Applications of molecular physics ‘biotechnology’ to the rational design of an improved phenytoin analogue

Seizure

1992;

1: 223-246

Applications of molecular physics the rational design of an improved analogue*

‘biotechnology’ phenytoin

to

DONALD F. WEAVER Department of Medicine (Neurology), Department of Chemistry, Queen ‘s University Epileptic Clinic, Queen’s University, Kingston, Ontario, Canada, K7L 3N6 Correspondence

to Donald

F. Weaver

at the above

address.

Tel:

(613)

548-1347.

Fax: (613)

545-6669

This study exploits molecular physics, in conjunction with a large scale computing environment, as a tool for understanding the clinical phenomenology of phenytoin (PHT) toxicology at a molecular level and for employing this understanding in an attempt to design improved drugs. The application of molecular physics techniques, such as quantum mechanics and molecular force field calculations, to the process of rational anticonvulsant drug design remains virtually unexplored. A 3-step strategy for applying these techniques to the design of an improved PHT molecule is presented. Step 1 employs quantitative structure-activity relationship calculations on 80 PHT analogues to ascertain the portion of the PHT molecule necessary for bioactivity (i.e. the ‘bioactive face’ of PHT); the N3-C4(0)-C5-R fragment of PHT was identified as the bioactive face. Step 2 employs molecular modelling studies to determine the portion of the PHT molecule necessary for the teratogenit, mutagenic and connective tissue toxicities of PHT (i.e. the ‘biotoxic face’); the C2(0)-N3 fragment of PHT was identified as the biotoxic face. Step 3 experiments design an ‘improved’ PHT analogue, which maintains the bioactive face while eliminating the integrity of the biotoxic face; 2-deoxy-5,5-diphenylhydantoin was designed and synthesized as the improved PHT analogue. This compound had biological activity equivalent to PHT, but was unable to bind to nucleic acids or to chelate metals involved in connective tissue metabolism. Key words: anticonvulsant;

phenytoin; teratogenesis; gingival hyperplasia; zinc; molecular mechanics.

INTRODUCTION

‘I am become Death, the Destroyer of Worlds’ (Bhagavad Gita 94:15; quoted by J. Robert Oppenheimer at the explosion of the first atom bomb, 16th July 1945, Alamogordo, New Mexico.) Since its conceptual inception, molecular and atomic physics, a discipline dedicated to the understanding of reality at an atomic and subatomic level, has been exploited for its destructive capabilities. Fortunately, recent political events have witnessed a reduction in the arms race and have heralded the existence of a socalled ‘peace dividend’. It is now imperative * Based on a paper presented at Epilepsy Europe 1992, in Glasgow, that was awarded the Gowers Young Physicians Prize by the Council of the British Branch of the International League Against Epilepsy. 1059-l

31 l/92/040223+24

$08.00/O

that this peace dividend must be not only financial but also intellectual. The understanding of disease aetiology and pathogenesis at an atomic or sub-atomic level is emerging as an important research endeavour, which may benefit from the recent advances of molecular physics. The result is a multidisciplinary approach combining physics with pharmacology to discover clinically useful new drugs and to improve drugs already in use. In clinical pharmacology, the development of new neuroactive drugs, especially anticonvulsants, is a continuing priority’-5. Although 1.2% of the general population experiences recurrent

seizures, only 65-70%

of these indi-

viduals enjoy reasonable control with currently available anticonvulsant drugs5. Moreover, the use of these drugs is associated with significant untoward effects, ranging from unpleasant cosmetic side-effects to serious hae@ 1992 Bailliere

Tindall

224

D.F. Weaver

matological or hepatic toxicities3s4. There remains, therefore, a need to design new and improved anticonvulsant drugs; in particular, there is a need to develop receptor-specific drugs with greater pharmacological efficacy and decreased toxicity5. Molecular physics has immense potential to facilitate this drug design process. Drug design is an evolving technology. The time-honoured ‘drug discovery techniques’ of screening proserendipity and massive grammes are inefficient; there is a need for ‘rational drug design’. In the past 5 years, molecular physics combined with supercomputerassisted molecular modelling have emerged as useful adjuncts to this process, thus giving rise to ‘computer-aided rational drug design’ (CARDD). Not surprisingly, molecular physics calculations have been identified as a ‘biotechnology of the future’. The use of molecular physics calculations in anticonvulsant drug design remains unexplored. This study employs molecular physics and large scale computing strategies in an attempt to improve the anticonvulsant drug phenytoin (PHT). This has been approached in a three-step process: Step I, the use of molecular physics/quantum pharmacology calculations combined with structure-activity relationship studies to ascertain the portion of the PHT molecule (‘the bioactive fragment’) responsible for pharmacological anticonvulsant activity; Step II, The use of molecular physics/quantum pharmacology calculations combined with molecular modelling studies to ascertain the portion of the PHT molecule (‘the biotoxic fragment’) responsible for clinical toxicity; Step III, The design and synthesis of a new PHT-like molecule in which the bioactive fragment is retained but in which the biotoxic fragment has been elimated. This study exploits molecular physics, in conjunction with a large scale computing environment, as a tool for understanding the clinical phenomenology of phenytoin toxicology at a molecular level and for employing this understanding in an attempt to design improved drugs.

METHODS AND MATERIALS A. General comments on methodology To employ a molecular improve anticonvulsant

physics approach to drugs, it is first

necessary to reduce the biology of anticonvulsant drug action to molecular terms and then to model these molecules with calculational techniques. This requires the application of molecular physics methodologies such as quantum mechanics and classical mechanical force field calculations. Until the beginning of the twentieth century, it was believed that all reality was describable by Newton’s Laws. These laws, or Newtonian (classical) mechanics, appeared able to treat natural objects as large as planets or as small as atoms. However, it was eventually realized that classical mechanics failed at the atomic level of reality. An alternative approach was needed for the quantitative evaluation of molecular phenomena. In the first three decades of the twentieth century, there occurred significant advances in physical philosophy. Planck showed that energy is emitted from heated bodies in the form of discrete particles or quanta; Einstein expanded upon this theory with the proposal that an atom emits radiant energy only in quanta, and that this energy is related to the mass and to the velocity of the light; Schrodinger incorporated these evolving ideas of the new quantum theory into an equation that described the wave behaviour of a particle. Since the Schrodinger equation (which lies at the mathematical heart of quantum mechanics) permitted quantitative agreement with experiment at the atomic level, the physicists of the 1930s predicted an end to the experimental sciences, suggesting that they would become a branch of applied mathematics lz8 . These hopes (or fears!) soon proved groundless. Although in principle the Schrodinger equation afforded a complete description of Nature, in practice it could not be solved for the large molecules of medical and pharmacological interest during the early days of quantum physics. Nevertheless, atomic physics flourished in the realm of military research. In the 1990s new conceptual/computational methodologies are permitting pharmacologically useful studies to be achieved using quantum mechanics and classical mechanics. Quantum mechanics is a non-empirical method in which the properties of a stationary state of a molecule are obtained by solution of the Schrodinger partial differential equation: H+ = E$, where H is the Hamiltonian differential operator and $ is the wave function. Molecular quantum mechanics calculations may be ab initio or semi-empirical. Semi-empirical calcu-

Design of an improved

phenytoin

analogue

lations use a simpler Hamiltonian than the correct molecular Hamiltonian and incorporate additional parameters designed to fit experimental data. In contrast, ab initio calculations use the full correct Hamiltonian and attempt a solution through a rigorous first principles treatment. Classical mechanics force field calculation is conceptually quite different: it is an empirical technique, which employs multiple classical equations of motion to provide a priori geometries for varying conformations of large molecules. The sum of the individual classical equations constitutes a multi-dimensional potential energy function (termed the ‘force field’), which expresses the restoration forces acting on a molecule when the minimum potential energy conformation is perturbed. By minimizing the force field equation, it is possible to determine the lowest energy native conformation of a large biomolecule. Before employing molecular physics for the task of designing drugs for people, it is first necessary to address the philosophical problem arising from applying ‘microscopic’ mathematical modelling studies at the level of the electron to the ‘macroscopic’ biological phenomena of human disease. That is, is it intellectually valid to conceptualize a disease that affects the entire organism (and even the interactions of this organism within society) at the molecular and submolecular level? This invokes a need to consider the practical issue of reductionism versus holism before embarking on the process of large scale computational modelling at one end of the structural hierarchy (i.e. the electron) to address problems at the other end of the hierarchy (i.e. the epileptic patient in society). Despite the obvious plurality of scientific explanation on various levels of description, there exists a definite bias towards theoretical monism, a bias which favours reductionism16’. In the context of medicine, reductionism is the view that all phenomena of life can be ultimately reduced to the laws of physics and chemistry. The resulting hypothesis is simple: since organisms are built of matter, biological problems must be conceptualized in terms of the fundamental theory of matter, without need for autochthonous biological laws. In philosophy, this approach has powerful proponents; for example, Bertrand Russell claimed, ‘There is no reason to suppose living matter subject to any laws other than those to which inanimate matter is subject, and con-

siderable reason to think that everything in the behaviour of living matter is theoretically explicable in terms of physics and chemistry’7. Holists, on the other hand, deny that the laws of physics and chemistry are completely sufficient to explain the phenomena of life; they emphasize the inherently global properties of ‘complexity’ and ‘organic wholeness’. In the context of medicine, holism is the view that a rigorous description of the organic whole cannot be derived from a comprehensive theory of its parts. When employing quantum mechanics and classical mechanics to discover drugs for epileptic people, the dilemma of reductionism versus holism becomes seemingly relevant. However, upon close inspection it becomes apparent that in the realm of drug design, the view that reductionism is opposite to (and hence incompatible with) holism must be rejected as naive. In quantum pharmacology, reductionism is in harmony with holism, and the emergence of essential novelty in a higher level description is merely a compelling consequence of this compatibility. Thus, the use of sophisticated molecular physics calculations, which invoke arguments at the electron level, to design drugs for people are justified. It is reasonable to apply the ‘biotechnology’ of molecular physics to the discovery of new antiepileptic drugs and to the improvement of existing drugs.

B. Molecular physics methods The classical mechanics calculations used in Steps I and II of this study were performed using a classical force field equation (given in Appendix 1) as implemented in the commercially available MM2(85) program113. This programme has been modified extensively by the author specifically for the modelling objectives described in this research; for example, the force field has been specifically parameterized using semi-empirical molecular orbital quantum mechanics calculations (employing the AM1 Hamiltonian) and ab initio molecular orbital quantum mechanics calculations (employing the Gaussian 86 package)1’0-138. Details of these modifications are provided in Appendix 1. Semi-empirical quantum mechanics calculations were performed using the AM1 Hamiltonian within the MOPAC 5.0 programme package using the keyword PRECISE. Calculations were performed on three com-

226

puters: an IBM 3081G computer operating under VM/CMS, an IBM RS/6000 550 RISC computer and an IBM RW6000 320H RISC computer. Additional molecular graphics were performed using BIOGRAF software from MOLECULAR SIMULATIONS INC. (Pasadena, California). Calculations were done in the Queer&/IBM Molecular Modelling Laboratory.

C. Drug synthesis techniques The goal of Step III of this research is to design, synthesize and test a new phenytoin-like compound developed to satisfy the criteria established in Steps I and II. All chemical reagents employed in these syntheses were obtained from commercial sources (Aldrich Chemical Co., Sigma Chemical Co., Fisher Scientific Inc.). Solvents were purified before use by established procedures. Proton nuclear magnetic resonance spectra were recorded on a Brucker 400 MHz FT Spectrometer; samples were dissolved in either CDCls or DMSO-ds (Merck Isotopes); chemical shifts were reported in 6 values relative to tetramethylsilane. Infrared spectra were recorded on a Bruker IFS-85 FTIR Spectrometer. Melting points were recorded on a Thomas Hoover Capillary Melting Point Apparatus. Thin layer chromatography was performed on pre-coated Brinkmann silica gel 60 F-254 plates; spots were visualized using ninhydrin spray, iodine vapour, and/or short-wave ultra-violet light. Additional details concerning syntheses are provided in Appendix 2.

D. Biological evaluation techniques The goal of Step III of this research is the design, synthesis and biological evaluation of a PHT-like molecule designed to satisfy the criteria of Steps I and II. A rodent maximal electroshock seizure model as described by Krall et al. was employed6.

D.F.

Weaver

molecular properties that impart anticonvulsant activity, whilst eliminating those molecular properties responsible for toxicity. Specifically, this will be achieved through a 3-step strategy: Step I, the use of molecular physics/ quantum pharmacology calculations combined with structure-activity relationship studies to ascertain the portion of the PHT molecule (‘the bioactive fragment’) responsible for pharmacologic anticonvulsant activity; Step II, the use of molecular physics/quantum pharmacology calculations combined with molecular modelling studies to ascertain the portion of the PHT molecule (‘the biotoxic fragment’) responsible for clinical toxicity; Step III, the design and synthesis of a new PHT-like molecule in which the bioactive fragment is retained but in which the biotoxic fragment has been eliminated.

STEP I CALCULATIONS Goal of Step I calculations The aim was to use molecular physics/quantum pharmacology calculations combined with structure-activity relationship studies to ascertain the portion of the PHT molecule (‘the bioactive fragment’) responsible for pharmacological anticonvulsant activity. This goal will be achieved by randomly selecting a series of hydantoin analogues with varying anticonvulsant activities. Each analogue is subjected to an extensive battery of molecular mechanics, quantum mechanics and graph theory calculations yielding a group of descriptors for each analogue, regardless of bioactivity; these descriptors describe the structural properties of each hydantoin analogue. A data array is then constructed with hydantoin analogues of varying activity along one axis and descriptors representing the structural properties of each analogue along the other axis. This data array is then searched to ascertain the minimal number of descriptors necessary to define bioactivity by distinguishing active from inactive analogues; as a corollary, the bioactive fragment is deduced.

E. Overall approach and design strategy Approach of Step I calculations The central goal of this research is to employ the evolving techniques of molecular physics for the task of improving the PHT drug molecule. In principle, this is achieved by re-engineering the PHT molecule to incorporate the

The correlation of biological activity with molecular structure is central to modern neuropharmacology and enables an enhanced mechanistic understanding of drug action at

Design of an improved

phenytoin

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the molecular level of reality. Accordingly, contemporary approaches to the study and design of bioactive molecules are based on the notion that bioactivity may be related quantitatively to molecular structure through the application of quantitative structure-activity relationship (QSAR) techniques%“. The establishment of QSAR relationships for anticonvulsants has been hindered by the chemical diversity of the molecules and by the complexity of the physiological and biochemical processes that intitiate and propagate seizure activity. Although the hydantoin anticonvulsants, such as PHT, have been the most successful class of anticonvulsants of the twentieth century, few studies have tackled the problem of identifying the common structural denominator which imparts biological activity. Accordingly, in Step I of this study, a QSAR technique, employing theoretically calculated descriptors, has been devised and used to evaluate the hydantoin class of anticonvulsant agents. A variety of QSAR techniques are available, including free energy models (Hansch equationll,“), mathematical models (FreeWilson equation13), statistical models’* (discriminant analysis, cluster analysis), quantum mechanics and molecular mechanics methods15, topological methods16 and pattern recognition methods 17-“. In recent years, the pattern recognition technique described by Stuper and Jurs18, Kirschner and Kolalskilg and Wold et uZ.~~,~’ has emerged as one of the most all-encompassing and versatile techniques. In Step I of this study, a modification of the pattern recognition method, which uses theoretical descriptors derived from quantum mechanics and molecular mechanics calculations, has been applied to the problem of hydantoin structure-activity relationships. The initial objective of this Step I theoretical pattern recognition study was to identify a theoretical ‘classification rule’ capable of correctly categorizing hydantoin compounds into pharmacologically active and inactive classes based solely upon an examination of their structural properties. This problem was approached in two stages. First, a set of hydantoins of known biological activity was selected and designated as the ‘training set’. A group of theoretical molecular ‘descriptors’ was then generated to describe each compound in this training set. Statistical techniques (regression analyses) were then employed to identify the minimal number of descriptors (i.e. the ‘essential descriptor set’) capable of distinguishing

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active from inactive hydantoins. This essential descriptor set constitutes a classification algorithm or rule. This classification rule was next applied to a second set of hydantoins, termed the ‘test set’, to evaluate the validity and predictability of the rule. As a corollary to developing a classification rule which designated activity from inactivity, it was possible, through complementarity arguments, to identify a molecular fragment (i.e. ‘the bioactive fragment’) which constituted the region of structural commonality among active hydantoin anticonvulsants. The selection of descriptors capable of completely encoding the significant and structural aspects of hydantoin molecular structure was crucial to the validity of Step I calculations. The hydantoin numbering scheme is shown in Fig. 1. The descriptors are listed in Table 1,

N3

2’ 07/

\

\n

R2

w

Fig. 1: Hydantoin

numbering

scheme.

and are categorized into four classes: geometric, electronic, topological and physiochemical. Geometric descriptors represent three-dimensional properties and reflect aspects of molecular shape and size. The geometric descriptors employed in this study were derived from theoretical quantum mechanics and molecular mechanics calculations. The hydantoin analogues under study were conformationally and geometrically optimized using the AM1 Hamiltonian after a preliminary scanning of their conformational space using multiple molecular mechanics force field optimizations. Electronic descriptors include descriptors such as charge densities, bond moments, orbital energies and frontier electron densities, which reflect molecular properties arising from variable electron distribution through a compound’s structural framework.

D.F. Weaver

228 Table 1: List of descriptors A. Geometric descriptors (from molecular mechanical calculations) Bond lengths Nl-C2, C2-N3, N3-C4, C4-C5, Nl-C5 Bond angles C5NlC2, NlC2N3, C2N3C4, N3C4C5, C4C5N1,

N3C406,N3C207 Dihedral angles C5NlC2N3, NlC2N3C4, C4C5NlC2 Substituent volumes Rl, R2, R3/R4

C2N3C4C5,

B. Electronic descriptors (from quantum calculations) 5. Atom charge densities Nl, C2, N3, C4, C5,06,07 6. Bond dipole moments Nl-C2, C2-N3, N3-C4, C4-C5, Nl-Rl, C2-07, C4-06

N3C4C5N1,

mechanical

C5-Nl,

N3-R2,

C. Topological descriptors (from graph theory calculations) 7. Number of atoms in molecule 8. Number of atoms in substituents (Rl, R2, R3/R4) 9. Number of rings in molecule 10. Number of rings in substituents (Rl, R2, R3/R4) 11. Molecular weight of molecule 12. Molecular weight of substituents (Rl, R2, FWR4) 13. Presence of heteroatom functionalities in substituents (Rl, R2, R3iR4) 14. Presence of aromatic groups in substituents (Rl, R2, R3/R4) 15. Zagreb topological indices (Ml, M2) 16. Randic topological index 17. Platt topological index D.

Physicochemical descriptors theoretical calculations) 18. Lipophilicity of molecule 19. Lipophilicity of substituents

(from (log P) (Rl,

empirical

R2, R3/R4)

The electronic descriptors used in this study were obtained from the Mulliken population analysis of theoretical AM1 semi-empirical quantum mechanics calculations. Topological descriptors encode aspects of molecular composition and connectivity, and describe patterns of interatomic interconnections that determine ultimate molecular architecture. The topological descriptors used in this study were derived from graph theory calculationszM1. In graph theory calculations, a graph G is a mathematical structure consisting of vertices (atoms) connected by edges (bonds). The mathematical structure that maps a certain molecule (molecular graph G) is the adjacency-matrix A[G]. For an N-atom molecule, A[G] is an N x N matrix with entries aij having values of either 1 or 0, since any two atoms in a given molecule are in binary relation being either connected or not connected. Physicochemical descriptors,

such as the partition coefficient (log P) describe molecular lipophilicity, representing the ability of a drug to traverse biological membrane barriers. In this study, molecular log P values were calculated using the empirical equation of Klopman, Namboodiri and Schochet32,33. Lipophilicities of molecular fragments were calculated using a fragment approach34,35. The hydantoin molecule is readily amenable to chemical modification and a literature review provided biological data for 839 analogues47-76. From this overall data base, a subgroup of 80 compounds with varying bioactivities, which had been tested using comparable electroshock techniques, was randomly selected. One-quarter of this subgroup (20 analogues) was used as the training set (Table 2); the remaining 60 analogues constituted the test set (Table 3). A selection of the training set structures are shown in Fig. 2. The Table 2: Training set hydantoin analogues 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Hydantoin 5-Methylhydantoin 5-Ethylhydantoin SPropylhydantoin 5-Isobutylhydantoin 5-Phenylhydantoin 5,5-Dimethylhydantoin 5-Hexyl-5-methylhydantoin 5-Ethyl-Sisopentylhydantoin 5,5-Diisopropylhydantoin 5-Ethyl-5-phenylhydantoin 5-Butyl-5-phenylhydantoin 5,5-Diphenylhydantoin 3-Isopropyl-5-phenylhydantoin 1-Ethyl-5-phenylhydantoin 1-Isopropyl-5-phenylhydantoin 3,5,5-Trimethylhydantoin 3,5,5-Triphenylhydantoin 1,5-Diethyl-5-phenylhydantoin 1,3,5,5Tetramethylhydantoin

Inactive Inactive Inactive Inactive Inactive Active Inactive Active Active Active Active Active Active Inactive Active Active Inactive Inactive Active Inactive

N analogues of the training set and the M descriptors of the descriptor set were arranged in a two-dimensional array. The rows and columns of this array were searched to identify the minimal descriptor set which separated the N analogues into active and inactive groups.

Results of Step I calculations The 20 analogues of the training set were structurally probed utilizing the 55 descriptors of the descriptor set. Table 4 depicts a sample

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229

Table 3: Test set hvdantoin analowes’ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Rl

R2

R3

R4

H H H H H H Me Et H H H H H H H H H H n-&H, n-&H, n-&H, CH&H,CcH, H H H H H H H H H C,H,OCO H H H H H H ‘XAGH, H H H H H H H H H H H Me H H H H H H Me H H

H H H H H H H H H H H H H H H H H H H H H H

H H Me i-C,Hs c-&J-b I c-‘&H, t Me Me n-&H7 i-C3H7 n-C4Hs i-&H, Et Et n-&H7 i-C3H, i-C,Hs n-W-I,1 Me Et n-&H, Ph H Me C-CsHB c-&H, Et H Ph Ph Ph Et Ph H Ph H Et Et H H Et Me Et Ph H i-&H,, Ph C,H,OCH, c-cc311 set-CIHc,OCHz Ph n-C,H,0CH2 P-BGH, Me&(Br)CH(Br) H c-C&H, Me 2-C,H,S i-C3H7SCH2 Me

i-&H7 set-C&H, c-W-L i-C,Hs c-‘XL c-&H7 Me Me Ph Ph Ph Ph l-W%

~-GJHT H Me H Me Ph Me CHCCHB CNCHzCHz H (Et)zNCH, H H CH2C6H4 Me Et H CH,CH(OH)CH(OH) HOCH,CHc H p-NH&&H, (Et)2NCH,CH, H H H H H H H Me H H CHs0CH2 H HO&CH&H, H H CHsOCHc

* A significant number of test-set analogues have satisfied, the presence of heteroatoms is compatible limit to the steric magnitude of the C5 substituents.

heteroatoms in their substituents. with bioactivity. As demonstrated (Note: I = inactive; A = active.)

I I A A I A I I A A A A A A A A A I A A A A I I A A A A A I I A I I A A I I A I I I I I I A A A A I A I A I I A I A A I

2-W-b l-C& l-G,H, l-GoH, P-CH-GH~ Ph Ph Ph H Ph U-I&C c-&H, Ph Ph H Ph Ph Ph Ph Ph 2-C4H3S 2-C4HSS H 2-C4HsS 2-C4HsS 2-C4HsS Ph Ph p-NH&&H4 Ph Ph CH,OCH* CHoOCHc CHsOCH, Ph i-CsH70CH2 c-W-L CzH50CHz Ph Et Me Ph 2-C,H,S Ph c-&H, Ph i-&H9 If the classification by entry 18, there

rule is otherwise may be an upper

D.F. Weaver

230

I

A

A

I

Fig. 2: Structural formulae of representative training set hydantoin analogues. Six compounds from Table 2 are shown. I, inactive; A, active; Me, methyl; Et, ethyl; Ph, phenyl. Table 4: Sample array describing training set hydantoins in terms of descriptors 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23 1.23

2

3

100.9 100.7 100.6 100.2 100.3 100.0 100.2 100.1 100.0 99.6 99.4 98.9 99.6 99.7 100.0 100.0 100.2 99.8 99.9 100.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

4 100 114 128 142 156 176 128 198 198 186 204 232 252 218 204 216 142 328 232 156

5 2 16 30 44 58 78 30 100 100 86 106 134 154 78 78 78 30 154 106 30

Descriptors: (1) C4-06 bond length; (2) NlC5C4 bond angle; molecular weight; (6) Rl molecular weight; (7) R2 molecular log P for molecule; (12) Ml Zagreb topological index.

array in which hydantoins (rows) are described in terms of descriptors (columns). Examination of the geometric descriptors failed to identify any significant variations in bond lengths, bond angles or dihedral angles capable of distinguishing activity from inacti-

6

7

8

9

10

11

12

1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 43 1 77 29 15

1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 15 1 1 15

28.27 85.38 142.3 199.3 256.3 268.3 142.3 427.3 427.3 352.3 371.2 476.2 486.1 257.2 257.2 257.2 142.3 486.1 371.2 142.3

3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 128.2 176.2 3.37 243.1 128.2 71.1

3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 3.37 71.1 3.37 3.37 71.1 3.37 3.37 71.1

-2.89 -2.42 -1.94 -1.47 -0.99 -0.62 -1.95 0.42 0.42 0.02 0.33 1.28 1.66 0.80 0.33 0.66 -1.47 3.93 1.28 -0.99

32 38 41 46 52 66 46 66 66 56 78 86 102 82 85 82 43 136 88 58

(3) NlC2N3C4 dihedral angle; (4) molecular weight; (5) R3/R4 weight; (8) R3/R4 volume; (9) Rl volume; (10) R2 volume; (11)

vity. It is noteworthy, however, that for all hydantoins the Nl-C2 and N3-C4 amides differ significantly from the C2-N3 amide in length. (The Nl-C2 and N3-C4 amides are 1.33A in length [similar to peptide backbone amides], whereas the C2-N3 amide is 1.39 A in

Design of an improved

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length [similar to nucleic acid amides]). A minor correlation between bioactivity and the magnitude of the Nl-C&C4 bond angle was noted. In active analogues this angle is less than 100”; in inactive analogues it is greater. This observation is a manifestation of the ‘Thorpe-Ingold Effect’36, and reflects the presence of bulky, sterically encumbering C5 substituents which cause, by mutual repulsion, a decrease in the Nl-C5-C4 angle. As revealed by the dihedral angle values, the hydantoin ring was planar in both active and inactive analogues. The volume descriptors, on the other hand, did demonstrate a definite correlation between antiepileptic activity and the N3 and C5 substituent volumes. Large volume, bulky substituents correlated with bioactivity positively at the C5 position and inversely at the N3 position. Bioactivity appeared relatively insensitive to Nl substitution. Inspection of electronic descriptors revealed no correlation between bioactivity and hydantoin atomic charge densities. The possibility of a ‘biologically active centre’ at C5, as postulated by previous workers37, was not confirmed. The correlation between bond dipole moments and anticonvulsant activity was equally poor. However, a slightly higher N3R2 bond moment did correlate weakly with bioactivity, suggesting that a hydrogen bonding capability by the N3 amide hydrogen may be significant in active analogues. Examination of the topological descriptors identified a number of structure-activity relationships. High atom counts, ring counts and substituent molecular weights correlated with bioactivity positively at the C5 position and negatively at the N3 position. The presence of aromaticity in the C5 substituents also correlated positively with enhanced activity. The topological indices, such as the Zagreb and Randic indices, confirmed the bioactivityenhancing value of bulky, branching C5 substituents. Inspection of the physicochemical descriptors failed to demonstrate any correlation with bioactivity when all 20 analogues in the training set were considered. Nevertheless, when the N3 substituted analogues were neglected, a positive correlation emerged. As demonstrated by fragment calculations, lipophilic substitutents at the C5 position enhanced anticonvulsant activity. By identifying the minimal descriptors necessary for describing bioactivity, it is possible to put forth a classification rule that dis-

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tinguishes activity from inactivity. The soidentified bioactive anticonvulsant molecular segment for hydantoins consists of a 1.33A long amide functionality (with an unsubstituted amide nitrogen capable of hydrogen bonding) bonded through a distance of 1.56A via the amide carbonyl (-C(O)-) to a tetravalent carbon atom which is substituted with bulky and preferably aromatic substituents (see Fig. 3).

0 l

t

N-H Fig. 3: Common structural denominator for hydantoin bioactivity. The classification rule permits the bioactive anticonvulsant segment to be identified. In phenytoin this consists of the N3-C4 amide connected to C5 and its phenyl substituents. In general this segment consists of an amide linkage (A) bonded through some intermediary atom (X) to a large and preferably aromatic substituent (L).

By applying this classification rule to the 60 compounds of the test set, all were classified correctly except two compounds (analogues 28 and 36, Table 3). Both of these molecules have large N3 substituents, which the classification rule developed on the training set suggests as being incompatible with anticonvulsant activity. However, if these two analogues are rotated such that the N3 atom occupies the

D.F. Weaver

location of the C5 atom and the Nl-C2 amide replaces the N3-C4 amide, then an acceptable bioactive segment is again identified (see Fig. 4). The resultant modified description of the

Q Ph

Fig. 4: Re-orientation of the 3-phenylhydantoin anticonvulsant. When the N3 atom is rotated into the C5 position, this molecule satisfies the classification as depicted in Fig. 3.

bioactive anticonvulsant molecular fragment consists of a 1.33 A long amide functionality (with an unsubstituted amide nitrogen capable of hydrogen bonding) bonded through a distance of 1.4-1.6 A via the carbonyl carbon to a tetravalent atom, which is substituted with bulky and preferably aromatic substituents. Therefore, in phenytoin the -N3-C4(=0)C5(-RI- fragment of the molecule defines the anticonvulsant ‘bioactive fragment’ of the hydantoin ring.

functions in relatively similar positions in space and proposed that these stereochemical features formed the common structural basis for antiepileptic action. Jones and Woodbury, however, have opposed this point of view and have demonstrated its failings when applied to other anticonvulsant chemicals42. More recently, experimental studies by Jones et ~1.~~ and Codding et ~1.~~ on PHT, carbamazepine, cyheptamide and phenacyloxindole have suggested that the region of antiepileptic structural commonality is an amide group connected to a substituted carbon atom. The results of this Step I theoretical QSAR study conclude that the antiepileptic hydantoin segment consists of a 1.33 A long amide functionality bonded through a distance of 1.41.6A via the carbonyl carbon to a heavily substituted tetravalent atom. To achieve this conclusion, 80 randomly selected hydantoins, a substantially larger number than previous studies, were studied. Moreover, both active and inactive compounds were evaluated. To investigate all possible structural influences, a complete set of geometric, electronic, topological and physicochemical descriptors was used. By considering only hydantoin analogues, the problems associated with studying molecules having different mechanisms of action and varying receptor sites have been eliminated.

Conclusion of Step I calculations Although PHT has been in clinical use since 1938, the structure-activity relationship properties of hydantoin anticonvulsants have yet to be elucidated clearly. Between 1938 and 1968, hundreds of hydantoins were synthesized and biologically tested; nonetheless, the structure-activity relationships remained unexplained. In 1969, molecular orbital calculations by Andrews37 on a series of barbiturates, hydantoins, succinimides and glutarimides failed to identify a biologically active centre. Subsequent studies in the 1970s by Lien et al. on a group of barbiturates, hydantoins, succinimides and oxazolidinediones suggested a correlation between bioactivity and the log P physicochemical constant38,3g. The first major structural study came from an extensive series of X-ray crystallogaphic analyses of PHT, diazepam, procyclidine, trihexylphenidyl, phenacemide, sulthiame21*40*41. ethylphenacemide and Camerman and Camerman40*41 discovered that these diverse compounds possessed certain hydrophobic regions and electrophilic

In conclusion, Step I calculations reveal that the -N3-C4(=0)-C5-phenyl segment in phenytoin defines the anticonvulsant bioactive fragment.

STEP II CALCULATIONS Goal of Step II calculations Step II calculations aimed to use molecular physics/quantum pharmacology calculations combined with molecular modelling studies to ascertain the portion of the PHT molecule (‘the biotoxic fragment’) responsible for clinical toxicity. This goal will be achieved using two quantum pharmacological molecular modelling studies. First, the interaction of PHT with DNA will be computationally modelled to address toxicities such as teratogenesis or mutagenesis. Second, the interaction of PHT with the Zn2+ ion will be computationally modelled to address connective tissue toxici-

Design of an improved

phenytoin

analogue

ties, such as gingival hyperplasia, which may arise from the toxic effects of PHT-induced Zn2+ chelation. The molecular modelling studies will identify the portion(s) of the PHT molecule necessary for interacting with DNA or complexing Zn2+, thus defining the ‘biotoxic fragment’.

Approach of Step II calculations The side-effects associated with PHT are many and diverse. Some, such as ataxia and encephalopathy, are dose-dependent and are common amongst neurologically active drugs; others, such as hirsutism and gingival hyperplasia, are uncommon amongst anticonvulsants and are relatively unique to PHT. Two major groups of PHT side-effects arising from specific undesirable molecular interactions have been identified: (i) PHT’s interference with nucleic acid biochemistry producing lymphadenopathy, teratogenesis, mutagenesis and adverse haematopoietic reactions; and (ii) PHT’s interference with connective tissue biochemistry producing gingival hyperplasia, generalized thickening of subcutaneous tissues, enlargement of lips and nose and other coarsening of facial features. In Step II of this study, molecular physics calculations combined with molecular modelling strategies have been used to examine these two groups of biochemical sideeffects at a molecular level of structural refinement.

Results of Step II calculations (i) Molecular modeling of the influence of PHT upon nucleic acid metabolism It has been postulated that PHT administration may rarely predispose to the development of cancer78. Various case reports have implicated PHT as a causative factor in malignant lymphoma8~g, multiple myelomagO, hairy cell leukaemiagl and other haematological malignanciesg2. In utero, PHT exposure has been associated with mesenchymomag3, neuroblastomag4*g6, ganglioneuroblastoma”, ependymoblastoma” and melanotic ectodermal tumourloo . The co-existence of foetal hydantoin syndrome and neoplasia has been notedg6pg7, and a possible link between teratogenesis and carcinogenesis has been discussed”‘. The foetal hydantoin syndrome, first described by

Loughnan et al. in 1973, is a much studied controversial entity, which includes a wide range of facial and skeletal abnormalities such as distal digital hypoplasia and hypertelorism153-156. In addition, phenytoin administered to pregnant mice induces abnormalities that persist through successive generations suggesting a structural alteration in the molecular genetic apparatuslo3. A variety of mechanistic explanations have been put forward to explain PHT-induced teratogenesis and mutagenesis. The first is the ‘free radical mechanism’. PHT, serving as an electron donor, is co-oxidized by thyroid peroxidase and prostaglandin synthetase producing an electron-deficient free radical species which can covalently bind to intracellular macromolecules such as nucleic acids157. This observation may be of mechanistic relevance in PHT mutagenesis/teratogenesis; for example, pretreatment of pregnant mice with ol-phenyl-N-tbutylnitrone; a free radical spin-trapping agent, reduces the number of cleft lip/palates secondary to PHT in the offspring158. A second possible mechanistic explanation for PHTrelated mutagenesis is the ‘arene epoxide mechanism’15g,160. Via microsomal monoxygenase catalysis, PHT is metabolized to an arene oxide, an electrophilic thermodynamically unstable aromatic epoxide, which may elicit mutagenesis through covalent binding to intracellular nucleic acids161,162. Despite the obvious strengths of the free radical and arene epoxide hypotheses, these mechanisms do not completely explain the phenomenology of PHT-related mutagenesis/teratogenesis163v164. For instance, mephytoin and ethotoin, which are not metabolized via arene oxide intermediates, produce teratogenic dysmorphisms and embryopathies similar to PHT165,166. Likewise, trimethadione, an anticonvulsant pentaatomic heterocycle, which is similar to the C2(0)-N3-C4(0) segment of PHT but which has no phenyl rings, is clearly teratogenic despite an entirely different biotransformation pathway164. From these deficiencies has emerged the need to study the direct interaction of PHT with nucleic acids. Since the molecular mechanism of PHTinduced mutagenic and teratogenic effects remains incompletely elucidated, there is a need to comprehend rigorously the interaction of PHT with DNA at an atomic level. In vitro studies have demonstrated that PHT inhibits DNA synthesis in lymphocytes104,105; in uiuo investigations have found that PHT inhibits

234

protein synthesis via transcriptional/translational interference106-10g. An interaction between adenine and PHT explains these biological effects by the disruption of the normal adenine-uracil or adenine-thymine associations. Considerable experimental evidence supports the existence of a specific PHT-adenine interaction in DNA. Subcellular distribution studies show selective accumulation of PHT in the nucleic acid rich fractions of mammalian brain77. Moreover, the aqueous solubility of PHT is enhanced by the addition of adenine or denatured DNA, while polymers not containing adenine have no effect upon solubility. Native DNA does not alter solubility suggesting that the nitrogenous bases have to be ‘exposed7’. Specific hydrogen bonding interactions between PHT and 9-ethyladenine, an adenine analogue, have been demonstrated by both NMR spectroscopy and X-ray techniques “g8’ . Despite these studies, the nature of the DNA-PHT interaction remains poorly understood at the molecular level. The intranuclear accumulation of PHT, which contributes to the teratogenic, mutagenic and carcinogenic toxicity, arises from a specific complexation between PHT and adenine components of nucleic acids. To explore the DNA-PHT interaction at a molecular level, a theoretical molecular mechanics approach has been devised in Step II of this study. The MM2 molecular mechanics force field equation has been selected and parameterized specifically for nucleic acids and hydantoins using rigorous quantum mechanics calculations (See Appendix 1). The resulting computational technique constitutes a theoretical biomimetic computer model which may be employed to evaluate possible interactions between PHT and DNA. Although the DNA polymer may assume a variety of theoretical conformations, the B form (the common conformation of native DNA) was used in this study. In accord with the nomenclature of Saenger82 and of Voet and Richs3, the furanose ring of the B form exists in a C3’-exe conformation, and its orientation with respect to the base is specified by a x = 127” torsional angle. An additional five torsional angles describe the polynucleotide backbone conformation: angles o, 6 and f3 are within the nucleotide; cpand IJJhave the same meaning as in polypeptide chains [N(H)-Ca-C’-N(H); C’-N(H)-Cd-C’]; u lies within the sugar ring. In the B form DNA, w = 155", 5 = 36, 8 = -146, cp = -96” and (I = -46”. The resulting DNA double helix is

D.F. Weaver

‘tight’, with a diameter

of 2.0 nm and a pitch of

3.4nm. In the DNA-PHT interaction, a hydantoin ring amide (-C(O)-N(H)-) hydrogen bonds to the acceptor surface of adenine in a manner analogous to thymine or uracil. Since PHT contains three amides [Nl-C2(0), N3-C2(0), N3-C4(0)1, the fundamental problem is not the energetics of the isolated adenine-PHT interaction, but rather the energetics of the overall intercalation of PHT into some feasible DNA docking position. In this regard, molecular mechanics is being used as a sophisticated form of molecular modelling and as a tool to identify prohibitive collisions during intermolecular interactions. To model the interaction, the starting geometries for PHT and DNA were obtained from X-ray data21,s1-s4. The DNA segment consisted of the trinucleotide guanine-adenine-guanine (GAG) and its complementary bases, thymine being replaced with PHT. Cytosines were appropriately hydrogen-bonded to the guanines, but PHT was located 4.5-5.081 from the adenine base. By adjusting this interaction distance, the planar hydantoin ring was advanced towards the adenine receptor site in incremental steps. The sugar-phosphate backbone was fixed to preserve the B form conformation of DNA. The bases and PHT structures were allowed ‘to float freely’. Energy optimizations were achieved via the Newton-Raphson second derivative minimization sub-routine within the MM2(85) force field. As depicted in Figs 5 and 6, all three possible interactions between PHT and DNA were explicitly considered. When the interactions with the N3-C4(0) and Nl-C2(0) amides were evaluated, calculated interaction energies were in excess of 1000 kcal/mol when the PHT molecule was still more than 3.5A from the adenine. These high energies arose from the steric interaction between the phenyl groups of PHT and the atoms of the bases of the adjacent nucleotides; that is, there were prohibitive steric intermolecular collisions because these approaches were not feasible. However, when the C2(0)-N3 amide of PHT was evaluated, it was possible to bring this amide to within 2.91A of the Nl-CG-NH2 group of adenine without the steric hindrance of colliding molecules. Moreover, the C2(0)-N3 bond length of PHT is 1.39 A (compared with 1.33 A for the Nl-C2(0) and N3-C4(0) amides), which compares favourably to the 1.39A length of the analogous amide bonds in uracil and thymine.

Design of an improved

phenytoin

analogue

235

-cc4

P\

I -N3-C2

Nl -H I

C

c

G

G

5’

3’

Fig. 5: Molecular modelling of the DNA-PHT interaction. All three possible interactions of PHT with DNA were considered. Interaction 2 (N3-C2 amide) was favoured because of steric interactions from the C5 phenyl groups.

Therefore, these energy calculations indicate that the C2(0)-N3 amide of PHT’s hydantoin ring hydrogen bonds to the Nl-CG-NH2 group of adenine in preference to the Nl-C2(0) and N3-C4(0) amides. This molecular modelling simulation permits the DNA-PHT interaction, and not simply the adenine-PHT interaction, to be evaluated while avoiding the crystal packing which effects and solubility problems influenced previous experimental studies. Arguably, the calculations of this study represent only the B form of DNA and other conformations are not considered; nevertheless, these calculations do evaluate the ability of PHT’s three amide ‘faces’ to replace thymine at its docking site in native DNA (B form). Although only the trinucleotide GAG was studied, this molecular modelling study indicates that the factors identified by the calculations are operative for other adenine containing nucleotide combinations.

(ii) Molecular modeling of PHT’s influence on connective tissue biochemistry Long-term treatment with PHT produces alterations in connective tissue metabolism: gingival hyperplasia, hirsutism and alteration of facies have been reported to occur with chronic therapy2-4. While the biochemical basis of PHT-induced connective tissue change is not well understood, this effect is probably augmented by the influence of PHT upon connective tissue trace metal biochemistry (although other factors may also contribute). Compounds that chelate Zn2+, thereby mobilizing it out of the intracellular pool and into the extracellular pool for subsequent excretion, produce phenytoin-like connective tissue changes by reducing the pool of available metal ions within the dermal cells and by altering the function of metal-dependent enzymes such as 5cY-reductase and lysyl oxidase167. Understanding the molecular basis of

D.F. Weaver

Zn2+ chelation by PHT may permit a partial understanding of the adverse influence of PHT on connective tissue biochemistry. Further to modelling the PHT-DNA interaction, additional Step II molecular modelling studies were performed to evaluate the molecular mechanism of PHT-Zn2+ chelation139-152. Zinc chelation depends on numerous factors. The divalent zinc cation, owing to its size, prefers a tetrahedral 4-coordinate complexation environment (although uncommon 2- and B-coordinate environments are known). The stereochemistry of the complexation is defined by the size, electrostatic forces and covalent bonding forces amongst the interacting groups. Ligands bond to Zn2+ through electron-rich donor atoms such as nitrogen (N), oxygen (0) and sulphur (S). Organometallochemistry has demonstrated that amides are powerful chelating moieties for Zn2+‘42. Amides are excellent ligands for this type of complexation since they possess two electronegative atoms, N and 0, in close geometric proximity142. Phenytoin contains multiple amide functionalities, which

enable it to complex Zn2+. Phenytoin-Zn2+ chelation has been demonstrated previously: Weismann et al. found that PHT increases 65Zn absorption in rats 147. Palm and Hallmans noted that PHT therapy produced low serum concentrations of Zn2+15’; PHT chelates Zn2+ and removes it from the bioavailable ~001’~~. To provide rigorous molecular modelling of the PHT-Zn2+ interaction, semi-empirical molecular orbital quantum mechanics calculations were performed using the AM1 Hamiltonian. For purposes of chelation, PHT may be considered to have four amide functionalities: Nl-C2(=0), C2(=0)-N3, N3-C4(=0) and the non-bonded Nl-C4(0) ‘amide’. Initial studies showed that a single PHT molecule was insufficient for adequate chelation and that two PHT molecules were required to form a stable zinc chelate. Crystal structure data of other transition metal-PHT complexes support the presence of two PHT molecules in the chelate geometry145,146. All possible orientations for a Zn(PHTj2 complex (in which an amide, NXCY(=O), from one PHT and an amide, NX’-

I

Fig. 6: Molecular

modelling

of the DNA-PHT

interaction.

The

interaction

of PHT

with the adenine

base

in DNA.

Design of an improved

Fig. 7: Molecular

modelling

phenytoin

analogue

of the Zinc-PHT

237

interaction.

A zinc ion interacting

CY’(=O), from a second PHT define the chelation environment) were considered explicitly; thus, ten conformations were calculationally optimized: Nl-CB(=O)/Nl’-C2’(=0), NlC2(=O)iN3’-C2’(=0), Nl-C2(=O)/N3’C4’(=0), Nl-C2(=O)/Nl’-C4’(=0), N3C2(=O)/N3’-C2’(=0), N3-C2(=O)/N3’C4’(=0), N3-C2(=O)/Nl’-C4’(=0), N3C4(=O)/N3’-C4’(=0), N3-C4(=O)/Nl’C4’(=0), and Nl-C4(=O)/Nl’-C4’(=0). The stable conformers were also optimized in hydrated forms using 2 to 5 water molecules clustered around hydrogen bonding centres. The calculations demonstrated that the most energetically favourable orientation for Zn2+ chelation involved the N3-C2(=0) amide of both PHTs involved in the chelation process (see Figs 7 and 8). Therefore, the N3-C2(=0) amide for PHT constitutes the portion of the PHT molecule which participates in Zn2+ chelation and which probably influences connective tissue biochemistry. Conclusion of Step II calculations Step II calculations reveal that the C2(=0)N3 amide is the ‘face’ of the PHT molecule

with the C2-N3

amide

of two PHT

molecules.

which is involved both in interactions with DNA (contributing to teratogenic/oncogenic side-effects) and in interactions with Zn2+ (augmenting connective tissue side-effects).

In conclusion, Step II calculations suggest that the -C2(=0)-N3(H)amide of phe-

STEP III EXPERIMENTS Goal of Step III experiments Step III experiments aimed to design and synthesize a new PHT-like molecule in which the bioactive fragment (as identified in Step I calculations) is retained but in which the biotoxic fragment (as identified in Step II calculations) has been eliminated. Since the central theme of this research study is the exploration of molecular physics calculations as a tool in anticonvulsant drug design (with an illustrative application to the

D.F. Weaver

238

redesign of phenytoin) the synthesis and biological testing will be presented in greater detail elsewhere.

Approach of Step III experiments Step I calculations reveal that the N3-C4-C5phenyl fragment of the PHT molecule defines the ‘bioactive fragment’ of the PHT molecule. Step II calculations reveal that the C2(01-N3 portion of the PHT molecule defines the ‘biotoxic fragment’ of the PHT molecule. Therefore, although the bioactive and biotoxic fragments overlap at the N3 atom, they are not congruent. Accordingly, in principle it is possible to design a molecule in which the bioactive fragment is retained, but in which the integrity of the biotoxic fragment is eliminated.

d.

Results of Step III experiments After additional modelling calculations, a PHT analogue in which the C2 carbon was changed from a -C(-O)carbonyl group to a -C(Hzlmethylene group was selected as the simplest congener satisfying the design criteria. The resulting 2-deoxy phenytoin analogue maintains the bioactive fragment, but does not possess the biotoxic -C2(=0)-N3(H)amide moiety. A synthesis for this new compound was devised (see Appendix 2). To synthesize this compound, the 2-thio analogue in which a -C2(=S)- thiocarbonyl replaced the -C2(=0)carbonyl of the 2,4-imidazolidinedione ring in PHT was initially prepared; the sulphur atom was then removed [-C2(=Sl+-C2(H&l employing Raney Nickel reagent to yield the desired product. With the new 2-deoxy-5,6-diphenylhydan-

-038

1064

-06

Fig. 8: Molecular modelling of the Zinc-PHT interaction. A hydrated molecular model of the Zinc-PHT interaction.

Design of an improved

phenytoin

analogue

239

Mechanical

stirrer

Thermometer

Protective

u

cap

Octanol

membrane

Divided

beaker

phase

U cell

0 I

J

o

-Water

bath

1 IT-

Heating

coil

Insulation

Fig. 9: Experimental biomimetic system. This system consists of two aqueous phases divided by an octanol membrane phase. This apparatus is employed to model the removal of zinc from the intracellular phase to the extraeellular phase.

toin compound available, it was next necessary to ascertain whether Step I and Step II criteria were satisfied experimentally. Biological testing demonstrated that anticonvulsant activity was equipotent to phenytoin (Step I criteria satisfied) in the maximal electroshock assay6. Chemical testing revealed no interaction between adenine and the deoxy-PHT analogue: adenine did not increase the aqueous solubility of the deoxy-PHT analogue and nuclear magnetic resonance studies showed no field shifts compatible with attractive (e.g. hydrogen bonding) interactions between adenine and the deoxy-PHT analogue. Chemical testing likewise demonstrated that Zn2+ chelation for the deoxy-PHT analogue was < 10% of PHT’s (Step II criteria satisfied). To measure Zn2+ chelation, an experimental biomimetic system (EBS) was designed and developed by the author (previously described”?. The EBS is shown in Fig. 9 and is designed to demonstrate the chelation and removal of cations from a

biological system. It consists of two aqueous phases (one ‘intracellular’; one ‘extracellular’) separated by a ‘membrane phase’. The area of surface contact between each aqueous phase and the membrane phase is 39.3cm2. All phases are stirred continuously to eliminate the effects of concentration gradients; multiple voltage regulators control the stirring devices ensuring experimental reproducability; the entire system is temperature controlled at 37” f 0.1 C. The extracellular aqueous phase was Zn-free, while the intracellular aqueous phase was 50m~ in Zn(SCNJ2. The latter compound was prepared by the reaction of zinc nitrate with potassium thiocyanate in methanol. Through spectrophotometric determination of zinc thiocyanate, the ability of PHT and deoxyPHT to chelate Zn2+ in the intracellular phase and transport it to the extracellular phase was measured. PHT chelated/transported Zn2+ at 0.023 pmol/h; deoxy-PHT chelated/transported at 0.002 p.mol/h.

D.F. Weaver

240

These chemical studies suggest that the deoxy-PHT does not bind adenine and does not chelate Zn2+. However, long term in vitro biological studies to ascertain the mutagenic and connective tissue effects of the deoxy analogue are indicated to determine whether these design modifications have meaningfully altered PHT’s toxicity profile.

Conclusion of Step III experiments

In conclusion, Step III experiments preliminarily suggest that a 2-deoxy congener of phenytoin possesses the bioactive face of PHT, but eliminates the biotoxic face.

the clinical phenomenology of phenytoin toxicity at a molecular level and for assisting in the design of an improved phenytoin analogue.

ACKNOWLEDGEMENTS The Medical Research Council of Canada, The Natural Sciences and Engineering Research Council of Canada, The Physicians Services Incorporated Foundation, the J.P. Bickell Foundation, The Botterell Foundation, and IBM Canada Inc. are acknowledged for financial support. D.F.W. acknowledges the financial support of an Ontario Ministry of Health Career Scientist Award.

REFERENCES C :ONCLUSIONS

The past decade has witnessed significant conceptual advances in molecular physics and in the practical application of these advances to the molecular modelling of biochemical processes. Indeed, the combined use of molecular physics calculations and large scale computing strategies has been identified as a ‘biotechnology of the future’. Although there exists a need for improved anticonvulsants, the application of molecular physics calculations to anticonvulsant drug design remains unexplored. Accordingly, a 3-step molecular physics calculational strategy, employing classical mechanics force field methods and quantum mechanics, has been applied to the task of improving the anticonvulsant drug phenytoin. Step I calculations revealed that the -N3-C4(X(R)- segment of phenytoin defined the anticonvulsant bioactive fragment; Step II calculations revealed that the -C2(=0)-N3(H)segment of phenytoin defined the biotoxic fragment; Step III calculations revealed that a 2deoxy-phenytoin analogue can be designed which maintains the bioactive fragment but partially eliminates the biotoxic fragment. Thus, this study demonstrated the design utility of molecular physics/quantum pharmacology calculations and emphasized the observation that drug efficacy and toxicity must be understood at a molecular/atomic level before meaningful improvement of drug structure can be considered. Moreover, this study exploited molecular physics as a tool for understanding

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APPENDIX 2: ORGANIC SYNTHESES AND EXPERIMENTAL PROCEDURES

APPENDIX 1: FORCE FIELD CALCULATIONS”~‘38 The classical mechanics calculations presented in this study were performed using the following force field equation:

E TOT=

CG

+

CJ%

+

CE,

ral angle); E,i represents electrostatic energies (D is dielectric constant; lo is bond dipole; r is interdipole distance; x is the interdipole angle; cxis the angle subtended by a dipole and a line joining the mid-points of two dipoles); Evdw is the non-bonded van der Waals energy (rc is atomic spherical radius; E is spherical hardness.) For the purpose of this study, equation [ll was ‘parameterized’ specifically for hydantoins and nucleic acids by the author. Ideal bond lengths and bond angles were taken from high quality neutron diffraction experimental data. Bond lengths and angles containing hydrogen atoms were taken from split-valence basis set ab initio quantum mechanics calculations on representative molecular fragments. Torsional angle energy profiles were taken from semiempirical quantum mechanics calculations using the AM1 Hamiltonian and then curve fitting by the method of Hopfinger to obtain the V, terms of the Fourier expansion. Atomic point charges were determined from a least squares fitting of calculated electrostatic potentials to quantum mechanically determined electrostatic potentials. The calculational validity of this force field was ascertained through a series of refining calculations.

+

CJL

(i) A three-stage synthesis of 2-deoxy-5,5diphenylhydantoin (a) Synthesis of Raney Nickel

+

r11

C&W

where

El = (1/2)K,(Z-Z,12

+ K1,(Z-Zo13

El

E8 = (1/2)K2(f3-80)2

+ K2,(e-e,)4

[31

E, = (VJ2)(1 + cosw) + (V2/2)(1-cos2w) (V3/2)(l + cos3w)

+

[41

E,z.i= (/.J+b)/(D.rij3) X (cosx-3cOs~iCOs~j)

151

E vdw = (2.90 X 105) E e-12.5(ro’r)-2.25 rF

[61

f (i-c/

El represents bond length energies (Ki and Ki, are stretching force constants); Ee represents bond angle energies (Kg and K2, are bending force constants; E, represents torsional energies (V, are torsional constants; w is the dihed-

A solution of sodium hydroxide (16O.Og, 4 mole) in water (600ml) was placed in a 2.01 flask equipped with a mechanical stirrer. After cooling to 10°C in an ice-bath, nickel aluminium alloy (125 g) was added in small portions over 90min. The resulting suspension was stirred at room temperature for 1 h and at 50 “C for an additional 8 h. The suspension was transferred to a graduate cylinder and the aqueous supernatant was decanted. The resulting slurry was shaken with a 2.5~ aqueous NaOH solution, which was removed by decanting. The nickel catalyst was washed 35 times by suspension in water (100ml) followed by decanting. The washing was repeated 3 times with absolute ethanol (100 ml), and the resulting Raney Nickel was stored under absolute ethanol.

D.F. Weaver

246

(b) Synthesis of 5,5-diphenyl-2- thiohydan toin

(ii) Zinc chelation and transport studies

Benzil(2.00 g, 0.0095 mol) and thiourea (1.22 g, 0.016 mol) were suspended in ethanol (20.0 ml). To this was added potassium hydroxide (2.64 g, 0.047mol) in water (5.0ml). The resulting solution was refluxed for 3 h and after cooling, filtered. The yellow filtrate was acidified by the addition of concentrated hydrochloric acid (12 M, 5.0 ml). The resulting solid was collected by suction filtration, dried under vacuum and recrystallized twice from ethanol to give a white crystalline solid: 1.21 g (45%); mp 235 “C; ‘Hmr (CDCls): 7.3 (lOH,m), 8.9 (lH,s), 10.7 (lH,s); tic: Rf 0.45 (methylene chloridelmethanol, 5/l).

(a) Synthesis of zinc thiocyanate A solution of KSCN (2.61 g, 0.0268mol) in methanol (120.0 ml) and a solution of Zn(NOs)z (4.00 g, 0.0134 mol) in methanol (80.0ml) were mixed. The resulting white precipitate was removed by vacuum filtration and discarded. The methanol filtrate was evaporated to dryness giving an oily solid which, upon trituration with anhydrous ethyl ether gave a quantitative yield of the desired product as a white crystalline solid which was used immediately.

(c) Synthesis of 2-deoxy-5,5-diphenylhydantoin

(b) Zinc chelation/transport

studies

5,5-diphenyl-2-thiohydantoin (5.6 g, 0.02 mol) in ethanol (100ml) was heated under reflux with Raney nickel (15.0ml of the settled slurry) for 6 h. The mixture was filtered through diatomaceous earth (Celite) while hot and the filtrate was concentrated under vacuum. After overnight storage at 4°C the residue crystallized and was recrystallized twice from ethanol giving a white crystalline product: 3.Og (63%); mp: 181-182°C; tic: Rf 0.65 (methylene chloride/acetone/acetic acid, 100/100/0.5). The compound was homogenous on tic. NMR spectroscopy provided appropriate peaks.

In the EBS, the intracellular phase contained Zn(SCN)z (50 mM) in water (80 ml); the extracellular phase was water (80ml); the membrane phase was octanol (40ml), containing either PHT or deoxy-PHT in a concentration of 10m~. The transport rate was determined by periodic sampling of the extracellular phase with subsequent spectrophotometric determination of the zinc thiocyanate. Aliquots (0.50 ml) were obtained from the extracellular phase and mixed with aqueous 0.0133 M FeCls (5.00 ml) producing a characteristic red colour. Unknown concentrations were determined from a Beer’s Law Plot.