version date: 1 December 2006 EXERC|sEⅢ2 A LABORATORY COURSE IN MEDICINAL CHEMISTRY INTRODUCING MOLECULAR MODELING Ivone Carvalho*, Monica T Pupo, Aurea D. L. Borges, and Lilian SCBernardes Departmento de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de s Paulo, Av. do cafe s/n 14040-903. Ribeirao preto sP. Brazil E-mail: carronal@usp. br Abstract: A laboratory course in medicinal chemistry introducing molecular modeling. Molecular modeling is an important and useful tool in drug design and for predicting biological activity in library compounds. A wide variety of computer programs and methods have been developed to visualize the 3D geometry and to calculate the physicochemical properties of drugs. In this paper, we describe a practical approach to molecular modeling as a powerful tool to study structure-activity relationship in drugs such as antibacterials, hormones, and cholinergic and adrenergic agents. Early in the course, the students learn how to draw 3d structures and to use them to perform conformational and molecular analyses. Thus, they may compare drugs with similar pharmacological activities by superimposing their structures and evaluating geometry and physical properties Keywords: molecular modeling; conformational analysis; structure-activity relationships INTRODUCTION Planning and selecting educational activities in the teaching of medicinal chemistry are ever constant and necessary tasks in adapting program contents to meet the challenges of a world in permanent change. Transformations should direct the course in medicinal chemistry to favor the use of new technological resources and contribute to develop both alternative ways and the students critical thinking. Some methodological strategies should be incorporated into the teaching of medicinal chemistry, thus promoting the teaching- learning processes The classical structure-activity relationship(SAR) studies implied the synthesis of several, structurally related analogs to a lead compound and successive biological activity tests. After decades of SAR research, some general rules on the influence of specific structural changes on biological activity could be drawn, including the size and shape of the carbon chain, the nature and rate of substitution, and stereochemistry of lead compounds SAR and the traditional techniques of molecular modifications are still important tools in
1 EXERCISE III.2 A LABORATORY COURSE IN MEDICINAL CHEMISTRY INTRODUCING MOLECULAR MODELING Ivone Carvalho*, Mônica T. Pupo, Áurea D. L. Borges, and Lilian S. C. Bernardes Departmento de Ciências Farmacêuticas de Ribeirão Preto, Universidade de S. Paulo, Av. do café s/n 14040-903, Ribeirão Preto, SP, Brazil E-mail: carronal@usp.br Abstract: A laboratory course in medicinal chemistry introducing molecular modeling. Molecular modeling is an important and useful tool in drug design and for predicting biological activity in library compounds. A wide variety of computer programs and methods have been developed to visualize the 3D geometry and to calculate the physicochemical properties of drugs. In this paper, we describe a practical approach to molecular modeling as a powerful tool to study structure–activity relationship in drugs such as antibacterials, hormones, and cholinergic and adrenergic agents. Early in the course, the students learn how to draw 3D structures and to use them to perform conformational and molecular analyses. Thus, they may compare drugs with similar pharmacological activities by superimposing their structures and evaluating geometry and physical properties. Keywords: molecular modeling; conformational analysis; structure–activity relationships. INTRODUCTION Planning and selecting educational activities in the teaching of medicinal chemistry are ever constant and necessary tasks in adapting program contents to meet the challenges of a world in permanent change. Transformations should direct the course in medicinal chemistry to favor the use of new technological resources and contribute to develop both alternative ways and the student’s critical thinking. Some methodological strategies should be incorporated into the teaching of medicinal chemistry, thus promoting the teachinglearning processes.1 The classical structure–activity relationship (SAR) studies implied the synthesis of several, structurally related analogs to a lead compound and successive biological activity tests. After decades of SAR research, some general rules on the influence of specific structural changes on biological activity could be drawn, including the size and shape of the carbon chain, the nature and rate of substitution, and stereochemistry of lead compounds. SAR and the traditional techniques of molecular modifications are still important tools in version date: 1 December 2006
version date: 1 December 2006 the search for new drugs, but they are expensive highly time-consuming, and eventually successful Chemical computer programs and the Web databases are important tools in the current search for and design of drugs. A series of interesting molecules can be rapidly screened as to their biological activity versus physicochemical properties. New therapeutic agents can be developed, analyzing theoretical data on structure-activity in the 3D form obtained through recent molecular modeling techniques. In a broad definition of medicinal chemistry, relating the invention, discovery, planning, identification, and preparation of biologically active compounds, to the study of its metabolism, mechanism of molecular action, and construction of SARs, it is highly important to insert and approach topics in molecular modeling in graduation courses on medicinal chemistry According to IUPAC, molecular modeling is an investigation of structures and molecular properties by using techniques of computational chemistry and graphic visualization aiming to obtain, under certain circumstances, a 3D representation Computer-assisted drug design( CADD)is described in many sites on the Internet, helping through tutorials, the investigation of receptor-ligant chemical interactions and the exploring of structural factors connected to biological effects. As a result, the integration of essential knowledge in organic chemistry, biochemistry, molecular biology, and pharmacology, contributes to the understanding of the mechanisms in drug molecular actions The laboratory course in medicinal chemistry is presented to the fifth-period Pharmacy students(30 h, 2 groups)in parallel to the theoretical course(60 h). After careful analysis of the different approaches, laboratory practices were directed to the study of geometry and properties of drugs, enabling the students to explore the chemical and molecular basis of the drug-receptor interaction, by employing computational techniques More specifically, the objectives are (i) conformational analysis of drugs by visualizing its 3D format (ii) analysis of the size and shape of the pharmacophore (ii) importance of the nature and rate of functional groups substitution (iv) stereochemical aspects of drugs and their relation to biological activity (v) to relate a single series of drugs trough structures and physical properties (vi) to predict molecular mechanisms involved in drug action Methods and computational resources employed in the drawing, accurate structural representation, and 3D visualization of drugs are initially presented in this paper; this is followed by showing the use of molecular modeling in the theoretical determination of physicochemical properties and comparison of data obtained with adrenergic and cholinergic drugs, active in the autonomic nervous system This approach significantly contributes both to an integration of theoretical and laboratory data in structure-activity of drugs, and to the implementation of practical courses in medicinal chemistry
2 the search for new drugs, but they are expensive, highly time-consuming, and eventually successful.2 Chemical computer programs and the Web databases are important tools in the current search for and design of drugs. A series of interesting molecules can be rapidly screened as to their biological activity versus physicochemical properties. New therapeutic agents can be developed, analyzing theoretical data on structure–activity in the 3D form, obtained through recent molecular modeling techniques. In a broad definition of medicinal chemistry, relating the invention, discovery, planning, identification, and preparation of biologically active compounds, to the study of its metabolism, mechanism of molecular action, and construction of SARs, it is highly important to insert and approach topics in molecular modeling in graduation courses on medicinal chemistry.3 According to IUPAC, molecular modeling is an investigation of structures and molecular properties by using techniques of computational chemistry and graphic visualization aiming to obtain, under certain circumstances, a 3D representation.4 Computer-assisted drug design (CADD) is described in many sites on the Internet, helping, through tutorials, the investigation of receptor-ligant chemical interactions and the exploring of structural factors connected to biological effects.5 As a result, the integration of essential knowledge in organic chemistry, biochemistry, molecular biology, and pharmacology, contributes to the understanding of the mechanisms in drug molecular actions. The laboratory course in medicinal chemistry is presented to the fifth-period Pharmacy students (30 h, 2 groups) in parallel to the theoretical course (60 h). After careful analysis of the different approaches, laboratory practices were directed to the study of the geometry and properties of drugs, enabling the students to explore the chemical and molecular basis of the drug–receptor interaction, by employing computational techniques. More specifically, the objectives are: (i) conformational analysis of drugs by visualizing its 3D format. (ii) analysis of the size and shape of the pharmacophore (iii) importance of the nature and rate of functional groups substitution (iv) stereochemical aspects of drugs and their relation to biological activity (v) to relate a single series of drugs trough structures and physical properties (vi) to predict molecular mechanisms involved in drug action Methods and computational resources employed in the drawing, accurate structural representation, and 3D visualization of drugs are initially presented in this paper; this is followed by showing the use of molecular modeling in the theoretical determination of physicochemical properties and comparison of data obtained with adrenergic and cholinergic drugs, active in the autonomic nervous system. This approach significantly contributes both to an integration of theoretical and laboratory data in structure–activity of drugs, and to the implementation of practical courses in medicinal chemistry. version date: 1 December 2006
version date: 1 December 2006 EXPERIMENTAL Drawing, conformational, and molecular analysis of drugs Drawing and 3D visualization Several easily utilized programs are available for building bidimensional molecules like Chem Window, Isis Draw, and Chem Draw. Accurate and high-quality figures and diagrams can be elaborated with the help of such programs that frequently contribute to documentation and communication in science The students learn the resources available in the main menu of chem draw and Chem3D and how to utilize the tool and template selection to design chemical structures The stereochemical aspects are discussed in depth and through exercises, they are able to correctly represent the asymmetrical carbons of drugs like benzylpenicillin(1)and estramustine(2), Fig. 1. Eventually, the structures can be drawn in perspective representing the molecules in the projections of Fisher, Newman, and Haworth 量昔 Benzylpenicillin Estramustine (1) C16HI8N2O4s Molecular weight. 334.39 C23H30CINNa2O6P C.5747:H.5.43:N.8.38 Molecular weight: 564.35 C.4895:H536:Cl.12.56:N,248 O.19.14:S.959 Na.8.15:O.17.01:P.549 Fig 1 Drug drawings showing relevant stereochemical features( ChemDraw) Several molecular properties can be calculated and/or represented in some of the programs as well as the molecular formulae, molecular weights and the theoretical elementary analysis. More sophisticated programs like ChemDraw UItra can, in addition predict H and C chemical shifts in NMR of chemical compounds, their freezing and melting points, log P, molar refractivity, and heat of formation, besides furnishing the correct chemical name (IUPAC) The students are trained to chemically recognize heterocyclic rings, frequently present in drugs, through the use of the main menu of Chem Draw by clicking on"Edit and Insert name as Structure". By introducing the English ring name in the dialog box, it is possible to visualize the corresponding chemical structure in the drawing window and the accepted IUPAC nomenclature, which helps the student build complex molecules In the Chem3D program, drugs are three-dimensionally visualized, by the grad building of bonds based on information on their length and position angles. More complex molecules can be obtained by alternating several of the available resources such as drawing
3 EXPERIMENTAL Drawing, conformational, and molecular analysis of drugs Drawing and 3D visualization Several easily utilized programs are available for building bidimensional molecules like ChemWindow, Isis Draw, and ChemDraw. Accurate and high-quality figures and diagrams can be elaborated with the help of such programs that frequently contribute to documentation and communication in science. The students learn the resources available in the main menu of ChemDraw6 and Chem3D7 and how to utilize the tool and template selection to design chemical structures. The stereochemical aspects are discussed in depth and through exercises, they are able to correctly represent the asymmetrical carbons of drugs like benzylpenicillin (1) and estramustine (2), Fig. 1. Eventually, the structures can be drawn in perspective, representing the molecules in the projections of Fisher, Newman, and Haworth. O O H N N S H H COOH N O CH3 OPO3Na2 O Cl Cl H H Benzylpenicillin (1) Estramustine phosphate (2) C16H18N2O4S Molecular weight.: 334.39 C, 57.47; H, 5.43; N, 8.38; O, 19.14; S, 9.59 C23H30Cl2NNa2O6P Molecular weight: 564.35 C, 48.95; H, 5.36; Cl, 12.56; N, 2.48; Na, 8.15; O, 17.01; P, 5.49 H Fig. 1 Drug drawings showing relevant stereochemical features (ChemDraw). Several molecular properties can be calculated and/or represented in some of the programs as well as the molecular formulae, molecular weights and the theoretical elementary analysis. More sophisticated programs like ChemDraw Ultra8 can, in addition, predict 1 H and 13C chemical shifts in NMR of chemical compounds, their freezing and melting points, log P, molar refractivity, and heat of formation, besides furnishing the correct chemical name (IUPAC). The students are trained to chemically recognize heterocyclic rings, frequently present in drugs, through the use of the main menu of ChemDraw by clicking on “Edit” and “Insert name as Structure”. By introducing the English ring name in the dialog box, it is possible to visualize the corresponding chemical structure in the drawing window and the accepted IUPAC nomenclature, which helps the student build complex molecules. In the Chem3D7 program, drugs are three-dimensionally visualized, by the gradual building of bonds based on information on their length and position angles. More complex molecules can be obtained by alternating several of the available resources such as drawing version date: 1 December 2006
version date: 1 December 2006 tools, whole substructures ready in the program and the dialog box, where formulae in linear representation are typed. In parallel, molecules generated in ChemDraw(copy)can be converted to the 3D model in Chem3D (paste), as shown in Fig. 2 for sulfamethoxazole(3) CH3 H2N (3) Fig. 2 Conversion of the 3D sulfamethoxazole (3)structure into the cylindrical bond 3D display( ChemDraw- Chem In the Chem3D program, the molecule can be drawn in different formats, such backbone, ball and stick, and space filling by using standard length and bond angle values, Fig 3 3c) Fig 3 Different representations of sulfamethoxazole: (3a)wire,(3b)cylinder and sphere, (3c)cylinder, and (3d) space filling( Chem3D) Handling 3D molecular models from Chem3D or Molecular Modeling Pro programs can assess relevant stereofeatures of drugs, allowing information about the size volume, and shape of the molecules The importance of the stereochemistry in the mechanism of action of drugs is illustrated by epinephrine (4)and propranolol (5), acting on B-adrenergic receptors, Fig. 4 Compounds 4 and 5 can be easily drawn in their active configurations, R and s, respectively, by rotating the molecules around the x,Y, z axis and attributing according to the classical rule of Cahn-Ingold-Prelog. The configurations of the asymmetrical carbon in the side chains are apparently opposite, due to r and s nomenclature, but in comparison the 3D representations show that the disposition and spatial orientation of the hydroxyl groups are similar, both directed to the same face. The difference in their naming, R and s, is due to the priority rule, the aryloxy group in the antagonist(5) has priority over the methylenamino group of the side chain, which is not the case in the epinephrine molecule
4 tools, whole substructures ready in the program and the dialog box, where formulae in linear representation are typed. In parallel, molecules generated in ChemDraw (“copy”) can be converted to the 3D model in Chem3D (“paste”), as shown in Fig. 2 for sulfamethoxazole (3). H2N N H S N O O O CH3 (3) Fig. 2 Conversion of the 3D sulfamethoxazole (3) structure into the cylindrical bond 3D display (ChemDrawChem3D). In the Chem3D program, the molecule can be drawn in different formats, such as backbone, ball and stick, and space filling by using standard length and bond angle values, Fig. 3. (3a) (3b) ( (3c) 3d) Fig. 3 Different representations of sulfamethoxazole: (3a) wire, (3b) cylinder and sphere, (3c) cylinder, and (3d) space filling (Chem3D). Handling 3D molecular models from Chem3D7 or Molecular Modeling Pro9 programs can assess relevant stereofeatures of drugs, allowing information about the size, volume, and shape of the molecules. The importance of the stereochemistry in the mechanism of action of drugs is illustrated by epinephrine (4) and propranolol (5), acting on β-adrenergic receptors, Fig. 4. Compounds 4 and 5 can be easily drawn in their active configurations, R and S, respectively, by rotating the molecules around the X, Y, Z axis and attributing according to the classical rule of Cahn–Ingold–Prelog. The configurations of the asymmetrical carbon in the side chains are apparently opposite, due to R and S nomenclature, but in comparison, the 3D representations show that the disposition and spatial orientation of the hydroxyl groups are similar, both directed to the same face. The difference in their naming, R and S, is due to the priority rule, the aryloxy group in the antagonist (5) has priority over the methylenamino group of the side chain, which is not the case in the epinephrine molecule (4).10 version date: 1 December 2006
version date: 1 December 2006 3D conversion active isomer r 3D conversion Propranolol(5) active isomer s Fig 4 Conformations of R-epinephrine(4)and S-propranolol (5)with distinct configuration descriptors Cahn-Ingold-Prelog priority rules), but with the stereogenic center in the same spatial disposition The importance of the shape and size of the molecule is illustrated by trans diethylstilbestrol (6)used to mimic estradiol, the natural hormone(7), Fig. 5. Comparing the interatomic distances in the natural and synthetic products, it can be seen that only the trans-isomer(6)has the desired distances between carbons containing hydroxyl groups around 9.0 A. The corresponding cis-isomer(8)shows distances of 5.9 A, quite different from the estradiol molecule(8.6A)
5 O N H OH 3D conversion Propranolol (5) active isomer S HO HO H N CH3 OH 3D conversion Epinephrine (4) active isomer R Fig. 4 Conformations of R-epinephrine (4) and S-propranolol (5) with distinct configuration descriptors (Cahn–Ingold–Prelog priority rules), but with the stereogenic center in the same spatial disposition. The importance of the shape and size of the molecule is illustrated by transdiethylstilbestrol (6) used to mimic estradiol, the natural hormone (7), Fig. 5. Comparing the interatomic distances in the natural and synthetic products, it can be seen that only the trans-isomer (6) has the desired distances between carbons containing hydroxyl groups, around 9.0 Å. The corresponding cis-isomer (8) shows distances of 5.9 Å, quite different from the estradiol molecule (8.6 Å).11 version date: 1 December 2006
version date: 1 December 2006 Estradiol (7) 8,6A trans-diethylstilbestrol(6 3A cis-diethylstilbestrol (8) 5,9A Fig 5 Comparing molecule format and interatomic distances in hormone estradiol(7)and its active and inactive derivatives, respectively, trans-diethylstilbestrol (6) and cis-diethy stilbestrol(8) Conformational analysis and minimal energy The Chem3D program was employed for these studies, but others like Molecular Modeling Pro, Chem Site, Alchemy, Sybyl, Hyperchem, ChemX, CAChe, and Weblab Viewer are also available In conformational analysis of molecules, the bond rotation changes the dihedral angles and, consequently, the corresponding steric energy due to spatial overlaying of non- linked atoms and rotation torsional barriers The molecules drawn three-dimensionally are not necessarily in the most stable conformation generating a certain structure causes molecular distortions with unfavorable lengths, angles, and dihedral angles. Non-linked atoms also interact in the same spatial regions, generating steric and electrostatic repulsion. Correction of the molecule distortions may be achieved by energy minimization through two mathematical models(i) molecular mechanics or(ii) quantum mechanics. Unpredictable interactions related to superimposing molecular orbitals, electronic density distribution, or steric influence can be solved by computational methods. The optimized geometry of a molecule results from the interaction
6 H CH3 OH H H HO HO OH trans-diethylstilbestrol (6) Estradiol (7) OH HO cis-diethylstilbestrol (8) 5,9 Å 9,3 Å 8,6 Å Fig. 5 Comparing molecule format and interatomic distances in hormone estradiol (7) and its active and inactive derivatives, respectively, trans-diethylstilbestrol (6) and cis-diethylstilbestrol (8). Conformational analysis and minimal energy The Chem3D program was employed for these studies, but others like Molecular Modeling Pro, Chem Site, Alchemy, Sybyl, Hyperchem, ChemX, CAChe, and Weblab Viewer are also available. In conformational analysis of molecules, the bond rotation changes the dihedral angles and, consequently, the corresponding steric energy due to spatial overlaying of nonlinked atoms and rotation torsional barriers. The molecules drawn three-dimensionally are not necessarily in the most stable conformation. Generating a certain structure causes molecular distortions with unfavorable lengths, angles, and dihedral angles. Non-linked atoms also interact in the same spatial regions, generating steric and electrostatic repulsion. Correction of the molecule distortions may be achieved by energy minimization through two mathematical models (i) molecular mechanics or (ii) quantum mechanics. Unpredictable interactions related to superimposing molecular orbitals, electronic density distribution, or steric influence can be solved by computational methods. The optimized geometry of a molecule results from the interaction version date: 1 December 2006
version date: 1 December 2006 of conformational analysis and energy minimization. The method of choice for the minimization of energy is dependent both on the size of the molecule and the availability of stored data and parameters, as well as computational resources. Computer-generated molecular models are based on mathematical equations that estimate positions and properties of electrons and nuclei; furthermore, the added calculations experimentally explore the structure, producing a molecule under new perspectives A Molecular mechanics Energy is calculated by comparing angles and distances bonds in a molecule, using values that are listed by the MM2 program. Molecular mechanic equations only consider atomic nuclei and do not include electrons in the calculations. The interactions due to stretching of bonds, angular torsional and spatial deformation are determined by the program, which also calculates the energy of the starting molecule comparing it with a standard, methane( KJ/mol). The program of molecular mechanics resulting in new conformations and the corresponding energy calculation modifies angles and lengths of the original atomic bonds The program also recognizes changes leading to more stable structures with lower steric energy, but the calculations are interrupted if the variation in energy in relation to the original molecule is not considerable. Although molecular mechanics predict energy associated to a particular conformation the quantities expressed are not absolute ones, but only differences between two or more conformations. 0 B. Quantum mechanics In this process, properties of the molecule are calculated by equations of quantum physics, involving interactions between electron and nuclei. Electron movements are more rapid and, since they rotate independently of the nucleus, it is possible to describe electronic energy separately from the nuclear one. Some approximations based on empirical data ar re made in calculations by this process, which are not exact and may be executed by two methods, ab initio and semi-empiric. The first one, is applied only to small molecules, and although more precise and not needing stored data, requires ample computer memory capacity and time. On the other hand, the semi-empiric method is faster and can be used to minimize energy and optimize molecules with 10 to 120 atoms, although less accurate Energy is calculated by the Schrodinger equation from stored parameters; MOPAC is the most frequently used semi-empiric method, subdivided into the following: AMI MINDO/3, MNDO, MNDO-d, and PM3. Figure 6 shows chlorpromazine(9)and indomethacin(10). In these drugs, the biologic effect is highly dependent on certain conformations for the receptor interactions. There is an obvious difference in spatial arrangements between non-optimized forms(9b and 10b)and the corresponding(9c and 10c), energetically optimized by the program MOPAC (AMI). The tricyclic system of the antipsychotic chlorpromazine(9a)in the planar form does not seem to react with the dopaminergic receptor, but it does in an angle of approximately 25 in the ring junction that coincides with structure(9c). Indomethacin(10a), however, shows relevant features for anti-inflammatory activity, such as the nonplanar or perpendicular orientation of the N-p chlorobenzylic group in relation to the indolic system. Structure 10c, closer to the bioactive conformation can be obtained by minimizing structure 10b, relatively planar. A more detailed visualization of 3D minimized molecules can be obtained by movements around axis X, Y, and Z, with the help of the"mouse
7 of conformational analysis and energy minimization.10 The method of choice for the minimization of energy is dependent both on the size of the molecule and the availability of stored data and parameters, as well as computational resources. Computer-generated molecular models are based on mathematical equations that estimate positions and properties of electrons and nuclei; furthermore, the added calculations experimentally explore the structure, producing a molecule under new perspectives. A. Molecular mechanics Energy is calculated by comparing angles and distances bonds in a molecule, using values that are listed by the MM2 program. Molecular mechanic equations only consider atomic nuclei and do not include electrons in the calculations. The interactions due to stretching of bonds, angular torsional and spatial deformation are determined by the program, which also calculates the energy of the starting molecule comparing it with a standard, methane (1 KJ/mol). The program of molecular mechanics resulting in new conformations and the corresponding energy calculation modifies angles and lengths of the original atomic bonds. The program also recognizes changes leading to more stable structures with lower steric energy, but the calculations are interrupted if the variation in energy in relation to the original molecule is not considerable. Although molecular mechanics predict energy associated to a particular conformation the quantities expressed are not absolute ones, but only differences between two or more conformations.10 B. Quantum mechanics In this process, properties of the molecule are calculated by equations of quantum physics, involving interactions between electron and nuclei. Electron movements are more rapid and, since they rotate independently of the nucleus, it is possible to describe electronic energy separately from the nuclear one. Some approximations based on empirical data are made in calculations by this process, which are not exact and may be executed by two methods, ab initio and semi-empiric. The first one, is applied only to small molecules, and although more precise and not needing stored data, requires ample computer memory capacity and time. On the other hand, the semi-empiric method is faster and can be used to minimize energy and optimize molecules with 10 to 120 atoms, although less accurate. Energy is calculated by the Schrödinger equation from stored parameters; MOPAC is the most frequently used semi-empiric method, subdivided into the following: AM1, MINDO/3, MNDO, MNDO-d, and PM3.10 Figure 6 shows chlorpromazine (9) and indomethacin (10). In these drugs, the biologic effect is highly dependent on certain conformations for the receptor interactions. There is an obvious difference in spatial arrangements between non-optimized forms (9b and 10b) and the corresponding (9c and 10c), energetically optimized by the program MOPAC (AM1). The tricyclic system of the antipsychotic chlorpromazine (9a) in the planar form does not seem to react with the dopaminergic receptor, but it does in an angle of approximately 25° in the ring junction, that coincides with structure (9c). Indomethacin (10a), however, shows relevant features for anti-inflammatory activity, such as the nonplanar or perpendicular orientation of the N-pchlorobenzylic group in relation to the indolic system.13 Structure 10c, closer to the bioactive conformation can be obtained by minimizing structure 10b, relatively planar. A more detailed visualization of 3D minimized molecules can be obtained by movements around axis X, Y, and Z, with the help of the “mouse”. version date: 1 December 2006
version date: 1 December 2006 Chlorpromazine(9a) (9b) (9c) CH2COOH Indomethacin(10a) (10b) (10c) Fig 6 Drug structures: 3D, nonoptimized, and optimized by the program MOPAC (Chem3D) Chlorpromazine(9a),(9b), nonoptimized and(9c)optimized; Indomethacin(10a), (10b), nonoptimized and (10c), optimized C Molecular dynami It was already stated that 3D conformations are not necessarily the most stable ones and that the energy minimization process is interrupted when structure variations imply small changes in energy. Graph I shows that this stable" conformation may be separated from another, even more stable that the minimizing process is unable to overcame. In this instance, the most stable conformation, with a minimal global energy, has to be identified by comparison of different conformations and the corresponding energy values
8 N S Cl N Chlorpromazine (9a) (9b) (9c) N CH3 O Cl CH2COOH Indomethacin (10a) (10b) (10c) Fig. 6 Drug structures: 3D, nonoptimized, and optimized by the program MOPAC (Chem3D). Chlorpromazine (9a), (9b), nonoptimized and (9c) optimized; Indomethacin (10a), (10b), nonoptimized and (10c), optimized. C. Molecular dynamics It was already stated that 3D conformations are not necessarily the most stable ones and that the energy minimization process is interrupted when structure variations imply small changes in energy. Graph 1 shows that this “stable” conformation may be separated from another, even more stable that the minimizing process is unable to overcame. In this instance, the most stable conformation, with a minimal global energy, has to be identified by comparison of different conformations and the corresponding energy values.10,14 version date: 1 December 2006
version date: 1 December 2006 Energy carting ener minimum global minimum dihedral angle Graph 1 Local and global minimal energy, respectively, obtained by the minimization process and by molecular dynamic Molecular dynamics can be used to determine the most stable conformation. In this process, the stretching of bonds and angular alterations mimic a procedure of heating the molecule, where the energy barriers between conformations are overcome. An important example is the boat-distorted conformation of cyclohexane as it is minimized by this procedure. Heating the molecule by molecular dynamics generates new conformations including the most stable one, such as the chair. Clonidine(11), the blood anti-hypertension drug, when converted from the 2D-conformation(1la)to the minimized form by molecular mechanics(MM2)(11b)and submitted to molecular dynamics(llc), is a good illustration of the several spatial dispositions. It is important to note the variations in the dihedral angle of the different conformations(11). Structure llc, obtained by molecular dynamics, has the imidazolidine ring closer to a perpendicular orientation toward the 2, 6-dichorophenyl group, which mimics the neurotransmitter, epinephrine, in the interactions with the a receptor(Fig. 7). Another more detailed and systematic procedure may obtain unidentified conformations in the Chem3D program, whereby new ones are gradually generated rotating a central bond and predetermining an angle alteration by the Newman projection. The steric energy in each conformation is determined and represented in energy versus angle graphs, to visualize the most stable ones. This procedure is shown and explored during the laboratory course using acetylcholine(12)as a model. Three techniques are usual in the studies of the conformational properties of (12): X-ray crystallography, nuclear magnetic resonance, and molecular modeling By interacting with different nicotinic and muscarinic receptors in the autonomic nervous system, acetylcholine triggers several biologic effects. Many derivatives in different conformations of the drug have been prepared, but there is still no assurance as to the right receptor-specific conformations. It has been verified, however, that the pharmacophore group should have distinct spatial arrangements in order to interact with nicotinic and muscarinic cholinergic receptors. In this respect, the versatility of the molecule can be explained by the differences in the interatomic distances, 5.9 and 4.4 A between the ester and quaternary ammonium groups, respectively, for the nicotinic and muscarinic receptors interaction. Interatomic distances are directly related to the
9 Energy dihedral angle local minimum global minimum starting energy Graph 1 Local and global minimal energy, respectively, obtained by the minimization process and by molecular dynamics. Molecular dynamics can be used to determine the most stable conformation. In this process, the stretching of bonds and angular alterations mimic a procedure of “heating” the molecule, where the energy barriers between conformations are overcome. An important example is the boat-distorted conformation of cyclohexane as it is minimized by this procedure. Heating the molecule by molecular dynamics generates new conformations including the most stable one, such as the chair. Clonidine (11), the blood anti-hypertension drug, when converted from the 2D-conformation (11a) to the minimized form by molecular mechanics (MM2) (11b) and submitted to molecular dynamics (11c), is a good illustration of the several spatial dispositions. It is important to note the variations in the dihedral angle of the different conformations (11). Structure 11c, obtained by molecular dynamics, has the imidazolidine ring closer to a perpendicular orientation toward the 2,6-dichorophenyl group, which mimics the neurotransmitter, epinephrine, in the interactions with the α2 receptor (Fig. 7).15 Another more detailed and systematic procedure may obtain unidentified conformations in the Chem3D program, whereby new ones are gradually generated rotating a central bond and predetermining an angle alteration by the Newman projection. The steric energy in each conformation is determined and represented in energy versus angle graphs, to visualize the most stable ones.10 This procedure is shown and explored during the laboratory course using acetylcholine (12) as a model. Three techniques are usual in the studies of the conformational properties of (12): X-ray crystallography, nuclear magnetic resonance, and molecular modeling. By interacting with different nicotinic and muscarinic receptors in the autonomic nervous system, acetylcholine triggers several biologic effects. Many derivatives in different conformations of the drug have been prepared, but there is still no assurance as to the right receptor-specific conformations. It has been verified, however, that the pharmacophore group should have distinct spatial arrangements in order to interact with nicotinic and muscarinic cholinergic receptors. In this respect, the versatility of the molecule can be explained by the differences in the interatomic distances, 5.9 and 4.4 Å, between the ester and quaternary ammonium groups, respectively, for the nicotinic and muscarinic receptors interaction.10 Interatomic distances are directly related to the version date: 1 December 2006
version date: 1 December 2006 conformation of acetylcholine, that is, to the disposition of the dihedral or torsional angles of the molecule NH (11) lla)τ0. (11b)τ60.7° (1le)τ73.5° Fig. 7 Spatial representation of clonidine(11) showing corresponding dihedral angles(t); (lla),structure drawn in program ChemDraw and converted to Chem3D; (11b), structure minimized by molecular mechanics, MM2 and (llc) structure showing systematic changes by molecular dynamics To improve the understanding of the different conformations of acetylcholine the students are trained to do the Newman projection of the central atoms of the drug, O(3)- C(4)-C(5)-N(6), in the Chem3D program and to gradually analyze the torsional angles of the molecule. The torsional or dihedral angle(t)can be considered as the one formed by two defined planes as A-X-Y and X-Y-B of four atoms linked in the A-X-Y-B order. The projection shows the spatial relative disposition of the ester and quaternary ammonium groups In the main"View"menu of Chem3D, it is possible to select"Settings"and then Movies"to manually rotate only bond C(4)-C(5)in the X-Y axis of the projection. After each 60 rotation, the dihedral angle is altered and a new conformation is generated totaling six different conformations. At each change of the dihedral angle, the steric (gauche ) and synplanar(eclipse-like)are calculated by the MM2 program, which only &2 energies corresponding to the forms antiperiplanar(star-like)synclinal(gauche ) anticlin considers the internuclear lengths and angles, Table 1. It is possible, in thi s experimen calculate the minimal global steric energy and predict the most stable preferential conformation of acetylcholine. The positive(0-180%)and negative(180-0%)rotation faces are really considered as a complete 360 movement in the X-Y axis, to facilitate graphic representation and interpretation of the results
10 conformation of acetylcholine, that is, to the disposition of the dihedral or torsional angles of the molecule. 2 1 3 4 2 4 3 1 2 4 3 1 (11a) τ 0.5 º (11b) τ 60.7 º (11c) τ 73.5 º HN NH N Cl Cl (11) Fig. 7 Spatial representation of clonidine (11) showing corresponding dihedral angles (τ); (11a), structure drawn in program ChemDraw and converted to Chem3D; (11b), structure minimized by molecular mechanics, MM2 and (11c) structure showing systematic changes by molecular dynamics. To improve the understanding of the different conformations of acetylcholine the students are trained to do the Newman projection of the central atoms of the drug, O(3)- C(4)-C(5)-N(6), in the Chem3D program and to gradually analyze the torsional angles of the molecule. The torsional or dihedral angle (τ) can be considered as the one formed by two defined planes as A-X-Y and X-Y-B of four atoms linked in the A-X-Y-B order. The projection shows the spatial relative disposition of the ester and quaternary ammonium groups. In the main “View” menu of Chem3D, it is possible to select “Settings” and then “Movies” to manually rotate only bond C(4)-C(5) in the X-Y axis of the projection. After each 60° rotation, the dihedral angle is altered and a new conformation is generated, totaling six different conformations. At each change of the dihedral angle, the steric energies corresponding to the forms antiperiplanar (star-like) synclinal (gauche), anticlinal (gauche), and synplanar (eclipse-like) are calculated by the MM2 program, which only considers the internuclear lengths and angles, Table 1.7 It is possible, in this experiment, to calculate the minimal global steric energy and predict the most stable preferential conformation of acetylcholine. The positive (0–180°) and negative (180–0°) rotation faces are really considered as a complete 360° movement in the X-Y axis, to facilitate graphic representation and interpretation of the results. version date: 1 December 2006
