J. Med. Chem. 2003, 46, 5619-5627
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Rational Design of an Indolebutanoic Acid Derivative as a Novel Aldose Reductase Inhibitor Based on Docking and 3D QSAR Studies of Phenethylamine Derivatives Won Suck Sun,† Yoon Sun Park,† Jakyung Yoo,‡ Ki Duk Park,† Sung Han Kim,† Jung-Han Kim,† and Hyun-Ju Park*,‡ Department of Biotechnology, College of Engineering and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea, and College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea Received November 26, 2002
A series of 45 phenethylamine derivatives were synthesized and evaluated for their inhibitory activity against pig kidney aldose reductase (ALR2, EC 1.1.1.21). Their IC50 values ranged from 400 µM to 24 µM. The binding modes of compounds at the active site of ALR2 were examined using flexible docking. The results indicated that phenethylamine derivatives nicely fit into the active pocket of ALR2 by forming various hydrogen bonding and hydrophobic interactions. 3D-QSAR analysis was also conducted using FlexX-docked alignment of the compounds. The best prediction was obtained by CoMSIA combined with hydrophobic and hydrogen bond donor/acceptor field (q2 ) 0.557, r2 ) 0.934). A new derivative, 4-oxo-4-(4hydroxyindole)butanoic acid, was designed, taking into account the CoMSIA field and the binding mode derived by FlexX docking. This rationally designed compound exhibits an ALR2 inhibition with an IC50 value of 7.4 µM, which compares favorably to that of a well-known ALR2 inhibitor, tolrestat (IC50 ) 16 µM) and represents a potency approximately 240-fold higher than that of an original phenethylamine lead compound, YUA001. Introduction Aldose reductase (ALR2, EC 1.1.1.21) is a NADPHspecific aldehyde reductase that catalyzes the conversion of glucose to sorbitol in the polyol pathway and is known to be involved in the onset of long-term diabetic complications such as cataract, neuropathy, retinopathy, and nephropathy.1-3 To date, studies to develop aldose reductase inhibitors (ARIs) to prevent those complications have yielded many structually diverse ARIs such as tolrestat, sorbinil, zopolrestat, and others.4-9 ALR2 contains a (R/β)8 barrel motif with a large hydrophobic pocket, approximately 4 × 15 Å wide and 15 Å deep, which favors aromatic and apolar substrates over highly polar monosaccharides. The cofactor NADPH is bound in an extended conformation across the barrel with the nicotinamide ring centered at the bottom of this cavity. Crystal structures of ALR2 complexed with NADPH and diverse ARIs revealed that this pocket is the only possible active site and is mainly composed of two regions: an anion binding site of hydrophilic residues (Tyr48, His110, Trp111, and the 4-pro-R hydrogen of the nicotineamide ring of NADPH) and a wall of hydrophobic residues (Trp20, Trp79, Trp111, Phe122, Leu300, etc.).10-13 Many studies have proposed the requisite properties of ARIs: (1) an aromatic ring system to form hydrophobic or π-π stacking interactions with the hydrophobic amino acid residues in the active site, and (2) ionizable groups such as carboxylic acids and spirohydantoins which can anchor the anionic binding site.14-16 * Corresponding author. Tel.: 82-31-290-7719. Fax: 82-31-290-5403. e-mail:
[email protected]. † Yonsei University. ‡ Sungkyunkwan University.
Since the early 1980s, along with progress in computer technology, a significant advance has been made in the application of computer modeling to medicinal chemistry.17-19 Although the predictive power of molecular modeling remains controversial, this methodology has proven useful in drug discovery and recently provides an alternative, so-called virtual screening. Virtual screening is a computing technology to analyze large databases of chemicals in order to discover possible drug candidates, and complement current advances in high-throughput chemical synthesis and biological assays.20-24 Recently, Costantino et al. reported a series of benzopyran-4-one derivatives as ARIs on the basis of binding mode, and enhanced their activity through SAR study.25,26 Rastelli et al. identified candidate ARIs having novel pharmacophoric groups such as sulfonic acids, nitro derivatives, sulfonamides, and carbonyl derivatives by performing database search and docking.27 Iwata et al. also discovered a series of indole-3acetic acids analogues as ARIs by screening the 3D chemical database (ACD3D).28 In our previous study, derivatives (1-11 in Figure 1) of YUA001 (N-2-methylbutanoyl tyramine from alkalophilic Corynebacterium sp. YUA25) were synthesized and evaluated for their inhibitory activity against pig kidney ALR2. Most of them showed more potent inhibitory activity than that of YUA001; however, the need for enhancing their activity and elucidating their inhibitory mechanism remained.29 In this study, we synthesized 45 phenethylamine derivatives and evaluated their inhibitory activity against ALR2. Then, a structure-based drug design approach was taken to find new derivatives with improved activity.30-33 Flexible docking and 3D-QSAR/CoMSIA
10.1021/jm0205346 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/20/2003
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Figure 1. Preparation of (A) phenethylamine derivatives, 1-31, (B) benzyl and phenylamine derivatives, 32-39, (C) indoline and indole derivatives, 40-46.
analysis were used to explore structure-activity relationships of the compounds we identified, and guided the design of the most potent derivative, 4-oxo-4-(4hydroxyindole)butanoic acid (46 in Figure 1). The present study demonstrates a successful example of rational drug design approaches: we used not only a CoMSIA model to decide the substituent properties of a new derivative, but also a binding conformation obtained from flexible docking analysis to define positions to be substituted. Results Synthesis and Biochemical Evaluation. A novel aldose reductase inhibitor, N-[2-(4-hydroxyphenyl)ethyl]-
2-methylbutanamide (YUA001), isolated from alkalophilic Corynebacterium sp. YUA25,29 was chosen as a lead, and various structural modifications were conducted to find new synthetic derivatives with improved activities. We modified it with an elongated aliphatic chain (1-6) and the introduction of aromatic rings (78) or terminal carboxyl acids (9-45), while maintaining phenethylamine as a framework. The syntheses of N-substituted tyramine derivatives 1-11 were reported previously.34,35 As outlined in Figure 1A and B, the parasubstituted tyramine derivatives 12-31 were prepared by treating each anhydride with para-substituted amine in acetonitrile (THF) at 40 °C. For benzyl and phenylamine derivatives 32-39, cyclic anhydride was reacted
Design of a Novel Aldose Reductase Inhibitor
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Table 1. Inhibitory Activities against Pig Kidney Aldose Reductase of Phenethylamine Derivatives 1-46 compounds YUA001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
IC50 value (M)a 10-3
1.8 × 1.6 × 10-4 1.3 × 10-4 1.2 × 10-4 1.1 × 10-4 1.1 × 10-4 1.5 × 10-4 1.6 × 10-4 1.0 × 10-4 3.9 × 10-4 0.8 × 10-4 3.4 × 10-4 0.78 × 10-4 0.73 × 10-4 0.88 × 10-4 1.04 × 10-4 1.7 × 10-4 1.55 × 10-4 1.77 × 10-4 1.85 × 10-4 0.94 × 10-4 0.75 × 10-4 0.88 × 10-4 0.76 × 10-4
compounds
IC50 value (M)a
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Tolrestatb
0.99 × 10-4 1.12 × 10-4 2.03 × 10-4 1.54 × 10-4 0.94 × 10-4 0.99 × 10-4 0.81 × 10-4 1.1× 10-4 1.92 × 10-4 1.82 × 10-4 2.27 × 10-4 4.4 × 10-4 1.41 × 10-4 1.5 × 10-4 1.8 × 10-4 1.33 × 10-4 1.02 × 10-4 0.4 × 10-4 0.95 × 10-4 0.24 × 10-4 0.37 × 10-4 0.3 × 10-4 0.74 × 10-5 0.16 × 10-4
a Evaluted in vitro against pig kidney aldose reductase. b N[[6-Methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-Nmethylglycine.
with benzyl or phenylamine for 10 h at 40 °C. The indoline and indole derivatives 40-46 were synthesized following the synthetic method shown in Figure 1C. The anhydrides were treated with indoline and indole in the presence of a large excess of triethylamine and vigorously stirred for 24 h at 80 °C. The inhibitory activities of the compounds (1-45) against ALR2 are evaluated and expressed as IC50 values in Table 1. Various modifications of phenethyl, phenyl, and benzylamine (1-39) did not improve the inhibitory activity significantly, exhibiting IC50 values in the low millimolar to micromolar range. However, replacement of the phenethylamine unit with indole or indoline ring (40-45) increased the activity significantly. Notably, 4-oxo-4-indolebutanoic acid (43) exhibited the strongest ALR2 inhibitory activity with an IC50 value of 24 µM, which is comparable to the activity of tolrestat (IC50 ) 16 µM). Docking Study. We conducted docking analysis using the FlexX algorithm36,37 not only to examine the binding mode of the compounds at the active site of aldose reductase (AR), but also to obtain a basis for further synthetic derivatization. The crystal structure of the ternary complex which includes cofactor NADPH and tolrestat was used in this study (pdb entry ) 1AH3).38 A COOH hydrophilic head of tolrestat and the compounds 9-45 (pKa between 3.5 and 5.0) is probably bound by AR in the form of carboxylate anion; the COOH group of those molecules was calculated in the anionic form which shares a negative charge between two oxygen atoms. The energy-minimized structure of tolrestat was preliminarily docked into ALR2 to examine how closely the FlexX algorithm can reproduce the experimentally determined binding conformation of tolrestat at the active site of AR. A superposition of docked tolrestat onto the crystallographic geometry yielded a root-mean-square (rms) deviation of 0.74 Å and
revealed that FlexX was successful in reproducing the binding conformation of tolrestat. To evaluate the relationship between relative ALR2 inhibitory activities and binding energy scores obtained from FlexX docking, we have used consensus scoring program, Cscore, that integrates multiple well-known scoring functions: a GOLD-like function, a DOCK-like function, ChemScore, a PMF function, and FlexX.39-42 The FlexX scoring function performs best, and the resulting ranking showed good agreement with the ALR2 inhibitory activities of the compounds. Therefore, we selected the top-scoring conformer of each ligand based on the FlexX scores calculated from FlexX, and positioned it onto the active site of ALR2. They fit well into the active site pocket of ALR2 occupied by tolrestat in the X-ray crystal structure. All the compounds primarily contain either a phenolic hydroxyl (YUA001 and 1-8) or carboxylate (9-45) group. Those groups anchor into the anionic binding site by forming numerous hydrogen bonds with NADP+ or the side chain of Trp111, His110, and Tyr48. The interaction between the ligand and amino acid residues of the active site were determined and are illustrated in Figure 2 for the three representative compounds, YUA001, 10, and 43, in comparison with the X-ray structure of tolrestat.38 An aliphatic chain of the acyl moiety of YUA001 is positioned close to Phe122 and Leu300, possibly forming a hydrophobic interaction. For compound 10, the phenolic hydroxyl group forms additional hydrogen bonding with the Ser302 residue. The indole moiety of compound 43 intercalates between Phe122 and Leu300 and is likely to play an important role in enhancing the affinity in the active site through the π-π interaction. 3D-QSAR/CoMSIA Modeling. 3D-QSAR methods, especially CoMFA(Comparative Molecular Field Analysis),43 are used widely in drug design, because they allow rapid generation of QSARs from which biological activity of newly designed molecules can be predicted. The basic assumption in CoMFA is that an appropriate sampling of the steric and electrostatic fields around a set of aligned molecules might provide all the information necessary for understanding their biological activities. A main difficulty in the application of 3D-QSAR methods such as CoMFA is that a spatial orientation of the ligands toward one another has to be found to yield a correct model. Therefore, the success of molecular field analysis largely determined by the quality of alignment of studied molecules. Another 3D-QSAR method, the CoMSIA (Comparative Molecular Similarity Indices Analysis)44,45 technique, is recently of particular interest since it is less alignment-sensitive than CoMFA and includes a hydrophobic field and two hydrogen bond fields. In this study, we performed 3DQSAR by CoMSIA on a select 42 compounds (except compound 46 in Table 2), by using hydrophobicity and hydrogen bond donor and acceptor as descriptors and attempted to obtain a reliable CoMSIA model to guide to the design of analogous AR inhibitors. For alignment of the ligands, we used a superimposition of the topscoring conformations derived from FlexX docking.46 As listed in Table 2, the biological activities of 42 ligands used for the 3D-QSAR study were expressed as pIC50 values. To derive a predictive relationship model, the partial least squares (PLS) method was used, and the
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Figure 2. Binding modes of phenethylamine derivatives obtained from the FlexX docking. Dotted lines indicate hydrogen bonding interactions (
