Struct Chem (2011) 22:635–648 DOI 10.1007/s11224-011-9741-z
ORIGINAL RESEARCH
Molecular structure, pKa, lipophilicity, solubility, absorption, polar surface area, and blood brain barrier penetration of some antiangiogenic agents Milan Remko • Andrej Boha´cˇ • Lucia Kova´cˇikova´
Received: 19 July 2010 / Accepted: 11 January 2011 / Published online: 23 January 2011 Springer Science+Business Media, LLC 2011
Abstract The methods of theoretical chemistry have been used to elucidate molecular properties of selected and novel antiangiogenic agents (semaxanib, sunitinib, N-methylsunitinib, sorafenib, motesanib, ABT-869, vatalanib, vandetanib, AEE 788, CP-547632, A-1, A-2, A-3, and A-4). 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. Wherever possible the most stable conformations of inhibitors studied are stabilized by means of intramolecular hydrogen bonds. Water has a remarkable effect on the geometry of the antiangiogenic agents studied. Computed partition coefficients (ALOGPS method) varied between 2.3 and 5. Compounds studied are described as lipophilic inhibitors. Semaxanib is inhibitor with lowest lipophilicity. The antiangiogenic agents studied are only slightly soluble in water; their computed solubility (log S) from interval between -3.4 and -5.4 is sufficient for fast absorption. Selection criteria for drug-like properties of VEGFR2 inhibitors investigated were designed. Based on these criteria, three compounds (A-2, A-3, and A-4) were
Electronic supplementary material The online version of this article (doi:10.1007/s11224-011-9741-z) contains supplementary material, which is available to authorized users. M. Remko (&) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University Bratislava, Odboja´rov 10, 83232 Bratislava, Slovakia e-mail:
[email protected] A. Boha´cˇ L. Kova´cˇikova´ Department of Organic Chemistry, Faculty of Natural Sciences, Comenius University Bratislava, Mlynska´ dolina, 84215 Bratislava, Slovakia
selected for synthesis and biological testing for antianiogenic activity on VEGFR2 receptor. Keywords Antiangiogenic agents Molecular structure Solvent effect Lipophilicity, solubility, absorption, blood brain barrier penetration VEGFR2
Introduction Angiogenesis, the growth of new blood vessels, is tightly regulated in development and adult life. Under normal physiological conditions, various proangiogenic and antiangiogenic factors exist in a state of equilibrium, ensuring that angiogenesis remains well controlled. Excessive angiogenesis is involved in numerous pathologies, namely cancer, diabetic retinopathy, arthritis, and atherosclerosis among others [1–3]. Several angiogenic factors regulate the angiogenesis process. A complex cascade of events follows the release of these proangiogenic molecules, culminating in activation of endothelial cells, release of proteolytic enzymes, followed by migration and proliferation of endothelial cells and ultimately, capillary tube formation [4]. Vascular endothelial growth factor (VEGF) is considered to be one of the most potent and specific proangiogenic factors [2–4]. Anti-VEGF agents have thus become considered as a well-defined and rational strategy for development of antitumor drugs. VEGF represents one of the best-characterized pro-angiogenic proteins. The extensive research programs resulted in the development of various vascular endothelial growth factor receptor inhibitors (VEGFR inhibitors), of which, the tyrosine kinase inhibitors, sunitinib and sorafenib, blocking VEGF receptors among other targets were recently approved by FDA for the treatment of cancer. A large amount of angiogenesis
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inhibitors targeting VEGF pathway are also under clinical trials or preclinical studies [2, 3, 5–8]. In spite of their interesting biological properties, the VEGFR inhibitors remain one of the structurally less wellcharacterized classes of drugs. There is no single experimental study concerned with the systematic comparative experimental investigation of the physicochemical and pharmacokinetic parameters of these medicinally useful new antiangiogenic agents. Quantitative structure activity relationships and docking of factor VEGFR inhibitors were discussed quite recently [9], and the X-ray crystal structures of sorafenib [10], chlorine derivative of sunitinib [11], motesanib [12], vandetanib [13], and AEE 788 [14] in complexes with protein kinases deposited in the Protein Data Bank [15] were used to clarify the binding mode of these ligands. The absence of experimental structural data of highly potent synthetic angiogenesis inhibitors targeting a number of VEGF, as well as platelet-derived growth factor (PDGF) receptors presents a challenge to the application of the molecular modeling methods in order to enhance our understanding of the biological activity of these compounds. In this paper, we carried out large-scale theoretical calculations with the aim to examine molecular structure, lipophilicity, solubility, absorption, and blood– brain barrier penetration of 14 inhibitors of VEGF and PDGF receptors. These data are used for design of selection criteria for drug-like properties of VEGFR inhibitors investigated. Based on those selection criteria and molecular modeling new highly potent inhibitors are designed and prepared. Our theoretical results are compared with the available experimental data and discussed with the present theories of molecular action of these inhibitors of angiogenesis.
Computational details Ab initio calculations of the semaxanib, sunitinib, N-methylsunitinib, sorafenib, motesanib, ABT-869, vatalanib, vandetanib, AEE 788, CP-547632, A-1, A-2, A-3, and A-4 (Fig. 1) were carried out with the Gaussian 03 computer code [16] at the ab initio SCF (HF [17]) and density functional theory (DFT, Becke3LYP [18–22]) levels of theory using the 6-31G(d) and 6-31??G(d,p) basis sets. The effect of solvent (water) on the energy and geometry of the species studied was evaluated using the PCM solvation model [23, 24]. The structures of all gasphase 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 calculations of the antiangiogenic agents, using the larger basis set 6-31??G(d,p)
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implemented in the Gaussian 03 package of computer codes [16, 17]. The energy of available biologically active conformations of the inhibitors was computed at the Becke3LYP/6-31??G(d,p) level of theory using the experimental geometry of individual compounds. The structures of all condensed-phase (SCRF) species were fully optimized without any geometrical constraint at the Becke3LYP/631G(d) level of theory applied. The calculations of the macroscopic pKa of antiangiogenic drugs investigated were performed using the program AB/pKa module implemented in the VCCLAB program [25]. Lipophilicity and water solubility calculations were carried out using web-based VCCLAB software [25, 26]. For calculations of molecular polar surface areas the fragment-based method of Ertl and coworkers [27, 28] incorporated in the Molinspiration Cheminformatics software [29] was used.
Results and discussion Molecular structures Quantum chemical calculations are now applied successfully in medicinal chemistry and drug design to determine accurately molecular structures and properties for use in a wide variety of CADD studies [30]. It is common in the computational study of drugs to use structural data obtained from X-ray crystallography or NMR spectroscopy as guides to the quality of theoretical computations. However, in the absence of the experimental published data about molecular conformations of the novel antiangiogenic agents studied, as an alternative to analyzing small molecule crystal structures we examined the conformations of these drugs bound to their protein targets. Initial conformations to use in the theoretical calculations were constructed by means of the Gauss View graphical interface of Gaussian. The relative orientation of individual functional groups in the inhibitors investigated (defined by dihedral angles a, b, c, d, e, f, g, h …) is shown in Fig. 1. Important geometric parameters of the molecules investigated are given in Table A of the Supporting Information. Initial calculations were carried out using the Hartree–Fock method in combination with the double-f basis set. Subsequent frequency calculations served to identify the nature of stationary points on the potential energy surface. An analysis of the harmonic vibrational frequencies of the optimized species proved that all of them are minima (zero number of imaginary frequencies). The geometries of drugs studied optimized at the B3LYP/6-31??G(d,p) level of theory are shown in Fig. 2. Wherever possible the most stable conformations are stabilized by means of intramolecular hydrogen bonds. Examination of the space models of the
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Fig. 1 Structure and atom labeling in the antiangiogenic agents studied
H3C 4 2
3
1
N H N H
CH3
O
11
CH3
10
Semaxanib
N CH3
9
6
4 F
2
1
5
R H3C
O
3
N H N
8
HN 7
H3C
CH3
O Sunitinib; R = H N-Methylsunitinib; R = CH 3
NH 14
O
N
13 12 11 10
8 7
O HN 1
3
2
4
5
9
N
O 6
6
H3C
5
N H
CH3
4
1N
F3C
11
O
3 NH 2
8 7
9
10
NH
N H
Cl Sorafenib
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 the PCM solvation method. The polarizable continuum model (PCM) defines the cavity as the union of a series of interlocking atomic spheres [23, 24]. Continuum-based methods of solvation are used extensively and successfully in a variety of problems [31, 32]. Water has a remarkable effect especially on the geometry of the structurally flexible polar parts of the drugs studied. Table A shows the results obtained for geometry calculations performed in both vacuum and that based on the PCM solvation method used. The energy difference between gas phase and solvated phase was significant.
Motesanib
Hydration results in an appreciable stabilization of drugs in solvated state (Table 1). Semaxanib Semaxanib, SU 5416 ((Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl) methylene)indolin-2-one), was the first synthetic inhibitor of VEGFR, which entered into clinical trials. Its clinical development was, after disappointing results of several phase II studies in different tumors, stopped [33]. Structurally semaxanib is a rather rigid molecule. The calculations showed that of the two possible conformations defined by the dihedral angle a[C(1)-N(2)-C(3)-C(4)], Table A, the conformer stabilized via intramolecular hydrogen bond of
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Fig. 1 continued
N NH
H2N
Cl 5 4
11 8
HN F
1
3
9
7
O
HN 3
10
2
1 5
4
N H
2
N
6
N 6
7 8 9
CH3
N
10
Vatalanib
ABT-869
H3C
11
N
10 9
O
8
6
7
N
N 1
O CH3
6 5 4
H3C
2 3
HN
4
5
Vandetanib
F
3 NH 2
7
1
11
10
N
8
12
9
13 14
N H
N
Br
15
N
N AEE 788
CH3
F
O
1 2
S
N
3
5
4
8
N H
7
O
6
Br F
9
10
NH2
O
CP 547632
R N 9 10
SO2Et 7 5O
1 2
3
N H
6
4
N
OMe A-1; R = H A-2; R = OH A-3; R = OCH 3 A-4; R = OCH 2OCH3
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8
11
14
12
H N
N 11
13
15
Struct Chem (2011) 22:635–648
Semaxanib
Sunitinib
639
Sorafenib
ABT-869
Motesanib
Vandetanib
AEE 788
CP-547632
A-1
Vatalanib
A-2
A-3
A-4
Fig. 2 B3LYP/6-31??G(d,p) optimized structures of the antiangiogenic agents investigated
˚ is the most the C=OH–N type with the length of 1.748 A stable one (Fig. 2). A second, non-planar conformer (a = 350) is by about 53 kJ/mol less stable. The same stability order is also preserved in water. The gas-phase geometry of semaxanib practically does not change in water. Sunitinib Sunitinib ((Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide) is, like semaxanib, an indolinone derivative able to
inhibit the tyrosine kinase activity of a number of receptors including VEGF(R-1, -2) [2, 5]. Sunitinib was approved in 2006 for treatment of gastrointestinal stromal tumors and advanced renal cell carcinoma. The indolinone and pyrrole moieties were, like by semaxanib, considered in a more stable conformation stabilized by the intramolecular hydrogen bond of the C=OH–N type (Fig. 2). Optimized conformation of the sunitinib provides an extended form in which the amide group is almost co-planar with the pyrrole ring. The connecting –C2H4– group is in a gauche arrangement. The relative orientation of individual functional groups
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Table 1 The Becke3LYP/6-31G(d) solvent stability of the angiogenesis inhibitors investigated DE (kJ/mol)
No.
Drug
1
Semaxanib
-42.73
2
Sunitinib
-66.48
2a
N-methylsunitinib
3
Sorafenib
4
Motesanib
-77.32
5
ABT-869
-105.43
6 7
Vatalanib Vandetanib (ZD6474)
-72.74 -48.75
8
AEE788
-77.39
9
CP547632
-83.14
10
A-1 (H)
-89.98
11
A-2 (OH)
-79.68
12
A-3 (OCH3)
13
A-4 (OCH2OCH3)
-44.96 -112.01a
-95.43 -106.51
Water as solvent a
Single-point PCM calculation
in the sunitinib is defined by dihedral angles a, b, c, d, e, and f is shown in Table A. At the both low and high level of the DFT theory calculated torsion angles of sunitinib are within &5 of each other. The solvent has an appreciable effect on the conformation of the pyrrole-3-carboxamide moiety only. Upon hydration the amide group is rotated out of the pyrrole ring plane by about 37. In the absence structural data for sunitinib our computed structure can be compared with the experimental data for chloro-substituted derivative of sunitinib. In this molecule, the indolinone moiety is 5-chloro substituted. The X-ray structure of this inhibitor corresponds to the bound molecule at the calmoduline-dependent protein kinase type 1G [11]. The largest differences between the gas-phase and bound conformations are observed for basic diethylaminoethyl moiety. N-methylation of the indolinone ring in the N-methylsunitinib does not change the optimized geometry of the bulky dimethyl-1H-pyrrole-3-carboxamide moiety.
phenylureidophenoxy part of sorafenib is practically planar (dihedral angles a, b, c, d, and e, respectively, Table A). The coplanar structure in the phenoxy and pyridine groups is unlikely because of steric hindrance. The most stable conformer of sorafenib was computed to be that where the pyridine ring is more or less coplanar to, and neighboring phenyl ring almost perpendicular with, the C–O–C plane (dihedral angles f and g). An inverse correlation between these two dihedral angles resulted also from the analysis of aryl–O–aryl systems, which are one of common fragments in medicinal chemistry [34]. The methylpyridine-2-carboxamide group is always in coplanar position with dihedral angle h of about 1. For reason of comparison, Table A also contains sorafenib bound to its protein target (pdb codes 3HEG and 3GCS). An analysis of these X-ray data shows that sorafenib in these complexes with the protein kinase p38a exists in two distinct thermodynamically stable conformations which differ in mutual position of individual planar subunits. The N,N0 -diaryl urea moiety is, on the contrary to the gas-phase and/or solution state conformation, not planar. Both phenyl rings are rotated out of the urea –HN–C(=O)–NH– group plane by about 30 (dihedral angles a and e). However, much larger structural deviation is observed in the phenoxypyridine region of this drug. Dihedral angles, f and g, were found to be quite different for the sorafenib bound to the same protein target in the 3HEG (blue) and 3GCS complexes (violet), Fig. 3. The biologically active conformation of sorafenib has a relatively high energy compared with the computed energy minimum (by about 88 kJ/mol and 196 kJ/mol for 3HEG and 3GCS complexes, respectively). These data are generally of lower resolution ˚ and 2.20 A ˚ for 3GCS and 3HEG, respectively). This (2.10 A difference may be, at least in part, due to a different intermolecular interaction present in two crystal forms and/or targeting different forms of protein kinase. A comparison of the fully optimized molecule and the bound sorafenib (pdb codes 3HEG and 3GCS) shows that the largest structural rearrangement resulting in the biologically active conformation refers to the dihedral angles a, e, f, and g.
Sorafenib Sorafenib (4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-N-methylpicolinamide) is a multikinase inhibitor targeting a number of serine/threonine and receptor tyrosine kinases, which was in 2005 approved for the treatment of advanced renal cell carcinoma [7, 8]. The B3LYP optimized structure of sorafenib possesses characteristic L-shaped form by two large structural units: phenylureidophenoxy and pyridine moieties (Fig. 2). The computed dihedral angles show that in both gas-phase and solvated state the
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Fig. 3 Molecular superimposition of the two conformations of sorafenib (pdb files 3GCS (violet) and 3HEG (blue) bound to the same protein target protein kinase 14 (Color figure online)
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Motesanib Motesanib (N-(3,3-dimethylindolin-6-yl)-2-(pyridin-4-ylmethylamino)nicotinamide) is a highly selective inhibitor of angiogenesis with direct antitumor activity [35]. Rigid pyridine and dihydroindol rings of this molecule connected by the amide group are in both gas-phase and solvated state almost coplanar, which is manifested by the calculated dihedral angles f and g of about 175, Table A. Hydration causes only small change in the structure of this rigid moiety. Both the gas-phase and solvated structures are stabilized by means of an intramolecular hydrogen bond of the C=OH–N type (Fig. 2). Larger conformational change in the geometry upon hydration exhibits the pyridin-4-ylmethylamino moiety (dihedral angles b, c, and d). The 3D structure of the bound motesanib at the vascular endothelial growth factor receptor 2 (pdb code 3EFL), the gas-phase structure and hydrated form are different. Largest difference was observed for the moiety containing pyridine and dihydroindol. When bound at the receptor, the dihydroindol group is rotated out of the plane containing pyridine-3-carboxamide by about 52, dihedral angle g. The DFT calculation suggests planar conformation with the dihedral angle g[C(8)-N(9)-C(10)-C(11)] of about -175. Comparison of the structures and relative energies of bound and unbound motesanib shows that the biologically active conformation is by about 99 kJ/mol less stable. ABT-869 (Linifanib) ABT-869 (1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea) is another promising multi-targeted tyrosine kinase inhibitor, which is active against vascular endothelial growth factor VEGFRs, as well as platelet-derived growth factor receptor (PDGFR) [36]. ABT-869, a 3-aminoindazole-based orally active drug, contains, like sorafenib, an N,N0 -diaryl urea moiety (Fig. 1) and is one of the most rigid molecules of the antiangiogenic agents studied. This structurally rigid moiety is in both isolated molecule and solvated state computed to be planar (Table A). The steric repulsion of the aminoimidazole ring and the bulky N,N0 -diaryl urea moiety force the rings about 50 out of the plane with each other (dihedral angle f, Table A). Vatalanib Vatalanib (N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine) is an oral anilinophtalazine that inhibits VEGFR-1-3, as well as PDGF receptor, c-Kit, and c-Fms [5, 7]. Conformational structure of this drug is governed by four dihedral angles a, b, c, and d, Table A and consists of two aryl–X–aryl systems. Two acyclic bonds via
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methylene functional group to the 4-pyridyl structural units link central phtalazine unit. 4-Chlorophenyl is linked by means of NH group also to the phtalazine moiety (Fig. 1). The NH group of vatalanib is calculated to be almost coplanar with the 4-chlorphenyl and twisted out of the phtalazine structural unit (dihedral angle a is about 57). For diarylmethane part of inhibitor, the most stable structure is where phtalazine and pyridine rings are positioned almost orthogonal to each other forming L-shaped conformation (dihedral angles c and d are about 75 and 130 for both isolated molecule and hydrated system, Table A). Our computed values for these two sets of aryl–X–aryl systems in vatalanib are in agreement with the dihedral angles distributions for those systems derived from the CSD statistics [34]. Vandetanib Vandetanib (N-(4-bromo-2-fluorophenyl)-6-methoxy-7[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine) is a dual inhibitor of VEGFR and epidermal growth factor receptor (EGFR) tyrosine kinases [37]. Its 3D structure is governed by five dihedral angles (a, b, c, d, and e, respectively, Table A). The 3D structure of the bound vandetanib at the RET tyrosine kinase domain (pdb code 2IVU) and the B3LYP optimized structure of vandetanib are close to each other indicating that only small structural changes are induced upon coordination of this inhibitor at its receptor. Computed structures of the drug, using two basis sets showed that an increase in the basis set gives essentially the same results. The relative insensitivity of the geometry optimization calculations by means of the DFT method using different basis sets was recently demonstrated for diverse sets of drug molecules [38–41]. The amino group and quinazoline moiety are practically coplanar (dihedral angle a is about 178). Bromofluorophenyl group is rotated out of the aminoquinazoline plane by about -120, dihedral angle b). The biologically active conformation of vandetanib is less stable by about 115 kJ/mol. AEE788 AEE788 ((S)-6-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)N-(1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine) is also a dual receptor tyrosine kinase inhibitor of the VEGFR and EGFR. It is currently in Phase I clinical trials in solid tumors [7, 8]. The pyrrolopyrimidine part and phenethylamine group are in the isolated state in mutual perpendicular position (dihedral angles b[C(2)-N(3)-C(4)-C(5)] and c[N(3)-C(4)-C(5)-C(6)] are about 90, Table A). The phenyl ring is rotated out of the phenethylamine plane by about 25. The piperazine ring plane makes angle of about
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140 with the neighboring phenyl group. Hydration causes larger conformational change in the arrangement of the phenethylamine moiety of the molecule only (dihedral angle c, Table A). The 3D structure of the isolated molecule and the 3D structure of AEE788 when bound to the EGFR kinase (pdb code 2J6M) are substantially different (Fig. 4). The coordination of the AEE788 leads to the more planar pyrrolopyrimidine and phenethylamine moieties. The basic nitrogen N3 atom of the pyrimidine interacts with the hydroxyl group of the Thr854 via a bridging water molecule [14]. The phenylethylamine unit fills the hydrophobic pocket of the receptor cavity. This hydrophobic interaction is accompanied with structural rearrangement of this part of molecule. The ethylpiperazine group of the bound AEE788 extends toward solvent at the edge of the active site and accommodates, in comparison with the free molecule, a different space, Fig. 4. The large conformational change of AEE788 upon binding is also manifested in substantial energy change. The biologically active conformer is 317 kJ/mol less stable than the isolated molecule. CP-547632 CP-547632 (3-(4-bromo-2,6-difluorobenzyloxy)-5-(3-(4(pyrrolidin-1-yl)butyl)ureido) isothiazole-4-carboxamide) is a potent, selective inhibitor of VEGFR-2 [5, 42]. This drug contains, like sorafenib and ABT-869, a urea linkage connecting a polar isothiazole-4-carboxamide moiety with the butylpyrrolidine group. The isothiazole–urea alignment is computed to be coplanar and stabilized by means of intramolecular hydrogen bond of the C=OH–N type (Fig. 2). The equilibrium structure of the CP-547632 is further stabilized via intramolecular nonbonded interaction between sulfur and oxygen atoms (Fig. 2). In this molecule, the ˚) Becke3LYP/6-311?G(d,p) computed distance (2.715 A between nonbonded SO atoms of the urea group and the isothiazole ring is clearly shorter than the sum of the corresponding van der Waals radii for oxygen and sulfur atoms ˚ ) [43]. This effect has been observed in a large number (3.32 A of organosulfur compounds controlling the conformation
Fig. 4 Molecular superimposition of the AEE788 from the cocrystal with EGFR kinase, pdb 2J6M (blue) and of the Becke3LYP/631??G(d,p) optimized molecular structure of AEE788 (violet) (Color figure online)
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of small and large molecules [41, 44]. The 4-bromo-2, 6-difluorophenyl substituent is, with respect to the polar isothiazole–urea moiety, in the mutual perpendicular arrangement (dihedral angle a, Table A). The connecting four-carbon chain is in gauche conformation and orients the pyrrolidine ring toward an extended arrangement of the molecule (Fig. 2). The solvent effect treated within a continuum PCM model did not change the equilibrium conformation. Experimental 3D structure of the CP-547632 corresponds to the bound molecule at the human p38 MAP kinase (pdb file 3L8S), 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 CP-547632–kinase complex are well interpreted by the corresponding angles computed for the isolated CP-547632. The alignment of the isothiazole–urea and phenyl substituents in the biologically active conformation does not change appreciably. The main difference in the molecular structure of bound and unbound CP-547632 arises from the position of the butylpyrrolidine group, dihedral angles h, k, l, and m. The extended conformation of this group, found for both the isolated molecule and water solvated system, is not preserved. When bound on the kinase, the conformation of the butylpyrrolidine group possesses typical L-shaped structure (Fig. 5). The biologically active conformation is about 195 kJ/mol less stable than the preferred conformation of the isolated molecule. A-1, A-2, A-3, and A-4 A-1 (N-(5-(ethylsulfonyl)-2-methoxyphenyl)-5-(3-(pyridin2-yl)phenyl)oxazol-2-amine) belongs to a new class of
Fig. 5 Molecular superimposition of the CP-547632 from the cocrystal with MAP kinase, pdb 3L8S (blue) and of the Becke3LYP/6-31??G(d,p) optimized molecular structure of CP-547632 (violet) (Color figure online)
Struct Chem (2011) 22:635–648
potent VEGFR2 kinase inhibitors [45]. Its 3D structure is governed by four dihedral angles a, b, c, and d, Fig. 1. The arylamine and 1,3-oxazole parts of this compound may exist in two equilibrium conformations. The conformer stabilized via the N–HO intramolecular hydrogen bond is, in comparison with the structure stabilized by the N–HN hydrogen bond, by about 3.1 kJ/mol more stable (B3LYP/6-31??G(d,p) calculation). Similarly, the amine and the 2-methoxy substituents both on the benzene ring are stabilized by five-membered intramolecular hydrogen bond of the N–HO type. All further calculations for A-1 and its congeners A-2, A-3, and A-4 were carried out for this more stable conformer displayed in Fig. 2. Optimized conformation of A-1 is stabilized by means of two intramolecular hydrogen bonds (Fig. 2). The extended 2-methoxyaniline–oxazole–phenyl system is practically coplanar, dihedral angles a, b, and c are about -4, 179, and 1, respectively, for both isolated molecule and hydrated system (Table A). The co-planarity of the 2-anilino-5-phenyloxazole moieties is also preserved in the 4-phenyl substituted derivatives A-2, A-3, and A-4. The m-2-pyrid-2-yl group in A-1 is oriented out of the phenyl group plane by about 23 (dihedral angle d). The 3D structure of bound A-1 at the VEGFR2 receptor [45] and the conformation of unbound A-1 are substantially different. The coordination of the A-1 to the VEGFR2 (pdb file 1Y6A) leads to the bent biologically active conformation in which the oxazole and 5-phenyl group on oxazol ring are almost coplanar (dihedral angle c[O(5)-C(6)-C(7)C(8)] = 1.5), and m-2-pyrid-2-yl group oriented perpendicularly to the oxazole group with the dihedral angle d[C(8)-C(9)-C(10)-C(11)] = -98.7. However, because no clear density is visible for this part of A-1, this group in the crystal structure with VEGFR2 was found to be oriented in multiple conformations [45]. The preferred conformation of A-1 that binds to its receptor is by 240 kJ/mol higher in energy than the conformation of unbound molecule. More interesting situation is with the A-2 molecule. The intramolecular hydrogen bond O–HN type between the 4-hydroxy substituent of the phenyl ring and nitrogen atom of the m-2-pyrid-2-yl group stabilizes planar conformation with the dihedral angle d[C(8)-C(9)-C(10)-C(11)] of about 0 (Fig. 2). The possible proton transfer from the phenolic O–H group to the m-2-pyrid-2-yl nitrogen atom was also investigated. The calculations indicate that the most stable structure is in both gas-phase and solvated state, conformer stabilized by a normal O–HN hydrogen bond. The Hartree–Fock level of theory shows the conformer containing proton-transfer O-H–N? hydrogen bond to be by 60.3 kJ/mol less stable. The DFT model chemistry predicts the existence of just conformer with normal O–HN hydrogen bond. In polar solvents like water, the conformer containing proton-transfer O-H–N? hydrogen bond
643
becomes much more favored. However, this conformer is, by comparison with the hydrogen-bonded one, still by 13.7 kJ/ mol less stable. In the absence of the O–HN hydrogen bond in A-3 and A-4 molecules the m-2-pyridine group is in the most stable conformer rotated out of the phenyl ring by about 50 (dihedral angle d[C(8)-C(9)-C(10)-C(11)]). Lipophilicity and solubility The computed log P values (P is the partition coefficient of the molecule in the water–octanol system), together with the experimental data, are shown in Table 2. The ALOGPS method is part of the ALOGPS 2.1 program [46] used to predict lipophilicity [47] and aqueous solubility [48] of compounds. The lipophilicity calculations within this program are based on the associative neural network approach and the efficient partition algorithm. The LogKow (KowWIN) program [49] is atom/fragment contribution method developed at Syracuse Research Corporation [50]. The XLOGP2 is atom-additive method applying corrections [51]. Available experimental log P values of sunitinib and sorafenib were extracted from the literature [52]. Of the three methods used the experimental lipophilicity of sunitinib and sorafenib is best reproduced by the XLOGP2 method (Table 2). Alike good performance of the XLOGP2 method to predict lipophilicity of aromatic drugs (hypoglycemic agents) was recently reported [39]. Computed partition coefficients (XLOGP2 method) for angiogenesis inhibitors studied varied between 2.3 and 5. Neutral inhibitors are described as highly lipophilic drugs. Semaxanib is a drug with lowest lipophilicity. Among the clinically useful inhibitors, sorafenib, compared with sunitinib, is substantially more lipophilic drug. The log P value of inhibitors studied is an important parameter for oral absorption because it governs the ability of a drug to pass trough intestine cell membranes [53]. Compounds that are too lipophilic (log P [ 5) are too insoluble in physiological solutions to be transported to the target cells [53]. All angiogenesis inhibitors investigated (Table 2) fulfill this lipophilicity criterion. Owing to high lipophilicity all compounds under study are only slightly soluble in water (Table 2). The computed log S values—intrinsic solubilities (S) of studied drugs in neutral state are also presented in Table 2. Experimentally these compounds are characterized as insoluble. Because of low water solubility sunitinib and sorafenib are used in clinical praxis in the form of their salts—orally bioavailable malate salt of sunitinib and the tosylate salt of sorafenib, which are more soluble in water. Dissociation constants Dissociation plays an important role in both partition and receptor binding processes of drug action. It is therefore
123
644
Struct Chem (2011) 22:635–648
Table 2 Calculated partition coefficients and solubility of the angiogenesis inhibitors studied No.
Drug
1
Semaxanib
2
Sunitinib
2a
N-methylsunitinib
3
Sorafenib
4
Motesanib
LogPa (exp.)
ALOGPs
KoWWIN
XLOGP2
2.64
3.06
2.29
2.5
3.24
3.15
2.47
2.96
2.98
2.62
3.8
4.12
5.30
3.75
3.59
3.86
2.99
Solubility (exp.)
ALOGpS -3.36 (0.10 g/L)
0.511 g/Lc
-3.47 (0.13 g/L) -3.99 (41.86 mg/L)
Insoluble
-5.43 (1.71 mg/L) -4.52 (11.22 mg/L)
5
ABT-869
4.20
3.80
4.65
-4.84 (5.46 mg/L)
6
Vatalanib
4.50
4.95
4.36
-5.29 (1.79 mg/L)
7
Vandetanib (ZD6474)
8
AEE788
5.0b
5.01
4.89
4.14
-4.67 (10.23 mg/L)
4.38
4.06
4.99
-4.43 (16.50 mg/L)
9
CP547632
3.04
4.00
3.26
-5.06 (4.66 mg/L)
10 11
A-1 (H) A-2 (OH)
3.89 3.89
3.36 2.88
4.08 3.67
-4.21 (26.87 mg/L) -3.91 (55.73 mg/L)
12
A-3 (OCH3)
4.19
3.44
3.99
-4.15 (33.10 mg/L)
13
A-4 (OCH2OCH3)
4.04
3.18
3.94
-4.09 (39.97 mg/L)
a
Ref. [52]
b
Ref. [37]
c
Ref. [66]
Table 3 The pKa values of the angiogenesis inhibitors investigated
No.
Compound
Acid function
% Ionized form (pH 7.4) Basic function
11.6
Acid function
Basic function
1
Semaxanib
2
Sunitinib
9.30
98.7
2a
N-methylsunitinib
9.30
98.7
3
Sorafenib
2.20
0
4
Motesanib
5.80
1.5
5
ABT-869
3.20
0
6
Vatalanib
5.80
1.5
7
Vandetanib (ZD6474)
10.30
99.9
8
AEE788
7.60
61.3
9
CP547632
10.40
100
10 11
A-1 (H) A-2 (OH)
4.40 4.40
8.50
0
0
0.1 0.1
12
A-3 (OCH3)
4.40
0.1
13
A-4 (OCH2OCH3)
4.40
0.1
important to know if drug molecules exist predominantly in the neutral or ionized forms. The angiogenesis inhibitors studied contain a basic nitrogen center and thus they may undergo protonation reactions. In solution dissociation constant or the pKa is a measure of the strength of an acid or a base. We used the pKa’s predictor implemented in the software AB/pKa [54] to compute the theoretical pKa values of studied drugs in condensed phase (water). The calculated macroscopic pKa values are listed in Table 3. Sunitinib (N-methylsunitinib), vandetanib, and CP-547632 contain highly basic nitrogen atom in the N,N-diethylaminoethyl, N-methylpiperidine and pyrrolidine functional
123
pKa
groups of these compounds. The computed pKa values for studied inhibitors are in the range of 9.3–10.4 and at pH 7.4 they exist as fully protonated bases (Table 3). The less basic ethylpiperazine moiety of the AEE788 is partially protonated at physiological pH 7.4. The rest of the drugs studied are weak bases and at pH 7.4 they exist in unionized form. Absorption, polar surface area, and ‘‘rule of five’’ properties High oral bioavailability is one of the very important properties for the development of bioactive molecules as
Struct Chem (2011) 22:635–648
645
ratio of the steady-state concentrations of the drug in the brain and in the blood:
therapeutic agents. Lipinski et al. [53] proposed ‘‘the rule of five’’ (the rule states that most molecules with good membrane permeability have logP B 5, molecular weight B 500, the number of hydrogen bond acceptors B 10, and the number of hydrogen bond donors B 5) for a preliminary estimation of good oral bioavailability. This rule is widely used as a filter for drug-like properties. All drugs studied satisfy Lipinski’s four rules of five exhibiting drug-like properties criteria with respect to their oral bioavailability. The primary barrier toward good bioavailability is human intestinal absorption. Table 4 contains calculated percentage of absorption (%ABS), molecular polar surface area (PSA), and Lipinski parameters of the drugs studied. Magnitude of absorption is expressed by the percentage of absorption. Absorption percent was calculated using the expression: %ABS = 109–0.345 PSA [55]. Polar surface area (PSA) was determined by the fragment-based method of Ertl and coworkers [26–29]. All compounds belong to the good-absorption class (%ABS [ 30%) [56]. Semaxanib, vatalanib, and vandetanib are drugs with low polar surface area and very good absorption (C80%). In general, indolinone derivatives exhibit better absorption than urea derivatives.
BB ¼
Cbrain Cblood
C is the concentration of the compound. BB of most prescribed CNS drugs is [0.3 (log BB [ -0.5), drugs with BB \ 0.1 (log BB \ -1) penetrate poorly into the brain [57]. Log BB was calculated using the Clark equation: Log BB = -0.0148 PSA ? 0.152 clogP ? 0.139 [58]. As it is well known, antiangiogenic compounds interfere with chemotherapy of brain tumors [59]. The limited survival advantage attributed to chemotherapy is partially due to low CNS penetration of current antineoplastic agents across the BBB [60]. It was shown experimentally that vandetanib selectively inhibited angiogenic growth aspects glioma and resorted the BBB of nude mice carrying intracerebral angiogenic melanoma metastases [61]. The possible application of the vascular endothelial growth factor receptor inhibitors under evaluation for treatment malignant gliomas patients has been reported [59]. Exploring beneficial effects of VEGF in neuredegenerative diseases (stroke, acute spinal cord ischemia, ischemic neuropathy) has been investigated [3]. VEGFR blockers like sorafenib and sunitinib readily penetrate the BBB. The chronic blockade of VEGFRs in the nervous system upon delivery of these drugs in cancer treatment might cause muscle weakness and fatigue, common side effects of sorafenib and sunitinib [62, 63]. Preclinical and clinical trials with VEGFRIs in brain with these drugs may have adverse effects as indicated. Thus, in order to design new VEGFRIs with improved delivery to the target site, it is
Blood brain barrier (BBB) penetration Blood brain barrier (BBB) permeability is a crucial factor, which needs careful examination in the process of drug discovery. Drugs targeted at the CNS must cross BBB to exhibit therapeutic effect whereas for non-CNS drugs passage through the BBB may lead to unwanted side effects. The degree of BBB penetration is defined as the
Table 4 Calculated absorption (%ABS), total polar surface area (TPSA) and Lipinski parameters of the angiogenesis inhibitors studied NROTB
n ON acceptors
n OHNH donors
Log P, calcda
Formula weight
48.65
1
3
2
2.29
238.29
80.98
7
6
3
2.47
398.48
No.
Drug
%ABS
Volume
1
Semaxanib
92.2
221.52
2
Sunitinib
81.1
370.95
2a
N-methylsunitinib
84.8
387.89
70.13
7
6
2
2.62
412.51
3
Sorafenib
77.1
368.25
92.35
6
7
3
3.75
464.83
4
Motesanib
81.8
347.12
78.94
5
6
3
2.99
373.50
5
ABT-869
75.9
328.25
95.83
3
6
5
4.65
375.41
6
Vatalanib
91.5
301.12
50.70
4
4
1
4.36
346.94
7
Vandetanib (ZD6474)
88.5
380.22
59.52
6
6
1
4.14
475.40
8
AEE788
88.3
425.41
60.08
7
6
2
4.99
440.65
9
CP547632
71.2
405.96
109.58
10
8
4
3.26
532.46
10
A-1 (H)
76.5
374.27
94.33
7
7
1
4.08
435.54
11
A-2 (OH)
69.5
382.29
114.56
7
8
2
3.67
451.54
12 13
A-3 (OCH3) A-4 (OCH2OCH3)
73.3 70.1
399.81 425.60
103.56 112.79
8 10
8 9
1 1
3.99 3.94
465.57 495.60
a
TPSA
XLOGP2 method
123
646 Table 5 Calculated blood brain barrier penetration of the drugs studied
Struct Chem (2011) 22:635–648
No.
Drug
Log BB Clark
%ABS
Volume
1
Semaxanib
-0.232
92.2
221.52
48.65
2.29
2
Sunitinib
-0.684
81.1
370.95
80.98
2.47
2a
N-methylsunitinib
-0.501
84.8
387.89
70.13
2.62
3
Sorafenib
-0.657
77.1
368.25
92.35
3.75
4
Motesanib
-0.574
81.8
347.12
78.94
2.99
5
ABT-869
-0.572
75.9
328.25
95.83
4.65
6
Vatalanib
0.051
91.5
301.12
50.70
4.36
7
Vandetanib (ZD6474)
-0.113
88.5
380.22
59.52
4.14
8
AEE788
0.008
88.3
425.41
60.08
4.99
9
CP547632
-0.987
71.2
405.96
109.58
3.26
10
A-1 (H)
-0.636
76.5
374.27
94.33
4.08
11
A-2 (OH)
-0.998
69.5
382.29
114.56
3.67
12
A-3 (OCH3)
-0.787
73.3
399.81
103.56
3.99
13
A-4 (OCH2OCH3)
-0.931
70.1
425.60
112.79
3.94
also necessary to appreciate BBB penetration issues of these compounds. Analysis of the computed log BB values of studied compounds has shown that semaxanib, vatalanib, vandetanib, and AEE788 penetrate through BBB most easily. On the other side, CP-547632, A-2, A-3, and A-4 with log BB close to -1 penetrate poorly into the brain (Table 5). Thus, these compounds may serve as selectively acting VEGFRIs with limited penetration to the CNS. Selection criteria for drug-like properties Table 6 contains selection criteria for drug-like properties of VEGFR inhibitors investigated. The first three properties are based on Lipinski’s ‘‘rule of five’’ and represent, together with the 3D structural data discussed in previous part of this paper, important physicochemical properties of drug-like VEGFR inhibitors. Polar surface area is an important molecular descriptor regarding to absorption and BBB penetration. Drugs like semaxanib, vatalanib, vandetanib, and AEE788 with lowest calculated PSA values (Table 4) exhibit largest absorption and also good BBB penetration (Table 5). Of the drugs studied, the compounds classified as non-CNS drugs have more hydrogen bond donors and acceptors, lower log P, higher PSA, and more rotatable bonds (Tables 4, 5). From the results of molecular modeling studies, three compounds (A-2, A-3, and A-4) were selected for synthesis and biological testing for antianiogenic activity on VEGFR2 receptor. Results of experimental in vitro testing of these compounds on the enzymatic VEGFR-2 activity [64] have been shown that three new derivatives are highly potent nanomolar inhibitors with the IC50 values of 12.8, 14.7, and 87.3 nM for A-2, A-3, and A-4 compounds, respectively (Table 7). The comparison of similar IC50 data
123
PSA
XLOGP2
Table 6 Drug-like properties of the angiogenesis inhibitors studied Molecular weight
230–530
Octanol/water partition coefficient (clog P)
2.6–4.6
Aqueous solubility (clog S) ˚ 2) Polar surface area (PSA, A
(-3)–(-5.5) 50–110
Percent of oral absorption (%ABS)
70–90
Brain/blood partition (log BB)
(-1)–(?0.1)
Table 7 Biochemical activity (IC50) of the angiogenesis inhibitors investigated Receptor kinase
IC50 (nmol/L)
Ref.
Semaxanib
VEGFR2
700
[65]
Sunitinib
VEGFR2
50
[66]
3 4
Sorafenib Motesanib
VEGFR2 VEGFR2
179.7 3
[67] [35]
5
ABT-869
VEGFR2
28.4
[68]
6
Vatalanib
VEGFR2
138
[69]
7
Vandetanib (ZD6474)
VEGFR2
45
[70]
8
AEE788
VEGFR2
Not published
9
CP547632
VEGFR2
11
[71]
10
A-1 (H)
VEGFR2
22
[45]
11
A-2 (OH)
VEGFR2
12.8
[64]
12
A-3 (OCH3)
VEGFR2
14.7
[64]
13
A-4 (OCH2OCH3)
VEGFR2
87.3
[64]
No.
Drug
1 2
for the rest of the inhibitors studied taken from the literature indicate that our new inhibitors perform a rather well with IC50 values even better than clinically useful sunitinib and sorafenib (Table 7).
Struct Chem (2011) 22:635–648
Conclusions
647
antiangiogenic activity on VEGFR2 receptor. Experimental testing of these compounds confirmed their high (nanomolar) in vitro biological activity.
This theoretical study set out to determine stable conformations, solvent effect, pKa, lipophilicity, solubility, absorption, polar surface area, and BBB penetration of 13 inhibitors of VEGFR2 for which a relatively small amount of experimental physicochemical data exist, considering its pharmacological importance. Using the theoretical methods the following conclusions can be drawn.
Acknowledgment This work was supported by the grants from the VEGA Granting Agency (Grants No. 1/0448/09 and 1/0084/10), Cost Action CM0602 and Biomagi, Ltd.
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Wherever possible the most stable conformations of compounds investigated are stabilized by means of intramolecular hydrogen bonds. Water has a remarkable effect especially on the geometry of the conformationally flexible polar parts of the inhibitors studied. The comparison of the conformations of unbound and available structures of drugs when bound on the receptor shows that these inhibitors may exist in structurally different biologically active conformations. Our calculations have been shown that the biologically active conformations of VEGFR2 inhibitors have a relatively high energy compared with the energy minimum for unbound inhibitor. The computed relative energies span a rather broad energy interval (from 100 to 300 kJ/mol). Computed partition coefficients (XLOGP2 method) for angiogenesis inhibitors studied varied between 2.3 and 5. Neutral inhibitors are described as highly lipophilic compounds. Semaxanib is inhibitor with lowest lipophilicity. Among the clinically useful inhibitors sorafenib compared with sunitinib is substantially more lipophilic drug. Owing to high lipophilicity all compounds under study are only slightly soluble in water. The most of the compounds studied are weak bases and at pH 7.4 they exist in unionized form. Sunitinib (N-methylsunitinib), vandetanib and CP-547632 with the computed pKa values in the range of 9.3–10.4 exist as fully protonated bases. All compounds belong to the good-absorption class. Semaxanib, vatalanib, and vandetanib are drugs with low polar surface area and very good absorption (C80%). In general, indolinone derivatives exhibit better absorption than urea derivatives. Semaxanib, vatalanib, vandetanib, and AEE788 penetrate through BBB most easily. On the other side, CP-547632, A-2, A-3, and A-4 with log BB close to -1 penetrate poorly into the brain. Thus, these compounds may serve as selectively acting VEGFRIs with limited penetration to the CNS. Selection criteria for drug-like properties of VEGFR inhibitors investigated were designed. Based on these criteria, three compounds (A-2, A-3, and A-4) were selected for synthesis and biological evaluation for
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