3LYP/6–
`31G(d) levels of theory without any geometrical constraint. In
`order to check the correctness of the B3LYP calculated relative
`energies using the double-f basis set, we also performed calcula-
`tions of the anticoagulant species, using the larger basis set
`6–31++G(d,p) implemented in the Gaussian 03 package of com-
`puter codes [14,15]. The structures of all condensed-phase (SCRF)
`species were fully optimized without any geometrical constraint
`at the DFT level of theory applied. Lipophilicity and water solubility
`calculations were carried out using web-based VCCLAB [28–30].
`For calculations of molecular polar surface areas the fragment-
`based method of Ertl and coworkers [31,32] incorporated in the
`Molinspiration Cheminformatics software [33] was used.
`
`3. Results and discussion
`
`3.1. Molecular structures
`
`Conformational search using theoretical methods for such large
`systems was in the past limited to use some of the available force-
`field methods [34]. Rapid advances in computer hardware and
`software and in quantum medicinal chemistry have brought
`high-performance computing and graphic tools within the reach
`of most academic and industrial laboratories, thus facilitating the
`development of useful approaches to rational drug design. Quan-
`tum chemical calculations are now applied successfully in medici-
`nal chemistry and drug design to determine accurately molecular
`structures and properties for use in a wide variety of CADD studies
`[35]. It is common in the computational study of drugs to use
`structural data obtained from X-ray crystallography or NMR spec-
`troscopy as guides to the quality of theoretical computations. In
`the absence of the experimental published data about molecular
`conformations of drugs, as an alternative to analyzing small mole-
`cule crystal structures the conformations of drugs bound to their
`protein targets can be examined [36]. Rivaroxaban, otamixaban
`and DX-9065a are chiral molecules and may be present as race-
`mates. However, under the development process of these drugs a
`clear preference for particular enantiomer was observed,
`
`indicating a specific interaction with fXa. The calculations for these
`drugs were carried out with the biologically most active enantio-
`mers only. In the absence of the small molecule crystal structures,
`we examined the conformations of ligands bound to their fXa and
`trombin targets by studying the macromolecular crystal structures
`deposited in the Protein Data Bank. In the context of drug design,
`the conformation a small molecule adopts when bound to a phar-
`maceutical target is of fundamental importance. The relative orien-
`tation of anticoagulant moiety defined by individual dihedral
`angles (a, b, c, d, e, f, g, and h, Fig. 1) was taken from the experimen-
`tal data for available X-ray data of the crystal structures deposited
`in the Protein Data Bank (PDB) [37] (complexes of the Xa factor
`with rivaroxaban (PDB: 2W26), apixaban (PDB: 2P16), otamixaban
`(PDB: 1KSN), razaxaban (PDB: 1Z6E), DX-9065a (PDB: 1FAX), and
`complex of the ethylester of dabigatran with trombin (PDB:
`1HTS). A particular ligand conformations observed in these com-
`plexes may be due to an intermolecular interaction that is not
`present in solution or vapor phases. All compounds, but apixaban
`possess amide functionality, which imparts certain conformational
`rigidity to the overall structure of molecules studied. The possible
`relaxation of the geometry of a ligand upon dissociation from the
`receptor may bring new information about the conformation of
`drug in isolated state. An analysis of the harmonic vibrational fre-
`quencies at the HF level of theory of the optimized species revealed
`that all the structures obtained were minima (no imaginary fre-
`quencies). The Cartesian coordinates (Å) of all gas-phase drugs
`investigated, optimized at the B3LYP/6–31++G(d,p) level of theory,
`are given in Table A of the electronic Supporting Information. The
`geometries optimized at the B3LYP/6–31++G(d,p) level of theory
`are shown in Fig. 2. In the case of the Xa inhibitors (rivaroxaban,
`apixaban, otamixaban, betrixaban, razaxaban, and DX-9065a) the
`fully optimized most stable conformers possess characteristic
`L-shape structure. Examination of the space models of the B3LYP
`computed structures using two basis sets of the drugs investigated
`shows that the increase of the basis set gives essentially the same
`results. The effect of bulk solvent is treated with two solvation
`methods (the Onsager [27] and PCM [25,26]) for comparison. Ini-
`tial calculations were carried out, for computational reasons, using
`the SCRF formalism of Wong et al. [38–41]. The radii of the cavities
`used in this approach were chosen after a volume calculation of
`each molecule, and the dielectric constant of water (e = 78.5) was
`used. The placing of the isolated molecules into a spherical cavity
`within a dielectric medium of the Onsager model of solvation does
`not represent the realistic situation in the biological medium; it
`seems helpful in revealing the main role of the solvent in intermo-
`lecular electrostatic interactions. The second, PCM (conductor-like
`polarizable model), defines the cavity by the envelope of spheres
`centered on the atoms or the atomic groups [25,26]. The whole
`concept of using such macroscopic properties as dielectric con-
`stants in microscopic computations has been criticized [42,43]. De-
`spite all these valid criticisms, continuum-based methods of
`solvation are used extensively and successfully in a variety of prob-
`lems [44,45]. It has been shown previously [46] that the conduc-
`tor-like polarizable method reproduces hydration energies with
`accuracy in the order of a few kcal/mol but mostly (70% of the
`cases) even better than one kcal/mol.
`The factor Xa inhibitors (rivaroxaban, apixaban, otamixaban,
`betrixaban, razaxaban, and DX-9065a) studied do not possess com-
`mon pharmacophore functionality. However, the intense research in
`this field accumulated new results, which have been summarized in
`a number of publications [1–12]. The binding of inhibitor to fXa is
`characterized by two general interaction sites S1 and S4 (Fig. 3).
`Based on the analysis of the binding in the S1 pocket fXa inhibitors
`are categorized into two classes. One class of early inhibitors mimics
`the natural interaction between arginine of prothrombin in the fXa
`active site with the Asp189 of the S1 pocket of fXa. Such compounds
`
`

`

`78
`
`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`CF3
`
`10
`
`N
`
`11
`
`N
`
`13
`
`12
`
`NH2
`
`9
`
`O
`
`8
`
`7
`
`NH
`
`6
`
`F
`
`CH3
`
`CH3
`
`N
`
`4
`
`5
`
`N
`
`1
`
`2 3
`
`N
`
`Razaxaban
`
`N
`
`O
`
`8
`
`7
`
`6
`
`O
`
`10
`
`9
`
`12
`
`Cl
`
`12
`
`14
`
`S
`
`15
`
`O
`13
`
`11
`
`NH
`
`MeO
`
`10
`
`9
`
`O
`
`O
`
`1
`
`2
`N
`
`3
`
`4
`
`5
`
`8
`
`6
`
`7
`N
`
`O
`
`Rivaroxaban
`
`O
`
`O
`
`1
`
`2
`N
`
`4
`
`5
`
`3
`
`6
`
`O
`
`8
`
`7
`
`N
`
`12
`
`N
`
`11
`
`10
`
`N
`
`9
`
`NH2
`
`O
`
`Apixaban
`
`OMe
`
`10
`
`CH3
`
`11
`
`O
`
`12
`
`13
`
`14
`
`O
`
`8 9
`
`NH
`
`1
`
`4
`
`5
`
`O
`
`+
`N
`
`2
`
`3
`
`6
`
`7
`
`11
`N
`
`13
`
`14
`
`N
`
`15
`
`16
`
`5
`
`O
`
`4
`
`NH
`
`1
`
`23
`
`N
`
`CH3
`
`NH
`
`LY-517717
`
`15
`
`16
`17
`
`18
`NH
`
`NH2
`
`Otamixaban
`
`NH
`16
`
`NC
`
`H3
`
`17
`
`13
`
`12
`
`15
`
`14
`
`11
`
`9
`
`CH3
`
`Betrixaban
`
`O
`10
`
`NH
`
`8
`
`7
`
`6
`
`45
`
`O
`
`3
`
`NH
`
`1 2
`
`N
`
`OMe
`
`OH
`
`O
`
`Cl
`
`15 16
`
`13
`
`14
`
`NH
`17
`
`NH2
`
`CH 3
`
`CH3
`
`11 12
`
`NH
`
`9
`
`10
`
`N
`
`N
`8
`
`Dabigatran
`
`N
`3
`
`4
`
`6
`
`7
`
`O
`
`5
`
`12
`
`N
`
`CH 3
`
`O
`
`O
`
`O
`
`O
`
`N
`17
`
`NH2
`
`15 16
`
`13
`
`14
`
`CH3
`
`11 12
`
`NH
`
`9
`
`10
`
`N
`
`N
`8
`
`Dabigatran Etexilate
`
`N
`3
`
`4
`
`6
`
`7
`
`O
`
`5
`
`12
`
`N
`
`Fig. 1. Structure and atom labeling in the anticoagulant drugs studied.
`
`often contain phenylamidine group mimicking this interaction (ota-
`mixaban, betrixaban, DX-9065a). The second category of com-
`pounds utilized neutral aryl groups bind into the S1 pocket (e.g.
`
`aminobenzisoxazole for razaxaban, chlorothiophene moiety of riva-
`roxaban). In addition to the S1 pocket, a second major binding site of
`fXa is a narrow hydrophobic channel (the S4 pocket) defined by the
`
`

`

`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`79
`
`Fig. 2. B3LYP/6–31++G(d,p) optimized structures of the anticoagulants investigated.
`
`Tyr 228
`Important
`interaction
`
`S
`
`Cl
`
`S1
`Pocket
`
`O
`
`NH
`
`Coplanar rings
`
`N
`
`O
`
`O
`
`2.0 Å
`
`3.3 Å
`
`Hydrogen bond
`
`Gly 219
`
`O
`
`N
`
`O
`
`Perpendicular arrangement
`
`S4
`Hydrophobic
`pocket
`
`Fig. 3. Binding model of rivaroxaban. S1 and S4 pocket represents binding sites typical for synthetic direct fXa inhibitors.
`
`aromatic rings of Tyr99, Phe174, and Trp215 [8]. Dabigatran is a no-
`vel reversible direct thrombin inhibitor. It interacts with the active
`site of thrombin composed of the specificity (S1), proximal (S2)
`
`and distal (D) pockets. The amidine group of dabigatran interacts
`with the aspartic acid (Asp189) of the S1 pocket. The central methyl-
`benzimidazole part is bound to thrombin by a hydrophobic
`
`

`

`80
`
`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`interaction with the S2 pocket, and the 2-pyridyl group is positioned
`between Leu99 and Ile174 in the D-pocket [47].
`The relative molecular orientation of individual drugs studied is
`described by different number of rotatable bonds. These dihedral
`angles of the fully optimized anticoagulants are given in Table 1 to-
`gether with the available X-ray structures of these drugs in the
`bound state (at the receptor). Values for these dihedral angles for
`individual drugs are different (Table 1), and no general conclusions
`about pharmacophore functionality can be deduced. Thus, the
`structure of drugs studied will be discussed individually. According
`to our calculations in the gas state, the equilibrium geometries
`computed at the HF level of theory are in general agreement with
`the DFT results obtained with the ‘‘standard” 6-31G(d) basis set.
`However, the ab initio SCF and DFT optimized dihedral angles of
`some drugs studied exhibit large differences (within the 10°–
`20°). In order to study the basis set effect on the geometry of the
`anticoagulants investigated within the DFT theory we also carried
`out calculations using the larger basis set 6–31++G(d,p). The exten-
`sion of the basis set in the DFT calculations resulted in only small
`changes in the equilibrium geometry of the drugs studied. The
`optimized dihedral angles using two basis sets within the DFT the-
`ory fit one another to within about 2°–5° (Table 1).
`Water has a remarkable effect on the geometry of the anticoag-
`ulants studied (Table 1). Table 2 shows the results obtained for cal-
`culations performed in both, vacuum and that based on the
`solvation method used. The anticoagulant drugs exhibit the largest
`stability in solvent as expected, since they hold considerable dipole
`moment (Table 2). The energy difference between gas phase and
`solvated phase was significant for the both solvation models em-
`ployed in this work. The solvated phase energies within the Onsag-
`er model were obtained after full geometry optimization in water.
`However, some anticoagulants failed to optimize geometries in
`water within the PCM formalism. Table 2 contains water stabiliza-
`tion energies using single-point PCM calculations and in vacuo
`fully optimized geometries. The comparison of the single-point
`PCM and available water stabilization energies obtained after full
`geometry optimization in water indicate that in the case of neutral
`drugs the optimization of geometry in aqueous medium does not
`significantly change the solvation energy. The difference in water
`stabilization energy is very small (around 5 kJ/mol). The PCM pro-
`vided substantially more stable structures than Osanger’s model.
`Experimentally, small molecule drug conformations are com-
`monly studied using X-ray crystallography. The absence of the
`published experimental X-ray structural data of anticoagulants
`studied presents a challenge to the application quantum chemical
`methods in order to obtain information about the stable conforma-
`tions of these drugs in the gas phase and in solution. A comparison
`of the ab initio SCF calculated conformational energies of drug mol-
`ecules with the conformer distribution in the solid state routinely
`show a good correlation [48]. Moreover, previous investigations of
`the protein–ligand complexes revealed similar torsion angles dis-
`tributions for fragments when the bound and unbound distribu-
`tions were compared [49]. For the reason of comparison and
`analysis of theoretically determined conformations and protein-
`bound conformations of anticoagulants studied we also present
`the available structural data for bound anticoagulants on the factor
`Xa receptor.
`
`3.1.1. Rivaroxaban
`The 1,3-oxazolidin-ring system of rivaroxaban (5-chloro-N-
`[[(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-
`yl]methyl]thiophene-2-carboxamide) has in position 5 a chiral
`carbon atom and possesses two enantiomers termed R and S with
`a clear preference for (S)-configuration [8]. Optimized molecular
`conformation of the (S)-rivaroxaban (Fig. 4) provides the L-shape,
`which is needed for factor Xa binding. Experimentally determined
`
`3D structure of the (S)-rivaroxaban studied corresponds to the
`bound molecule at the protein, therefore the general structural
`motifs of drug can be compared with results for isolated molecule
`from theoretical methods only. The experimental values for the
`dihedral angles in the rivaroxaban–fXa complex are well inter-
`preted by the corresponding angles computed for the solvated
`(S)-rivaroxaban (Fig. 4). The main difference in the molecular
`structure of bound and unbound rivaroxaban arises from the posi-
`tion of the morpholinone end moiety, dihedral angle a[C(1)–N(2)–
`C(3)–C(4)]. The carbonyl group of this moiety effects mainly a
`planarization of the morpholinone ring and brings it into a rather
`perpendicular arrangement to the aryl ring [8]. The DFT calcula-
`tions suggest more planar arrangement of the morpholine and aryl
`rings. The dihedral angle a[C(1)–N(2)–C(3)–C(4)] is about 51°–55°
`(DFT method), and for (S)-rivaroxaban in water solution, and/or at
`receptor increases to about 67°–77° (Table 1). Oxazolidone and
`aryl rings are almost coplanar (the dihedral angle b[C(5)–C(6)–
`N(7)–C(8)]) (Table 1).
`
`3.1.2. Apixaban
`Apixaban (1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-
`1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-carboxamide)
`is one of the most rigid molecules of the anticoagulants studied.
`Its 3D structure (Fig. 2) is governed by three dihedral angles (a,
`b, and c), Table 1. The 3D structure of the bound apixaban at the
`factor Xa receptor, the gas-phase structure and solvated apix-
`aban are substantially different. In general, the coordination of
`the apixaban to its fXa receptor (PDB file 2P16) leads to the per-
`pendicular arrangement of the phenyllactam moiety (dihedral
`angle a[C(1)–N(2)–C(3)–C(4)]). The same perpendicular arrange-
`ment forms also bicyclic pyrazole scaffold and p-methoxyphenyl
`moiety of bound apixaban (dihedral angle c[C(9)–N(10)–C(11)–
`C(12)]. The phenyllactam and the bicyclic pyrazole scaffold of
`bound apixaban are also in almost perpendicular arrangement
`(dihedral angle b[C(5)-C(6)-N(7)-C(8)]), Table 1. Values of these
`dihedral angles in the gas-phase structure and solvated apixaban
`are quite different. The unbound apixaban is in gas phase and in
`water solution substantially more planar, the planarization effect
`is especially considerable for the moiety containing phenyl ring,
`bicyclic pyrazole scaffold and p-methoxyphenyl group (dihedral
`angles b, and c). The central part in the planarization of this
`apixaban moiety plays the carbonyl group of the central lactam
`group of the bicyclic pyrazole scaffold. This group effects a plan-
`arization of whole moiety via electrostatic intramolecular inter-
`action with acidic hydrogen atoms of the neighboring rings
`(Fig. 2). The C-3 carboxamido substituent and the pyrazole scaf-
`fold are in mutual planar arrangement.
`
`3.1.3. Otamixaban
`(R,R)-Otamixaban (methyl (2R,3R)-2-(3-carbamimidoylbenzyl)-
`3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate) is structur-
`ally very flexible molecule, and its space arrangement is defined
`by 8 dihedral angles (Fig. 1). The P4 phenylpyridyl-N-oxide and
`benzamidine groups, responsible for the interaction of otamixaban
`with the fXa receptor, are connected by the system of single bonds
`enable large structural flexibility of this drug on receptor. A com-
`parison of the fully optimized isolated molecule and the bound
`otaximaban (PDB file 1K3N) indicates that the largest structural
`rearrangement resulting in the biologically active conformation is
`related with the dihedral angles e, f, and g (Table 1). The gas-phase
`conformations of the phenylpyridyl-N-oxide and benzamidine
`groups of otamixaban are also preserved in bound drug. The ami-
`dine group of the benzamidine moiety is twisted out of the aro-
`matic ring with the dihedral angle l[C(15)–C(16)–C(17)–N(18)]
`from a relatively narrow interval of 155°–160° (Table 1).
`
`

`

`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`81
`
`Table 1
`Optimized dihedral anglesa of the drugs studied.
`
`Dihedral angle
`
`X-ray
`
`HF/6–31g(d)
`
`B3LYP/6–31g(d)
`
`B3LYP/6–31++g(p,d)
`
`DFT – Onsager
`
`DFT – CPCM
`
`a[C(1)–N(2)–C(3)–C(4)]
`b[C(5)–C(6)–N(7)–C(8)]
`c[C(8)–C(9)–C(10)–N(11)]
`d[C(9)–C(10)–N(11)–C(12)]
`e[C(9)–C(10)–N(11)–O(13)]
`f[C(10)–N(11)–C(12)–C(14)]
`g[N(11)–C(12)–C(14)–C(15)]
`
`a[C(1)–N(2)–C(3)–C(4)]
`b[C(5)–C(6)–N(7)–C(8)]
`c[C(9)–N(10))–C(11)–C(12)]
`
`a[C(1)–C(2)–C(3)–C(4)]
`b[C(5)–C(6)–C(7)–O(8)]
`c[C(5)–C(6)–C(7)–N(9)]
`d[C(6)–C(7)–N(9)–C(10)]
`e[C(7)–N(9)–C(10)–C(11)]
`f[N(9)–C(10)–C(11)–C(12)]
`g[N(9)–C(10)–C(11)–C(13)]
`h[C(10)–C(11)–C(13)–C(14)]
`l[C(15)–C(16)–C(17)–N(18)]
`
`a[C(1)–C(2)–N(3)–C(4)]
`b[C(2)–N(3)–C(4)–O(5)]
`c[C(2)–N(3)–C(4)–C(6)]
`d[N(3)–C(4)–C(6)–C(7)]
`e[C(6)–C(7)–N(8)–C(9)]
`f[C(7)–N(8)–C(9)–O(10)]
`g[C(7)–N(8)–C(9)–C(11)]
`h[N(8)–C(9)–C(11)]–C(12)]
`l[C(13)–C(14)–C(15)–N(16)]
`m[C(13)–C(14)–C(15)–N(17)]
`
`a[C(1)–N(2)–C(3)–C(4)]
`b[C(5)–C(6)–N(7)–C(8)]
`c[C(6)–N(7)–C(8)–O(9)]
`d[C(6)–N(7)–C(8)–C(10)]
`e[N(7)–C(8)–C(10)–C(11)]
`f [C(10)–N(11)–C(12)–C(13)]
`
`a[C(1)–C(2)–C(3)–C(4)]
`b[C(2)–C(3)–C(4)–C(5)]
`c[C(3)–C(4)–C(5)–C(6)]
`d[C(7)–C(8)–O(9)–C(10)]
`e[C(8)–O(9)–C(10)–C(11)]
`f[C(11)–N(12)–C(13)–N(14)]
`g[C(a)–C(b)–C(c)–N(d)]
`
`a[C(1)–C(2)–N(3)–C(4)]
`b[C(2)–N(3)–C(4)–O(5)]
`c[C(2)–N(3)–C(4)–C(6)]
`d[N(3)–C(4)–C(6)–C(7)]
`e[N(8)–C(9)–C(10)–N(11)]
`f[C(9)–C(10)–N(11)–C(12)]
`g[C(10)–N(11)–C(12)–C(13)]
`h[C(14)–C(15)–C(16)]–N(17)]
`
`a[C(1)–C(2)–N(3)–C(4)]
`b[C(2)–N(3)–C(4)–O(5)]
`c[C(2)–N(3)–C(4)–C(6)]
`d[N(3)–C(4)–C(6)–C(7)]
`e[N(8)–C(9)–C(10)–N(11)]
`f[C(9)–C(10)–N(11)–C(12)]
`g[C(10)–N(11)–C(12)–C(13)]
`h[C(14)–C(15)–C(16)]–N(17)]
`
`2w26
`77.9
`19.7
`63.2
`96.3
`0.5
`178.4
`1.3
`2p16
`84.2
`116.6
`94.1
`
`1k3n
`36.7
`20.7
`158.2
`177.9
`127.9
`179.5
`54.6
`159.9
`156.5
`
`1z6e
`83.5
`115.3
`6.7
`172.7
`163.8
`81.4
`
`1fax
`44.0
`55.6
`57.2
`127.4
`41,4
`169.2
`179.8
`1KTSb
`83.0
`170.8
`4.5
`132.6
`31.1
`79.3
`33.3
`176.7
`
`74.7
`15.7
`61.9
`105.8
`3.8
`176.7
`8.7
`
`104.5
`70.5
`62.3
`
`42.7
`25.2
`154.7
`176.3
`79.1
`170.4
`64.8
`162.6
`153.2
`
`5.7
`2.1
`178.2
`145.6
`155.0
`1.1
`179.7
`22.6
`35.9
`147.8
`
`66.3
`115.8
`2.4
`176.3
`145.2
`69.3
`
`77.6
`68.6
`62.2
`177.5
`81.9
`175.2
`154.2
`
`142.3
`145.6
`39.2
`33.5
`125.7
`82.7
`18.2
`156.0
`
`142.8
`146.1
`38.8
`33.8
`124.6
`82.4
`18.2
`155.7
`
`a For definition of dihedral angles see Fig. 1.
`b Dabigatran ethylester.
`
`51.1
`4.3
`62.1
`103.9
`1.7
`178.2
`5.6
`
`125.4
`50.0
`52.8
`
`32.9
`22.9
`157.2
`176.9
`77.3
`171.2
`64.4
`162.8
`158.0
`
`3.8
`2.8
`178.1
`155.7
`172.1
`3.7
`176.9
`17.9
`35.2
`149.3
`
`56.9
`127.5
`0.2
`178.4
`147.7
`58.1
`
`80.7
`67.6
`65.9
`176.0
`82.1
`177.1
`159.0
`
`146.9
`152.0
`33.5
`31.6
`123.4
`80.8
`18.4
`160.3
`
`147.5
`152.4
`33.0
`32.8
`122.2
`80.9
`18.5
`160.8
`
`(S)-Rivaroxaban
`57.7
`4.9
`59.7
`106.3
`2.1
`178.1
`0.6
`Apixaban
`116.8
`55.0
`57.8
`
`(R,R)-Otamixaban
`35.3
`21.9
`157.9
`177.7
`76.8
`170.3
`65.1
`162.6
`154.9
`
`Betrixaban
`2.4
`3.9
`176.9
`154.8
`170.1
`3.2
`177.3
`15.5
`38.8
`145.5
`
`Razaxaban
`58.2
`122.8
`2.3
`176.3
`145.6
`62.7
`
`(S,S)-DX9065a
`79.4
`67.9
`62.0
`177.9
`81.5
`178.1
`156.3
`
`Dabigatran
`144.7
`152.7
`32.6
`34.2
`125.7
`78.1
`16.3
`158.1
`
`Dabigatran etexilate
`145.5
`153.5
`31.6
`35.0
`122.9
`79.6
`16.9
`160.7
`
`66.9
`7.8
`59.2
`106.7
`5.0
`175.3
`2.6
`
`112.4
`58.7
`62.3
`
`7.8
`2.4
`178.0
`152.3
`160.8
`1.8
`178.7
`24.7
`43.3
`141.4
`
`57.9
`128.2
`0.6
`178.0
`154.2
`74.2
`
`75.2
`66.2
`62.4
`178.0
`81.1
`178.2
`156.9
`
`54.7
`2.4
`60.9
`100.9
`5.5
`174.8
`9.4
`
`129.6
`54.3
`53.2
`
`33.6
`24.9
`156.3
`177.4
`59.4
`177.5
`55.8
`144.7
`160.5
`
`2.2
`3.0
`177.8
`156.4
`173.7
`1.9
`179.0
`17.9
`36.4
`148.8
`
`58.2
`127.8
`0.3
`178.3
`148.9
`59.0
`
`78.6
`66.3
`64.7
`178.2
`81.0
`178.2
`157.3
`
`146.4
`150.6
`34.7
`35.6
`122.4
`77.4
`15.7
`161.1
`
`146.2
`151.3
`33.7
`37.4
`118.7
`78.2
`15.6
`162.7
`
`

`

`82
`
`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`Table 2
`The Becke3LYP/6–31G(d) solvent stability of the anticoagulant agents investigateda.
`
`Cavity valueb (a0)
`5.59
`5.92
`5.98
`5.44
`5.63
`5.58
`6.06
`6.64
`
`DEc Onsager
`2.9
`5.3
`7.6
`1.2
`1.9
`3.5
`4.1
`7.8
`
`DEc,d PCM
`93.8 (95.5)e
`93.3 (97.8)e
`122.2
`72.6 (76.1)e
`113.1 (119.2)e
`123.7
`148.8
`130.1
`
`Gas-phase dipole momentf
`
`3.29
`7.49
`3.96
`2.19
`2.84
`3.70
`4.39
`6.87
`
`No.
`
`Drug
`
`1
`2
`3
`4
`5
`6
`7
`8
`
`(S)-Rivaroxaban
`Apixaban
`(R,R)-Otamixaban
`Betrixaban
`Razaxaban
`(S,S)-DX-9065a
`Dabigatran
`Dabigatran etexilate
`
`a Water as solvent.
`b Ångström (Å).
`c kJ/mol.
`d Single-point PCM calculation.
`e Full geometry optimization in the solvent.
`f Debye (D).
`
`er stabilization effect of intramolecular hydrogen bond of the
`NAH. . .O@C type (Fig. 2). The N,N-dimethylamidine group is
`twisted out of the aromatic ring by about 35°. Water solvent does
`not have pronounced effect on the overall shape of betrixaban (Ta-
`ble 1).
`
`3.1.5. Razaxaban
`Razaxaban (2-(3-amino-1,2-benzoxazol-5-yl)-N-[4-[2-(dimeth-
`ylaminomethyl)imidazol-1-yl]-2-fluorophenyl]-5-(trifluorometh-
`yl) pyrazole-3-carboxamide) is a predecessor of apixaban that was
`discontinued based on less that optimal pharmacological properties
`[51]. The discovery of the pyrazole scaffold was an important mile-
`stone in search for new molecules targeting fXa and proved to be cru-
`cial in the evolution of orally bioavailable fXa inhibitors such as
`razaxaban and apixaban [10,12]. The dimethylaminomethylimidaz-
`ole moiety and proximal phenyl ring in vapor-phase razaxaban has a
`dihedral angle a of about 57° (DFT calculation). The amide group is
`planar and rotated out of the phenyl ring by about 122° (DFT, angle
`b). The DFT calculated angle between the plane of the pyrazole moi-
`ety and the aminobenzisoxazole plane of razaxaban is about 60° (Ta-
`ble 1). Water has only slight effect on the geometry of the razaxaban.
`The biologically active conformation of the fXa bound razaxaban
`(PDB file 1Z6E) differs from the gas-phase structure and/or solvated
`molecule in two points. The imidazole ring of the phenylimidazole
`moiety is at the receptor in a perpendicular arrangement to the prox-
`imal phenyl ring (dihedral angle a[C(1)–N(2)–C(3)–C(4)] = 83.5°).
`Almost perpendicular arrangement is also observed for the moiety
`containing the pyrazole scaffold and the aminobenzisoxazole group
`(dihedral angle f [C(10)–N(11)–C(12)–C(13)] = 81.4°).
`
`3.1.6. DX-9065a
`((+)-2S-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidi-
`(S,S)-DX-9065a
`nyl]oxy]phenyl]-3-[7-a midino-2-naphthyl]propanoic acid) be-
`longs to the class of early inhibitors mimicked the natural
`interaction of the arginine of prothrombin and Asp189 of the S1
`specificity pocket in the fXa active site. The pyrrolidine ring of this
`drug binds to the aryl binding site S4 of fXa. In the gas-phase struc-
`ture the amidine group of the naphthamidine moiety is rotated out
`of the naphthalene ring system by about 160°, dihedral angle
`g[C(a)–C(b)–C(c)–N(d)], Fig. 1. The connecting carbon chain of
`the propanoic acid moiety orients the second pharmacophoric
`group containing phenyl and pyrrolidine almost perpendicularly
`to the naphthalene ring plane allowing L-shape conformation
`needed for good fXa inhibitory activity. The pyrrolidine ring is in
`perpendicular position with respect to the plane of the phenyl
`group (dihedral angle e[C(8)–O(9)–C(10)–C(11)] is about 82°,
`Table 1). The biologically active conformation of the bound
`DX-9065a at the fXa receptor (PDB file 1FAX) is quite different.
`
`Fig. 4. (A) Molecular superimposition of the Becke3LYP optimized molecular
`structure of (S)-rivaroxaban (green) and (S)-rivaroxaban from the cocrystal with
`coagulation factor Xa, PDB 2W26 (blue). (B) Molecular superimposition of the
`Becke3LYP optimized molecular structure of (S)-rivaroxaban (green) and in solution
`optimized (S)-rivaroxaban (violet). (C) Molecular superimposition of the (S)-
`rivaroxaban from the cocrystal with coagulation factor Xa, PDB 2W26 (blue) and
`in solution optimized (S)-rivaroxaban (violet). For simplicity the hydrogen atoms
`are omitted. (For interpretation of color mentioned in this figure legend the reader
`is referred to the web version of the article.)
`
`3.1.4. Betrixaban
`Betrixaban (N-(5-chloropyridin-2-yl)-2-[[4-(N,N-dimethylcar-
`bamimidoyl)benzoyl]amino]-5-methoxybenzamide)
`[50]
`is an-
`other
`fXa
`inhibitor
`containing
`benzamidine moiety.
`Its
`conformational structure is, like in otamixaban, determined by
`eight dihedral angles a, b, d, e, f, h, l, and m (Fig. 1). Both amide
`groups are practically planar (dihedral angles b, c, f, and g). HF
`and DFT methods describe differently the conformation of the
`amide groups on the central methoxyphenyl ring. The DFT method
`prefers more planar arrangement, which is apparently due by high-
`
`

`

`M. Remko / Journal of Molecular Structure: THEOCHEM 916 (2009) 76–85
`
`83
`
`Largest changes upon interaction with the active site are observed
`in the flexible region of substituted propionic acid (dihedral angles
`a, and b) and around ether bond (dihedral angles d, and e). Amidine
`group in the bound conformation of DX-9065a is situated in the
`plane of the naphthalene moiety (Table 1).
`
`3.1.7. Dabigartan and dabigatran etexilate
`Dabigatran (3-[[2-[[(4-carbamimidoylphenyl)amino]methyl]-1-
`methylbenzimidazole-5-carbonyl]-pyridin-2-ylamino]propanoic
`acid) is direct inhibitor