WO1990007111A2 - Method for predicting biological activity of antibiotics, and novel non beta-lactam antibacterial agents derived therefrom - Google Patents

Abstract

A molecular model of the interaction of various non β-lactam antibiotics with the active site of a penicillin receptor has been developed so that it is now possible to predict the ''fit'' and ''reactivity'' of potential antibacterial compounds with this receptor. Novel structural types and compounds are defined.

Description

Method for predicting biological activity of antibiotics, and novel non beta-lactam antibacterial agents derived therefrom

This invention relates to novel antibacterial agents and a method for predicting the activity thereof relative to penicillin. More particularly, this application describes a molecular modelling technique for determining the fit and reactivity of candidate compounds with bacterial cell wall receptors, and hence a method for predicting structural types that exhibit activity.

It has been known since the 1940's that β-lactam

antibiotics, such as the penicillins and cephalosporins, are effective by reason of their interference with the integrity of bacterial cell walls. It has also been discovered that the interference is effected by covalent bonding to the active site serine residue of one or more of a group of enzymes termed penicillin binding proteins (PBP's). These enzymes serve to complete bacterial cell wall synthesis by a cross linking of peptidoglycan chains, and are essential to the cells. All known PBP's include a sequence -Ser-X-X-Lys- and the simplest kinetic description of the reaction between a PBP and a β-lactam antibiotic is given in equation 1, below, where A is a generalized structure. Since the PBP is regenerated in the deacylation step, useful antibacterial activity is considered to require k₃/K ≥ 1000 M^-1 sec^-1 and k₄ ≤ 1 x 10^-4 sec^-1. 3 U (

The question is, therefore, what is the correlation, if any, between antibacterial activity and the "lock-and-key" interactions which take place between the PBP and the antibiotic.

It is an object of the present invention to determine the correlation between antibacterial activity and the lock-and-key interactions between PBP's and selected antibiotics and thus provide a means by which the "fit" (Step 1) and

"reactivity" (Step 2) of any selected candidate structure relative to the fit and reactivity of penicillin may be predicted with some degree of quantitative accuracy.

It is another object of this invention to design with this model novel non β-lactam compounds having antibacterial activity.

One aspect of the present invention provides a method of determining the molecular structure of large molecules wherein the strain energy of the molecule is minimized in terms of molecular parameters, characterized in that in order to identify starting parameters for the minimization procedure the one-point energies of a large number of most probable random structures are first calculated, and a predetermined number of said random structures having the lowest energies are selected for said minimization

procedure. Thus by another aspect of this invention there is provided a method for determining fit and reactivity of any selected candidate antibacterial compound comprising (a) simulating the reaction of said compound with a model of a penicillin binding protein which includes a serine-lysine active site, by determining the relative ease of formation of a four-centred relationship between OH of said serine and a

reactive site of said compound; and (b) determining the activation energy for the four-centred reaction of the chemically active functional group of said compound with methanol relative to the activation energy of the

corresponding reaction of methanol with N-methylazetidinone.

Another aspect of this invention provides a non-β-lactam containing compound characterized in that said compound is capable of forming a four-centred transition structure which includes a serine OH group contained in a model of a

penicillin binding protein, reacted therewith; said compound having an activation energy for reaction with methanol not greater than 3 kcal/mol higher than the activation energy exhibited by N-methyl- azetidinone. Another aspect of this invention provides compounds of the formula: Z R

where

X is selected from S, O, CH₂, NH, NR₇, and Se

Y is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, are each hydrogen, alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

β-lactam active side chains are side chains known to be active in β-lactam antibiotics. As used herein, the

substituents acceptable in beta-lactam antibiotics may be any of the wide range of permissible substituents disclosed in the literature pertaining to penicillin and cephalosporin compounds. Such substituents may, for example, comprise a group of the formula

-XQ wherein X represents oxygen or sulfur and Q represents C_1-4 alkyl (e.g., methyl or ethyl), C_2-4 alkenyl (e.g. vinyl or propenyl) or aryl C_1-4 alkyl (e.g., phenyl C_1-4 alkyl such as benzyl).

Such substituents also may be, for example, an unsaturated organic group, for example, a group of the formula

wherein R₁ and R₂ which may be the same or different, and are each selected from hydrogen, carboxy, cyano, C_2-7 alkoxycarbonyl (e.g., methoxycarbonyl or ethoxycarbonyl), and substituted or unsubstituted aliphatic (e.g., alkyl, preferably C₁-C₆ alkyl such as methyl, ethyl, isopropyl or n-propyl). Specific substituted vinyl groups of the above formula include 2-carboxyvinyl, 2-methoxycarbonylvinyl, 2-ethoxycarbonylvinyl and 2-cyanovinyl. Alternatively, the β-lactam acceptable substituent may also be an unsubstituted or substituted methyl group depicted by the formula

-CH₂Y wherein Y is a hydrogen atom or a nucleophilic atom or group, e.g., the residue of a nucleophile or a derivative of a residue of a nucleophile. Y may thus, for example, be derived from the wide range of nucleophilic substances characterized by possessing a nucleophilic nitrogen, carbon, sulfur or oxygen atom. Such nucleophiles have been widely described in the patent and technical literature respecting β-lactam chemistry and are exemplified, for example, in Foxton et al U.S. Patent No. 4,385,177 granted May 24, 1983, at column 4, line 42 - column 8, line 24 and column 34, line 51 - column 36, line 17, the disclosure of which is

incorporated by this reference herein.

Yet another aspect of this invention provides compounds of the formula:

where

X is selected from S, O, CH₂, NH, NR₈, and Se

Y is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ are each hydrogen, alkyl, or aryl, and

R₉ is a a β-lactam active side chain,

and pharmaceutically acceptable salts thereof A further aspect of this invention provides compounds of the formula:

where

X-Y is selected from S-S, CH₂CH₂, S-CH₂, CH₂-S, S-NR₈, NR₈-S ,

CH₂H-O, O-CH₂, O-NR₈, NR₈-O, Se-Se, CH₂-CH₂, and Se-CH₂

Z is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₈ are each hydrogen, alkyl, aryl

R₇ is alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof

A still further aspect of the invention provides compounds of the formula:

where

X is selected from S, O, CH₂, NH, NR₆, and Se

Y is selected from N, CH, and CR₇

Z is OH, NH₂, SH, or NHCOR₉ (when Y=N)

Z is R₁₀ (when Y=CH, or CR₇)

R₁=R₂=R₃=R₄=R₅=R₆=R₇= are each hydrogen, alkyl, or aryl, and

R₉ is a β-lactam active side chain

^R10 ^{= R}11

where R₁₁ is alkyl, or aryl, and

R₁₂=OH, NH₂, NHCOR₉, SH

and pharmaceutically acceptable salts thereof. Another aspect of the invention provides compounds of the formula:

where

X is selected from S, O, CH₂, NH, NR₅, and Se

Y is NR₆- Z, and

R₁, R₂, R₃, R₄, R₅, and R₆ are each H, alkyl, or aryl

Z is OH, SH, NH₂, or NHCOR₇

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof. Preferably,

R₆ is hydrogen and Z is NHCOR₉ where R₉ is lower alkyl and particularly benzyl.

As used herein, the term "alkyl" includes alkyl groups containing up to twenty carbon atoms, preferably C_1-6 alkyl groups, which can optionally be monosubstituted, distributed or polysubstituted by functional groups, for example by free, etherified is esterified hydroxyl or mercapto groups, such as lower alkoxy or lower alkylthio; optionally

substituted lower alkoxycarbonyloxy or lower alkanoyloxy; halogen; oxo; nitro; optionally substituted amino, for example lower alkylamino, di-lower alkylamino, lower

alkyleneamino, oxo-lower alkyleneamino or aza-lower alkyleneamino, as well as acylamino, such as lower

alkanoylamino, lower alkoxycarbonylamino, halogeno-lower alkoxycarbonylamino, optionally substituted phenyl-lower alkoxycarbonylamino, optionally substitutedcarbamoylamino, ureidocarbonylamino or guanidinocarbonylamino, and also sulfoamino which is optionally present in the form of a salt, such as in the form of an alkali metal salt, azido, or acyl, such as lower alkanoyl or benzoyl;

Optionally functionally modified carboxyl, such as carboxyl present in the form of a salt, esterified carboxyl, such as lower alkoxycarbonyl, optionally substituted carbamoyl, such as N-lower alkylcarbamoyl or N, N-di-lower alkylcarbamoyl and also optionally substituted ureidocarbonyl or

guanidinocarbonyl; nitrile; optionally functionally modified sulfo, such as sulfamoyl or sulfo present in the form of a salt; or optionally O-monosubstituted or O, O-disubstituted phosphono, which may be substituted, for example, by

optionally substituted lower alkyl, phenyl or phenyl-lower alkyl, it also being possible for O-unsubstituted or O-monosubstituted phosphono to be in the form of a salt, such as in the form of an alkali metal salt.

As used herein, the term "aryl" includes carbocyclic, hetrocyclic aryl. The carbocyclic aryl includes phenyl and naphthyl, optionally substituted with up to three halogen, C_1-6 alkyl, C_1-6 alkoxy, halo (C_1-6) alkyl, hydroxy, amino, carboxy, C_1-6 alkoxycarbonyl, C_1-6 alkoxycarbonyl-(C_1-6)-alkyl, nitro, sulfonamido, C_1-6 alkylcarbonyl, amido (-CONH₂), or C_{1- 6} alkylamino groups.

The term "heterocyclic" includes single or fused rings comprising up to four hetro atoms in the ring selected from oxygen, nitrogen and sulphur and optionally substituted with up to three three halogen C_3-6 alkyl, C_1-6 alkoxy, halo (C_1-6) alkyl, hydroxy, amino, carboxy, C_1-6 alkoxycarbonyl, C_1-6 alkoxycarbonyl (C_1-6) alkyl, aryl, oxo, nitro, sulphonamido, C_1-6 alkyl-carbonyl, amido or C_1-6 alkylamino groups.

Suitable C_1-6 alkyl groups may be straight or branched chain and include methyl, ethyl n- or iso-propyl, n-, sec-, iso-, or tert-butyl. In those cases where the C_1-6 alkyl group carries a substituent the preferred C_1-6 alkyl groups include methyl, ethyl and n-propyl.

The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows the structure of a model of a penicillin receptor whose docking to penicillins and

cephalosporins leads uniformly to four-centered

interactions between C-O-H of serine and (o) C-N of the penicillin or cephalosporin;

Figure 2 is a stereoscopic view of penicillin V docked to the peptide of Figure 1;

Figure 3 is a stereoscopic view of a ▲³-cephalosporin docked to the peptide of Figure 1;

Figure 4 is a stereoscopic view of a ▲²-cephalosporin docked to the peptide of Figure 1; Figure 5 is a stereoscopic view of a 4-epi-▲²- cephalosporin docked to the peptide of Figure 1; Figure 6 is a close-up view of the four-centred

interaction between C-O-H of serine and (O)C-N of the β-lactam ring which exists in Figure 2;

Figure 7 shows the N-protonated transition structure for the attack of methanol upon the exo face of N- methylazetidinone (ab initio calculation);

Figure 8 is the O-protonated transition structure for the attack of methanol upon the exo face of N- methylazetidinone (ab initio calculation). Figure 9 is a stereoscopic view of the transition structure calculated using MINDO/3 for the reaction of methanol with a penicillin via an N-protonated pathway.

Figure 10 is a stereoscopic view of the transition structure calculated using MINDO/3 for the reaction of methanol with a penicillin via an O-protonated pathway;

Figure 11 is a stereoscopic view of the transition structure for the reaction of methanol with penam via endo-attack;

Figure 12 is a stereoscopic view of the complexation of 5 to the peptide of Figure 1; and

Figure 13 is a stereoscopic view showing the

interaction of a cyclic structure with a model of a penicillin receptor.

Possible structures for peptides (e.g., enzymes), penicillins and cephalosporins were examined using the computer program MMP2(85), which is available from the

Quantum Chemistry Program Exchange (QCPE) at the University of Indiana, Bloomington, Indiana, U.S.A. This program calculates the strain energy of a molecule in terms of the contributions to this energy associated with stretching of bonds, bending of bond angles, torsion about bonds, and electrostatic and van der Waals interactions of non-bonded atoms. To carry out the calculation, the Cartesian

coordinates of all atoms must be entered, and lists of connected and attached atoms defined. If the types of atoms present in the molecule of interest are known, the strain energy can be minimized by the application of the Newton-Raphson procedure to an unconstrained multivariable non-linear function that includes all of the individual

contributions noted above. This function is termed the force field. For the minimization to proceed in a reliable manner it is important that the geometry entered at the beginning of the calculation be reasonably accurate, and close to the bottom of an energy well.

For each different molecule to be examined with MMP2(85), it is first necessary to determine the parameters associated with the types of atoms present within this molecule. These parameters include, inter alia, standard bond lengths and bond angles, and stretching and bending force constants.

Bond lengths and angles are available from compilations of vibrational data, and others can be calculated by molecular orbital (MO) procedures. The general strategy for parameter development can be found in the monograph "Molecular

Mechanics", by U. Burkert and N. L. Allinger, published by the American Chemical Society, Washington, 1982. Since the parameters for peptides (e.g., enzymes), penicillins and cephalosporins to establish the force field required

MMP2(85) were previously unknown, these were first

determined and tested for their ability to reproduce known experimental crystal structures, and known effects of solvent upon the conformations (three-dimensional

structures) of the different structural types. The

parameters are termed PEPCON (Appendix 1) (for peptides), PENCON (Appendix 2) (for penicillins), and CEPARAM (Appendix 3) (for cephalosporins). A second necessary requirement for the use of MMP2(85) is the provision of the initial set of Cartesian coordinates. For small molecules, such as penicillins and cephalosporins, the coordinates of an experimental crystal structure can be used. Minimization with the appropriate parameters then leads to a calculated structure that reproduces the

experimental structure. From this structure it is possible to proceed to other conformations and to the global minimum of the molecule by a series of dihedral drives around each of the dihedral angles of the molecule. This is an option available in MMP2(85), and it works well. However, such a strategy is impractical for the analysis of a peptide because of the very large number of dihedral angles that would have to be examined for any such molecule which contains more than two or three amino acid residues. Therefore, the computer programme ECEPP (Empirical

Conformational Energy Program for Peptides), which is available from QCPE, was modified to allow a random number generator to calculate the one-point energies of 200,000 initial structures containing permutations of the most probable backbone and dihedral angles. The fifty lowest energy structures identified in this manner were read out, minimized using a quadratic minimization procedure, and then converted to MMP2(85) format for final minimization by the Newton-Raphson procedure. The objective of this initial search was to identify suitable starting parameters. This strategy has been tested extensively, works well, and has been applied to the treatment of a PBP, as described below.

More particulalry, in this procedure the strain energy of the molecule is minimized as a function of its dihedral angles with bond lengths and bond angles held constant. The minimization is preceded by a consideration of a subset of the parameters which form a basis for a specific subset of the complete parameter space, and the subset is comprised of the values 0, ±90, 180 degrees for the ∅ and

U dihedral angles of the backbone, and the values -60 and 180 degrees for the first dihedral angle of the side chains. The w dihedral angle and all other side chain dihedral angles are maintained at 180 degrees. Each of the infinite number of points in this parametric subspace corresponds to an

associated molecular strain energy. The subspace is then subjected to a sufficiently rich discrete randomly

distributed uniform mapping so that there is an arbitrarily large probability that, some points (r) are found in a convex neighbourhood of local energy minima, and this set of points (r) is then used for the initialization of the minimization procedure. A reasonable number of points for the randomly chosen discrete subset described above is 200,000 in the case of a polypeptide containing up to 10 amino acid

residues, and the set of points (r) preferably numbers 50. With these procedures in place, an initial series of nine penicillins (1a - 1i) was examined. Of these nine compounds, 1a - 1d are highly active antibiotics widely used in medicine (ampicillin, syncillin, penicillin G, penicillin V) , 1e - 1f are significantly less active, and 1g - 1i are biologically inactive. The conformational analyses of these compounds revealed that antibacterial activity is associated specifically with a three dimensional structure in which the carboxyl group and side chain N-H project onto the convex face, and engage in hydrogen bonding lock-and-key

interactions with the receptor, i.e., the PBP.

1c: R = PhCH₂CO-

1d: R = PhOCH₂CO-

I

Next a conformational analysis was performed on the cephalosporins 2a - 2c. Each of these has the

phenoxyacetyl side chain, and can therefore be compared to penicillin V (1d). The ▲³-isomer 2a is biologically active, but undergoes a facile equilibration with the ▲²-isomer 2b, which is biologically inactive. The reason for the lack of activity of 2b has not previously been established, but it has been suggested that the 4-epi-▲²-isomer 2c would exhibit a better fit to the PBP receptor, and possess antibacterial activity. However, such compounds are also inactive. The reason for this lack of activity is, therefore, also unknown.

Each of 2a - 2c, like the penicillins 1a - 1d, is found to prefer a conformation in which the side chain N-H occupies the convex face of the molecule. As with the penicillins, it can thus be postulated that lock-and-key interactions with the receptor involve primary binding by the carboxyl group and this side chain N-H.

The active site serine D-alanyl carboxypeptidase- transpeptidase of Streptomyces R61 has been crystallized with incorporation of β-lactam compounds, and the crystal structure has been partially solved. The pH-dependence of the same enzyme has also been examined. Both kinds of studies suggest that the carboxyl group of a penicillin is closely associated with the protonated terminal amino group of the lysine residue of X-X-Lys. The crystal structure confirms that, in the complex, the β -lactam ring of penicillin is in close proximity to the active site serine. The pH-dependence study rules out

involvement of a histidine residue in the chemical process, in contrast to the behaviour of chymotrypsin and related serine proteases. This result means that the serine O-H participates in the chemical reaction with the substrate.

These observations suggest that a valid model of the active site of a PBP can be obtained in terms of the amino acids that surround the unique serine residue, i.e., in this case, Val-Gly-Ser-Val-Thr-Lys.

Accordingly, the peptide Ac-Val-Gly-Ser-Val-Thr-Lys-NH-CH₃ was subjected to an ECEPP search of 200,000 initial structures, followed by MMP2(85) refinement of 50 low energy structures identified in this search. One low energy structure having the lysine and serine side chains in proximity was found. This structure is characterized by the set of dihedral angles summarized in Table 1, and is shown as Figure 1.

Table 1 Dihedral angles of the model of the active site of the PBP of Streptomyces R61

The structure of Figure 1 has several features of

interest. The convex face is mainly hydrophobic, and the concave face, which includes the serine and lysine side chains, is mainly hydrophilic. The concave face also contains the amide oxygen of the N-terminal acetyl group. These three sites are noted on Figure 1 as S (serine), L (lysine) and A (acetyl). The existence of a lock-and-key relationship between the concave face of Figure 1 and the previously determined convex face of penicillin and cephalosporin now seems clear. In terms of such a relationship, contact is required between the carboxyl group of the antibiotic and the terminal amino group of lysine, and also between the side chain N-H of the antibiotic and the acetyl oxygen.

The construction of a supermolecule in which the receptor is docked to a substrate through NH₃+...-O₂C and N-H...O=C hydrogen bonds is, therefore, desirable. To obtain the structure and energy of such a supermolecule using the program MMP2(85), it is necessary to devise a procedure for the generation of a starting set of Cartesian

coordinates.

A computer program has been written based on the

following approach to the problem. Let A refer to a receptor molecule containing N, atoms, and B a substrate molecule containing N₂ atoms, which is to be docked to A. It is assumed that the geometries of A and B are known in Cartesian or internal coordinates, and that

transformation between the two types of coordinate systems is possible. A start is thus made with (3N₁-6) and (3N₂-6) predetermined internal coordinates. To describe the geometry of the supermolecule containing (N₁ + N₂) atoms requires 3 (N₁ + N₂) - 6 internal coordinates, i.e., six new internal coordinates must be determined and minimized. These comprise, typically, one bond length, two bond angles, and three dihedral angles, and they may be termed "intermolecular" internal coordinates.

To use the computer program, for which the source code listing is given in Appendix 4, one of the desired hydrogen bonding interactions is selected, and its distance set at 1.7-2.5 Å, a typical intermolecular hydrogen bonding distance. Initial values are then given to the five remaining variables, and the energy is minimized, with the second hydrogen bond distance as a probe. The geometry of the resulting supermolecule, now expressed in Cartesian coordinates, is considered

appropriate for MMP2(85) minimization when the second hydrogen bond distance is 1.7-2.5 Å.

Figures 2-5 show stereoscopic views of the results of docking of the receptor model with, respectively,

penicillin V, ▲³-cephalosporin V, ▲²-cephalosporin V and 4-epi-▲²- cephalosporin V. It can be seen that, in each case, the serine O-H sits on the convex face of the β-lactam compound, in such a manner as to create a four-centred interaction between O-H and (O)C-N. This four-centred interaction is shown in closer detail for

penicillin V in Figure 6.

From the Cartesian coordinates of C-O-H and (O)N-C of the optimized complexes, it is possible to compute the root mean square deviations (rms) in Å of the different four centred interactions, relative to a standard substrate, in this case penicillin V. When this is done for the series of penicillins 1a - 1i, it is found that all active penicillins have rms less than 0.2 Å, and all inactive penicillins have rms greater than 0.4 Å. For the series shown in Figures 2-5, the rms deviations are 0.000, 0.149, 0.338 and 0.148 Å.

This implies that the "fits" of the biologically active ▲³- cephalosporin and the biologically inactive 4-epi- ▲²-cephalosporin to the penicillin receptor are identical. The biologically inactive ▲²-cephalosporin has a poorer fit.

The biological activity of a drug depends not only on its ability to fit to a receptor, i.e., Step 1 of equation 1, but also on its ability to react chemically with the receptor, i.e., Step 2 of equation 1. The chemical reaction suggested by Figures 2-6 is a four centred process in which C7-O(Ser) (see A) and N-H(Ser) bond formation are concerted. This is an unprecedented chemical mechanism.

The hydrolysis and alcoholysis of β-lactam compounds has received much experimental and theoretical attention. In water above pH 8, the rate-determining step is addition to the carbonyl group to form a tetrahedral intermediate; below pH 6, there is rate-determining proton transfer to the β-lactam nitrogen, from the convex face of the molecule. Hydrolysis is extremely slow in the

biologically relevant pH range 6-8, and the possible existence of a molecular (four-centred) mechanism in this region has not been established. Likewise, all previous theoretical studies of β-lactam hydrolysis have emphasized anionic addition to the β-lactam carbonyl group.

Molecular orbital (MO) calculations of the ab initio type represent an accepted and well established procedure for the probing of the mechanisms of chemical reactions.

Such calculations can be performed using low level (STO-3G) and high level (3-21G) basis sets using the computer programs GAUSSIAN 82 and GAUSSIAN 86, available from GAUSSIAN Inc., Pittsburgh, PA, U.S.A. Molecular orbital calculations of the semi-empirical type can be performed on relatively large molecular systems, and are valid once they have been calibrated with respect to an ab initio calculation on the same system. The semi empirical procedures AMI, MNDO and MINDO/3 are available in the computer program AMPAC, available from QCPE.

Table 2 summarizes the ab initio data (

, kcal/mol) for the reactions of N-methylazetidinone with water and with methanol via exo-oriented N- and O- protonated structures. For the hydrolysis reactions, the O-protonated structure is favoured by 1.75 kcal/mol at the lower STO-3G level (STO- 3G//STO-3G). One point

calculations at the more appropriate 3-21G level (3-21G//3-21G) increases the preference for the N-protonated transition structure to 5.66 kcal/mol. Analogous results are seen for methanolysis of N- methylazetidinone. These results prove that the four- centred interaction seen in Figures 2-6 reflects a genuine chemical process and, indeed, the energetically preferred chemical process.

The N- and O-protonated methanolysis transition

structures are shown in Figures 7 and 8, respectively.

Table 2 also summarizes the semi-empirical results for the hydrolysis and methanolysis of N-methylazetidinone, and it is evident that only MINDO/3 correctly reproduces the preference for the N-protonated transition structure. Accordingly, MINDO/3 was used to examine the activation energies for the reactions of a large number of bicyclic azetidinones with methanol. These are summarized in Table 3.

Table 2

Relative

for the Hydrolysis and Methanolysis of N- Methylazetidinone via N- and O-Protonated Transition

Structures . ^a

Within each row of Table 3, the reactions of the

different structural types are compared to that of the parent penam ring system of penicillin, and the data are discussed row-by-row:

Table 3

Calculated

(kcal/mol, MINDO/3) relative to N- Methylazetidinone for the Methanolysis of β-Lactam

Compounds via Exo Formation of a Four-Centred N- Protonated Transition Structure .

(1) the relative reactivities are carbapenam > penem > oxapenam > penam. Oxapenicillins and penems having the C3 and C6 substituents of penicillins are known to have antibacterial activity. Although the carbapenam ring system is known, carbapenicillins have not yet been prepared. (2) in the comparison of the penam and cephem ring systems, the relative reactivities are penam > ▲³-cephem > ▲²-cephem, acetoxymethyl- ▲³-cephem. With a common acylamino side chain, penicillins are an order of

magnitude more active than acetoxymethyl-▲³-cephalosporins and the latter are, in general, an order of magnitude more active than 3-methyl-▲³-cephems; ▲²-cephems are inactive.

(3) introduction of the C3 -carboxyl group enhances the reactivity. It is believed that the carboxyl group assists the methanolysis through hydrogen bonding, because epimerization (C3β) decreases the reactivity significantly.

(4) introduction of C2-methyl substituents decreases the reactivity, unless a C3 -carboxyl group is present.

(5) the 6β-acylamino substituent has almost no effect on the reactivity. Consequently, the chemical reactivity of a penicillin differs only slightly from that of the parent penam. Figures 9 to 11 show, respectively, stereoscopic views of the N- and O-protonated transition structures for exomethanolysis of a penicillin- and O-protonated endomethanolysis of penam. Such endo-oriented transition structures are ca 1 kcal/mol higher in energy than the O-protonated exo-structures and 5-6 kcal/mol higher in energy than the N-protonated exo-structures.

Table 4 summarizes the "fits" of penicillin V and 2a - 2c mentioned above, as well as the "reactivities" of the different ring systems, as given by

for the reaction of methanol with the carboxylated substrates shown. The product rms x

represents a combination of fit and reactivity, and is seen to order correctly the different classes of antibiotics in the order of their biological activities. Based on this quantity, 2b is inactive because of its poorer fit to the receptor, and 2c is inactive because of its decreased reactivity.

The difference between 2b and 2c can be compared to the differences seen in Row 3 of Table 3. That difference is attributed to facilitation of the chemical process by hydrogen bonding of the attacking alcohol to the carboxyl group when this group is on the convex face of the molecule. Thus 2c recovers the fit lost in 2b but concomitantly becomes less reactive. These

considerations suggest that the attachment of a hydrogen bonding donor substituent on the convex face of 2c will restore the chemical reactivity while retaining the acceptable fit to the receptor. Possible sites for the attachment of the required substituent are sulfur, C4 and C7 (see Table 4^d for numbering). Attachment of F, CH₃O and CH₂OH to C4 and C7 in the required manner does not enhance the reactivity of 2c, but an alpha-oriented sulfoxide (3) exhibits reactivity superior to that of penicillin.

Although a malonic acid derivative which combines the favourable properties of 2b and 2c (4) exhibits somewhat reduced reactivity compared to penicillin (

= 3.51 kcal/mol), the product rms x ± is intermediate between

the active and inactive entries of Table 4. Accordingly, 3 and 4 are novel β-lactam containing structural types of potential biological interest. Table 4

Root Mean Square (rms) Difference (A) , relative to

Penicillin V, of the Cartesian Coordinates of the C-O-H Atoms of Serine and the N-C=O atoms of the Azetidinone Ring in the Complexes of β-Lactam Compounds with a Model of the Penicillin Receptor; Activation Energies

(kcal/mol) for the Reaction of Azetidinones with

Methanol, relative to the Penam Nucleus; and the Product rms x

a Refers to MINDO/3 calculations on

b Refers to MINDO/3 calculations on

c Refers to MINDO/3 calculations on

d Refers to MINDO/3 calculations on

It is also possible to design entirely new structural types compatible with the combination of fit and

reactivity developed here. Based on the dihedral angles of penicillin V, a carboxyl group oriented so that it makes a dihedral angle of 150-160° with a "reactive site", and a hydrogen bonding donor such as N-H or O-H oriented so that it makes a dihedral angle of -150 to -160° with the "reactive site" is required. The reactive site should be one that reacts with methanol via a four-centred transition structure, and with E no greater than 3-4 kcal/mol higher than that for the reaction with an azetidinone.

Systematic calculation of activation energies has

identified

the imino moiety

as a functional group possessing the required reactivity, and incorporation of this moiety into a cyclic structure possessing dihedral angles of the required magnitude has identified structure 5 as a

candidate structure having antibacterial activity by a penicillin-cephalosporin mechanism. The result is shown in Figure 12. EXAMPLE 1

Application of PEPCON to the Calculation of the

Polypeptide Crambin

This polypeptide contains 46 amino acid residues, 327 heavy atoms, and 636 atoms including hydrogens. The published crystal structure includes diffraction data refined to 1.5 Å. The Cartesian coordinates of the heavy (non-hydrogen) atoms of this crystal structure were used as input to MMP2(85), hydrogens were added using an option available in MMP2(85), and Newton-Raphson

minimization was performed using PEPCON. The calculated structure shows an rms deviation from the experimental structure of 0.291 Å for the heavy atoms of the backbone, and 0.310 Å for all heavy atoms. EXAMPLE 2

Application of PENCON to the Calculation of Penicillin V

Repetition of the experiment of Example 1, with the

Cartesian coordinates of the crystal structure of

penicillin V and the PENCON parameters leads to an rms deviation of 0.1 Å for all atoms.

EXAMPLE 3

Application of CEPARAM to the Calculation of

Cephalosporin The Cartesian coordinates of the crystal structure of a ▲²-cephalosporin having the phenoxyacetyl side chain were entered, and the energy was minimized using MMP2(85) in conjunction with the CEPARAM

parameters. The resulting rms deviation was 0.35 Å. EXAMPLE 4

Application of the Random Number Strategy and ECEPP to the Conformational Analysis of a Peptide

The peptide Gly-Trp-Met-Asp-Phe-NH₂ was entered into ECEPP, and an initial search was performed on 200,000 initial conformations of this molecule. The fifty lowest energy structures identified in this manner were

minimized in ECEPP using a quadratic minimization

procedure, and then refined using the PEPCON parameters of MMP2(85). One structure was strongly preferred, and the dihedral angles of this structure are identical to those of the gastrin tetrapeptide, which contains the Trp-Met-Asp-Phe-NH₂ moiety of the above compound.

EXAMPLE 5 Calculation of the Structure of a Penicillin Receptor.

The peptide Ac-Val-Gly-Ser-Val-Thr-Lys-NHCH₃ was treated as described in Example 4, and the fifty final structures were examined. Only one structure possessed lysine and serine side chains on the same side of the molecule.

This structure is shown in Figure 1, and its dihedral angles are summarized in Table 1.

EXAMPLE 6

Docking of Penicillin V to a Model of the Penicillin Receptor The receptor model of Example 5 was docked to penicillin V using the computer program of Appendix 4. Several conformations of the penicillin were examined, and the final lowest energy complex is shown in Figure 2 The compounds identified in this manner may thereafter be synthesized in accordance with standard chemical

procedures known to persons skilled in the art.

The invention will be further illustrated by way of the following specific examples of compounds that have been prepared:

EXAMPLE 7: Synthesis of 3-Carboxy-5-Hydroxymethyl-6, 6-Dimethyl-▲⁴-1, 4-Thiazine

In formula I, X = S; Y = OH; R₁ = R₂ = CH₃, R₃ = R₄ = R₅ = R₆ = H. Both D- and L- configurations at C₃ are prepared.

STEP 1

Methyl isopropyl ketone (15 mL, 140 mmoles) was added to a solution of potassium chloride (1.1 g, 14.8 mmoles) in water (9.6 mL). The mixture was stirred, warmed to 60 C, and illuminated with a 350 watt tungsten lamp mounted beside the flask. Bromine (11.9 g, 74.4 mmoles) was then added dropwise. When the colour of the first few drops had disappeared, the heating bath was replaced by a cold water bath, and the 350 watt bulb was replaced by a 60 watt bulb. Addition of bromine was continued at a rate sufficient to maintain the internal temperature at 40- 45ºC. When the addition was complete (25 min) the reaction mixture was allowed to stand for 2h and the organic phase was then separated, washed with water- magnesium oxide and dried over anhydrous calcium chloride. Fractional distillation afforded 7 g of Al, b.p. 82-86°/145 torr. NMR (CDCl₃) 2.36 (3H, s),

1.77 (6H, s).

STEP 2

The bromeketone Al (4.65 g, 28 mmoles) was dissolved in glacial acetic acid (40ml), and freshly recrystallized lead tetraacetate (12.5 g, 28.2 mmoles) was added. The mixture was heated at 100°C, with stirring, for 2 h and cooled to room temperature. Ethylene glycol (2 mL) was then added to destroy unreacted lead tetraacetate. The reaction mixture was diluted with ether (100 mL), washed successively with 10% sodium carbonate, water and saturated sodium chloride, dried and evaporated. The residue was distilled, and the fraction boiling at 57-60°C/120 torr was further purified by chromatography (silica gel, 5% > 10% -> 15% ether-hexane) to give the bromoketoacetate B1. NMR (CDCl₃: 5.16 (2H, s), 2.13 (3H, s), 1.87 (6H, s).

STEP 3

Triethylamine (140 mL) was added to methylene chloride (3 mL). The solution was cooled to -20°C, and gaseous hydrogen sulfide was introduced during 10 min. Then the bromoketoacetate B1 (200 mg), in methylene chloride (1.0 mL), was added dropwise with stirring during 10 min. The yellow solution was diluted with methylene chloride (30 mL), washed successively with 2N hydrochloric acid, waterand saturated sodium chloride, dried over anhydrous sodium sulfate and evaporated to yield the

mercaptoketoacetate C1. NMR (CDCl₃) 5.16 (2H, s), 2.18 (3H, s), 1.57 (6H, s), 1.55(1H, s) .

STEP 4

To triphenylphosphine (258 mg, 0.98 ramole) in dry tetrahydrofuran ( 1 .0 mL) , at -78°C under a nitrogen atmosphere, was added dropwise with stirring a solution of dimethylacetylenedicarboxylate (144 mg, 0.99 mmole) in tetrahydrofuran (1.0 mL). The white slurry as maintained at -78°C for 10 min, and a solution of Boc-L (or D-)-serine (184 mg, 0.90 mole) in tetrahydrofuran (1.0 mL) was added dropwise. The temperature was maintained at - 78°C for 20 min and the reaction mixture was then allowed to warm to room temperature (2h). The solvent was removed and the residue was chromatographed on silica gel. Elution with 15% -> 22% -> 30% -> 35% ethyl

acetate-hexane afforded the beta-lactone D1. NMR (CDCl₃) 5.29 (1H, br), 4.92 (1H, br), 4.34 (2H, br), 1.07 (9H, s).

STEP 5

To a solution of C1 (79.6 mg, 0.45 mmole) in dry degassed dimethylformamide (1.5 mL) was added dropwise a solution of lithium diisopropylamide (0.8 mmole) in

tetrahydrofuran (1.5 mL). The addition was carried out under nitrogen at -60°C. The reaction mixture was allowed to warm to -25°C during 50 min, cooled again to -55°C, and a solution of D1 (56.4 mg, 0.30 mmole) in dry degassed dimethylformamide (0.5 mL) was added dropwise. When the addition was complete, the mixture was warmed to -20°C, stirred for 25 min and then diluted with ethyl acetate (30 mL) and washed with 0.5N hydrochloric acid (2 mL). The aqueous layer was extracted with ethyl acetate (2 x 10 mL) and the combined organic extracts were washed with water (2 x 5 mL) and saturated sodium chloride (1 x 5 mL), dried and evaporated. The oily residue was purified by preparative layer chromatography on a 10 x 20 cm plate coated with silica gel, using methylene chloride-ethyl acetate acetic acid (1.7:0.3:0.05) as eluant to give E1 (77 mg, 70.3%). NMR (CDCl₃) 5.43 (1H, br), 5.20 (1H, d, 18 Hz), 5.04 (1H, d, 18 Hz), 4.46 (1H, br), 2.97 (1H, br), 2.78, 2.74 (1H, dd, 4.5, 9.0Hz), 2.17 (3H, s),

1.48(3H, s), 1.47 (3H, s), 1.44 (9H, s).

STEP 6

The acid E1 (77mg) was dissolved in methylene chloride (10 mL) and treated at 0°C with an ethereal solution of diazomethane. The solvent was removed and the residue was purified on a 5 x 10 cm silica gel plate using hexane-ethyl acetate (1.4:0.6) as eluant to give the ester F1 (48. 2 mg). NMR (CDCl₃) 5.32 (1H, br d), 5.15 (1H, d, 11Hz), 5.07 (1H, d, 11Hz), 4.48 (1H, br, q), 3.76 (3H, s), 2.91 (1H, q, 4, 12Hz), 2.74 (1H, q, 5.5, 12 Hz), 1.47 (6H, d), 1.44 (9H, s).

STEP 7

The ester Fl (46 mg), in tetrahydrofuran (1 mL) was treated at room temperature with 0.25 M lithium hydroxide (0.4 mL). After 25 min an additional 0.4mL of lithium hydroxide was added. The mixture was stirred for 35 min and then diluted with ethyl acetate (10 mL) and washed with 0.5 N hydrochloric acid (2 x 5 mL). The aqueous layer was extracted with ethyl acetate (2 x 5 mL) and the combined organic extracts were washed with water (1 x 5 mL), followed by saturated sodium chloride (1 x 5 mL), dried and evaporated. The residue was dissolved in the minimum of methylene chloride, treated with ethereal diazomethane, concentrated, and the residue was purified on a 10 x 20 cm silica gel plate. Elution with hexaneethyl acetate (1.4 : 0.6) gave Gl (14.4 mg). NMR (CDCl₃) 5.22 (1H, br), 4.58 (2H, d), 4.48 (1H, br), 3.75 (3H, s), 3.06 (1H, br), 2.92 (1H, br), 2.74 (1H, dd, 5, 11Hz), 1.46 (9H, s), 1.44 (6H, s).

STEP 8

To a solution of G1 (5 mg, 0.015 mmole) in freshly dried pyridine (0.2 mL) were added successively silver nitrate (3.4 mg, 0.02 mmole) and t-butyldiphenylchlorosilane (6.3 mg, 0.023 mmole). The solution was stirred for 15 min at room temperature under nitrogen. The solvent was then removed and the product was purified by preparative layer chromatography to give H1 (5.5 mg). NMR (CDCl₃) 7.69 (4H, m), 7.41 (6H, m), 5.07 (1H, br), 4.70 (2H, s), 4.41 (1H, br), 3.72 (3H, s), 2.70 (1H, dd), 2.55(1H, dd), 1.43(9H, s), 1.28(3H, s), 1.26(3H, s), 1.10 (9H, s).

STEP 9

The silyated ester H1 (5 mg) was treated at room

temperature with formic acid (0.2 mL). After 33 min the reaction mixture was frozen and the solvent was removed by lyophilization to yield the enamine II. NMR

(CDCl₃):7.69 (4H, m), 7.40 (6H, m), 5.90 (1H, s), 4.65 (1H, br), 3.79 (3H, s), 3.76 (1H, br), 3.17 (1H, dd, 10, 15Hz), 3.00(1H, dd,3,15 Hz), 1.49(3H, s), 1.31(3H, s), 1.08(9H, s).

STEP 10

The thiazine I1 was treated with lithium hydroxide, as described in Step 7, to remove the ester protecting group. The silylated protecting group was also removed in part to afford a reaction mixture which contained 3-carboxy-5-hydroxymethyl-6,6-dimethyl ▲⁴-1,4-thiazine. EXAMPLE 8: Synthesis of 3-Carboxy-5-(2-Hydroxypropyl)-6,6-Dimethyl- ▲⁴-1,4-Thiazine

In formula II, X=S; Y=OH; R₁=R₂=CH₃; R₃=R₄=R₅=R₆=H; R₇=CH₃. Both D- and L- configuration at C3 are prepared, but the R- and S- epimers at C8 have not been separated; the D-isomer is active.

STEP 1

A solution of ethyl 2-methylcyclopropanecarboxylate (5.0 g, 38.9 mmoles) in dry ether (5 mL) was added dropwise, with stirring under nitrogen, to the Grignard reagent prepared from magnesium turnings (1.935 g, 0.080 g-atom) and methyl iodide (12.43 g, 87.6 mmoles) in dry ether (42mL). The addition required 30 min; stirring was continued for 2.75 h at room temperature and then for 2 h under reflux. The reaction mixture was cooled in an ice- bath and saturated ammonium chloride (10mL) was added, with stirring. The layers were separated and the aqueous layer was extracted with ether (2 x 20mL). The combined organic phase was dried, evaporated and the residue distilled at 132-136°C to give the tertiary alcohol A2 (4.24 g, 95%)

STEP 2

To the alcohol A2 (4.24 g, 37 mmoles) cooled in an ice-bath, was added ice-cold 48% hydrobromic acid (15 mL). The mixture was shaken vigorously in the ice-bath for 30 min. The two layers were then separated, the aqueous layer extracted with hexane (2 x 20 mL), and the combined organic phase was washed successively with saturated bicarbonate (2 x 10 mL), water (2 x 10 mL) and saturated sodium chloride (2 x 10 mL), dried over anhydrous sodium sulfate, and evaporated. Distillation afforded 3.72 g (60%) of the bromide B2, b.p. 46-54°C/10 torr.

STEP 3

To a solution of the bromide B2 (3.72 g, 21 mmoles) in glacial acetic acid (20mL) was added potassium acetate (3.1g, 31.6 mmoles). The mixture was heated under reflux for 12 h, cooled, and poured into water (30mL).

Extraction with ether (3 x 30 mL), followed by successive washing of the organic phase with saturated sodium carbonate, water and saturated sodium chloride, drying, and evaporation at room temperature yielded the acetate C2, 2.82 g (85%). NMR CDCl₃) 5.10 (1H, brt), 4.88 (1H, q, 6Hz), 2.30 (1H, m), 2.19 (1H, m), 2.02 (3H, s), 1.71 (3H, br s), 1.62 (3H, br s), 8.00 (3H, d, 6Hz).

Step 4

The acetate C2 (320 mg, 2.05 mmoles) was dissolved in methanol (2mL) and treated dropwise with a 1.5 M solution of potassium hydroxide in methanol (1.38 mL). The reaction mixture was allowed to stand for 6h and was then neutralized with 1.5 M methanolic hydrogen chloride, and the solvent was removed. The residue was dissolved in methylene chloride, and this solution was washed

successively with water and saturated sodium chloride, dried and evaporated to give the alcohol D2 (208 mg, 99%).

STEP 5A

The alcohol D2 (312 mg, 2.73 mmoles) was dissolved in dimethylformamide (2mL) and to this solution were added successively t-butyl dimethylchlorosilane (535mg, 3.55 mmoles). The mixture was stirred for 2h and then

filtered. The insoluble material was triturated with ether (20mL) and the combined organic material was washed successively with saturated sodium bicarbonate, water and saturated sodium chloride, dried and evaporated to give the silyated compound E2A (620 mg, 100%).

STEP 5B

The alcohol D2 (25mg, 0.22 mmole) was dissolved in dimethylformamide (0.2mL), and the solution was treated successively with pyridine (27 μl, 0.33 mole), t-butyldiphenylchlorosilane (90 μL, 0.35 mmole) and silver nitrate (56mg, 0.33 mmole). The mixture was stirred at room temperature for 4 h, and the product was then isolated, as described in Step 5A, to yield E2B.

STEP 6A

The olefin E2A (624mg, 2.73 mmoles) was dissolved in acetone (3mL) and 18-crown-6 (100mg, 0.27 mmole) and acetic acid (0.16mL) were added successively followed, dropwise, by a solution of potassium permanganate (603mg, 3.82 mmoles) in water (7.5mL). The mixture was stirred for 1 hr and then diluted with methylene chloride (50mL). The organic phase was washed successively with 20% sodium bisulfite, 0.5 N hydrochloric acid, saturated sodium bicarbonate, water and saturated sodium chloride, dried and evaporated. The residue was subjected to flash chromatography on silica gel (7g). Elution with 4 -> 15% ethyl acetate-hexane gave 479 mg (70%) of the ketol F2A.

STEP 6B

The olefin E2B (77.5mg, 0.22 mole) was oxidized with potassium permanganate, as described in Step 6A, to yield the ketol F2B. NMR (CDCl₃) : 7.72 (4H, m), 7.43 (6H, m),

4.43 (1H, q, 6Hz), 3.81 (1H,S), 2.81 (1H, dd, 5, 16Hz),

2.58 (1H, dd, 7, 16Hz), 1.31 (3H, s), 1.29 (3H, s), 1.10

(3H, d, 5Hz), 1.04 (9H, s)

STEP 7A

To a solution of the ketol F2A (478 mg, 1.83 mmoles) in methylene chloride (6mL) were added successively

triethylamine (0.76 mL, 4.0 mmoles) and methanesulfonyl chloride (0.24mL, 3.1 mmoles). The reaction mixture was stirred for 5h at room temperature and then diluted with methylene chloride (80mL). The solution was washed successively with water, 0.5 N hydrochloric acid,

saturated sodium bicarbonate, water and saturated sodium chloride, dried and evaporated. Flash chromatography on silica gel (3g) and elution with 7% -> 8% -> 9% -> 10% ethyl acetate-hexane gave G2A (432 mg, 70%).

STEP 7B

The ketol F2B (277mg, 0.72 mmole) was converted into the mesylate G2B (233mg), as described in Step 7A. NMR

(CDC1₃) 7.71 (4H, m), 7.41 (6H, m), 4.44 (1H, dd), 3.08 (3H, s), 2.95 (1H, dd, 6, 18Hz), 2.27 (1H, dd, 7, 18Hz), 1.63 (3H, s), 1.61 (3H, s), 1.15 (3H, d, 6Hz), 1.06 (9H, s )

STEP 8A

Methylene chloride (5mL) was saturated with hydrogen sulfide at -20°C, and triethylamine (0.14 mL, 1 mmole) and a solution of the mesylate G2A (233mg, 0.5 mmole) were added successively. The solution was stirred for 10 min at -20°C and for 45 min at -20°C -> 0°C, and was then diluted with methylene chloride (30mL), washed

successively with 0.5 N hydrochloric acid, water and saturated sodium chloride, dried and evaporated to give, after drying at 0.1 torr, the mercaptan H2A (170mg, 85%). NMR(CDCl₃) 4.37 (1H, m), 2.98 (1H, dd, 5, 11Hz), 2.63 (1H, dd, 4, 11Hz), 1.98 (1H, s), 1.49 (3H, s), 1.48 (3H, s), 1.17 (3H, d, 5Hz), 0.84 (9H, s), 0.05 (3H, s), 0.01 (3H, s)

STEP 8B

The mesylate G2B was converted into the mercaptan H2B as described in Step 8A. NMR (CDCl₃) 7.71 (4H, m), 7.40 (6H, m), 3.00 (1H, dd, 6, 16Hz), 2.75 (1H, dd, 7, 16Hz), 1.93 (1H, s), 1.46 (3H, s), 1.45 (3H, s), 1.13 (3H, d, 6Hz), 1.05 (9H, s)

STEP 9

Under nitrogen, the raercaptan H2A ( 100mg, 0.36 mole ) was dissolved in degassed dimethylorraamide ( 1 .0 mL) . The solution was cooled to -55°C and treated with 0.45mL of a solution of lithium diisopropylamide prepared from n-butyllithium (0.8mL of a 1.6M hexane solution) and diisopropylamine (0.36mL, 0.259g, 2.56 mmoles) in

degassed tetrahydrofuran (0.8mL). The reaction mixture was stirred at -45°C for 30 min, and a solution of the beta-lactone D1 (D- or L) (56.8mg, 0.30 mole) in degassed dimethylformamide (0.8mL) was added. The mixture was stirred at -30°C for 20 min and then diluted with

methylene chloride (10mL) and washed with 0.5N

hydrochloric acid. The aqueous layer was extracted with methylene chloride (2 x 5mL) and the combined organic extracts were washed with water, then saturated sodium chloride, dried and evaporated. The residue was dried under high vacuum and purified by flash chromatography (silica gel, 4g; 0% -> 8% ethyl acetate-methylene

chloride (1% acetic acid)) to give the coupled product I2D or I2L I2D (88.6%, [ ]_D -2.27 (c 0.1, chloroform)). NMR (CDCl₃) (one isomer) 5.28 (1H, br t), 4.48 (1H, br) 4.32 (1H, m), 2.83, 2.71 (2H, m), 2.71, 2.62 (2H, m),

1.44 (9H, s), 1.43 (6H, s), 1.16 (3H, d, 6Hz), 0.85 (9H, ), 0.05 (3H, s), 0.00 (3H, s). The nmr spectrum shows a 1:1 mixture of epimers in the 2-hydroxypropyl side chain.

I2L ( 83% , [ ] _D +2 .33 (c 0. 1 , chloroform) ) . NMR (CDCl₃) (one isomer) 5 .28 ( 1H, br t) , 4.48 ( 1H, br) , 4.32 ( 1H, m), 2.86, 2.79 (2H, m), 2.70, 2.61 (2H, m), 1.43 (9H, s), 1.42 (6H, s), 1.16 (3H, d, 6Hz), 0.83 (9H,s), 0.04 (3H, s), 0.00 (3H, s). The nmr spectrum shows a 1:1 mixture of epimers in the 2-hydroxypropyl side chain.

STEP 10A

To I2D (22.7 mg, 0.049 mmole) was added formic acid

(0.3mL). The solution was shaken for 20min at room temperature and the solvent was then removed by

lyophilization. The residue was dissolved in a mixture of ether (3 mL) and water (lmL). The ether phase was extracted with water (lmL), and the combined aqueous phase was neutralized with 5% sodium bicarbonate and lyophilized to give 2 (5mg, 40%) having the D-configuration at C3, as a mixture of epimers in the 2-hydroxypropyl side chain. NMR (D20) 4.23 (1H, m), 3.80 (1H, m), 3.30 (1H, q), 2.70-2.85 (3H, m), 1.40 (6H, s), 1.15(3H, d).

STEP 10B

The procedure of Step 10A was repeated on 12L to give 2 having the L-configuration at C3

EXAMPLE 9 Bioassy of 2.-D

The compound was assayed for antibacterial activity on plates inoculated either with Sarcina lutea or

Escherichia coli. In the former case, penicillin G was employed as a standard. In the latter case, Cephalexin was employed as the standard. The compound was found to be 800 times less active than penicillin G, and 10 times less active than Cephalexin. The L-isomer of 2. was found to be inactive in both assays. EXAMPLE 10 Synthesis of 2-Thia-4-Carboxy-6-(2-Hydroxypropyl)-7,7-Dimethyl- 5-1,5-Thiazepine. In formula III, X-Y = S-S; Z = OH; R₁=R₂=R₇=CH₃;

R₃=R₄=R₅=R₆. Both D- and L- configurations of C4 are prepared, but the R- and S - isomers at C9 have not been separated. The L-isomer is active (figure 13)

L-Cysteine hydrochloride (4.1mg, 0.026 mmole) was dissolved in 90% methanol-water (0.35mL), and a solution of the mercaptan H2A (Example 2, Step 8A) (7.1 mg, 0.026 mmole) in methanol (0.35mL) was added, followed by iodine (6.5mg, 0.026 mmole) and triethylamine (7 μL, 0.050 mmole). The reaction mixture was left for 30 min at room temperature and the solvent was then removed under reduced pressure. The residue was partitioned between pH 7 phosphate buffer (containing one drop of 10% sodium thiosulfate) and methylene chloride. The aqueous layer was extracted with ethyl acetate (1 x 5 mL) and

lyophilized. The residue was triturated with methanol, and the methanol extract was combined with the methylene chloride and ethyl acetate extracts and evaporated. The product was purified on a 10 x 15 cm alumina plate using methylene chloride-methanol-water (1.8 : 0.2 : 0.15) as eluant to give the disulfide A4-L (8.9mg, 80%). NMR (D₂O) : 4.19 (1H, m), 3.92 (1H, dd, 3.7 Hz), 3.18 (1H, m), 3.04 (1H, m), 2.92 (1H, m), 2.76 (1H, m), 1.43 (6H, s), 1.12 (3H, d, 7Hz). The compound is a mixture of epimers in the 2-hydroxypropyl chain.

Repetition of this experiment using D-cysteine in place of L-cysteine gave A4-D.

The acids A4-D and A4-L were dissolved in water

containing sodium bicarbonate and assayed for

antibacterial activity by plate assay using S. lutea. A zone of inhibition was observed with the L-isomer, but not with the D-isomer. The inhibition is ascribed to the formation of the cyclic structure 3L, whose interaction with the model of the penicillin receptor is shown in Figure 13.

EXAMPLE 11: Synthesis of 3-Carboxy-5-Oximino-1,4-Thiazine In formula IV, X=S; R₁= R₂=H; R₃=R₄=R₉=H; X=N; Z =OH.

Both D- and L- isomers are described.

STEP 1

To a solution of D-cysteine (605.8mg, 5 mmoles) in methanol (10mL) were added successively ethyl

bromoacetate (0.99g, 5.95 mmoles) and triethylamine

(1.4mL, 1.02g, 10 mmoles). The solution was stirred for 20 min at room temperature and ether (20mL) was then added. The product was collected by filtration, washed with ether and dried. Five hundred mg of this material were suspended in dimethylformamide (5mL), and p- toluenesulfonic acid (458mg, 2.41 mmoles) was added. The resulting solution was treated portionwise with

diphenyldiazomethane until the color of the diazo

compound persisted, and the reaction mixture was stirred overnight. It was then diluted with ether (20mL) and extracted with water (2 x 10mL). The aqueous extract was made alkaline by addition of saturated sodium carbonate, and was then extracted with ethyl acetate (3 x 10mL). The combined organic extracts were washed with water and saturated sodium chloride, dried and evaporated. A 370- mg portion of the residue (0.99 mmole) was dissolved in 1,4-dioxane (8mL), 2-pyridone (47 mg, 0.49 mole) was added, and the solution was heated under nitrogen at 102°C for 7 h. Additional 2-pyridone (23.5 mg, 0.25 mmole) was then added and heating was continued for 4 h. At this time the solvent was removed under reduced pressure and the residue was purified on 15 g of silica gel. Elution with 8% ethyl acetate-hexane afforded 252 mg (78%) of the thiazinone benzhydryl ester A5-D. NMR (CDCl₃) 7.34 (10H, m), 6.96 (1H, s), 6.48 (1H, s), 4.46 (1H, m), 3.33 (2H, s), 3.21 (1H, dd, 4, 15Hz), 2.98 (1H, dd, 9, 15Hz).

STEP 2

The thiazinone ester A5-D (252 mg, 0.77 mmole) was dissolved in dry tetrahydrofuran (5mL) under nitrogen, and the reagent prepared from phosphorous pentasulfide and diphenyl ether according to Tetrahedron Letters 3815 (1983) (244mg, 0.46 mole) was added. The solution was stirred for 35 min, concentrated, and the residue was purified on silica gel (8g). Elution with 15% ethyl acetate-hexane afforded 214 mg (81%) of the thioamide B5- D. NMR (CDCl₃) 8.59 (1H, s), 7.35 (10H, m), 6.98 (1H, s), 4.39 (1H, m), 3.79 (2H, s), 3.32 (1H, dd, 4, 15Hz), 3.02 (1H, dd, 8, 15Hz).

STEP 3

The thioamide B5-D (80mg, 0.23 mmole) was dissolved with stirring in ice-cold dry tetrahydrofuran (92mL) under nitrogen and sodium hydride (80%, 8.4mg, 0.28 mmole) was added. After 5 min stirring in an ice-bath, the reaction mixture was treated with 30 μL (0.48 mmole) of methyl iodide. Reaction was complete after 25 min. Dilution with ether, followed by successive extraction with water, saturated sodium bicarbonate and saturated sodium

chloride, drying and evaporation gave a product which was purified on silica gel (3g). Elution with 10% ethyl acetate-hexane afforded 59.1mg (75%) of the

thiomethylimine C5-D. NMR (CDCl₃) 7.35 (10H, m), 6.96 (1H, s), 4.53 (1H, m), 3.27 (1H, dd, 5, 18Hz), 3.15 (1H, dd, 5, 18Hz), 2.99 (1H, dd, 3, 13Hz), 2.81 (1H, dd, 4, 13Hz), 2.37 (3H, s).

STEP 4

The thiomethylimine C5-D (59 mg, 0.165 mole) was

dissolved in tetrahydrofuran (0.5mL) and added to a solution prepared under nitrogen from hydroxylamine hydrochloride (68.8mg, 0.99 mmole) and 1.65 M methanolic sodium methylate (0.3 mL, 0.5 mmole) in methanol (0.7mL). The reaction was complete in 10 min. The mixture was diluted with methylene chloride (10mL), washed

successively with saturated sodium bicarbonate, water and saturated sodium chloride, dried and evaporated.

Chromatography on silica gel (1.5g) and elution with 12% ethyl acetate-methylene chloride gave 52.7 mg (94%) of the oximino ester D5-D. NMR (CDCl₃) 7.34 (11H, m), 6.93. (1H, s), 5.97 (1H, s), 4.28 (1H, m), 3.30 (1H, d, 13Hz), 3.21 (1H, dd, 3, 13Hz), 3.16 (1H, d, 13Hz), 3.08 (1H, dd, 7, 13Hz).

STEP 5

The ester D5-D (47 mg) was dissolved in formic acid

(lmL). After 5h at room temperature the reaction mixture was frozen and the solvent removed by lyophilization.

The residue was partitioned between ether and water, the ether layer was extracted once with water, and the combined aqueous extracts were lyophilized again to yield 4-D. NMR (D₂O) : 4.15 (1H, m), 3.50 (1H, d, 14Hz), 3.34 (1H, d, 14Hz), 3.15 (1H, dd, 6, 15Hz), 3.02 (1H, dd, 6,

15 Hz)

The L-enantioraer of 4 was prepared as described above, but starting with L-cysteine in place of D-cysteine .

Antibacterial activity was observed on the D-isomer.

EXAMPLE 12: Synthesis of 3D-Carboxy-5-Phenylacetylhydrazil-▲⁴-Thiazine.

A solution of thiomethylimine C5-D (11 mg, 0.031 mmole) and phenylacetic hydrazide (9.2 mg, 0.062 mmole) was stirred overnight under nitrogen in methylene chloride (0.6 mL). The reaction mixture was purified by

preparative layer chromatography on silica gel to give the adduct P (14mg, 98%). NMR (CDCL₃): 7.34 (1H, m), 6.89 (1H, s), 6.55 (1H, br s), 4.24 (1H, br s), 3.78 (2H, s) 3.55 (1H, br, s), 3.33 (1H, d, 15 Hz), 3.17 (1H, d, 15 Hz), 3.05 (2H, br).

The adduct P (10 mg, 0.022 mmole) was treated with formic acid (0.4 mL). The solution was allowed to stand at room temperature for 5 h and the solvent was then removed by lyophilization. The residue was partitioned between ether (0.2 mL) and water (0.2 mL). The ether layer was extracted once with water (0.2 mL), and then the combined aqueous phase was freeze dried to give the product Q. (3 mg, 47%). NMR (D₂O, NaHCO₃) : 8.33 (1H, s), 7.28 (5H, m), 3.98 (1H, m), 3.55 (2H, s) 3.35 (1H, d, 17.5 Hz), 3.12 (1H, d, 17.5 Hz), 3.11 (1H, br d, 15 Hz), 2.83 (1H, br d, 15 Hz), 2.83 (1H, s).

The L-isomer QL was prepared in the same way, starting with C5-L.

APPENDIX I

PEPCON

159 39 9 123 39 78.50

5 6 1 5 0.00 0.00 0.30

5 3 9 1 0.00 15.00 0.00

3 9 1 6 0.00 0.00 0.00

9 1 6 5- 2.50 3.00 -1.00

6 1 9 14 0.00 0.00 0.00

1 1 1 1 0.20 0.27 0.09

1 1 1 3 0.26 0.00 0.06

1 1 1 5 0.00 0.00 0.27

1 1 1 8 0.00 0.00 0.06

1 1 1 9 0.00 0.00 0.06

1 1 1 13 0.00 0.00 0.40

1 1 1 15 0.00 0.00 0.80

1 1 1 19 0.00 0.00 0.60

1 1 2 2 0.00 0.00 0.10

1 1 3 7 0.00 0.00 -0.04

1 1 3 9 0.00 0.00 -0.04

1 1 3 11 0.00 0.00 -0.09

1 1 3 12 0.00 0.00 -0.20

1 1 4 4 0.00 0.00 0.10

1 1 4 26 0.00 0.00 0.10

1 1 4 27 0.00 0.00 0.10

1 1 6 21 0.00 0.00 0.30

1 1 8 23 0.00 0.00 0.30

1 1 9 1 0.00 0.00 0.12

1 1 9 3 0.00 0.00 0.06

1 1 9 14 0.00 0.00 0.06

1 1 13 23 0.00 0.00 0.20

1 1 15 1 0.00 0.00 0.70

1 1 15 15 0.00 0.00 0.80

1 1 15 25 0.00 0.00 0.20

1 1 19 2 0.00 0.00 0.05

1 1 19 23 0.00 0.00 0.05

1 2 2 2 0.00 3.50 0.00

1 2 2 5 0.00 3.50 0.00

1 3 9 1 1.80 6.49 0.00

1 3 9 1 1.80 19.00 0.00

1 3 9 14 0.00 2.66 0.00

1 3 12 24 0.60 0.00 3.20

1 4 4 5-0.30 3.80 0.00

1 4 4 26 0.00 3.50 0.00

1 4 26 4 0.00 5.00 0.00

1 4 26 23 0.00 5.00 0.00

1 4 27 2-0.30 3.80 0.00

1 4 27 27 0.00 3.50 0.00

1 9 1 3 0.00 0.00 0.12

1 9 1 5 0.00 0.00 0.12

1 9 3 7 0.00 7.19 0.00

1 15 1 5 0.00 0.00 0.70

1 15 15 1 0.00 -7.60 1.70 1 19 2 8 0.00 4.50 0.00 1 19 2 19 0.00 4.50 0.00 2 1 1 3 0.26 0.00 0.06 2 1 1 5 0.00 0.00 0.27 2 1 1 8 0.00 0.00 0.06 2 1 1 9 0.00 0.00 0.06 2 1 1 13 0.00 0.00 0.06 2 1 3 7 0.00 0.00 -0.35 2 1 3 9 0.00 -0.50 -1.70 2 2 1 3 0.00 0.00 0.90 2 2 1 5 0.00 0.00 0.05 2 2 2 2- 0. 30 3.80 0.00 2 2 2 5 0.00 3.50 0.00 2 2 2 6 0.00 3.80 0.00 2 2 2 27 -0 .30 3 . 80 0. 00 2 2 6 21 0.00 1.80 0.00 2 2 27 4 0.00 3.50 0.00 2 2 27 26 0.00 3.50 0.00 2 2 27 27-0.30 3.80 0.00 2 19 1 5 0.00 0.00 0.05 2 27 4 4 0.00 3.50 0.00 2 27 26 4 0.00 5.00 0.00 2 27 26 23 0.00 5.00 0.00 2 27 27 2- 0.30 3.80 0.00 2 27 27 4 0.00 3.50 0.00 2 27 27 26 0.00 3.50 0.00 3 1 1 3 0.26 0.00 0.06 3 1 1 4 0.26 0.00 0.06 3 1 1 5 0.00 0.00 0.16 3 1 1 6 0.26 0.00 0.06 3 1 1 8 0.00 0.00 0.06 3 1 1 9 0.00 0.00 0.08 3 1 1 13 0.00 0.00 0.06 3 1 1 15 0.26 0.00 0.06 3 1 8 23 0.00 0.00 0.303 1 9 3 0.00 0.00 0.06 3 1 9 14 0.00 0.00 0.06 3 1 13 23 0.00 0.00 0.30 3 9 1 5 0.00 0.00 0.064 1 1 5 0.00 0.00 0.274 1 1 8 0.00 0.00 0.06 4 1 1 9 0.00 0.00 0.06 4 1 1 13 0.00 0.00 0.06 4 44 1 5 0.00 0.00 0.104 44 26 4 0.00 5.00 0.00 4 44 26 23 0.00 5.00 0.004 4 4 26 27 0.00 5.00 0.004 44 27 27- 0.30 3.80 0.004 26 4 5 0.00 5.00 0.004 26 4 26 0.00 5.00 0.004 26 27 27 0.00 5.00 0.004 27 2 5- 0. 30 3.80 0.00 4 27 27 26- 0 .30 3.80 0.005 1 1 5 0.00 0.00 0.24 5 1 1 6 0.00 0.00 0.50 5 1 1 8 0.00 0.49 0.16 5 1 1 9 0.00 0.49 0.16 5 1 1 13 0.00 0.00 0.40 5 1 1 15 0.00 0.00 0.40 5 1 1 19 0.00 0.00 0.30 5 1 3 7 0.00 0.00 -0.04 5 1 3 9 0.00 0.00 -0.04 5 1 3 11 0.00 0.00 -0.09 5 1 3 12 0.00 0.00 -0.06 5 1 4 26 0.00 0.00 0.10 5 1 4 27 0.00 0.00 0.10 5 1 6 21 0.00 0.00 0.30 5 1 8 23 0.00 0.00 0.30 5 1 9 14 0.00 0.00 0.06 5 1 13 23 0.00 0.00 0.20 5 1 15 15 0.00 0.00 0.80 5 1 15 25 0.00 0.00 0.50 5 1 19 23 0.00 0.00 0.05 5 2 2 5- 0.30 3.80 0.00 5 2 2 6- 0.30 3.80 0.00 5 2 2 27 0.00 3.50 0.00 5 2 27 26 0.00 3.50 0.00 5 2 27 27 0.00 3.50 0.00 5 3 9 14 0.00 2.33 0.0 5 4 4 26 0.00 3.50 0.00 5 4 4 27 0.00 3.50 0.00 5 4 26 23 0.00 5.00 0.00 5 4 26 27 0.00 5.00 0.00 6 1 1 8 0.00 0.00 0.06 6 1 1 9 0.00 0.00 0.06 6 1 1 13 0.00 0.00 0.06 7 3 1 8 0.00 0.00 -0.04 7 3 1 9 0.00 0.00 -0.04 7 3 1 13 0.00 0.00 -0.04 7 3 9 14 0.00 2.66 0.00 7 3 12 24 0.00 0.00 3.10 8 1 1 15 0.00 0.00 0.06 8 1 3 9 0.00 0.00 -0.09 8 1 3 11 0.00 0.00 -0.09 8 1 3 12 0.00 0.00 -0.06 9 1 1 15 0.00 0.00 0.06 9 1 3 9 0.00 0.00 -0.04 9 1 3 11 0.00 0.00 -0.09 9 1 3 12 0.00 0.00 -0.06 9 3 1 13 0.00 0.00 -0.0611 3 1 13 0.00 0.00 -0.0912 3 1 13 0.00 0.00 -0.06 13 1 1 15 0.15 0.00 0.1019 2 19 23 0.00 4.50 0.00 19 2 8 23 0.00 4.50 0.00 23 19 2 8 0.00 4.50 0.00 23 26 4 26 0.00 5.00 0.0023 26 27 27 0.00 5.00 0.00 26 4 4 26-0 30 3.80 0.0026 4 4 27 0 00 3.80 0.0 1 1 4.40 1.525 1.510

1 2 4.48 1.504

1 3 4.75 1.526

1 4 4.40 1.504

1 5 4.60 1.081

1 6 5.36 1.425

1 8 5.10 1.461

1 9 5.47 1.452

1 13 5.15 1.472

1 15 3.21 1.815

1 19 5.27 1.460

5 6 4.60 0.968

2 2 9.60 1.382

2 5 4.60 1.101

2 6 6.20 1.381

2 8 5.10 1.331

2 19 5.10 1.331

2 27 6.51 1.400

3 5 4.8 1.103

3 6 5.05 1.330

3 7 10.01 1.229

3 9 7.74 1.330

3 11 5.11 1.250

3 12 5.05 1.328

6 21 4.60 0.968

4 4 7.19 1.371

4 5 4.60 1.101

4 26 5.69 1.394

4 27 5.39 1.459

8 23 6.10 1.015

9 14 5.78 0.991

12 24 7.20 0.972

13 23 6.03 1.023

15 15 3.10 2.024

15 25 3.80 1.354

19 23 5.95 1.007

23 26 6.05 1.010

26 27 5.94 1.380

27 27 6.21 1.419

1 1 0.000

1 2 0.100

1 3 -1.020

1 4 -0.180

1 5 0.000

1 6 2.530

1 8 3.980

1 9 2.650

5 6 -1.960

1 13 1.720

1 15 -0.671

1 19 2.058

2 2 0.000 2 5 -0.058

2 6 0.810

2 8 4.524

2 19 3.260

2 27 -0.700

3 5 0.0

3 6 0.000

3 7 3.010

3 9 3.320

3 11 3.950

3 12 1.850

6 21 -1.960

4 4 0.000

4 5 0.000

4 26 2.120

4 27 1.120

8 23 -1.410

9 14 -1.810

12 24 0.000

13 23. -1.350

15 15 0.000

15 25 0.000

19 23 -2.270

23 26 1.430

26 27 -1.030

27 27 0.000

4 0.044 1.940

11 0.066 1.780

12 0.050 1.740

13 0.030 1.900

14 0.017 0.930

19 0.055 1.820

5 0.036 1.250

6 0.055 1.820

7 0.044 1.940

1 1 1 0.45 110.30 1 1 1 1 0.45 111.20 2 1 1 1 0.45 112.40 3 1 1 2 0.58 114.00

1 1 3 0.67 107.80 1 1 1 3 0.67 110.80 2 1 1 3 0.67 112.20 3 1 1 4 0.71 113.10

1 1 5 0.36 109.39

1 1 6 0.56 109.10 1 1 1 6 0.56 104.10 2 1 1 6 0.56 109.40 3 1 1 8 0.57 109.47

1 1 9 0.56 109.40 1 1 1 9 0.56 109.60 2 1 1 9 0.85 111.10 3 1 1 13 0.90 111.20

1 1 15 0.63 108.80

1 1 19 0.75 111.20 1 2 2 0.55 121.40

1 2 5 0.36 118.20

1 3 7 0.86 120.60

1 3 9 0.78 116.40

1 3 11 0.64 117.00

1 3 12 0.70 115.00

1 4 4 0.80 129.80

1 4 26 0.80 121.70

1 4 27 0.80 128.60

1 6 21 0.35 108.40

1 8 23 0.48 109.50

1 9 1 0.45 111.90

1 9 2 0.75 123.20

1 9 3 0.49 120.60 11 9 3 0.35 121.70 2 1 9 14 0.54 124.00

1 13 23 0.40 109.50

1 15 1 0.78 97.60

1 15 15 1.17 103.90

1 15 25 0.48 96.00

1 19 2 0.56 123.20

1 19 23 0.38 118.40

2 1 3 0.47 110.2

2 1 5 0.36 109.40

2 2 2 0.43 120.00

2 2 5 0.36 120.00

2 2 6 0.75 121.00

2 2 27 0.96 120.00

2 6 21 0.35 113.00

2 8 23 0.50 120.00

2 9 23 0.50 120.00

2 19 23 0.38 120.00

2 27 4 0.90 134.90

2 27 26 0.35 132.80

2 27 27 0.90 122.70

3 1 5 0.37 107.90

3 1 8 0.82 110.74

3 1 9 0.44 110.00 13 1 9 0.47 109.70 2 3 1 9 0.56 110.80 33 1 13 0.90 110.74

3 9 14 0.50 122.50

3 12 24 0.74 106.10

4 1 5 0.38 109.50

4 4 5 0.36 126.30

4 4 26 0.80 107.90

4 4 27 0.43 106.40

4 26 4 0.80 106.30

4 26 23 0.40 126.40

4 26 27 0.80 111.60

4 27 27 0.95 108.80

5 1 5 0.32 109.40

5 1 6 0.43 103.10

5 1 8 0.46 108.80 5 1 9 0.36 109.39 1 5 1 9 0.36 109.41 2 5 1 9 0.36 110.00 3 5 1 13 0.50 108.80

5 1 15 0.36 112.00

5 1 19 0.38 109.00

5 2 27 0.36 120.00

5 3 7 0.37 112.0

5 3 9 0.40 122.3

5 4 26 0.36 120.00

7 3 9 0.85 124.10

7 3 12 1.13 124.50

8 2 19 0.80 120.00

11 3 11 0.85 126.00

14 9 14 0.50 120.00

19 2 19 0.80 120.00

23 8 23 0.50 106.80

23 19 23 0.50 120.00

23 13 23 0.50 109.50

23 26 27 0.40 124.20

26 4 26 0.90 110.90

26 27 27 0.90 104.40

1 6 5 0.35 108.40

9 1 6 0.62 111.00

3 11 0.8

3 12 0.8

9 14 0.05

2 27 0.05

2 1 0.05 A

2 2 0.05

2 5 0.05

2 6 0.05

2 8 0.05

2 19 0.05

3 1 0.8

3 5 0.8

3 7 0.8

3 9 0.8

9 1 0.05

9 3 0.05

4 26 0.05

4 4 0.05

27 27 0.05

27 2 0.05

27 4 0.05

26 27 0.05

4 1 0.05

4 5 0.05

4 27 0.05

27 26 0.05 APPENDIX 2

PENCON

117 25 6 70 25

1 1 3 6 0.40 -0.30 -0.07 1 3 9 20 1.80 6.49 -6.23 1 6 2 2 3.53 2.30 -2.53 1 6 3 7-1.66 8.98 0.00 1 6 3 16-2.50 1.39 0.00 1 8 20 5 0.00 0.00 0.52 1 8 20 22-0.20 0.73 0.80 1 8 20 26 0.00 0.00 0.00 1 16 1 5 0.00 0.00 0.27 1 16 15 22-1.00 3.01 1.86 1 16 16 3-0.26 1.00 -0.80 1 16 16 5 0.00 0.00 -0.90 1 16 16 27-0.26 0.70 -0.06 1 25 6 19 0.00 0.00 0.00 1 25 25 25 0.00 3.50 0.00

2 1 3 9 0.00 -0.50 -1.70

2 2 1 3 0.00 0.00 0.50

2 2 2 6 0.00 3.80 0.00

2 2 2 25-0.30 3.50 0.00

2 2 25 19 0.00 1.00 0.30

2 2 25 25 0.00 1.00 0.30

2 6 1 3 0.00 0.00 -0.60

2 6 1 5 0.00 0.00 0.53

2 25 19 6 0.00 4.50 0.00

2 25 25 3 0.00 3.50 0.00

3 6 1 6-1.00 -5.00 0.00

3 9 20 5 0.00 0.00 0.51

3 9 20 22 0.00 0.00 0.01

3 9 20 26-3.50 -0.05 -4.30

3 16 16 15-1.75 0.60 1.50

3 16 27 22-1.00 -0.80 0.40

3 16 27 26-1.00 -0.53 1.13

3 25 25 1 0.00 5.00 0.00

3 25 25 6 0.00 3.80 0.00

3 25 25 19 0.00 3.80 0.00

5 1 16 15 0.00 0.00 0.40

5 1 16 16 0.00 0.00 0.27

5 1 25 6 0.00 0.00 0.54

5 1 25 25 0.00 0.00 0.05

5 2 2 6-0.30 3.80 0.00

5 2 2 25 0.00 3.50 0.00

5 16 3 6 0.00 0.00 -0.02

5 16 3 7 0.00 0.00 -0.04

5 16 3 29 0.00 0.00 -0.04

5 16 3 30 0.00 0.00 -0.09

5 16 16 15 0.00 0.00 -0.80

5 16 27 22 0.00 0.00 0.40

5 16 27 26 0.00 0.00 0.04 5 20 22 5 0.00 0.00 1.30 5 20 22 15 0.00 0.00 0.04 5 20 22 27 0.00 0.00 0.10 5 20 26 27 0.00 0.00 -0.09 5 20 26 28 0.00 0.00 0.21 5 22 15 16 0.00 0.00 0.00 5 22 20 8 0.00 0.00 0.20 5 22 20 9 0.00 0.00 0.20 5 22 20 12 0.00 3.00 0.40 5 22 20 13 0.00 5.00 0.20 5 22 20 26 0.00 0.00 0.87 5 22 27 16 0.00 0.00 0.00 5 22 27 26 0.00 0.00 0.98 6 1 3 7 0.00 0.00 -0.04 6 1 3 9 0.00 0.00 -0.04 6 3 16 16 0.40 -0.30 -0.07 6 3 16 27 0.00 0.00 0.50 6 19 25 25 0.00 4.50 0.00 6 25 25 25 0.00 3.80 0.00 7 3 9 20 0.00 7.19 0.00 7 3 16 16 0.00 0.00 -0.04 7 3 16 27 0.00 0.00 -0.04 7 3 25 25 4.00 0.40 2.40 9 3 25 25 4.30 0.40 2.90 8 20 22 27 0.00 0.00 0.40 8 20 26 27 -4.30 5.00 -1.50 8 20 26 28 -3.50 3.00 8.00 9 20 22 15 0.50 0.00 1.00 9 20 22 27 0.00 0.00 0.40 9 20 26 27 -4.30 5.00 -1.50 9 20 26 28 -3.50 3.00 8.0012 20 22 15 0.00 0.00 0.0412 20 22 27 0.00 0.00 0.5012 20 26 27 0.00 0.00 -0.09 12 20 26 28 0.00 0.00 0.21 13 20 22 15 0.50 -2.75 3.00 13 20 22 27 0.00 0.00 1.4013 20 26 27 -4.30 5.00 -1.50 13 20 26 28 -1.50 5.00 8.0014 9 3 25 0.00 2.66 0.00 14 9 20 22 0.00 0.00 0.01 14 9 20 26 0.00 0.00 0.01 15 16 16 27 -0.75 2.00 -0.90 15 22 20 26 0.00 1.00 1.00 15 22 27 16 -3.00 7.00 0.00 15 22 27 26 0.00 1.00 0.30 16 15 22 20 -2.50 3 .60 -3.00 16 15 22 27 -2.50 3 . 00 -1. 00 16 16 3 29 0.00 0.00 -0.20 16 16 3 30 0.00 0.00 -0.09 16 16 15 22 0.00 2.50 0.40 16 16 27 22 -1.00 -2 .50 0.40 16 16 27 26 -0.50 0 .50 2.50 16 27 22 20 0.00 1.00 -0.50 16 27 26 20 -4.00 3.00 0.40

16 27 26 28 -1 .00 3.00 2.00

19 6 25 25 0 .00 3.50 0.00

19 25 25 25 0 .00 3.80 0.00

20 9 3 25 1 .80 6.49 -5.234 20 22 27 26 1 .50 10.00 1.004 20 26 27 22 -1 .00 8.50 5.00 4 22 20 26 27 -1 .00 9.00 3.00

22 20 26 28 0 .20 3.50 -2.50

22 27 26 28 0 .20 8.00 -4.70

25 6 19 25 0 .00 3.50 0.00 4 26 20 22 27 -2 .65 6.10 0.20

27 16 3 29 0 .00 0.00 -0.06

27 16 3 30 0 .00 0.00 -0.09

1 16 5.26 1.525

1 25 4.48 1.512

2 25 9.60 1.526

3 16 4.45 1.550

3 25 9.60 1.332

5 16 4.60 1.070

5 20 4.39 1.080

5 22 4.38 1.090

6 19 4.32 1.410

6 25 4.09 1.350

8 20 5.10 1.510

9 20 5.47 1.449

12 20 3.23 1.793

13 20 2.30 1.926

15 16 3.98 1.851

15 22 3.98 1.810

16 16 4.50 1.565

16 27 4.30 1.476

19 25 6.50 1.310

20 22 2.56 1.553

20 26 2.58 1.527

22 27 4.30 1.484

25 25 5.83 1.380

26 27 4.79 1.393

26 28 8.65 1.201

1 16 0.000

1 25 0.100

2 25 0.000

3 16 5.872

3 25 0.000

5 16 0.000

5 20 0.000

5 22 0.000

6 19 0.081

6 25 0.212

8 20 3.980

9 20 0.000

12 20 1.940

13 20 1.790

15 16 4.464 15 22 3.653

16 16 0 .000

16 27 2 .304

19 25 0 .330

25 25 0.000

20 22 -0 .062

20 26 1 .547

22 27 0 .712

26 27 2 .280

26 28 1 .533

16 0. 044 1 .920

20 0. 044 1 .920

25 0. 044 1 .920

26 0. 044 1 .920

27 0. 055 1 .820

28 0. 066 1 .740

1 6 2 0.77 117.00

1 8 20 0.63 110.00

1 16 1 0.45 110.90 1 1 16 1 0.45 111.20 2 1 16 1 0.45 112.40 3 1 16 5 0.36 109.40

1 16 15 0.63 109.80

1 16 16 0.45 110.30 1 1 16 16 0.45 111.20 2 1 16 16 0.45 112.40 3 1 25 6 0.50 117.00

1 25 25 0.55 134.00

2 2 25 0.43 120.00

2 25 19 0.43 120.00

2 25 25 0.43 128.00

3 1 6 0.70 106.00

3 9 20 0.49 121.20

3 16 5 0.37 107.90

3 16 16 0.67 113.90

3 16 27 0.44 111.30

3 25 25 0.60 129.00

5 1 16 0.36 109.40

5 1 25 0.36 109.40

5 16 15 0.36 112.00

5 16 16 0.36 113.36

5 16 27 0.50 100.00

5 20 8 0.36 98.90

5 20 9 0.36 98.90

5 20 22 0.33 113.50

5 20 26 0.63 113.50

5 22 15 0.36 112.00

5 22 20 0.45 119.30

5 22 27 0.37 112.40

6 3 16 0.65 107.10

6 19 25 0.90 106.00

6 25 25 1.38 109.00

7 3 25 0.50 118.00

7 3 16 0.86 110.60 8 20 22 0.56 117.30

8 20 26 0.56 115.00

9 3 25 0.50 115.00

9 20 22 0.56 118.70

9 20 26 0.56 116.50

12 20 13 1.03 111.11

12 20 22 0.35 115.30

12 20 26 0.35 114.70

13 20 22 0.36 117.70

13 20 26 0.36 110.50

14 9 20 0.54 137.90

15 16 16 0.95 104.10

15 22 20 0.63 119.50

15 22 27 0.95 104.10

16 15 22 1.10 93.00

16 16 27 0.95 105.70

16 27 22 0.70 117.40

16 27 26 0.95 126.10

19 6 25 0.71 109.00

19 25 25 1.46 111.004 20 22 27 0.27 87.504 20 26 27 0.52 92.20

20 26 28 -0.02 136.804 22 20 26 0.30 85.204 22 27 26 0.35 93.40

27 26 28 1.59 130.60

2 25 0.05

3 16 0.80

3 25 0.80

9 20 0.05

20 26 0.80

26 27 0.80

APPENDIX 3

CEPARAN

125 19 6 68 18 78.5 1 3 16 16 0.34 11.10 0.00 1 3 16 32 0.00 0.00 -0.09 1 3 9 20 1.80 6.49 -6.23 1 6 2 2 3.53 2.30 -2.53 1 8 20 5 0.00 0.00 0.52 1 8 20 22-0.20 0.73 0.80 1 8 20 31 0.00 0.00 0.00 1 15 22 5 0.00 0.00 0.00 1 15 22 20 2.50 3.60 0.00 1 15 22 32 2.50 3.00 1.00 1 16 1 5 0.00 0.00 0.27 1 16 1 15 1.68 1.11 -0.20 1 16 1 32 0.00 0.00 0.00 1 16 15 22 -1.00 3.01 1.86 1 16 16 3 0.00 15.00 0.00 1 16 16 5 0.00 0.00 -0.90 1 16 16 15 -0.45 27.10 -0.78 1 16 16 32 0.00 15.00 0.00 1 20 9 3 0.00 0.00 0.06 1 20 22 5 0.0 0.0 2.0 1 20 22 27 0.00 0.00 0.10 1 20 26 27 0.0 0.00 -0.09 1 20 26 28 0.0 0.00 0.27 1 32 22 15 4.97 6.13 5.47 1 32 22 20 0.0 1.00 -0.56 1 32 22 5 0.0 0.0 0.0 1 32 31 20 -4.00 3.00 0.40 1 32 31 28 -1.00 3.00 2.00 2 1 3 9 0.00 -0.50 -1.70 2 2 1 3 0.00 0.00 0.50 2 6 1 3 0.00 0.00 -0.60 2 6 1 5 0.00 0.00 0.53 3 1 16 1 0.00 0.00 0.00 3 1 32 22 -1.00 -0.08 0.40 3 1 32 31 -1.00 -0.53 1.13 3 9 20 5 0.00 0.00 0.51 3 9 20 22 0.00 0.00 0.01 3 9 20 31 - 3.50 -0.05 -4.30 3 16 16 15 -1.75 0.30 1.50 3 16 32 22 -1.00 -0.80 0.40 3 16 32 31 -1.00 -0.53 1.13 5 1 3 16 0.00 0.00 0.00 5 1 3 30 0.00 0.00 -0.09 5 1 15 22 0.00 0.00 0.00 5 1 16 15 0.00 0.00 0.40 5 1 16 16 0.00 0.00 0.27 5 1 20 9 0.00 0.49 0.16 5 1 20 22 0.00 0.00 0.27 5 1 20 26 0.00 0.00 0.16 5 1 32 22 0.00 0.00 0.40 5 1 32 31 0.00 0.00 0.04 5 16 3 7 0.00 0.00 -0.04 5 16 3 29 0.00 0.00 -0.04 5 16 3 30 0.00 0.00 -0.09 5 16 15 22 0.00 0.00 -0.10 5 16 16 15 0.00 0.00 -0.80 5 16 32 22 0.00 0.00 0.40 5 16 32 31 0.00 0.00 0.04 5 20 9 14 0.00 0.00 0.00 5 20 22 5 0.00 0.00 1.30 5 20 22 15 0.00 0.00 0.04 5 20 22 32 0.00 0.00 0.10 5 20 31 32 0.00 0.00 -0.09 5 20 31 28 0.00 0.00 0.21 5 22 15 16 0.00 0.00 0.00 5 22 20 8 0.00 0.00 0.20 5 22 20 9 0.00 0.00 0.20 5 22 20 12 0.00 3.00 0.40 5 22. 20 13 0.00 5.00 0.20 5 22 20 31 0.00 0.00 0.87 5 22 32 16 0.00 0.00 0.00 5 22 32 31 0.00 0.00 0.98 6 1 3 7 0.00 0.00 -0.04 6 1 3 9 0.00 0.00 -0.04 7 3 9 20 0.00 7.19 0.00 7 3 16 16 0.00 0.00 -0.04 7 3 16 32 0.00 0.00 -0.04 8 20 22 32 0.00 0.00 0.40 8 20 31 32-4.30 5.00 -1.50 8 20 31 28-3.50 3.00 8.00 9 20 22 15 0.50 0.00 1.00 9 20 22 32 0.00 0.00 0.40 9 20 31 32-4.30 5.00 -1.50 9 20 31 28-3.50 3.00 8.0012 20 22 15 0.00 0.00 0.0412 20 22 32 0.00 0.00 0.5012 20 31 32 0.00 0.00 -0.0912 20 31 .28 COO 0.00 0.2113 20 22 15 0.50 2.75 3.0013 20 22 32 0.00 0.00 1.4013 20 31 32-4.30 5.00 -1.5013 20 31 28-1.50 5.00 8.0014 9 20 22 0.00 0.00 0.01 14 9 20 26 0.00 0.00 0.0114 9 20 31 0.00 0.00 0.0115 1 16 16 1.68 1.10 -0.33 15 16 16 32-0.75 2.00 -0.9015 22 20 31 0.00 1.00 1.0015 22 30 16-3.00 7.00 0.0015 22 32 31 0.00 1.00 0.3016 1 3 30 0.00 0.00 -0.3516 1 15 22 1.67 1.11 -0.1816 1 32 22-1.00 2.50 0.40 16 1 32 31-0.50 0.50 2.50

16 15 22 20- 2.50 3.60 -3.00

16 15 22 32- 2.50 3.00 -1.00

16 16 1 3 0 .00 0.00 0.90

16 16 1 32 0. 0. 0.

16 16 3 29 0.00 10.00 0.00

16 16 3 30 0.00 10.00 0.00

16 16 15 22 0.00 2.50 0.40

16 16 32 22 6.34 8.05 3.16

16 16 32 31- 3.34 6.00 0.00

16 32 22 20 0.00 1.00 -0.50

16 32 31 20- 4.00 3.00 0.40

16 32 31 28-1.00 3.00 2.004 20 22 32 31 1.50 10.00 1.004 20 31 32 22-1.00 8.50 5.00 4 22 20 31 32-1.00 9.00 3.00

22 20 31 28 0.20 3.50 -2.50

22 32 31 28 0.20 8.00 -4.704 31 20 22 32 -2 .65 6.10 0.20

32 16. 3 29 0 .00 0.00 -0.06

32 1 3 30 0 .00 0.00 -0.09

32 16 3 30 0 .00 0.00 -0.09

1 15 3.02 1.806

1 32 4.10 1.449

15 22 2.72 1.752

1 16 4.55 1.486

15 16 3.45 1.790

16 16 10.10 1.310

16 32 5.34 1.413

3 16 3.05 1.550

3 30 10.01 1.240

5 16 5.18 1.101

22 32 4.55 1.413

31 32 4.57 1.396

20 22 4.58 1.564

9 20 5.70 1.449

20 31 4.53 1.527

28 31 12.48 1.199

1 5 4.39 1.081

5 22 4.20 1.081

5 20 4.53 1.081

1 16 0.000

3 16 5.872

3 30 3.950

5 20 0.000

5 22 0.000

9 20 0.000

1 15 - 1.347

15 22 0.672

15 16 0.755

15 22 0.448

1 32 0.756

16 16 0.000

16 32 1.192 20 22 -0.343

20 31 -1.628

22 32 0.977

31 32 2.117

28 31 -1.659

16 0. 044 1.920

20 0. 044 1.920

30 0. 066 1.740

31 0. 044 1.920

32 0. 055 1.820

28 0. 066 1.740

1 3 16 0.40 115.00

1 3 30 0.64 116.50

1 6 2 0.77 110.80

1 8 20 0.63 110.00

1 15 22 0.76 96.00

1 16 1 0.74 113.90 1 1 16 1 0.45 111.20 2 1 16 1 0.45 112.40 3 1 16 . 5 0.36 109.40

1 16 15 0.22 109.80

1 16 16 1.05 122.00

1 32 22 0.97 127.00

1 32 31 0.95 136.70

3 1 6 0.70 106.00

3 1 32 0.83 108.40

3 9 20 0.53 121.20

3 16 5 0.45 107.90

3 16 16 0.59 113.90

3 16 32 0.83 111.30

5 1 16 0.36 109.40

5 1 5 0.39 110.60

5 1 15 0.44 108.30

5 1 16 0.46 106.00

5 1 32 0.36 110.00

5 16 15 0.47 112.00

5 16 16 0.47 113.36

5 16 32 0.58 100.00

5 20 8 0.36 98.90

5 20 9 0.54 98.90

5 20 22 0.50 115.10

5 20 31 0.38 112.20

5 22 15 0.36 107.90

5 22 20 0.49 114.10

5 22 32 0.55 118.00

7 3 16 0.86 110.60

8 20 22 0.56 117.30

8 20 31 0.56 115.00

9 20 22 0.74 118.70

9 20 31 0.73 116.50

12 20 13 1.03 111.11

12 20 22 0.35 115.30

12 20 31 0.35 114.70

13 20 22 0.36 117.70 13 20 31 0.36 110.50

14 9 20 0.53 137.90

15 1 16 1.09 117.00

15 16 16 1.07 131.10

15 22 20 0.39 116.00

15 22 32 0.62 111.00

16 1 3 0.67 112.70

16 1 32 1.04 110.60

16 3 30 1.17 116.50

16 15 22 1.04 97.60

16 16 32 1.16 120.00

16 32 22 0.97 126.10

16 32 31 0.41 134.004 20 22 32 1.17 88.304 20 31 32 0.94 90.30

20 31 28 0.69 138.004 22 20 31 1.24 85.804 22 32 31 1.01 94.60

30 3 30 0.85 126.00

32 31 28 0.67 132.00

3 16 0.80

3 30 0.80

9 20 0.05

20 31 0.80

31 32 0.80

APPENDIX 4

PROGRAMME FOR DOCKING OF TWO MOLECULES

PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM , TITLE

INTEGER TYPEA,TYPEB ,TYP

COMMON/COOD / COORD(3,NG), CHARGE (NG)

COMMON/TYPE / TYPEA(NT),TYPEB(NT), TYP(NG)

COMMON/SYMM / ASYM(NG), TITLE (40)

COMMON/PARAM/ VEP (NG) ,VRA(NG)

COMMON/CORD / XA(NT),YA(NT), ZA(NT) ,XB (NT) ,YB

COMMON/FINAL/ TXB (NT) , TYB (NT) , TZB (NT) , CA(NT)

COMMON/INFO / NA,NB, IP1, IP2, IP3, IP4, R1,R2, Cl

COMMON/PATH/MYWAY

DIMENSION X(5) ,E(5)

DATA E /5*0.01/

READ (8, 10 ) TITLE

10 FORMAT(40A2)

READ(8,*) IP1,IP2,IP3,IP4 ,MYWAY, Rl, R2, SC C MYWAY=1 : DISTANCE

C MYWAY=2 : ENERGY

C SC = SPECIAL CHARGE

CALL COMBIN(NA,NB)

READ(8,*) THETA,PI,XROT,YROT,ZROT , SCALE, JCO

E(l) = EE1

E(2) = EE1

E(3) = EE1

E(4) = EE1

E(5) = EE1

C READ(5,*) (E(I),I=1,5)

C SPECIAL CHARGE FOR IP2 AND IP4

CIP2= SC

CIP4= -SC

IPRINT=1

ICON = 1

C CONVERT DEGREE TO RADIAN

DEGREE=57.29577951D0

X(l)= THETA/DEGREE

X(2)= PI/DEGREE

X(3)= XROT/DEGREE

X(4)= YROT/DEGREE

X(5)= ZROT/DEGREE

NVAR=5

C C READ COORDINATE WITH MM FORMAT

READ(4,20) (XA(I),YA(I),ZA(I),TYPEA(I),I=1,N 20 FORMAT(2(3F10.5,I5,5X))

READ(5,20) (XB(I),YB(I),ZB(I),TYPEB(I),1=1,N CALL SYMBOL

CALL IWRITE

CALL PARM

CALL CHARG WRITE(6,22) (CHARGE(I),I=1,12)

22 FORMAT(3X,6F10.4)

C

CALL OPTIM(X,NVAR, SCALE, IPRINT, ICON,E)

C C CONVERT RADIAN TO DEGREE

DO 30 J=1,5

30 X(J)= X(J)*DEGREE

WRITE (6, 35) X

35 FORMAT(4X,' OPTIMIZED THETA-PI-X-Y-Z ANGLES C WRITE BOND DISTANCE BETWEEN IP2 AND IP4

X1=XA(IP2)

Y1=YA(IP2)

Z1=ZA(IP2)

X2=TXB(IP4)

Y2=TYB(IP4)

Z2=TZB(IP4)

R12=DIST(X1,Y1,Z1,X2,Y2,Z2)

WRITE (6, 40) IP2 ,IP4 ,R12

DO 50 K=1,NA

COORD(1,K)=XA(K)

COORD(2,K)=YA(K)

COORD(3,K)=ZA(K)

50 CONTINUE

NTOT=NA+NB

DO 60 KK=1,NB

COORD( 1,NA+KK) =TXB (KK)

COORD(2,NA+KK) =TYB (KK)

COORD(3,NA+KK) =TZB (KK)

60 CONTINUE

40 FORMAT(//,' BOND LENGTH BETWEEN ',I3,' OF A 1,F10.5,' ANGSTROM',//)

CALL CHEMG(NTOT)

CALL MMDATA C WRITE FINAL CARTESIAN COORDINATE FOR CHEMGR

5 WRITE (6,' (//lOX, ' 'FINAL CARTESIAN COORDINA

WRITE(6,' (4X,''NO.'',7X,''ATOM'',9X,'*X'', 1 9X, ''Y'',9X,*'Z'',/)')

WRITE(6,' (I6,8X,A2,4X,3F10.5)')

1 (I,ASYM(I), (COORD(J,I),J=1,3),I=1,NTOT) C

STOP

END C ROUTINE FOR OUTPUT IF INITIAL COORDINATES

SUBROUTINE IWRITE

PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM ,TITLE

INTEGER TYPEA,TYPEB ,TYP

COMMON/COOD/ COORD(3,NG) ,CHARGE (NG)

COMMON/TYPE/TYPEA(NT) ,TYPEB(NT) ,TYP(NG)

COMMON/SYMM/ASYM(NG) ,TITLE (40) COMMON/CORD/XA(NT) ,YA(NT) , ZA(NT) ,XB(NT) ,YB(N COMMON/FINAL/TXB(NT) ,TYB(NT) ,TZB(NT) ,CA(NT) , COMMON/INFO / NA,NB, IP1, IP2, IP3, IP4,R1,R2,CI WRITE (6, 15) TITLE

15 FORMAT(1H1, /////,

1 10X, ' *************************************

2 10x , ' COORDINATES OF SUPERMOLECULE

3 15X, 40A2 , /,

4 10X , ' *************************************

C WRITE CARTESIAN COORDINATE

WRITE (6, ' (//10X, ' 'INITIAL CARTESIAN COORDI

WRITE (6, ' (4X, ' 'NO. ' ' , 7X, ' 'ATOM' ' , 9X, ' 'X' ', 1 9X, ' 'Y' ',9X, ' 'Z' ',/)')

WRITE (6, ' (I6,8X,A2,4X,3F10.5)')

1 (I,ASYM(I),XA(I),YA(I),ZA(I),I=1,NA)

C

WRITE (6,' (////10X, ' 'INITIAL CARTESIAN COOR

WRITE(6, ' (4X, ' 'NO. ' ' , 7X, ' 'ATOM' ',9X, ' 'X'', 1 9X, ' 'Y' ',9X, ' 'Z' ',/)')

WRITE(6, ' (I6,8X,A2,4X,3F10.5)')

1 (I,ASYM(NA+I), XB(I),YB(I),ZB(I),I=1,NB)

WRITE (6, ' (///)')

RETURN

END

SUBROUTINE OPTIM (X,N,ESCALE, IPRINT, ICON,E)

PARAMETER (NT=150)

DIMENSION W( 1000) ,X(5) ,E (5)

MAXIT=100

DDMAG=0.1*ESCALE

SCER=0.05/ESCALE

JJ=N*N+N

JJJ=JJ+N

K=N+1

NFCC=1

IND=1

INN=1

DO 1 I=1,N

DO 2 J=1,N

W(K)=0.

IF(I-J)4,3,4

3 W(K)=ABS(E(I))

W(I)=ESCALE

4 K=K+1

2 CONTINUE

1 CONTINUE

ITERC=1

ISGRAD=2

CALL CALCFX(N,X,F,EW,ECO,EIDS)

FKEEP=ABS (F) +ABS (F)

5 ITONE=l

FP=F

SUM=0.

IXP=JJ

DO 6 I=1,N IXP=IXP+1

W(IXP) =X(I)

6 CONTINUE

IDIRN=N+1

ILINE=1

7 DMAX=W(ILINE)

DACC=DMAX*SCER

DMAG=MIN (DDMAG,0.1*DMAX)

DMAG=MAX(DMAG, 20.*DACC)

DDMAX=10.*DMAG

GO TO (70,70,71),ITONE

70 DL=0.

D = DMAG

FPREV=F

IS=5

FA=F

DA=DL

8 DD=D-DL

DL=D

58 K=IDIRN

DO 9 1=1,N

X(I)=X(I)+DD*W(K)

K=K+1

9 CONTINUE

CALL CALCFX(N,X,F,EW,ECO,EDIS)

NFCC=NFCC+1

GO TO (10, 11, 12,13, 14, 96),IS

14 IF(F-FA)15,16,24

16 IF (ABS (D) -DMAX) 17,17,18

17 D=D+D

GO TO 8

18 WRITE (6, 19)

19 FORMAT(5X,44HVA04A MAXIMUM CHANGE DOES NOT A GO TO 500

15 FB=F

DB=D

GO TO 21

24 FB=FA

DB=DA

FA=F

DA=D

21 GO TO (83,23),ISGRAD

23 D=DB+DB-DA

IS=1

GO TO 8

83 D=0.5*(DA+DB-(FA-FB)/(DA-DB))

IS=4

IF((DA-D)*(D-DB))25,8,8

25 IS=1

IF (ABS (D-DB) -DDMAX) 8,8,26

26 D=DB+SIGN(DDMAX,DB-DA)

IS=1

DDMAX=DDMAX+DDMAX

DDMAG=DDMAG+DDMAG IF (DDMAX-DMAX) 8,8,27

27 DDMAX=DMAX

GO TO 8

13 IF(F-FA)28,23,23

28 FC=FB

DC=DB

29 FB=F

DB=D

GO TO 30

12 IF(F-FB)28,28,31

31 FA=F

DA=D

GO TO 30

11 IF(F-FB)32,10,10

32 FA=FB

DA=DB

GO TO 29

71 DL=1.

DDMAX=5.

FA=FP

DA=-1.

FB=FHOLD

DB=0.

D=l.

10 FC=F

DC=D

30 A=(DB-DC)*(FA-FC)

B= (DC-DA) *(FB-FC)

IF( (A+B)* (DA-DC)) 33,33,34

33 FA=FB

DA=DB

FB=FC

DB=DC

GO TO 26

34 D=0.5* (A*(DB+DC)+B*(DA+DC))/(A+B) DI=DB

FI=FB

IF(FB-FC)44,44,43

43 DI=DC

FI=FC

44 GO TO (86,86,85),ITONE

85 ITONE=2

GO TO 45

86 IF (ABS(D-DI)-DACC) 41,41,93

93 IF (ABS(D-DI)-0.03*ABS(D)) 41,41,45 45 IF ( (DA-DC) * (DC-D) ) 47,46,46

46 FA=FB

DA=DB

FB=FC

DB=DC

GO TO 25

47 IS=2

IF ( (DB-D)*(D-DC)) 48,8,8

48 IS=3 GO TO 8

41 F=FI

D=DI-DL

DD=SQRT((DC-DB)*(DC-DA)*(DA-DB)/(A+B))

DO 49 1=1,N

X(I)=X(I)+D*W(IDIRN)

W(IDIRN) =DD*W(IDIRN)

IDIRN=IDIRN+1

49 CONTINUE

W(ILINE) =W(ILINE)/DD

ILINE=ILINE+1

IF(IPRINT-1)51,50,51

50 WRITE(6,52)ITERC,NFCC,F

WRITE (7, 52) ITERC,NFCC,F

52 FORMAT (3X, ' ITERATION' ,15,19,' FUNCTION VAL WRITE (6, 68) EW,ECO,EDIS

WRITE (7, 68) EW,ECO,EDIS

68 FORMAT (3X, 'REP. = ',F12.5,' ATT. =',F12.5, GO TO(51,53),IPRINT

51 GO TO (55,38),ITONE

55 IF (FPREV-F-SUM) 94,95,95

95 SUM=FPREV-F

JIL=ILINE

94 IF (IDIRN-JJ) 7,7,84

84 GO TO (92,72),IND

92 FHOLD=F

IS=6

IXP=JJ

DO 59 1=1,N

IXP=IXP+1

W(IXP)=X(I)-W(IXP)

59 CONTINUE

DD=1.

GO TO 58

96 GO TO (112,87),IND

112 IF (FP-F) 37,91,91

91 D=2. *(FP+F-2.*FHOLD)/(FP-F)**2

IF (D*(FP-FHOLD-SUM)**2-SUM) 87,37,37

87 J=JIL*N+1

IF (J-JJ) 60,60,61

60 DO 62 I=J,JJ

K=I-N

W(K)=W(I)

62 CONTINUE

DO 97 I=JIL,N

W(I-1)=W(I)

97 CONTINUE

61 IDIRN=IDIRN-N

ITONE=3

K=IDIRN

IXP=JJ

AAA=0.

DO 65 1=1,N

IXP=IXP+1 W(K)=W(IXP)

IF (AAA-ABS(W(K)/E(I))) 66,67,67

66 AAA=ABS(W(K)/E(I))

67 K=K+1

65 CONTINUE

DDMAG=1.

W(N)=ESCALE/AAA

ILINE=N

GO TO 7

37 IXP=JJ

AAA=0.

F=FHOLD

DO 99 I=1,N

IXP=IXP+1

X(I)=X(I)-W(IXP)

IF (AAA*ABS(E(I))-ABS(W(IXP))) 98,99,99 98 AAA=ABS(W(IXP)/E(I))

99 CONTINUE

GO TO 72

38 AAA=AAA*(1.+DI)

GO TO (72,106),IND

72 IF (IPRINT-2) 53,50,50

53 GO TO (109,88),IND

109 IF (AAA-0.1) 89,89,76

89 GO TO (20, 116), ICON

116 IND=2

GO TO (100, 101), INN

100 INN=2

K=JJJ

DO 102 I=1,N

K=K+1

W(K)=X(I)

X(I)=X(I)+10.*E(I)

102 CONTINUE

FKEEP=F

CALL CALCFX(N,X,F,EW,ECO,EDIS)

NFCC=NFCC+1

DDMAG=0.

GO TO 108

76 IF (F-FP) 35,78,78

78 WRITE (6, 80)

80 FORMAT (5X,37HVA04A ACCURACY LIMITED BY ERRO GO TO 500

88 IND=1

35 DDMAG=0.4*SQRT(FP-F)

IF(DDMAG.GE.1.0E60) DDMAG=1.0E60

ISGRAD=1

108 ITERC=ITERC+1

IF (ITERC-MAXIT) 5,5,81

81 WRITE(6,82)MAXIT

82 FORMAT(15, 30H ITERATIONS COMPLETED BY VA04A) IF (F-FKEEP) 500,500,110

110 F=FKEEP

DO 111 I=1,N JJJ=JJJ+1

X(I)=W(JJJ)

111 CONTINUE

GO TO 20

101 JIL=1

FP=FKEEP

IF (F-FKEEP) 105,78,104

104 JIL=2

FP=F

F=FKEEP

105 IXP=JJ

DO 113 I=1,N

IXP=IXP+1

K=IXP+N

GO TO (114,115),JIL

114 W(IXP)=W(K)

GO TO 113

115 W(IXP)=X(I)

X(I)=W(K)

113 CONTINUE

JIL=2

GO TO 92

106 IF (AAA-0.1) 20,20,107

20 WRITE (6, 200)

WRITE(6,201)

WRITE (7, 201)

201 FORMAT(5X,' THE FUNCTION VALUE HAS BEEN MIN

WRITE (6, 200)

200 FORMAT(/1X, '******************************** 500 RETURN

107 INN=1

GO TO 35

ENDC

SUBROUTINE PARM

PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM ,TITLE

INTEGER TYPEA,TYPEB ,TYP

COMMON/PARAM/ VEP(NG) ,VRA(NG)

COMMON/INFO / NA,NB,IP1,IP2,IP3,IP4,R1,R2,CI

COMMON/TYPE / TYPEA(NT),TYPEB(NT),TYP(NG)

DIMENSION VEPS (60),VRAD(60)

C

DATA VRAD/

1 1.900, 1.940, 1.940, 1.940, 1.500, 1.740, 1

2 -1.0 , 1.780, 1.740, 1.900, 0.930, 2.110, 1

3 1.820, 1.920, 1.200, 1.920, 1.325, 0.900, 1

4 1.740, 1.90 , 1.780 , 1.92, 1.82, -1.0 , -1.0

5 -1.0 , -1.0 , -1.0 , -1.0 , -1.0 , -1.0 , 1 DATA VEPS/

1 0.044, 0.044, 0.044, 0.044, 0.047, 0.050, 0

2 -1.0 , 0.066, 0.050, 0.030, 0.017, 0.202, 0

3 0.055, 0.044, 0.036, 0.044, 0.034, 0.015, 0 4 0.066, 0.044, 0.066, 0.044, 0.055,-1.0 , -1.

5 -1.0 , -1.0 , -1.0 , -1.0 , -1.0 , -1.0 , IF(JCON.GT.O) THEN

DO 10 J=1,JCON

READ(5,*) ITYPE, EPS, RAD

VEPS(ITYPE) = EPS

VRAD(ITYPE) = RAD

10 CONTINUE

ENDIF

DO 20 1=1,NA

VEP(I) = VEPS(TYPEA(I))

IF(VEP(I).LE.0.0) THEN

WRITE (7, 25) TYPEA(I)

ENDIF

VRA(I) = VRAD(TYPEA(I))

IF(VRA(I).LE.0.0) THEN

WRITE (7, 25) TYPEA(I)

25 FORMAT(4X,' CHECK YOUR VAN DER WAAL DATA

ENDIF

20 CONTINUE

C

DO 30 I=1,NB

VEP(NA+I) = VEPS(TYPEB(I))

IF(VEP(NA+I).LE.0.0) THEN

WRITE (7, 25) TYPEB(I)

ENDIF

VRA(NA+I) = VRAD(TYPEB(I))

IF(VRA(NA+I).LE.0.0) THEN

WRITE (7, 25) TYPEB(I)

ENDIF

30 CONTINUE

RETURN

END

SUBROUTINE ENERGY(ETOT,EV,ETOTl,EDIS)

C FUNCTION PROGRAM FOR SUPER-MOLECULE

PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM , TITLE

INTEGER TYPEA,TYPEB , TYP

COMMON/COOD/ COORD(3,NG) , CHARGE (NG) _Λ

COMMON/TYPE/TYPEA(NT), TYPEB (NT) , TYP (NG)

COMMON/CORD/XA(NT), YA(NT) ,ZA(NT) ,XB(NT) ,YB(N

COMMON/FINAL/TXB (NT), TYB (NT) , TZB (NT) , CA(NT) ,

COMMON/PARAM/ VEP(NG) ,VRA(NG)

COMMON/INFO / NA,NB, IP1,IP2,IP3,IP4,R1,R2,CI

COMMON/PATH/MYWAY

C CALCULATION OF VAN DER WALLS ENERGY(ONLY 1-5

DIELC=78.5

C GO TO (1 ,2), MYWAY

X1=XA(IP2)

Y1=YA(IP2)

Z1=ZA(IP2)

X2=TXB(IP4)

Y2=TYB(IP4) Z2=TZB(IP4)

ETOT1=DIST(X1,Y1,Z1,X2,Y2,Z2)

ETOT1= ABS(ETOT1-R2) *500.0

IF(MYWAY.EQ.1) THEN

ETOT=ET0T1

RETURN ENDIF

2 EV=0.0

EC=0.0

DO 500 I=1,NA

XI=XA(I)

YI=YA(I)

ZI=ZA(I)

DO 500 K=1,NB

XK=TXB(K)

YK=TYB(K)

ZK=TZB (K)

RIK=DIST(XI,YI,ZI,XK,YK,ZK)

ECOUL=332.0*CHARGE (I) *CHARGE (NA+K) / (DIELC*RI

VEPI=VEP(I)

VEPK=VEP (NA+K)

VRAI=VRA(I)

VRAK=VRA(NA+K)

EPS=SQRT(VEPI*VEPK)

RV=VRAI+VRAK

P=RV/RIK

IF(P.GT.3.31) GO TO 30

IF (P.LT.0.072) THEN

E=EPS*(-2.25*P**6)

GO TO 35

ENDIF

E=EPS* (290000.0*EXP ( -12.5/P) -2.25*P**6)

GO TO 40

30 E=EPS*336.176*P*P

35 CONTINUE

40 EV = EV+E

EC = EC +ECOUL

500 CONTINUE

ETOT=EV + ETOT1 + EC

C ETOT=EV + ETOT1

X1=XA(IP2)

Y1=YA(IP2)

Z1=ZA(IP2)

X2=TXB(IP4)

Y2=TYB(IP4)

Z2=TZB(IP4)

EDIS=DIST (X1 , Y1 , Z 1 , X2 , Y2 , Z2 )

RETURN

END C FUNCTION DIST

FUNCTION DIST (X1 , Y1 , Z 1 , X2 , Y2 , Z2 )

X=X1 -X2

Y=Y1-Y2 Z=Z1-Z2

DIST=SQRT (X*X+Y*Y+Z*Z)

RETURN

END

SUBROUTINE CALCFX(NVAR, X, ETOT, EV, EC, EDIS)

PARAMETER (NT=150)

PARAMETER (NG=300)

COMMON/CORD/XA(NT), YA(NT), ZA(NT), XB(NT), YB(N

COMMON/FINAL/TXB (NT), TYB (NT), TZB (NT), CA(NT),

COMMON/INFO / NA,NB, IP1, IP2, IP3, IP4, R1, R2 CI

DIMENSION X(5),TX(150), TY ( 150), TZ ( 150),XROT(

DIMENSION CTX(150), CTY(150), CTZ(150)

C CONVERSION OF POLAR COORDINATE TO CARTECIAN COOR

DX=R1*SIN(X(1))*COS(X(2))

DY=R1*SIN(X(1))*SIN(X(2))

DX=R1*COS(X(1))

C FIXING OF PROBE 2 APART FROM PROBE 1 BY R1 ANG.

PX=XA(IP1)+DX

PY=YA(IP1)+DY

PZ=ZA(IP1)+DZ

C CALCULATE DISTANCE VECTORS BETWEEN PROBE P(PX,P

DVX=PX-XB(IP3)

DVY=PY-YB(IP3)

DVZ=PZ-ZB(IP3)

C PARALLEL MOVEMENT OF B MOLECULE BY (DVX,DVY,DVZ

DO 10 IM=1,NB

TX(IM) = XB(IM) + DVX

TY(IM) = YB(IM) + DVY

TZ(IM) = ZB(IM) + DVZ

10 CONTINUE

C MOVE TO MAKE AN ORIGIN OF PROBE3(IP3) IN

DO 20 10=1,NB

IF(IO.EQ.IP3) GO TO 20

TX(IO)=TX(IO)-TX(IP3)

TY(IO)=TY(IO)-TY(IP3)

TZ(IO)=TZ(IO)-TZ(IP3)

20 CONTINUE

TX(IP3) = 0.0D0

TY(IP3) = 0.0D0

TZ(IP3) = 0.0D0

C ROTATION

CSX= COS(X(3))

SSX= SIN(X(3))

CSY= COS(X(4))

SSY= SIN(X(4))

CSZ= COS(X(5))

SSZ= SIN(X(5))

C X ROTATION

DO 30 I=1,9

30 XROT(I) = 0.0

XROT(l) = 1.0

XROT(5) = CSX

XROT(6) =-SSX

XROT(8) = SSX XROT(9) = CSX

DO 40 1=1,9

40 YROT(I) = 0.0

YROT(1) = CSY

YROT(3) = SSY

YROT(5)= 1.0

YROT(7)= -SSY

YROT(9) = CSY

DO 50 1=1,9

50 ZROT(I)= 0.0

ZROT(1) = CSZ

ZROT(2) = SSZ

ZROT(4) =-SSZ

ZROT(5) = CSZ

ZROT(9) = 1.0

DO 60 J=1,NB

COXX = XROT(l)*TX (J ) +XROT(2)* TY(J ) +X COXY = XROT(4)*TX (J ) +XROT(5)* TY(J ) +X COXZ = XROT(7)*TX (J ) +XROT(8)* TY(J ) +X COYX. = YROT(1)*COXX +YROT(2)*COXY +YROT(3)*C COYY = YROT(4)*COXX +YROT(5)*COXY +YROT(6)*C COYZ = YROT(7)*COXX +YROT(8)*COXY +YROT(9)*C CTX(J) =ZROT(1)*COYX +ZROT(2)*COYY +ZROT(3)*C CTY(J)=ZROT(4)*COYX +ZROT(5)*COYY +ZROT(6)*C CTZ (J) =ZROT(7)*COYX +ZROT(8)*COYY +ZROT(9)*C

60 CONTINUE

C RETURN TO POINT P

DO 70 1= 1,NB

TXB(I) = CTX(I) + PX

TYB(I) = CTY(I) + PY

TZB(I) = CTZ (I) + PZ

70 CONTINUE

C

CALL ENERGY(ETOT,EV,EC,EDIS)

RETURN

END

SUBROUTINE COUL (ETOT,ER,EA)

PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM ,TITLE

COMMON/CORD/XA(NT) ,YA(NT) , ZA(NT) ,XB(NT) ,YB(N

COMMON/FINAL/TXB (NT) ,TYB (NT) ,TZB (NT) ,CA(NT) ,

COMMON/INFO / NA,NB,IP1,IP2,IP3,IP4,R1,R2,CI

ER=0.0

DO 10 I=1 , NA

X1=XA(I)

Y1=YA(I)

Z1=ZA(I)

DO 20 J=1,NB

X2=TXB(I)

Y2=TYB(I)

Z2=TZB(I)

ER =ER +(CA(I)*CB(J))/DIST(X1,Y1,Z1,X2,Y2,Z2 20 CONTINUE

10 CONTINUE

EA =0.0

X1=XA(IP2)

Y1=YA(IP2)

Z1=ZA(IP2)

X2=TXB(IP4)

Y2=TYB(IP4)

Z2=TZB(IP4)

EA =EA +(CIP2*CIP4)/DIST(X1,Y1,Z1,X2,Y2,Z2)

ETOT=ER + EA

RETURN

END C SUBPROGRAM TO GENERATE ATOM TYPE AND NET ATO SUBROUTINE CHARG PARAMETER (NG=300)

PARAMETER (NT=150)

INTEGER TYPEA,TYPEB , TYP

COMMON/COOD / COORD (3 ,NG) , CHARGE (NG)

COMMON/TYPE / TYPEA(NT),TYPEB(NT), TYP(NG) COMMON/INFO / NA,NB, IPl, IP2, IP3, IP4, R1,R2,CI DIMENSION DCHB(35)

DATA DCHB/ 0.241, 0.0 , 0.515, 0.0 , 0.0

1 -0.267, -0.509, 0.0 , -0.692 -0 .5

2 -0.135, 0.0 , 0.0 , 0.0 -0 . 6

3 0.0 , 0.243, 0.15 , 0.131 -0. 5

4 0.0 , -0.622, 0.515, 0.0 0 .0 DO 20 I=1,NA

CHARGE (I) =DCHB(TYPEA(I))

20 CONTINUE

C SPECIAL SIDE CHAIN FOR CARBONE C CHARGES FOR HYDANTON

DO 30 I=1,NB

CHARGE (NA+I) =DCHB (TYPEB (I))

30 CONTINUE

C CHARGE (NA+1) = -0.36

C CHARGE (NA+2) = 0.44

C CHARGE (NA+3) = -0.41

C CHARGE (NA+4) = 0.58

C CHARGE (NA+5) = -0.31

C CHARGE (NA+6) = 0.03

C CHARGE (NA+7) = -0.41

C CHARGE (NA+20)= 0.19

C CHARGE (NA+21) = 0.20

40 CHARGE (IP2)= CIP2

CHARGE (NA+IP4) = CIP4

WRITE (6, 22) (CHARGE (I), I=1, 12)

22 FORMAT(3X,6F10.4)

RETURN

END C

SUBROUTINE SYMBOL

PARAMETER (NT=150) PARAMETER (NG=300)

CHARACTER*2 ASYM , TITLE

INTEGER TYPEA,TYPEB, TYP, HH,NN,OO, CC

COMMON/COOD/ COORD(3,NG) , CHARGE (NG)

COMMON/TYPE/TYPEA(NT) , TYPEB (NT) ,TYP (NG)

COMMON/SYMM/ASYM(NG) , TITLE (40)

COMMON/INFO / NA,NB, IP1, IP2,IP3,IP4,R1,R2,CI

DIMENSION HH(6),NN(7),00(6),CC(10)

DATA HH/5,14,23,21,24,25/

DATA NN/8,9,13,19,26,32,28/

DATA 00/6,7,11,12,28,30/

DATA CC/1,2,3,4,16,20,22,27,29,31/

NTOT=NA+NB

DO 1 I=1,NA

TYP(I)=TYPEA(I)

1 CONTINUE

DO 2 I=1,NB

TYP (NA+I) =TYPEB (I)

2 CONTINUE

1=0

10 CONTINUE

I=I+1

IF(I.GT.NTOT) GO TO 9

DO 20 K1=1,6

IF (TYP(I).EQ.HH(K1)) THEN

ASYM(I)=' H'

GO TO 10

ENDIF

20 CONTINUE

DO 301l=1,7

IF (TYP(I).EQ.NN(Kl)) THEN

ASYM(I)=' N'

GO TO 10

ENDIF

30 CONTINUE

DO 40 K1=1,6

IF(TYP(I).EQ.OO(Kl)) THEN

ASYM(I)=' 0'

GO TO 10

ENDIF

40 CONTINUE

DO 50 K1=1,10

IF(TYP(I).EQ.CC(K1)) THEN

ASYM(I)=' C'

GO TO 10

ENDIF

50 CONTINUE

IF (TYP(I).EQ.15) THEN

ASYM(I)= ' S'

GO TO 10

ELSE

WRITE (6, 100) TYP (I) , I

100 FORMAT(3X,' UNDEFINED ATOM TYPE. : ',13,' O

ENDIF 9 CONTINUE

RETURN END SUBROUTINE CHEMG(NTOT)

C

CHARACTER NAME1*2, NAME2*3 ,CTEMP*80 ,TEMP*

PARAMETER (NG=300)

PARAMETER (NT=150)

INTEGER IH, IN, IC, IO, IS, HH,NN, CC,OO, SS

COMMON/COOD/ COORD(3,NG), CHARGE (NG)

COMMON/SYMM/ASYM(NG), TITLE (40)

C

J=0

IONE=1

WRITE (10,1) NTOT,IONE,IONE

1 FORMAT(//,I3,I5,/,I6)

DO 33 I = l,NTOT

C READ(4,' (1X,3F10.4,5X,A1)' ,END=999) X,Y,Z,NA

X=COORD(1,I)

Y=COORD(2,I)

Z=COORD(3,I)

NAME1=ASYM(I)

IF( NAME1.EQ.' F') GO TO 33

J=J+1

IF (NAME1.EQ. ' H') THEN

IH = IH + 1

WRITE (NAME2,' (13)') IH

ELSEIF (NAME1.EQ.' N') THEN

IN = IN + 1

WRITE (NAME2,' (13)') IN

ELSEIF (NAME1.EQ.' C) THEN

IC = IC + 1

WRITE (NAME2,' (13)') IC

ELSEIF (NAME1.EQ. ' 0') THEN

10 = 10 + 1

WRITE (NAME2,'(13)') 10

ELSEIF (NAME1.EQ.' S') THEN

IS = IS + 1

WRITE (NAME2, ' (13)') IS

ELSE

WRITE (6, ' (' 'You have a problem on line' ',14, &')I

ENDIF

IF (NAME2(1:1).EQ.' ') THEN

NAME2(1:1) = NAME2(2:2)

NAME2(2:2) = NAME2 (3 : 3 )

NAME2 (3 : 3 ) = ' '

IF (NAME2(1:1).EQ.' ') THEN

NAME2(1:1) = NAME2(2:2)

NAME2(2:2) = ' '

ENDIF ENDIF WRITE(10,' (I4,A2,A3,1X,3F10.4)') J,NAME1,NAM 33 CONTINUE

C ENDIF RETURN END SUBROUTINE WRIT(X,Y,Z)

DIMENSION X(150),Y(150),Z(150) DO 5 1=1,6

5 WRITE(6,10) X(I),Y(I),Z(I)

10 FORMAT(4X,3F12.5)

RETURN END C

SUBROUTINE COMBIN (NA,NB)

DIMENSION ICON(16),IAT1(150),IAT2(150) CHARACTER*2 TT(30)

READ (4, 10) NA

10 FORMAT (62X, I3)

READ (5, 10) NB

NTOT=NA+NB

IONE=1

IFOUR =4

TIME=100.0

WRITE (16,20) NTOT,IFOUR,IONE,TIME 20 FORMAT(60X,I5,I2,3X,I5,F5.0)

READ(4,30) NCONA,NATA,NSPA

30 FORMAT(I5,20X,I5,15X,I5)

READ(5,30) NCONB,NATB,NSPB

NCOT=NCONA+NCONB

NATT=NATA+NATB

NSPT=NSPA+NSPB

WRITE (16, 30) NCOT,NATT,NSPT

IF(NSPA.NE.O) THEN

DO 50 I=1,NSPA

READ(4,40) TT

40 FORMAT(30A2)

WRITE (16,40) TT

50 CONTINUE

ENDIF

IF(NSPB.NE.0) THEN

DO 60 I=1,NSPB

READ (5, 40) TT

WRITE (16, 40) TT

60 CONTINUE

ENDIF

DO 70 IA=1,NCONA

READ(4,75) (ICON(I),I=1,16)

75 FORMAT(1615)

DO 80 IZ=1 , 16

ISZ=16

IF( ICON(IZ).EQ.0) THEN

ISZ=IZ -1

GO TO 85

ENDIF

80 CONTINUE 85 WRITE(16,75) (ICON(I), I=1, ISZ)

70 CONTINUE

DO 90 IB=1,NCONB

READ(5,75) (ICON(I), I=1, 16)

DO 100 IZ=1,16

ISZ=16

IF( ICON(IZ).EQ.0) THEN

ISZ=IZ -1

GO TO 95

ENDIF

100 CONTINUE

95 DO 110 I=1, ISZ

110 ICON(I)= ICON(I)+NA

WRITE(16,75) (ICON(I), I=1, ISZ)

90 CONTINUE

READ(4,75) (IAT1(I), IAT2 (I), I=1,NATA) NATA1=NATA+1

READ(5,75) (IAT1(I), IAT2 (I), I=NATA1,NATT) DO 120 IL=NATA1,NATT

IATl(IL) = IAT1(IL)+NA

IAT2(IL) = IAT2(IL)+NA

120 CONTINUE

WRITE(16,75) (IAT1 (I), IAT2 (I), I=1,NATT) RETURN END C SUBROUTINE FOR MM INPUT SUBROUTINE MMDATA PARAMETER (NT=150)

PARAMETER (NG=300)

CHARACTER*2 ASYM ,TITLE

INTEGER TYPEA, TYPEB ,TYP

COMMON/COOD / COORD(3,NG), CHARGE (NG)

COMMON/TYPE / TYPEA(NT), TYPEB (NT), TYP (NG) COMMON/SYMM / ASYM(NG), TITLE (40)

COMMON/PARAM/ VEP(NG) ,VRA(NG)

COMMON/CORD / XA(NT),YA(NT), ZA(NT),XB(NT),YB COMMON/FINAL/ TXB (NT) ,TYB (NT) ,TZB(NT), CA(NT) COMMON/INFO / NA,NB,IPl,IP2,IP3,IP4,R1,R2,CI NTOT=NA+NB

WRITE (16,20) (COORD(1,I), COORD(2,I), COORD(3, 20 FORMAT(2(3F10.5,I5,5X))

RETURN END

Claims

Claims :

1. A method of determining the molecular structure of a polypeptide characterized in that the strain energy of the molecule is minimized as a function of its dihedral angles with bond lengths and bond angles held constant, said minimization is preceded by a consideration of a subset of the parameters which form a basis for a

specific subset of the complete parameter space, said subset is comprised of the values 0, ±90, 180 degrees for the ∅ and dihedral angles of the backbone, and the

values -60 and 180 degrees for the first dihedral angle of the side chains, the w dihedral angle and all other side chain dihedral angles are maintained at 180 degrees, each of the infinite number of points in this parametric subspace corresponding to an associated molecular strain energy, the subspace is then subjected to a sufficiently rich discrete randomly distributed uniform mapping so that there is an arbitrarily large probability that some points (r) are found in a convex neighbourhood of local energy minima, and this set of points (r) is then used for the initialization of the minimization procedure.

2. A method as claimed in claim 1, characterized in that a reasonable number of points for the randomly chosen discrete subset described above is 200,000 in the case of a polypeptide containing up to 10 amino acid residues, and the set of points (r) numbers 50.

3. A method of identifying compounds with antibacterial activity characterized in that the active site of a penicillin binding protein (PBP) is modelled as a peptide containing the sequence Ac-Val-Gly-Ser-Val-Thr-Lys-NHCH₃ having the conformation set forth in the table:

and candidate molecules are identified by calculating their ability to dock with said peptide.

4. A method as claimed in claim 3, characterized in that the ability of candidate molecules to dock with said peptide is calculated assuming hydrogen bonding

interactions between a carboxyl and N-H (or O-H) of the substrate and, respectively, the terminal amino group of the lysine residue and acetyl oxygen of the receptor peptide.

5. A method as claimed in claim 4, characterized in that the intrinsic reactivity of the compounds with said peptide is predicted by determining intrinsic reactivity thereof with methanol, relative to the reactivity of a penam ring system of penicillin.

6. A method of identifying compounds with antibacterial activity, characterized in that the product of rms differences for C-O-H of serine and an appropriate functional group of a candidate compound via a four- centred complex, relative to penicillin V is determined; and the intrinsic reactivity of said functional group in reaction with methanol, relative to the reactivity of a penam ring system of penicillin is determined.

7. A method as claimed in claim 5, characterized in that said functional group is

8. A method as claimed in claim 6, characterized in that said functional group is

9. A method of determining fit and reactivity of any selected candidate antibacterial compound with a PBP characterized in that it comprises (a) simulating the reaction of said compound with a model of a penicillin binding protein which includes a serine-lysine active site, by determining the relative ease of formation of a four-centred relationship between OH of said serine and a reactive site of said compound; and (b) determining the activation energy for the four-centred reaction of the chemically active functional group of said compound with methanol relative to the activation energy of the

corresponding reaction of methanol with N- methylazetidinone.

10. A non-β-lactam. containing compound characterized in that said compound is capable of forming a four-centred transition structure which includes a serine OH group contained in a model of a penicillin binding protein, reacted therewith; said compound having an activation energy for reaction with methanol not greater than 3 kcal/mol higher than the activation energy exhibited by N-methyl-azetidinone.

11. An antibacterial agent characterized in that it includes a structure which makes a dihedral angle of 150- 160° with a reactive site thereof, has a hydrogen bonding donor oriented so that it makes a dihedral angle of -150 to -160° with the reactive site, and said the reactive site is such that it reacts with methanol via a four- centred transition structure, and with the activation energy no greater than 3-4 kcal/mol higher than that

for the reaction with an azetidinone.

12. An antibacterial agent as claimed in claim 11, characterized in that said hydrogen bonding donor is N-H or O-H.

13. An antibacterial agent as claimed in claim 12, characterized in that said structure has an imino moiety

as a functional group with the required

reactivity.

14. An antibacterial agent characterized in that it has as a nucleus

15. An antibacterial agent characterized in that it has as a nucleus

16. An antibacterial agent characterized in that it has as a nucleus

-

17. An antibacterial agent as claimed in claim 16, characterized in that said nucleus has an imino moiety

as a functional group to provide the required reactivity.

18. An anitbacterial compound selected from

(a) a compound of the formula I

where

X is selected from S, O, CH₂, NH, NR₇, and Se

Y is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, are each hydrogen, alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof, (b) a compound of the formula II

where

X is selected from S, O, CH₂, NH, NR₈, and Se

Y is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ are each hydrogen, alkyl, or aryl, and

R₉ is a a β-lactam active side chain,

and pharmaceutically acceptable salts thereof,

(c) a compound of the formula III

where

X-Y is selected from S-S, CH₂CH₂, S-CH₂, CH₂-S, S-NR₈, NR₈- S, CH₂H-O, O-CH₂, O-NR₈, NR₈-O, Se-Se, CH₂-CH₂, and Se-CH₂ Z is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₈ are each hydrogen, alkyl, aryl R₇ is alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof, (d) a compound of the formula IV

where

X is selected from S, O, CH₂, NH, NR₆, and Se

Y is selected from N, CH, and CR₇

Z is OH, NH₂, SH, or NHCOR9 (when Y=N)

Z is R₁₀ (when Y=CH, or CR₇)

R₁=R₂=R₃=R₄=R₅=R₆=R₇= are each hydrogen, alkyl, or aryl, and R₉ is a β-lactam active side chain

R₁₀ - R₁₁ -

where R₁₁ is alkyl, or aryl, and

R₁₂=OH, NH₂, KHCOR₉, SH

and pharmaceutically acceptable salts thereof, and (e) a compound of the formula V

where

X is selected from S, O, CH₂, NH, NR₅, and Se

Y is NR₆- Z, and

R₁, R₂, R₃, R₄, R₅, and R₆ are each H, alkyl, or aryl

Z is OH, SH, NH₂, or NHCOR₇

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

19. A novel antibacterial compound as claimed in claim 18 characterized by the formula: I

where

X is selected from S, O, CH₂, NH, NR₇, and Se

Y is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, are each hydrogen, alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

20. A compound as claimed in claim 19 characterized in that X is S.

21. A compound as claimed in claim 20 characterized in that R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen or lower alkyl.

22. A compound as claimed in claim 21 characterized in that the lower alkyl groups are methyl groups.

23. A novel antibacterial compound as claimed in claim 18 characterized by the formula: II

where

X is selected from S, O, CH₂, NH, NR₈, and Se

Y is selected from OH, NH₂, NHCOR_g, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ are each hydrogen, alkyl, or aryl, and

R₉ is a a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

24. A compound as claimed in claim 23 characterized in that X is S.

25. A compound as claimed in claim 24 characterized in that R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are each hydrogen or lower alkyl.

26. A compound as claimed in claim 25 characterized in that the lower alkyl groups are methyl groups .

27. A novel antibacterial compound as claimed in claim 18 characterized by the formula: III

where

X-Y is selected from S-S, CH₂CH₂, S-CH₂, CH₂-S, S-NR₈, NR₈- S, CH₂H-O, O-CH₂, O-NR₈, NR₈-O, Se-Se, CH₂-CH₂, and Se-CH₂ Z is selected from OH, NH₂, NHCOR₉, and SH

R₁, R₂, R₃, R₄, R₅, R₆, R₈ are each hydrogen, alkyl, aryl R₇ is alkyl, or aryl, and

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

28. A compound as claimed in claim 27 characterized in that -X-Y- is -S-S-.

29. A compound as claimed in claim 28 characterized in that R₁, R₂, R₃, R_4, R₅, R₆, and R₈ are each hydrogen or lower alkyl and R₇ is lower alkyl.

30. A compound as claimed in claim 29 characterized in that the lower alkyl groups are methyl groups.

31. A novel antibacterial compound as claimed in claim 18 characterized by the formula:

where

X is selected from S, O, CH₂, NH, NR₆, and Se

Y is selected from N, CH, and CR₇

Z is OH, NH₂, SH, or NHCOR₉ (when Y=N)

Z is R₁₀ (when Y=CH, or CR₇)

R₁=R₂=R₃=R₄=R₅=R₆=R₇= are each hydrogen, alkyl, or aryl, and

R₉ is a β-lactam active side chain

R₁₀ is R11

where R₁₁ is alkyl, or aryl, and

R₁₂ is OH, NH₂, NHCOR₉, or SH

and pharmaceutically acceptable salts thereof.

32. A compound as claimed in claim 31 characterized in that X is S.

33. A compound as claimed in claim 32 characterized in that R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are hydrogen or lower alkyl.

34. A compound as claimed in claim 33 characterized in that the lower alkyl groups are methyl groups.

35. A compound as claimed in claim 31 through 34 characterized in that Z is OH and Y is N.

36. A novel antibacterial compound as claimed in claim 18 characterized by the formula: V

where

X is selected from S, O, CH₂, NH, NR₅, and Se

Y is NR₆- Z, and

R₁, R₂, R₃, R₄, R₅, and R₆ are each H, alkyl, or aryl

Z is OH, SH, NH₂, or NHCOR₇

R₉ is a β-lactam active side chain,

and pharmaceutically acceptable salts thereof.

37. A compound as claimed in claim 36 characterized in that Z is NHCOR₃ where R₇ is phenyl or lower alkyl.

38. A compound as claimed in 37 characterized in that R₇ is benzyl.

39. A compound as claimed in claims 36, 37 or 38

characterized in that R₁, R₂, R₃, R₄, R₅, and R₆ are each hydrogen or lower alkyl.

40. A compound as claimed in claims 36, 37 or 38

characterized in that R₁, R₂, R₃, R₄, R₅, and R₆ are each hydrogen.

41. A compound as claimed in claim 36 characterized in that X is S, R₁, R₂, R₃, R₄, and R₆ and Z is NHCO.benzyl.

42. 3-Carboxy-5-Hydroxymethyl-6, 6-Dimethyl- ▲⁴-1, 4-Thiazine, which is a compound as claimed in claim 18.

43. 3-Carboxy-5-(2-Hydroxypropyl)-6,6-Dimethyl-▲⁴-1,4-Thiazine, which is a compound as claimed in claim 18

44. 2-Thia-4-Carboxy-6-(2-Hydroxypropyl)-7,7-Dimethyl- ▲⁵-1,5-Thiazepine, which is a compound as claimed in claim 18.

45. 3-Carboxy-5-Oximino-1,4-Thiazine, which is a

compound as claimed in claim 18.

46. 3D-Carboxy-5-Phenylacetylhydrazil-▲⁴-Thiazine, which is a compound as claimed in claim 18.